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dolma-pes2o-chemistry

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2020-08-13T15:28:18.510Z	2020-08-13T00:00:00.000	221110110	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.nature.com/articles/s41467-020-17862-6.pdf", "pdf_hash": "09060fb073fe545d861ddf3264af691c2fb603ac", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:3", "s2fieldsofstudy": [ "Biology" ], "sha1": "09060fb073fe545d861ddf3264af691c2fb603ac", "year": 2020 }	pes2o/s2orc	ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex 2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)—the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism. T he ability to sense and respond to nutrient abundance is a fundamental requirement for cell survival, and to achieve this, cells have evolved several strategies that link metabolic function to transcriptional adaptation. One such strategy is the coupling of 2-oxoglutarate (2-OG) metabolism to gene transcription, whereby 2-OG, a key component of TCA cycle, can facilitate cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia inducible factor (HIF) response, DNA methylation, and histone modifications 1 . The relevance of 2-OG in modulating the activity of these dioxygenases is exemplified by changes in the relative abundance of cellular 2-OG. An increased 2-OG/succinate ratio promotes embryonic stem cell pluripotency 2 , and antagonises the growth of solid organ tumours 3 through increased hydroxymethylation of DNA (5hmC) and histone demethylation. Conversely, elevated cellular 2-OG can drive its own reduction to L-2-hydroxyglutarate (L-2-HG), which counterintuitively inhibits 2-OG dependent dioxygenases, leading to decreased DNA hydroxymethylation and histone demethylation, activation of the HIF response, altered T cell fate, and haematopoietic cell differentiation [4][5][6][7][8][9] . Consequently, understanding how 2-OG metabolism is regulated has broad biological implications. Central to maintaining cellular 2-OG homeostasis is the 2oxoglutarate dehydrogenase complex (OGDHc, also known as the α-ketoglutarate dehydrogenase complex), the rate-limiting enzyme within the TCA cycle that oxidatively decarboxylates 2oxoglutarate to succinyl-CoA. This evolutionarily conserved enzyme also requires lipoic acid, a redox sensitive cofactor that is synthesised within the mitochondria and conjugated to a single lysine within the OGDHc E2 subunit, dihydrolipoamide Ssuccinyltransferase (DLST) [10][11][12] . The cyclical reduction and oxidation of the two thiols of conjugated lipoic acid (lipoamide to dihydrolipoamide) serves as a redox intermediate, coupling the formation of succinyl CoA to generation of NADH. The importance of DLST and its lipoylation is highlighted by the recent identification of genetic mutations leading to human disease. Patients with germline mutations in lipoic acid synthesis genes develop a severe variant of the neurological condition, Leigh syndrome 13 , and loss of heterozygosity mutations in the OGDHc lead to angiogenic tumours (pheochromocytomas and paragangliomas), similar to other hereditary cancer syndromes activating the HIF pathway 14 . However, how OGDHc function and 2-OG abundance is regulated is unclear. Here, we use the sensitivity of the HIF pathway to 2-OG abundance to gain insights into how 2-OG metabolism is controlled. Using genome-wide CRISPR/Cas9 mutagenesis screens, we identify an uncharacterised protein, αβ-hydrolase domaincontaining 11 (ABHD11), as a mitochondrial enzyme that impairs OGDHc activity when depleted or inhibited. ABHD11 loss leads to the accumulation of 2-OG and formation of L-2-HG, which inhibits 2-OG dependent dioxygenases involved in the HIF response and DNA hydroxymethylation, similarly to genetic disruption of the OGDHc. ABHD11 also associates with the OGDHc and is required for catalytic activity and TCA cycle function. However, ABHD11 does not alter the constituent levels of the OGDHc. Instead, ABHD11 maintains functional lipoylation of the OGDHc, preserving the catalytic activity of DLST. Together, these studies identify a key role for ABHD11 in 2-OG metabolism, and demonstrate that lipoylation provides a previously unappreciated mechanism for mediating an adaptive transcriptional response to changes in OGDHc function. ABHD11 mediates activity of 2-OG dependent dioxygenases. To find genes involved in 2-OG metabolism we utilised the sensitivity of the HIF response to 2-OG availability, and carried out CRISPR/Cas9 mutagenesis screens in human cells using a fluorescent HIF reporter we developed 4,15 . This reporter encodes the consensus HIF responsive element (HRE) in triplicate that drives the expression of GFP fused to the oxygen and 2-OG sensitive region of HIF-1α ( Supplementary Fig. 1a) 4,15 . Therefore, reporter stability is dependent on 2-OG dependent dioxygenase activity of the prolyl hydroxylases (PHDs or EGLNs) 16,17 , which was confirmed with treatment with the PHD inhibitor dimethyloxalylglycine (DMOG), cell permeable 2-OG (dimethyl 2-OG) or incubation in 1% oxygen ( Supplementary Fig. 1b-d) 4,7,15 . Two genome-wide CRISPR sgRNA libraries were used to identify genes that when mutated activated the HIF reporter: the Brunello human genome-wide library (containing 76,441 sgRNA) 18 , and the Toronto genome-wide knockout library (containing 176,500 sgRNA) 19 . HeLa cells stably expressing the HRE-GFP ODD reporter and Cas9 were transduced with each genome-wide library and iteratively sorted for GFP HIGH cells by fluorescence-activated cell sorting (FACS) at day 10 and day 18 (Fig. 1a). SgRNAs enriched by FACS were identified by Illumina HiSeq and compared to a population of mutagenized cells that had not undergone phenotypic selection (Fig. 1a, b) (Supplementary Dataset 1). All screens were conducted in aerobic conditions (21% oxygen), thereby preventing oxygen availability limiting PHD function. Both screens identified genes involved in the canonical pathway for HIF stability (VHL, EGLN1 (PHD2)) and 2-OG metabolism (OGDHc components, lipoic acid synthesis pathway), validating the approach (Fig. 1b, c). Other biological processes that were significantly enriched for sgRNA included intracellular iron metabolism, the mTOR pathway, and transcriptional regulation (Fig. 1b, c). The reliance of the HIF pathway on these processes is well substantiated and in line with our prior studies using gene-trap mutagenesis in haploid cells 4,15 . In addition to these known pathways, we identified an uncharacterised α/β hydrolase, ABHD11, that was highly enriched for sgRNA in both screens (Fig. 1b, c). We next asked if ABHD11 loss resulted in HIF-1α stabilisation through impaired 2-OG dependent dioxygenase activity. PHD function can be readily assessed by measuring HIF-1α prolyl hydroxylation using a HIF prolyl hydroxy-specific antibody. Mixed knockout populations of ABHD11 stabilised HIF-1α in a non-hydroxylated form, similar to the HIF-1α stabilisation with DMOG ( Supplementary Fig. 1g). In contrast, inhibition of the VHL E3 ligase with VH298 20 , which stabilises HIF-1α by preventing ubiquitination and proteasome-mediated degradation showed high levels of hydroxylated HIF-1α ( Supplementary Fig. 1g). To verify that the decreased prolyl hydroxylation was due to impaired PHD activity, we directly measured prolyl hydroxylation of a recombinant HIF-1α protein in control or ABHD11 deficient lysates 4 . Rapid prolyl hydroxylation was observed with a HeLa control lysate but this was markedly reduced in the ABHD11 depleted cells, similarly to loss of OGDHc function 4 ( Supplementary Fig. 1h, i). This PHD inhibition activated a transcriptional HIF response, promoting activation of HIF-1α target genes, VEGF and carbonic anhydrase 9, similarly to loss of VHL or OGDH (Fig. 1h, i). We also explored whether ABHD11 loss altered the activity of other 2-OG dependent dioxygenases involved in transcription. ABHD11 KO cells showed a marked decrease in total DNA 5hydroxymethylcytosine (5hmC) levels, similar to those observed when OGDHc function is impaired 4 (Fig. 1j, Supplementary Fig. 1j), indicating that Ten-eleven translocation (TET) activity was impaired. However, the steady state levels of selected histone marks were not altered by ABHD11 depletion ( Supplementary Fig. 1k). As levels of methylation depend on transferase activity, demethylation and nucleosome turnover, lysine demethylases (KDM) may still be affected by ABHD11 loss. Despite these differences between TET and KDM activity, these studies suggested that ABHD11 loss had broader implications for 2-OG dependent dioxygenase function, aside from PHDs. that ABHD11 may be involved in 2-OG metabolism. Therefore, we first examined the consequences of ABHD11 loss on 2-OG levels and other TCA cycle intermediates. HeLa cells were depleted of ABHD11 and small molecule metabolites traced by incubating cells with uniformly 13 C labelled ([U-13 C 5 ]) glutamine, followed by liquid chromatography mass spectrometry (LC-MS) (Fig. 2a). Cells deficient in OGDH were used as a control to measure perturbations of 2-OG metabolism. ABHD11 depletion resulted in 2-OG accumulation, similarly to OGDH loss (Fig. 2b). This increase in 2-OG was not due to activation of the HIF response, as we previously demonstrated that PHD2 deficiency does not perturb 2-OG levels 4 . 13 C tracing confirmed that ABHD11 depletion impaired OGDHc function, as TCA cycle metabolites downstream of the OGDHc were decreased (succinate, fumarate and malate) (Fig. 2c-e), and cells adapted by showing a shift from oxidative metabolism to reductive carboxylation 4,21 , with a relative decrease in m + 4 and m + 2 citrate, and an increase in m + 5 and m + 3 citrate isotopologues (Fig. 2f). To substantiate that ABHD11 levels altered OGDHc function, we measured OGDHc enzymatic activity in isolated mitochondria, using a colorimetric assay which detects oxidation of exogenous 2-OG with a redox sensitive probe (Fig. 2g). OGDHc activity was decreased in ABHD11 deficient mitochondria, similarly to levels observed with depletion of the OGDH subunit (Fig. 2g). Loss of OGDHc function was not due to HIF stabilisation, as VHL depletion had no effect on OGDHc activity (Fig. 2g). Bioenergetic profiling also showed that ABHD11 depletion impaired oxygen consumption rates (Fig. 2h, i), consistent with a major defect in the TCA cycle and oxidative phosphorylation. Three enzymes are implicated in the formation of L-2-HG from 2-OG: lactate dehydrogenase A (LDHA), malate dehydrogenase 1 and malate dehydrogenase 2 6,8,9 . Reductive carboxylation and an acidic environment potentiate the reduction of 2-OG to L-2-HG and inhibition of LDHA alone is sufficient to prevent L-2-HG formation 4,7,8 . Therefore, to confirm that L-2-HG was responsible for decreased 2-OG dependent dioxygenase activity, we treated cells with sodium oxamate, which inhibits LDHA as well as decreasing 2-OG formation from glutamine 4,7 , or the selective LDHA inhibitor GSK-2837808A, and measured HIF-1α levels by immunoblot (Fig. 2l, m). Both treatments restored HIF-1α turnover in ABHD11 deficient HeLa cells. Together, these experiments confirmed that impaired OGDHc function and L-2-HG accumulation was responsible for the decreased PHD activity and activation of the HIF response. ABHD11 is a mitochondrial hydrolase. ABHD11 is a member of the alpha-beta hydrolase family, which contains 19 known genes, and encodes an α/β hydrolase fold ( Supplementary Fig. 2a), typical of many proteases and lipases 22 . Unlike most alpha-beta hydrolase family members, ABHD11 is predicted to localise to the mitochondria through a classical mitochondrial targeting sequence (Fig. 3a, Supplementary Fig. 2a). Therefore, we used immunofluorescence microscopy to determine whether ABHD11 resided within mitochondria. Endogenous ABHD11 could not be readily detected by immunofluorescence, but exogenously expressed ABHD11 fused to GFP (ABHD11-GFP), which still retained function (see Fig. 4f), colocalised with MitoTracker DeepRed (Fig. 3b, c). We biochemically confirmed ABHD11's endogenous localisation using isolated mitochondria and a Proteinase K protection assay. Cytoskeletal and outer membrane proteins were rapidly lost with the addition of Proteinase K (30 min at 37°C), but ABHD11 levels were unaffected, suggesting localisation inside of the outer membrane (Fig. 3d). Furthermore, ABHD11 was still retained in mitoplasts, irrespective of proteinase K treatment, consistent with its localisation to the mitochondrial matrix (Fig. 3d). As the stable isotope tracing demonstrated that ABHD11 loss altered OGDHc activity, we determined if ABHD11 associated with components of the complex. Both OGDH and DLST immunoprecipitated with HA conjugated ABHD11 (Fig. 3e), and ABHD11-GFP colocalised with the OGDH (Fig. 3f). We also subjected immunoprecipitated ABHD11-HA to mass spectrometry, which confirmed the association with OGDH and DLST (Supplementary Table 1). Furthermore, our findings were consistent with a prior unbiased mass spectrometry analysis of interactions between mitochondrial proteins, which identified that ABHD11 associated with OGDH with high confidence 23 . We next examined if ABHD11 enzymatic activity was required for its effect on HIF-1α stability. Structural modelling of ABHD11 predicted a typical α/β hydrolase fold with two catalytic motifs (Fig. 3a, g). Hydrolase activity is predicted to arise from the serine nucleophile motif (GXSXG), but ABHD11 also encodes a putative Fig. 1 Identification of ABHD11 as a mediator of 2-OG dependent dioxygenase activity. a HeLa HRE-GFP ODD cells were transduced and mutagenised with genome-wide sgRNA libraries (Brunello and Toronto KO). GFP HIGH cells were selected by iterative FACS and sgRNA identified by Illumina HiSeq. b, c Comparative bubble plot (b) and table (c) of sgRNA enriched in the GFP HIGH cells between the two genome-wide sgRNA libraries compared to a mutagenised population of HRE-GFP ODD cells that had not been phenotypically selected. Genes enriched for sgRNA clustered into six main groups: (1) the canonical HIF pathway, (2) OGDHc, Lipoylation (Lp) and 2-HG related pathways, (3) intracellular iron metabolism (iron, lysosomal), (4) mTOR, (5) Transcription and (6) Uncharacterised. Unadjusted p value calculated using MaGECK robust rank aggregation (RRA); FDR = Benjamini-Hochberg false discovery rate (multiple hypothesis adjustment of RRA p value). EGLN1 = PHD2, EGLN3 = PHD3. d-f HeLa HRE-GFP ODD (d), MCF-7 (e) and Hep G2 (f) cells stably expressing Cas9 were transduced with up to three different sgRNA targeting ABHD11. Reporter GFP or endogenous HIF-1α levels were measured by flow cytometry (d) or immunoblot (e, f) respectively after 10-13 days. Endogenous ABHD11 levels were measured by immunoblot and β-actin served as a loading control. g Reconstitution of mixed KO population of ABHD11 with exogenous ABHD11. HeLa cells expressing Cas9 were transduced with sgRNA targeting ABHD11 as described. Targeted cells were also transduced with exogenous ABHD11 with the PAM site mutated. Cells depleted of PHD2 served as a control for ABHD11 reconstitution. h-i Quantitative PCR (qPCR) of the HIF-1α target genes (VEGF and CAIX) in HeLa cells following ABHD11 depletion by sgRNA (n = 3, SEM, p < 0.028, p < 0.0065, two-tailed one-sample t test of ratio). sgRNA targeting OGDH and VHL were used as control for HIF-1α activation. j Genomic DNA was extracted from Hela control or mixed KO populations of ABHD11, LIAS or VHL, and 5hmC levels measured by immunoblot relative to total DNA content ( Supplementary Fig. 1j). 5hmC levels were quantified using ImageJ. n = 3, Mean ± SEM p = 0.010; ns:p = 0.39, VHL compared to control; two-tailed t test (not adjusted for multiple comparison). Ct = control. To confirm that these mutations were altering ABHD11 enzymatic activity, we purified wildtype and S141A ABHD11 and measured hydrolysis of p-nitrophenyl ester, a substrate validated for generic α/β hydrolase activity 22,24 . Wildtype ABHD11 protein and the S141A mutant were isolated by Fig. 4). ABHD11 predominantly migrated as a single species but a slower migrating form was apparent in the cell extract and purified protein, consistent with an immature form prior to mitochondrial insertion ( Supplementary Fig. 4a). Mass spectrometry analysis confirmed ABHD11's identity and demonstrated that the mitochondrial targeting sequence was lost in the predominantly expressed form (Supplementary Fig. 4b) (the slower migrating species was of too low abundance). Size exclusion chromatography identified two peaks but full length ABHD11 was only detected in the second peak, at an elution volume consistent with a monomeric species ( Supplementary Fig. 4c, d). Hydrolysis of the p-nitrophenyl ester confirmed ABHD11 enzymatic activity, but this was lost with the S141A mutant and following heat treatment (Fig. 3i). Thus, ABHD11 is a mitochondrial hydrolase that associates with the OGDHc, and loss of its enzymatic activity leads to HIF-1α accumulation. ABHD11 loss impairs lipoylation of the OGDHc. Conversion of 2-OG to succinyl-CoA by the OGDHc requires decarboxylation and the formation of succinyl intermediate (succinyl-dihydrolipoate), dependent on the cyclical reduction and oxidation of the lipoylated DLST subunit (Fig. 4a). Therefore, to understand how ABHD11 is required for OGDHc function, we first examined whether protein levels of core OGDHc components or its lipoylation were altered. ABHD11 depletion did not alter total levels of the OGDHc subunits (OGDH, DLST or DLD) in HeLa cells ( Fig. 4a, b). However, using a specific anti-lipoate antibody that detects conjugated lipoamide, we observed a reproducible loss of the faster migrating lipoylated protein species, attributed to the lipoylated DLST subunit of the OGDHc (Fig. 4b, c). Immunoprecipitation of endogenous DLST confirmed loss of lipoylation following ABHD11 depletion, without altering total DLST levels ( Supplementary Fig. 5a), and this decreased DLST lipoylation was observed in several cell types (Fig. 4d, e). Furthermore, in contrast to complete disruption of lipoic acid synthesis by LIAS depletion, ABHD11 loss preferentially decreased DLST lipoylation, without altering the other abundantly lipoylated protein within the mitochondria, the DLAT (dihydrolipoamide acetyltransferase) subunit of the pyruvate dehydrogenase complex (PDHc) ( Fig. 4b-e, Supplementary Fig. 5b). Indeed, PDHc function, as measured by [U-13 C 6 ] glucose stable isotope tracing, was not impaired in the ABHD11 deficient HeLa cells (Supplementary Fig. 6a-g), and lactate production was not increased compared to control HeLa cells ( Supplementary Fig. 6h). Complementation studies were used to determine whether the enzymatic activity of ABHD11 was required for lipoylation of the OGDHc. Exogenous wildtype or mutant ABHD11 were expressed in mixed ABHD11 KO populations and lipoylation levels measured by immunoblot. DLST lipoylation was restored with the wildtype ABHD11 but not with the S141A or H296A mutants (Fig. 4f). HIF-1α levels were only reduced to basal levels by reconstituting with wildtype ABHD11 but not the nucleophile mutants ( Fig. 4f), as previously shown. ABHD11 maintains functional lipoylation of DLST. The finding that ABHD11 loss showed a selective loss of DLST lipoylation was unexpected, as prior genetic studies of lipoate conjugation had not shown a requirement for an additional enzyme 26 . Furthermore, we confirmed that ABHD11 loss differed to depletion of other components of the lipoic acid synthesis pathway by generating CRISPR/Cas9 mixed KO populations of the key enzymes involved ( Supplementary Fig. 7a, b). Lipoyl(octanoyl) transferase 2 (LIPT2), LIAS, and lipoyltransferase 1 (LIPT1) all reduced DLAT and DLST lipoylation in HeLa cells to a similar level, but only ABHD11 showed a selective loss of DLST lipoylation ( Supplementary Fig. 7b). This preferential decrease in DLST lipoylation following ABHD11 loss argued against a general role for ABHD11 in lipoyl synthesis, and while it remained possible that ABHD11 was required for the final catalysis of DLST lipoylation, prior genetic studies suggested that LIPT1 was sufficient for this step [26][27][28] . Rather than acting as a conjugating enzyme, we hypothesized that ABHD11 may directly or indirectly be involved in maintaining a functional lipoate moiety on the OGDHc complex. We observed that ABHD11 loss in HeLa cells led to decreased cell viability after prolonged passage for three weeks. However, it was unlikely that general growth inhibition was responsible for the lipoylation phenotype as ML226 treatment, which efficiently inhibited ABHD11, did not alter cell growth ( Supplementary Fig. 8a). We also examined whether ABHD11 activity altered the mitochondrial redox environment, which could influence the reduction and oxidation of lipoylated DLST. Stable isotope tracing showed no overall change in cellular glutathione (GSH) Fig. 2 ABHD11 is required for OGDHc function. a Schematic of the TCA cycle (oxidative metabolism) and reductive carboxylation (reductive metabolism), illustrating the fate of 13 C carbons upon incubation with [U-13 C 5 ]-glutamine. b-g Stable isotope tracing of control HeLa cells compared to mixed CRISPR KO populations (sgRNA) of ABHD11 or OGDH incubated with [U-13 C 5 ] glutamine. 2-oxoglutarate (b), succinate (c), fumarate (d), malate (e) and citrate (f), divided by metabolite isotopologues (m + 0 to m + 5) are indicated. Two biologically independent replicates, n = 5 technical replicates per sample, mean ± SD (g) OGDHc activity in isolated mitochondria. Mitochondria were extracted from control or mixed CRISPR KO populations of ABHD11, OGDH or VHL HeLa cells and OGDHc activity measured by a redox sensitive colorimetric probe for 2-OG oxidation. n = 3 biologically independent samples, mean ± SEM, p = 0.0082, two-tailed t test. h Bioenergetic assays of oxygen consumption rates (OCR) in control, ABHD11 deficient or OGDH deficient HeLa cells (mixed KO populations). ABHD11 and OGDH were depleted as described, and analysed by using a Seahorse XF e 24 Extracellular Flux Analyzer (n = 4 technical replicates per sample, mean ± SD). Three basal measurements were made at 9 min intervals followed by three measurements per treatment (1 μM oligomycin, 1 μM FCCP and 1 μM antimycin/rotenone). OCR was normalised to total cell number. i Comparison technical repeats at first basal measurement from (h); *p = 6.3 × 10 −5 , two-tailed t test. j Measurement of 2-hydroxyglutarate (2-HG) levels following [U-13 C 5 ] glutamine stable isotope tracing in control HeLa cells compared to mixed CRISPR KO populations (sgRNA) of ABHD11 or OGDH. Metabolite isotopologues (m + 0 to m + 5) are indicated. Two biologically independent samples are shown; n = 5 technical replicates per sample, mean ± SD. k Relative quantification of 2-HG enantiomers upon derivatisation with diacetyl-L-tartaric anhydride and LC-MS analysis. l, m Inhibition of lactate dehydrogenase A (LDHA) in ABHD11 deficient HeLa cells. Mixed CRISPR KO ABHD11, VHL or PHD2 cells were treated with sodium oxamate (Ox) (l) or GSK-2837808A (m) as indicated for 24 h. HIF-1α levels were measured by immunoblot. levels ( Supplementary Fig. 8b). Small changes in mitochondrial ROS were observed with ABHD11 loss, using MitoSOX Red, similarly to OGDH or LIAS depletion ( Supplementary Fig. 8c, d). However, ML226 treatment, showed no change MitoSOX Red levels, and importantly, Antimycin A, which increased mitochondrial ROS to higher levels than ABHD11 inhibition, was not sufficient to activate the HIF reporter ( Supplementary Fig. 8e). Thus, alterations in mitochondrial ROS were unlikely to account for the HIF stabilisation or altered lipoylation following ABHD11 loss or inactivation of the OGDHc. To explore further how ABHD11 activity altered DLST lipoylation we used mass spectrometry analysis of the lipoate ARTICLE moiety. Immunoprecipitated DLST was treated with a reducing agent and then incubated with N-ethylmaleimide (NEM), forming an NEM-lipoyl conjugate, which had previously been shown to aid detection of the lipoate moiety 29 (Fig. 5a, Supplementary Fig. 9a). Interestingly, NEM treatment prevented detection of immunoprecipitated lipoylated DLST by immunoblot ( Supplementary Fig. 9a), demonstrating that the anti-lipoate antibody only detected the functional lipoate and not the NEMmodified form, suggesting that the apparent loss of DLST lipoylation in ABHD11 deficient cells may be due to modification of the lipoate moiety. We next measured levels of DLST lipoylation (NEM-lipoyl) by label-free quantification on immunoprecipitated DLST from wildtype HeLa cells or those deficient in LIAS or ABHD11 (Fig. 5a, b, Supplementary Fig. 9b). To account for potential differences in DLST protein abundance around the lipoylated region (DKTSVQVPSPA), we normalised these peptides to the sum of all DLST peptide label-free quantification values. Approximately 50% of the DKTSVQVPSPA DLST peptide in wildtype cells was modified with lipoate compared to the unmodified form and as expected, nearly all the lipoate detected was modified with NEM (Fig. 5b). DLST lipoylation was nearly completely lost in the LIAS deficient cells, with the majority of the DLST lipoylated peptide region found to be unmodified (Fig. 5b), confirming that this approach could readily identify a defect in lipoyl synthesis and conjugation. However, ABHD11 deficiency did not result in an accumulation of the unmodified DKTSVQVPSPA peptide, which could have been expected with a defect in conjugation. Instead, both the unconjugated or NEM-lipoyl DKTSVQVPSPA peptide were barely detectable, with a 10-fold decrease in abundance compared to the control or LIAS null cells (Fig. 5b). This decrease was not due to less total DLST, as DKTSVQVPSPA levels were normalised to other DLST peptides upstream or downstream of the lipoylated region. Therefore, a modification of the DKTSVQVPSPA peptide of undefined mass accounted for the apparent decrease in peptide abundance. Common posttranslational modifications (e.g., ubiquitination, phosphorylation or acetylation), combinations of modifications, or known DLST intermediates (e.g., succinyl-dihydrolipoamide, acyldihydrolipoamide or S-glutathionylation) (Supplementary Dataset 2) did not account for the peptide loss of the lipoylated DLST region, suggesting the formation of lipoyl adducts that were not detectable by mass spectrometry. The thiols within the lipoamide moiety are sensitive to attack by lipid peroxidation products, which disrupt OGDHc catalysis by preventing the cyclical oxidation and reduction of the lipoyl conjugate 30,31 (Fig. 5c). We measured whether common lipid peroxidation products formed in cells (4-hydroxy-2-nonenal (4-HNE) or 4-oxononenal (4-ONE)) 30-32 modified the lipoate moiety on immunoprecipitated DLST, but these modifications did not account for the unassigned mass of the DLST peptide (Supplementary Dataset 2). However, the complex nature of lipid based adducts of undefined and variable lengths may preclude their detection. While the exact nature of the lipoyl adduct formed in ABHD11 deficient cells was unclear, we examined whether exogenous treatment with 4-HNE could alter DLST lipoylation, similarly to ABHD11 depletion. 4-HNE treatment of cell lysates preferentially decreased detection of DLST lipoylation by immunoblot (Fig. 5d), consistent with the formation of lipoyl adducts preventing binding to the antibody. DLAT lipoylation was only affected at high concentrations (5 mM) of 4-HNE (Fig. 5d), suggesting that DLAT may be more resistant to lipoyl adduct formation than DLST, and consistent with our findings that ABHD11 loss preferentially effects the OGDHc. Finally, to explore whether ABHD11 protected against to the formation lipoyl adducts, such as those formed by 4-HNE, we measured if ABHD11 loss or inhibition made the OGDHc more susceptible to lipid peroxidation damage. Control or ABHD11 depleted HeLa cells or lysates were treated with 4-HNE, and lipoylation detected by immunoblot. 4-HNE decreased the detection of DLST preferentially to DLAT within cell lysates (Fig. 5d), consistent with lipoyl adducts preventing detection of the lipoyl moiety by immunoblot. 4-HNE treatment of cells also decreased DLST functional lipoylation preferentially to DLAT, and ABHD11 deficient cells were more susceptible to 4-HNE treatment compared to the control cells (Fig. 5e). Overexpression of ABHD11 inactive mutants competed with endogenous ABHD11 to also show an increase lipoyl-adduct formation following 4-HNE treatment (Supplementary Fig. 9c). ABHD11 overexpression did not increase DLST lipoylation compared to control cells (Supplementary Fig. 9c) but this finding is consistent with exogenous ABHD11 not increasing total lipoylation levels and reflect that OGDHc lipoylation is tightly regulated, with only 50% of DLST modified by lipoylation. While these studies demonstrated that 4-HNE could disrupt functional lipoylation, we were concerned that the concentrations required were higher than prior reports 33,34 , and considered that this may be due to the presence of L-cysteine within media. Therefore, we repeated these assays using lower concentrations of 4-HNE in media without Lcysteine (Fig. 5f). We now observed impaired DLST lipoylation with low concentrations of 4-HNE (40 µM) in control HeLa cells, Fig. 3 ABHD11 is a serine hydrolase that associates with the OGDHc. a Schematic of ABHD11 with the putative mitochondrial targeting sequence (MTS), mitochondrial processing peptidase (MPP) cleavage site and catalytic residues indicated (Serine 141, and Histidine 296). Modelled from UniProtKB-Q8NFV4, and MitoFates prediction tool 55 . b Confocal micrograph of HeLa cells lentivirally transduced with ABHD11-GFP. Mitochondria were visualised with MitoTracker Deep Red FM (MitoDeepRed); Scale = 10 μm, representative example from two biologically independent experiments and 14 images (c) Pearson correlation coefficient (Pearson r) comparing colocalisation of GFP and MitoTracker in HeLa cells expressing ABHD11-GFP (n = 14). HeLa cells expressing GFP under an SFFV promoter without a localisation signal served as a cytosolic control (pSFFV-GFP; n = 14) p = 7.0 × 10 −6 , two-tailed Mann-Whitney U test. d Mitochondrial protease protection assay. Mitochondria were extracted using the Qproteome Mitochondria Isolation Kit (Qiagen). Proteinase K was added to the final concentrations indicated, and incubation at 37°C for 30 min. e Immunoprecipitation of ABHD11-HA with endogenous OGDHc components. ABHD11-HA or the inactive mutants (S141A and H296A) were transduced into HeLa cells, lysed and immunoprecipitated using the HA tag. TMEM199, a membrane bound protein tagged with HA (TM-HA) was used as a control. f Colocalisation of ABHD11 with the mitochondrial matrix protein, OGDH. HeLa cells expressing ABHD11-GFP were fixed in paraformaldehyde. ABHD11 and OGDH subcellular localisation was visualised by immunofluorescence confocal microscopy. Scale = 10 μm, representative image of five technical repeats. g In silico modelling of ABHD11 with putative catalytic site and key residues S141 and H296 (Phyre2 structural prediction against a template of murine epoxide hydrolase, PDB: 1cr6, and visualised using PyMOL 2.3). h Reconstitution of mixed KO population of ABHD11 with exogenous ABHD11, or enzymatic inactive mutants. i p-nitrophenyl esterase activity of purified ABHD11-FLAG. Purified wildtype or S141A ABHD11-FLAG were incubated with p-nitrophenyl acetate and hydrolysis measured by rate of increase in absorbance at 405 nm (37°C for 40 min). An empty FLAG vector (EV), that had undergone affinity purification, was used as a control. ABHD11 enzymatic activity was also measured following heat inactivation of the protein (90°C for 5 min). n = 4, Mean ± SEM, p = 0.0006, two-tailed t test. with complete loss of DLST in ABHD11 deficient cells at 20 µM 4-HNE (Fig. 5f). Furthermore, while 4-HNE preferentially altered DLST, DLAT lipoylation was also decreased in the ABHD11 null cells compared to the controls (Fig. 5f). Similar findings were observed with ABHD11 inhibition, consistent with a requirement for ABHD11 to maintain functional lipoylation in the context of lipid peroxidation products. In conclusion, while the nature of adducts formed on lipoylated DLST remain to be fully determined, these studies demonstrate that ABHD11 is required for functional lipoylation of the OGDHc, and may protect against the formation of lipoyl adducts, such as those formed by 4-HNE (Fig. 6). Discussion This study identifies ABHD11 as a mitochondrial enzyme required for OGDHc function, and to our knowledge, is the first example of a mitochondrial pathway that maintains TCA cycle integrity by preserving functional OGDHc lipoylation (Fig. 6). Moreover, we demonstrate that ABHD11 inhibition allows 2-OG metabolism to be modulated in multiple cells and in a reversible manner, with potential broad implications for altering cell fatedecisions and manipulating 2-OG abundance in tumours. The selective loss of lipoylated DLST following ABHD11 depletion initially suggested that it may be necessary for OGDHc lipoate conjugation. However, a requirement for ABHD11 in lipoate synthesis had not been previously observed 13,26,35 , and LIPT1 deletion or human loss of function mutations prevent PDHc and OGDHc lipoylation [26][27][28] . It was possible that ABHD11 transfers lipoate moieties between 2-oxoacid dehydrogenases, but our mass spectrometry findings argued against this. If ABHD11 was required for lipoate transfer, the unmodified DLST peptide should accumulate in ABHD11 depleted cells. Instead, we found an absence of both the modified and unmodified lipoylated region of DLST by mass spectrometry (Fig. 5b, c). Similar coverage of DLST peptides upstream and downstream of the lipoylated region confirmed that there was no change in total DLST levels following ABHD11 loss. Therefore, a peptide of undefined mass must account for the apparent loss of this region, indicating a posttranslational modification other than lipoylation or the formation of a lipoyl adduct. Common post-translational modifications (e.g. ubiquitination, phosphorylation or acetylation), combinations of modifications, or known DLST intermediates (e.g. succinyl-dihydrolipoamide, acyl-dihydrolipoamide or S-glutathionylation) (Supplementary Dataset 2) did not account for the peptide loss of the lipoylated DLST region, suggesting that this DLST peptide was not uniformly modified. Lipid peroxidation products (hydroxyalkenals, such as 4-HNE), arise from free radical propagation through phospholipids 31 , and can easily react with thiol groups, inactivating 2-oxoacid dehydrogenases by forming lipoyl adducts 30 . We did not observe 4-HNE lipoyl adducts on immunoprecipitated DLST, but the hydrophobicity and complex nature of these adducts 32 may preclude their detection by mass spectrometry, accounting for the apparent loss of the DLST DKTSVQVPSPA peptide that we observed (Fig. 5b). We also required high concentrations of 4-HNE to decrease detectable lipoylation in immunoprecipitated DLST, and 0.1 mM 4-HNE in cells incubated in serum-free media. High levels or prolonged treatment of 4HNE may lead to depletion of cellular antioxidant levels and apoptosis 36,37 , but we did not observe changes in DLAT lipoylation at 0.1 mM consistent with a selective effect on DLST, as previously observed 38 . Furthermore, treatment of HeLa cells with 4-HNE in media without L-cysteine resulted in selective loss of DLST lipoylation at lower, biologically relevant 4-HNE concentrations 33,34 . The apparent specificity of ABHD11 for DLST may relate to the preponderance of lipoyl adducts formed by the OGDHc compared to other lipoylated proteins, or selectively binding to the OGDHc, rather than all 2-oxoacid dehydrogenases. It is possible that ABHD11 may regulate DLST indirectly, but this is unlikely to be due to altered mitochondrial ROS ( Supplementary Fig. 8). The association with DLST and OGDH (Fig. 3e), and marked accumulation of 2-OG (Fig. 2) are also consistent with a direct role on OGDHc function. These findings are also supported by prior mass spectrometry interactome studies, showing an association of ABHD11 with OGDH 23 . Whether ABHD11 interacts with other 2-oxoacid dehydrogenases will be of interest to explore further. We observed that ABHD11 can interact with DLAT by mass spectrometry (Supplementary Table 1), and a small decrease in DLAT lipoylation following ABHD11 loss was observed. ABHD11 deficient cells were also more susceptible to impaired lipoylation following 4-HNE treatment. However, ABHD11 loss did not increase pyruvate levels in HeLa cells ( Supplementary Fig. 5), which would be expected if PDHc activity was significantly impaired. ABHD11 is one of a family of alpha-beta hydrolase domaincontaining enzymes, which as a group are poorly characterised. Of these, only ABHD10 and ABHD11 are known to be mitochondrial 39 . ABHD10 is has recently been shown to be an acyl protein thioesterase, with S-depalmitoylase activity against the anti-oxidant protein peroxiredoxin-5 40 . These findings would be consistent with our observations for ABHD11 regulating the thiol-containing lipoate moiety on DLST. However, ABHD11 has less sequence identity with ABHD10 than other ABHD family members ( Supplementary Fig. 2), and is not inhibited by ML226 25 . This work provides insights into the functional role of ABHD11, for which no physiological substrate or role has been identified previously. ABHD11 is one of~26 genes included in the 7q11.23 hemizygous deletion of Williams-Beuren syndrome, a rare multisystem disorder often characterised by developmental and cardiac abnormalities 41 . While the phenotype of cardiac and soft tissue disease is felt largely due to loss of Tropoelastin 1 (ELN1) 41 , a functional understanding of ABHD11 may offer some insights into aspects of this syndrome. It will also be of interest to explore further the biological consequences of ABHD11 loss compared to other lipoate enzymes. ABHD11 loss disrupts the TCA cycle, impairing oxidative phosphorylation and promoting reductive metabolism, but ABHD11 has a distinct metabolic phenotype compared to loss of other lipoic acid pathway enzymes. Germline mutations in LIAS, LIPT2 and LIPT1 result in impaired PDHc and OGDHc activity 13,27,42,43 , Fig. 4 ABHD11 loss impairs lipoylation of the OGDHc. a OGDHc compromises 3 subunits: OGDH (E1) catalyses the oxidation of 2-OG to form the succinyl moiety, releasing carbon dioxide (CO 2 ) and reducing the lipoate to form a succinyl-dihydrolipoate intermediate. DLST (E2) catalyses transfer of the succinyl moiety to Coenzyme A (CoA), forming succinyl CoA and releasing dihydrolipoylated DLST. Dihydrolipoate is oxidised by DLD (E3) to reform lipoate, with the free electrons reducing NAD + to NADH. Thus 2-OG oxidation is coupled to cyclical lipoate reduction/oxidation and NADH formation. b, c Immunoblot and quantification of OGDHc subunits and lipoylation in ABHD11 or LIAS deficient cells. HeLa cells were transduced with sgRNA targeting ABHD11 or LIAS to generate mixed KO populations, and probed for OGDHc components (b). Lipoate (Lp) antibody detects lipoylated proteins. The predominant lipoylated proteins, DLAT and DLST, are indicated. ImageJ quantification of lipoyl-DLST (left) and DLST (right), n = 5 biologically independent samples (c). Mean ± SEM, ***p = 0.0004, ns:p = 0.19, two-tailed one sample t test. Ct = control (d, e) Immunoblot OGDHc subunits and lipoylation in ABHD11 or LIAS deficient THP-1 or MCF-7 cells. THP-1 (d) or MCF-7 (e) cells were transduced with sgRNA targeting ABHD11 or LIAS to generate mixed KO populations, and probed for OGDHc components or lipoate. β-actin served as a loading control. f Reconstitution of mixed KO population of ABHD11 with exogenous ABHD11-GFP, or enzymatic inactive mutants. g ML226 treatment in vitro. Purified wildtype ABHD11-FLAG was incubated with p-nitrophenyl acetate and hydrolysis measured by rate of increase in absorbance at 405 nm (37°C for 30 min), with addition of ML226 at the indicated concentration. Hydrolysis activity was subtracted from a background control without ABHD11-FLAG, and is normalised to the activity of ABHD11-FLAG with vehicle control. ϕ = vehicle control; n = 3. h, i ML266 treatment in HeLa cells (h) and C2C12 myoblasts (i). Cells were treated for 24 h with ML226 at the indicated concentrations and lipoylation measured by immunoblot. j HeLa cells were treated with 1μM ML226, or 0.075% DMSO as a vehicle (veh) control. ML226 was washed out after 24 h and lipoylation recovery measured by immunoblot. but ABHD11 loss did not significantly alter DLAT lipoylation in several cancer lines. PDHc activity is required for lipogenesis, by providing acetyl-CoA, and LIPT1 mutations result in defective lipid synthesis 44 . As ABHD11 acts predominantly on the OGDHc, it is possible that the preserved activity of PDHc fuels lipogenesis, and cell survival, and it will be important to determine whether ABHD11 activity, and the resulting HIF activation, feeds back on lipid synthesis and the mitochondrial fatty acid pathway. This study and prior reports demonstrate that human mutations in lipoylation enzymes increase 2-OG levels and promote L-2-HG formation 4,42 . However, how L-2-HG inhibits 2-OG sensitive enzymes when 2-OG is abundant remains to be fully resolved. It is possible that L-2-HG may allosterically inhibit the enzymes, or that additional factors aside from L-2-HG result in decreased dioxygenase activity. The reasons for the propensity to form L-2-HG under certain conditions of 2-OG accumulation also remains to be determined, as in other cellular responses, such as embryonic stem cell pluripotency 2 , 2-OG treatment does not result in inhibition of 2-OG dependent dioxygenases. These discrepancies may relate to the reductive environment that occurs when OGDHc activity is impaired (which is likely to occur with Lipoic acid has been traditionally described as an essential cofactor for 2-oxoacid dehydrogenases, but only 50% of DLST in HeLa cells was observed to be lipoylated in resting cells (Fig. 5b), and OGDHc lipoylation was rapidly stored after washout of ML226, suggesting a reserve capacity to alter lipoate levels and increase OGDHc activity (Fig. 4j). These findings show that lipoylation is a dynamic modification that must be maintained, which is further supported by recent observations that SIRT4 act as a lipoamidase, altering PDHc function 29 , and that increased lipoylation can enhance brown adipose tissue function, decreasing age-associated obesity 45 . Therefore, modulating lipoylation through ABHD11 activity provides an attractive approach to manipulating 2-OG metabolism. Moreover, these studies extend the role of lipoylation beyond an enzymatic cofactor, to a dynamic modification that couples the mitochondria to a transcriptional adaptive response mediated by 2-OG and oxygen sensitive enzymes. Full details of reagents and antibodies used are shown in Supplementary Table 2. Constructs. CRISPR sgRNAs were cloned into a lentiviral sgRNA expression vector pKLV-U6gRNA(BbsI)-PGKpuro2ABFP 46 . All sgRNA sequences are detailed in Supplementary Table 3. ABHD11 constructs were generated from the I.M.A.G.E. cDNA clone (IRATp970F0688D, Source Bioscience), cloned into the pHRSIN pSFFV backbone with pGK-blasticidin resistance (a gift from Paul Lehner), using NEBuilder HiFi (NEB). Prior to assembly, silent mutations were introduced inside the sequence targeted by ABHD11 sgRNA 2, using PCR primers detailed in Supplementary Table 3. Mutations of catalytic residues serine 141 to alanine (S141A) and histidine 296 to alanine (H296A) of ABHD11 was created using NEBuilder HiFi, with primers detailed in Supplementary Table 3. Lentiviral expression vectors (pHRSIN) for ABHD11, S141A ABHD11 and H296A ABHD11 with C-terminal eGFP tags or HA tags were created using NEBuilder HiFi (NEB). ABHD11 was also cloned into a transfection vector, pCEFL 3xFLAG mCherry vector, encoding a C-terminal 3X FLAG tag and mCherry under a separate promoter (a gift from David Ron) 47 , using Gibson Assembly (NEB) and NEBuilder HiFi. 250 µl of virus with 5 × 10 4 cells in a 24-well plate made up to 1 ml media. For the screens, a titration of increasing volumes of virus was used, with 10 6 HeLa cells in a 6-well plate. Cell plates were centrifuged for 1 h at 37°C at 750 × g immediately after addition of virus. Whole-genome CRISPR/Cas9 forward genetic screens. HeLa HRE-GFP ODD cells were transduced with Streptococcus pyogenes Cas9 (pHRSIN-FLAG-NLS-CAS9-NLS-pGK-Hygro) 49 and selected for Cas9 expression using hygromycin. 5 × 10 7 (Brunello) or 10 8 (TKO) HeLa HRE-GFP ODD cells were transduced with the appropriate volume of pooled sgRNA virus (multiplicity of infection (MOI) of~0.3), maintaining at least 150-fold sgRNA coverage. After 30 h, cells were treated with puromycin 1 µg/ml for 5 days. Representation was maintained throughout the screen such that no selection event occurred where the library was cultured at fewer than 200 times the number of sgRNA sequences in the library. The library was pooled immediately before any selection event. FACS was performed by harvesting 10 8 cells, washing the cells in PBS, and then resuspending them in PBS containing 2% foetal calf serum and 10 mM HEPES (Sigma H0887). Cells were sorted using an Influx cell sorter (BD); GFP-high cells were chosen in a gate set at one log 10 unit above the mode of the untreated population. Genomic DNA was extracted using a Gentra Puregene Core kit (Qiagen). Lentiviral sgRNA inserts were amplified in a two-step PCR (with Illumina adapters added on the second PCR), as previously described 49 . For the TKO screen, the forward inner PCR and sequencing primers were modified (Supplemental Table 3). Sequencing analysis was performed by first extracting the raw sequencing reads, trimming the first 20 bp (FASTX-toolkit), and aligning against the appropriate sgRNA library using Bowtie 50 . Read counts for each sgRNA were compared between conditions, and Benjamini-Hochberg false discovery rates for each gene calculated, using MAGeCK 51 (Supplementary Dataset 1). The analysis presented compares DNA extracted following the second sort to an unsorted DNA library taken at the same timepoint. Immunoblotting and immunoprecipitation. Cells were lysed in an SDS lysis buffer containing 2% SDS, 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol and 1:200 Benzonase nuclease (Sigma), for 15 min at room temperature, then heated at 90°C for 5 min. Proteins were separated with SDS-PAGE electrophoresis, transferred to a PVDF membrane, and probed using appropriate primary antibodies and a secondary with HRP conjugate. Densitometry measurements were made using ImageJ 52 . To identify protein interactions with ABHD11, HeLa cells lentivirally transduced with ABHD11 with a C-terminal HA tag were lysed in a buffer containing 100 mM Tris pH 8.0, 140 mM NaCl, 1% IGEPAL CA-630 (Sigma), 1 mM PMSF (Sigma P7626) and cOmplete Protease Inhibitor Cocktail (Roche). After centrifugation at 17,000 × g, the supernatant was pre-cleared using Sepharose CL-4B (GE Heathcare) and then incubated with EZView HA Red Anti-HA Affinity Gel (Sigma E6779) overnight on a rotator. Resins were washed with Tris-buffered saline containing 0.1% IGEPAL CA-630, and a further two washes with Trisbuffered saline. Proteins were eluted using an SDS lysis buffer (4% SDS, 100 mM Tris pH 7.4, 300 mM NaCl, 2 mM dithiothreitol, and 20% glycerol) heated at 90°C for 5 min, and separated using SDS-PAGE. Confocal microscopy. Mitochondrial labelling was performed using MitoTracker Deep Red FM (Thermo M22426). Cells were cultured overnight on a 1 cm glass coverslip, incubated with 250 nM Mitotracker Deep Red FM for 40 min, and after washing with PBS fixed with 4% paraformaldehyde for 20 min. Cells were mounted to slides using ProLong Gold Antifade Mountant with DAPI (Thermo). Quantitative PCR. Total RNA was extracted using the RNeasy Plus minikit (Qiagen) and reverse transcribed using Super RT reverse transcriptase (HT Biotechnology Ltd). PCR was performed on the ABI 7900HT Real-Time PCR system (Applied Biosystems; software: Quantstudio 1.3) using SYBR Green Master mix (Applied Biosystems). Reactions were performed with 125 ng of template cDNA. Transcript levels of genes were normalised to GAPDH. Measurements of 2-OG dependent dioxygenase activity. The in vitro HIF-1α prolyl hydroxylation activity of HeLa cell lysates against a His-tagged protein corresponding to residues 530-652 of human HIF-1α protein 4,15 was performed by incubating 10 μM HIF-1α 530-652 with 50 μl HeLa cell extracts in 20 mM HEPES (pH 7.5, 150 mM NaCl and 1 mM DTT) for 15 min at 37°C. Reactions were terminated by the addition of SDS loading buffer, and proteins visualised by SDS-PAGE. Images were quantified using ImageJ, and are presented as the ratio of densitometry of hydroxylated to total HIF-1α ODD peptide at the 15 min timepoint, normalised to PHD2. For the 5hmC dot blot assay, genomic DNA was extracted from HeLa cells using a Gentra Puregene kit (Qiagen), and dot blotting for 5hmC levels performed by serial dilutions of denatured genomic DNA on Hybond NX membranes 4 . Following UV crosslinking the membranes were blocked with 1% bovine serum albumin and 5% milk powder prior to probing with a rabbit polyclonal antibody to 5hmC (Active Motif). Total DNA levels were evaluated by methylene blue staining, and relative densitometry measured using ImageJ. For the KDM panel, cells were lysed in SDS lysis buffer and probed for selected H3 methylation marks. Mitochondrial protease protection assay. Mitochondria from 10 7 HeLa cells were extracted using a Qproteome Mitochondria Isolation Kit (Qiagen). After the final wash with mitochondria storage buffer, mitochondria were divided into tubes, pelleted by centrifugation at 6000 × g and resuspended in either 10 mM Tris-HCl pH 8.0 with 250 mM sucrose (for whole mitochondria), or 10 mM Tris-HCl pH 8.0 (for mitoplasts), to a final protein concentration of 1 mg/ml. Proteinase K (Sigma P2308) was added to a final concentration of 12 or 24 µg/ml, based on methods previously described 53 . Following incubation at 37°C for 30 min, the reaction was quenched with 1 mM PMSF. Mitochondria or mitoplasts were then pelleted again by centrifugation at 6000 × g, lysed in SDS buffer and analysed by immunoblot. Purification of ABHD11 from HEK293T cells. HEK293T cells were transfected with the pCEFL-ABHD11 -3XFLAG tag plasmid. In brief, cells were seeded in a 14 cm dish at 70% confluency and transfected using 270 μg polyethylenimine (Sigma) with 22.5 µg DNA in 6 ml Opti-MEM. Cells were harvested after 48 h, and lysed in TBS buffer (100 mM Tris-HCl pH 8.0, 140 mM NaCl) with 1% Triton X-100 and cOmplete Protease Inhibitor Cocktail (Roche). After centrifugation at 17,000 × g, the supernatant was pre-cleared using Sepharose CL-4B (GE Healthcare) and incubated overnight with FLAG M2 antibody conjugated beads (Sigma). Following five washes with TBS, ABHD11-FLAG was eluted using 100 mg/l 3xFLAG peptide (Sigma F4799), filtered using a 0.22 µm PVDF filter and separated using a Superdex 75 10/300 column on an Äkta-Pure liquid chromatography system (software: Unicorn 6.3). 500 µl fractions were collected and protein content visualised by SDS-PAGE and Coomassie staining. Protein identity was confirmed by LC-MS/MS. Liquid chromatography mass spectrometry. Samples were reduced, alkylated and digested in-gel using either trypsin, GluC or AspN. The resulting peptides were analysed by LC-MS/MS using an Orbitrap Fusion Lumos coupled to an Ultimate 3000 RSLC nano UHPLC equipped with a 100 µm ID × 2 cm Acclaim PepMap Precolumn (Thermo Fisher Scientific) and a 75 µm ID × 50 cm, 2 µm particle Acclaim PepMap RSLC analytical column. Loading solvent was 0.1% formic acid with analytical solvents A: 0.1% formic acid and B: 80% acetonitrile + 0.1% formic acid. Samples were loaded at 5 µl/min loading solvent for 5 min before beginning the analytical gradient. The analytical gradient was 3-40% B over 42 min rising to 95% B by 45 min followed by a 4 min wash at 95% B and equilibration at 3% solvent B for 10 min. Columns were held at 40°C. Data were acquired in a DDA fashion with the following settings: MS1: 375-1500 Th, 120,000 resolution, 4 × 10 5 AGC target, 50 ms maximum injection time. MS2: Quadrupole isolation at an isolation width of m/z 1.6, HCD fragmentation (NCE 30) with fragment ions scanning in the Orbitrap from m/z 110, 5 × 10 4 AGC target, 100 ms maximum injection time. Dynamic exclusion was set to +/−10 ppm for 60 s. MS2 fragmentation was trigged on precursors 5 × 10 4 counts and above. Raw files were processed using PEAKS Studio (version 8.0, Bioinformatics Solutions Inc.). Searches were performed with either trypsin, GluC or AspN against a Homo sapiens database (UniProt reference proteome downloaded 26/01/18 containing 25,813 sequences) and an additional contaminant database (containing 246 common contaminants). Variable modifications at PEAKS DB stage included oxidation (M) and carbamidomethylatation with 479 built in modifications included at PEAKS PTM stage. p-Nitrophenyl ester hydrolysis assay. Hydrolase activity of ABHD11-FLAG (or a heat-inactivated control made by incubation at 90°C for 5 min) was assayed by incubation in an assay buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.01% bovine serum albumin, 1.4% methanol, and 500 µM p-nitrophenyl acetate (Sigma N8130). 1.5 µg enzyme was added to 200 µl assay buffer, and the formation of p-nitrophenol assayed using a Clariostar plate reader (BMG Labtech), recording absorbance at 405 nm while incubating at 37°C for 40 min. The rate of formation of p-nitrophenol was calculated from the slope of the absorbance curve, subtracting the slope of a blank containing only assay buffer and substrate, and calibrated against a standard curve of p-nitrophenol (software: Microsoft Excel for Mac 16). OGDHc activity assay. OGDHc activity was measured in whole cell lysates using a Biovision ketoglutarate dehydrogenase activity assay (Biovision K678), according to the manufacturer's protocol. HeLa cells were lysed by three freeze-thaw cycles followed by passing 10 times through a 26-gauge needle. Activity was subtracted from a background control containing cell lysate but no substrate. Mitochondrial bioenergetics assay. Dynamic measurements of oxygen consumption rate and extracellular acidification were recorded using a Seahorse XFe24 (Agilent; software: Wave 2.3). HeLa cells were seeded 24 h beforehand at 1.5 × 10 4 cells/well, and assayed using the manufacturer's Mito Stress Test protocol. Stable isotope tracing by LC-MS. HeLa cells were seeded in five replicates in 6well plates 27 h prior to metabolite extraction, with a sixth well per condition used for cell count. Twenty-four hours prior to extraction, media was changed to either DMEM without L-glutamine (Sigma D6546) supplemented with 10% FCS and 4 mM [U-13 C 5 ]L-glutamine (Cambridge Isotopes CLM-1822), or DMEM without glucose (Gibco 11966-025) supplemented with 10% FCS, 1 mM sodium pyruvate and 25 mM [U-13 C 6 ]D-glucose (Cambridge Isotopes CLM-1396). Two biological replicates were undertaken, with independent lentiviral transductions of the same cell line on different days. Metabolites were extracted on dry ice, after washing with ice cold PBS, with 1 ml per 10 6 cells of extraction buffer, containing 50% methanol, 30% acetonitrile, 20% water and 100 ng/ml HEPES. To quantify the two enantiomers of hydroxyglutarate, a subset was derivatised using 50 mg/ml diacetyl-Ltartaric anhydride in 20% acetic acid/80% acetonitrile 4 . For mass spectrometry analysis, in-gel AspN digest and sample analysis were performed as previously described. To identify possible modifications of DLST K110, raw files were processed using PEAKS Studio Label-free quantitation values were obtained by processing raw files with MaxQuant (version 1.6.6.0) with the following parameters: specific AspN digestion; Human database (UniProt reference proteome downloaded 18 Dec 2018 containing 21066 proteins); oxidation, lipoylation, 2x NEM lipoylation, N terminal acetylation as variable modifications; carbamidomethylation as a fixed modification; label-free quantification enabled. Label-free quantification values were normalised to the sum of DLST peptides label-free quantification values. ABHD11 structural prediction. A structural model of ABHD11 was obtained using the NCBI reference sequence for ABHD11 transcript variant 1 (NP_683710.1), modelled with Phyre2 against a template of murine epoxide hydrolase (PDB: 1cr6) 54 and visualised using PyMOL 2.3 (Schrödinger, LLC). The mitochondrial targeting sequence was mapped with the MitoFates prediction tool 55 . Phylogenetic analysis and multiple sequence alignment of ABHD family members was performed with protein sequences obtained from Uniprot (canonical transcript variant) and aligned using Clustal Omega (EBI) 56 . Statistics and reproducibility. Statistical analysis of the screens was performed using MAGeCK version 0.5.5 51 , testing the sgRNA read counts obtained following the second sort against sgRNA read counts obtained from unsorted cells lysed at the same timepoint. Quantification and data analysis of other experiments are expressed as mean ± SEM and P values were calculated using two-tailed Student's ttest for pairwise comparisons, unless otherwise stated, and were calculated using Graphpad Prism version 8. Metabolomic samples were blinded and randomised prior to their evaluation. Qualitative experiments were repeated independently to confirm accuracy. Specifically, Figs. 1e, f, g; 3e, h; 4f, i, j; 5d, f; Supplementary Figs. 1e, g, k, 5a, b; 9c were repeated twice with similar findings. Figs. 2l, m; 3d; 4d, e, h; 5e; Supplementary Figs. 1f; 4a, d; 7b and 9a were performed independently at least 3 times. Representative data are shown in the figures. Uncropped original scans of all immunoblots are displayed in Supplementary Fig. 11.	v3-fos-license
2020-11-19T09:13:07.488Z	2020-11-01T00:00:00.000	227066197	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2073-4409/9/11/2491/pdf", "pdf_hash": "c225312c396bd88a8ce6b134e7f4026b5a230a4a", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:14", "s2fieldsofstudy": [ "Biology" ], "sha1": "b5e41dcf6e7e180d584af30fc8da10680cdbea45", "year": 2020 }	pes2o/s2orc	Methionine Supplementation Affects Metabolism and Reduces Tumor Aggressiveness in Liver Cancer Cells Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti-cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP-activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression. Introduction Liver cancer is the second leading cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and is associated with chronic liver damage, which can be caused by viral infections, such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infection or by alcoholic liver diseases and non-alcoholic steatohepatitis (NASH). Potentially curative treatments for very early/early stage HCC patients include surgical resection, liver transplantation and percutaneous ablation. At an advanced/late stage surgery is no longer applicable, and the currently available therapies are effective only in small groups of patients [1][2][3]. Very few therapeutic options, with unsatisfactory antitumor effects and toxicity, are nowadays available, thus, prognosis remains very poor. In 2007 Sorafenib was the first VEGFR TKI (vascular endothelial growth factor receptor Thus, since methionine/SAM administration in liver seems to have an opposite role than in other cancer types, more studies are necessary to support a therapeutic role of methionine or SAM supplementation in patients. Here, we investigate the effect of methionine supplementation in liver cancer cells in vitro, to explore the possibility that alterations in methionine dietary consumption could help treatment of liver cancer. HepG2, Huh7, SW480 and A549 cells were cultured using RPMI1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. MCF7 were cultured using EMEM/NEAA supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 • C in a humidified 5% CO 2 incubator. Methionine was dissolved in water at 45 mg/mL and added at a final concentration of 1.5 mg/mL to RPMI1640 medium; in the control medium, the same amount of water was added. Compound C (Calbiochem, San Diego, CA, USA) was dissolved in DMSO and added to the medium at a final concentration of 2 µM for HepG2, SW480, A549, and MCF7 cells, and at 1.5 µM for Huh7 cells. Growth Curves 1.5 × 10 5 cells were plated in 6 well plates, the day after the medium was changed (control medium, high methionine, Compound C or High methionine and Compound C). Cells were counted at t = 0, 48 h and 72 h. Migration Assay Cell migration was assessed using transwell permeable supports (Costar) with 8.0 µm filter membranes. Cells were treated with high methionine and/or Compound C for 24 h, and then serum starved for 24 h. 5 × 10 4 HepG2 cells and 3.5 × 10 4 Huh7 cells were resuspended in 100 µL of serum free medium (always in the presence or absence of high methionine and/or Compound C), plated onto each filter and 500 µL of complete medium (containing 10% FBS) were placed in the lower chamber. After 24 h, filters were washed, fixed and stained with 0.5% Coomassie brilliant blue (in 10% acetic acid, 45% methanol). Cells on the upper surface of the filters were removed with cotton swabs. Cells that had invaded to the lower surface of the filter were counted under the microscope. Clonogenic Assay A total of 2500 cells were plated in a 6 well plates, treated with high methionine and/or Compound C for 10-15 days (the medium was changed every 3-4 days). Then, colonies were fixed with 70% ethanol for 5 min, stained with 0.5% crystal violet in 10% ethanol for 15 min, finally, washed with water and manually counted. Shotgun Mass Spectrometry and Label Free Quantification Four technical replicates were performed for each HepG2 sample, grown for 48 h in the presence or absence of high methionine and/or Compound C. Proteins were lysed in RapiGest 0.1% (RG, Waters Corporation, Milford, MA, USA), reduced with 13 mM DTE (30 min at 55 • C) and alkylated with 26 mM iodoacetamide (30 min at 23 • C). Protein digestion was performed using sequence-grade trypsin (Roche) for 16 h at 37 • C using a protein/trypsin ratio of 20:1. The proteolytic digested was desalted using Zip-Tip C18 (Millipore, Burlington, MA, USA) before MS analysis [27]. LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System with a PicoFrit ProteoPrep C18 column (200 mm, internal diameter of 75 µm). Gradient: 2% ACN in 0.1% formic acid for 10 min, 2-4% ACN in 0.1% formic acid for 6 min, 4-30% ACN in 0.1% formic acid for 147 min, and 30-50% ACN in 0.1% formic for 3 min, at a flow rate of 0.3 µL/min. The eluate was electrosprayed into an LTQ OrbitrapVelos (Thermo Fisher Scientific, Bremen, Germany) through a Proxeon nanoelectrospray ion source (Thermo Fisher Scientific), as reported in [28]. The LTQ-Orbitrap was operated in positive mode in data-dependent acquisition mode to automatically alternate between a full scan (m/z 350-2000) in the Orbitrap (at resolution 60,000, AGC target 1,000,000) and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan (normalized collision energy of 35%). Data acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software (Thermo Fisher Scientific). A database search was conducted against the Homo Sapiens Uniprot sequence database (release 6 March 2019) with MaxQuant (version 1.6.0.1) software, using the following parameters: the initial maximum allowed mass deviation of 15 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks, trypsin enzyme specificity, a maximum of 2 missed cleavages, carbamidomethyl cysteine as fixed modification, N-terminal acetylation, methionine oxidation, asparagine/glutamine deamidation and serine/threonine/tyrosine phosphorylation as variable modifications. Quantification was performed using the built in XIC-based label-free quantification (LFQ) algorithm using fast LFQ [29]. False protein identifications (1%) were estimated by searching MS/MS spectra against the corresponding reversed-sequence (decoy) database. Statistical analysis was performed using the Perseus software (version 1.5.5.3, https://maxquant.net/perseus/). Only proteins present and quantified in at least 75% of the repeats were positively identified and used for statistical analysis. An ANOVA test (cut-off at 0.05 p-value) was carried out to identify proteins differentially expressed among the four conditions. Focusing on specific comparisons, proteins were considered differentially expressed if they were present only in one condition or showed significant t-test difference (Student's t-test p value ≤ 0.05) [30]. Bioinformatic analyses were carried out by Ingenuity ® Pathway Analysis software (IPA ® -QIAGEN) to cluster enriched annotation groups of Biological Processes, Pathways, and Networks within the set of identified proteins. Functional grouping was based on Fischer's exact test p value ≤ 0.05 (i.e., −log10 ≥ 1.3) and at least 3 counts. Comparison between the proteomic and metabolomic data was performed by IPA and by MetaboAnalyst software R3.0 [31]. Enrichment analysis aimed to evaluate whether the observed genes and metabolites in a particular pathway are significantly enriched (Fisher's exact test 0.05), while the topology analysis aimed to evaluate whether a given gene or metabolite plays an important role in a biological response, based on its position within a pathway (pathway impact). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier 7MlKNZqC. Chemicals for Metabolomics Analysis All chemicals and solvents used for extraction buffer and for liquid chromatography were LC-MS Chromasolv purity grade. Acetonitrile, methanol, 2-Propanol, and water was purchased from Honeywell, while chloroform and formic acid were purchased from Sigma-Aldrich. Metabolites Extraction for GC-MS Analysis Cells were quickly rinsed with 0.9% NaCl and quenched with 800 µL of 1:1 ice-cold methanol:water and collected by scraping. Cells were sonicated 5 s for 5 pulses at 70% power twice and then 400 µL of chloroform were added. Samples were vortexed at 4 • C for 20 min, and then centrifuged at 12,000 g for 10 min at 4 • C. The aqueous phase was collected in a glass insert for solvent evaporation in a centrifugal vacuum concentrator (Concentrator plus/ Vacufuge ® plus, Eppendorf) at 30 • C for about 2.5 h. Metabolites Extraction for LC-MS Analysis Cells were quickly rinsed with 0.9% NaCl and quenched with 500 µL ice-cold 70:30 acetonitrile-water. Plates were placed at −80 • C for 10 min, then the cells were collected by scraping. Cells were sonicated as above and then centrifuged at 12,000 g for 10 min at 4 • C. The supernatant was collected in a glass insert and dried as above. Samples were then resuspended with 150 µL of H 2 O prior to analyses. GC-MS Metabolic Profiling Derivatization was performed using automated sample prep WorkBench instrument (Agilent Technologies, Santa Clara, CA, USA). Dried polar metabolites were dissolved in 60 µL of 2% methoxyamine hydrochloride in pyridine (ThermoFisher) and held at 40 • C for 6 h. After the reaction, 90 µL of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamid) was added, and samples were incubated at 60 • C for 1 h. Derivatized samples were analyzed by GC-MS using a DB-35MS column (30 m × 0.25 mm i.d. × 0.25 µm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 7200 Accurate-Mass Quadrupole Time-of-Flight (QTOF) mass spectrometer (MS), operating under electron impact (EI) ionization at 70 eV. Samples (1 µL) were injected in a splitless mode at 250 • C, using helium as the carrier gas at a flow rate of 1 mL/min. The GC oven temperature was held at 100 • C for 2 min and increased to 325 • C at 10 • C/min. GC/MS data processing was performed using Agilent Muss Hunter software. Relative metabolites abundance was carried out after normalization to internal standard d27 Myristic acid and cell number and statistical analyses were performed using MetaboAnalyst 4.0 [32]. Metabolites Quantification in the Media Samples Absolute quantification of glucose, lactate, glutamine, and glutamate in spent media was determined enzymatically using YSI2950 bioanalyzer (YSI Incorporated, Yellow Springs, OH, USA). Bioenergetics by Seahorse Technology Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined by using Seahorse XFe96 Analyzer (Agilent Technologies). HepG2 and Huh7 cells were seeded in Agilent Seahorse cell culture microplates at density of 2 × 10 4 cells/well for HepG2 or 1 × 10 4 cell/well for Huh7 72 h prior to the assay. A total of 24 h after seeding cells were treated with high methionine (1.5 g/L) and/or Compound C (2 µM for HepG2 and at 1.5 µM for Huh7 cells) for 48 h, and then analyzed by using the Seahorse XF ATP rate assay kit (Agilent Technologies), according to manufacturer instructions. Three measurements of OCR and ECAR were taken for the baseline and after the sequential injection of mitochondrial inhibitors (1.5 µM oligomycin and 1.5 µM rotenone/antimycin A). OCR and ECAR from each well were normalized by protein content by using the Bradford assay. High Methionine and Compound C Induce Proteomic Changes We recently published findings that methionine activates AMPK and increases mitochondrial metabolism and respiration in the model organism Saccharomyces cerevisiae, especially in cells lacking Snf1/AMPK activity [33]. To investigate whether this is a conserved mechanism triggered by methionine, we explored the effect of high methionine concentrations in liver cancer cells, in the presence or absence of Compound C, which mimics AMPK inhibition. We performed a proteomic analysis on HepG2 cells grown for 48 h (CTR), in the presence of Compound C (CTRCC), in the presence of high methionine (MET), and in double treated cells (METCC) by a quantitative shotgun label free strategy. The comparison of the four data sets by an ANOVA test (p-value 0.05) showed proteins exclusively expressed in each condition, as well as 717 proteins common to all data sets, among which, 46 were statistically different. IPA analysis carried out on these proteins to find possible interactions highlighted that 25 out of 46 proteins differentially expressed belonged to a network classified as: cancer, protein synthesis, RNA damage and repair ( Figure 1A). The upstream regulator analysis, based on prior knowledge of expected effects between transcriptional regulators and their target genes in IPA, suggested that two transcription regulators were probably involved, hepatocyte nuclear factor 1 (HNF1A, p-value 0.0005) and hepatocyte nuclear factor 4 (HNF4A, p-value 0.001) ( Figure 1B), which target 6/46 and 12/46 of the proteins differentially expressed, respectively. This result suggests that the presence of methionine and Compound C significantly affects transcription, in keeping with Wang and coworkers who reported an impact on methionine metabolism by HNF4A [34]. Specific analyses were then carried out by comparing MET vs. CTR, METCC vs. MET, CTRCC vs. CTR. A summary of the results obtained is reported in Figure 3, while Table S2-S4 report the list of the proteins differentially expressed (either increased or decreased), or exclusively expressed in one condition in the three comparisons. The corresponding volcano plots are shown in Figure 1C. These proteins were further analyzed to find possible enrichment in GO Biological Processes ( Figure 2) and pathways (Figure 3), in the comparison between control and treatments with high methionine or/and Compound C. Differentially expressed proteins mainly belonged to the classes of metabolic processes, mitochondrion, and mitochondrial dysfunction, translation, RNA and protein processing and response to oxidative stress. As shown in Figure 2A,B and in Table S1, the expression of proteins related to oxidation-reduction events were altered, either up or downregulated, indicating a general impact on these processes in the presence of methionine and/or Compound C compared to the control. Many proteins involved in mitochondrial respiratory chain were upregulated by the double treatment with high methionine and Compound C. In particular, high methionine induced an increase of proteins involved in the mitochondrial electron transport and in complex I assembly, while Compound C treatment mainly increased the level of tricarboxylic acid (TCA) cycle and lipid metabolic processes (Figure 2A). Methionine and Compound C presence upregulated proteins involved in the cellular response to oxidative stress, manly at the endoplasmic reticulum level (Figure 2A, Figure 3), while Compound C alone induced the expression of proteins involved in membrane organization, transmembrane transport, and cell-cell adhesion (Figure 2A). Interestingly, methionine and Compound C altered sirtuin signaling pathway ( Figure 3). This pathway is a master regulator of several cellular processes and known to both extend lifespan and regulate spontaneous tumor development. It is well known that S-adenosyl-methionine, sirtuin, and mTOR pathway are strictly related [35]. In keeping with these data and with results reported above, the proteomic analysis suggested that mTOR signaling was altered mainly in response to increased methionine in the medium (Figure 3). Among the processes more altered in HepG2 cells treated with methionine and Compound C, RNA processing and mitochondrial translation were downregulated mainly by methionine, while Compound C had more impact on general translation ( Figure 2B). Overall, the double treatment with high methionine and Compound C induced a decrease in the level of ribosomal proteins RPS9, RPL23A, RPL34, mitochondrial ribosomal proteins MRPL37, MRPS18C, and translational initiation factors, such as EIF1 and EIF3M, as well as proteins of the SRP (signal-recognition-particle) co-translational process (SRP14, SSR3, SRP9), suggesting a general reduction of translation in this condition (Table S4). High Methionine and Compound C Induce a Metabolic Rewiring Since methionine supplementation induced deep changes of proteins associated with cellular metabolism, we further investigated this feature by performing metabolomics analysis in cells grown with high methionine and/or Compound C. Extracellular metabolites were analyzed by YSI biochemical analyzer. In HepG2 cells, only the double treatment (high methionine and Compound C) showed an increase of glucose and glutamine consumption, with a proportional increase in lactate and glutamate secretion ( Figure 4A). However, no changes in extracellular metabolites were observed in Huh7 cells in any condition. This difference could be due to the lower growth rate of Huh7 cells, or to their higher basal metabolism compared to HepG2 cells; indeed, their basal glucose uptake and their lactate secretion were three times higher than those of HepG2 cells ( Figure 4A,B). We then measured relative abundance of intracellular metabolites in HepG2 cells treated for 48 h with high methionine and/or Compound C, by an untargeted metabolomic GC/MS and LC/MS analysis. This analysis revealed a strong metabolic change due to high methionine, but also to Compound C addition (Supplementary Figure S1, Figure 4C). The most affected pathways were those related to amino acids metabolism, like Cys-Met (as expected upon methionine supplementation), Ala-Asp-Glu, Arg-Pro, and Gly-Ser-Thr ( Figure 4C), but also those related to aminoacyl-tRNA biosynthesis, coherently with the downregulation of translation associated proteins ( Figure 2B). In addition, alterations in the pathways of glutathione metabolism (which is directly associated to methionine metabolism), of the sulfur-containing molecules taurine and hypotaurine, in the pathways of purine and nucleotide metabolism and of fatty acid metabolism were identified ( Figure 4C). As expected, methionine supplementation in the medium (either in the presence or absence of Compound C) increased the intracellular level of methionine, but also of its derived metabolites, such as S-adenosyl-methionine and S-adenosyl-homocysteine, which are part of the methionine cycle ( Figure 4D). In addition, high methionine treatment induced an increase of cysteine, which can derive from homocysteine and serine, through conversion to cystathionine, and of glutathione, which is synthesized from Cys, Glu, and Gly. In high methionine condition there was also an increase of two direct methionine derivatives, the regulatory metabolite methylthioadenosine (MTA) and the toxic compound methionine sulfoxide ( Figure 4D). Remarkably, the MTA level negatively correlates with growth potential in the liver [36,37]. In fact, it has been reported that MTA decreases after partial hepatectomy, when the replicative response of hepatocytes is higher [38], and MTA administration in vitro reduces liver cell growth [39]. Furthermore, MTA inhibits the synthesis of polyamines [36], whose level is correlated with proliferation and which were indeed decreased in high methionine condition (see spermidine and putrescine, Figure 4E). On the contrary, the MTA derivatives adenine and adenosine increased in high methionine condition ( Figure 4E). High methionine also induced an increase of metabolites related to the urea cycle, such as arginine, ornithine, uric acid ( Figure 4E), which was probably increased to convert excess nitrogen (given by the high methionine supplied in the medium) into urea. High Methionine and Compound C Increase Mitochondrial Functionality The metabolomics analysis revealed also that many metabolites of the TCA cycle were modulated by high methionine, with higher levels of cis-aconitate, citrate, isocitrate, α-ketoglutarate, succinate and malate and lower levels of fumarate and oxaloacetate compared to the control ( Figure 4F,G). Since the metabolomics and proteomics data suggested an increase of the TCA cycle, we measured the rate of ATP production from glycolysis and from mitochondrial respiration in HepG2 and Huh7 cells, through Agilent Seahorse technology ( Figure 4H). Although the amount of ATP deriving from glycolysis was only slightly altered by treatments, cells grown in high methionine condition (with or without Compound C) produced a higher fraction of ATP through mitochondrial respiration ( Figure 4H). The increase of mitochondrial-derived ATP was clearly evident in HepG2 cells and it was also confirmed, albeit with a lower increase, in Huh7 cells. According to systems biology, the phenotype of a cell results from the interaction of several component, in which the emergent behavior is wider than the sum of their parts [40]. As a result of this, we decided to integrate the results from metabolomic and proteomic analysis, using the IPA and MetaboAnalyst softwares. This integration better highlighted that TCA cycle was one of the most affected process in all three comparisons (MET versus CTR, CTRCC versus CTR, METCC versus CTR) ( Figure 5A-C). However, as reported in Figure 5D,E, focusing on the first 10 more significant pathways in terms of enrichment (p-values) and topology (pathway impact) in the three comparisons, the integration analysis suggested that citrate and TCA cycle were more affected by the presence of Compound C than methionine, which, in turn, had a major impact on amino acid metabolism, especially of those amino acids directly linked to the nitrogen cycle, such as Arg, Gln, and Glu. In keeping with the effect on TCA cycle, pyruvate metabolism and the synthesis and degradation of ketone bodies prevailed in the presence of Compound C if compared to the supplementation of methionine ( Figure 5D,E). Strikingly, the pentose phosphate pathway, Arg/Pro metabolism, glyoxylate, and dicarboxylate metabolism were altered only upon double treatment ( Figure 5D). In addition, glutathione metabolism and metabolism of xenobiotics, which were affected by single treatments, were no more altered upon double treatment ( Figure 5D). These results indicate that the combination of Compound C and high methionine does not result in a simple synergistic effect of the single treatments, but rather leads to the emergence of new features in liver cancer cell metabolism. High Methionine Activates AMPK and mTOR Pathways Since we previously reported that methionine can activate Snf1/AMPK in budding yeast, we then investigated the effect of methionine supplementation in liver cancer cells. AMPK showed a dose-dependent activation after 24 h in HepG2 cells ( Figure 6A), detectable as increased phosphorylation on T172, as well as increased phosphorylation of Acc1-S79, the main target of AMPK, often used as reporter of its activity. It is known that AMPK activation is mainly due to an energy reduction [41], although it can also be activated without any ATP decrease [42]. Since data presented above ( Figure 4H) indicate that ATP level did not decreased, but rather increased after methionine treatment, we can speculate that AMPK activation was not a result of energy deficiency. (A) HepG2 were treated with methionine for 24 h and AMPK activation state was assayed by Western analysis, using the pT172-AMPK antibody (against pT172 in the activation loop) and using the anti-pS79-Acc1 antibody (against the target site of AMPK on Acc1). An anti-AMPK total antibody and an anti-vinculin antibody were used as controls. (B) HepG2 and Huh7cells were gown in control medium and 1.5 g/L methionine was added to the cultures at time 0. Samples were collected at the indicated time points to evaluate AMPK activation, using an anti-pT172-AMPK antibody and an anti-pS79-Acc1 antibody. An anti-AMPK total antibody and an anti-tubulin antibody were used as controls. (C) HepG2 and Huh7 cells were gown for 48 h in the absence or presence of Compound C. Then 1.5 g/L methionine was added to the cultures, and samples were collected at the indicated time points to evaluate mTOR activation, using anti-pS6K antibody and Akt activation using anti-pS473-Akt antibody. Anti-Akt total antibody and anti-tubulin antibody were used as controls. To better investigate AMPK activation, we performed time-course experiments in both HepG2 and Huh7 cell lines. A total of 1.5 g/L of methionine was added to cells growing in regular medium and phosphorylation of AMPK-T172 and of Acc1-S79 were detected by Western blot analysis ( Figure 6B). AMPK phosphorylation, as well as the phosphorylation of its target Acc1, increased in both cell lines, with a peak at 0.5 h and 1 h after methionine addition, for HepG2 and Huh7 respectively ( Figure 6B). Since amino acids can activate mTOR, the master regulator of cell growth [43] and, since mTOR involvement was suggested by our proteomics analysis (Figure 3), we investigated the activation of the mTOR pathway. mTOR was activated in response to high methionine in the medium, as observed by increased pS6K phosphorylation ( Figure 6C), in keeping with previously reported data [44], both in HepG2 and in Huh7 cells. Phosphorylation increased after 30-min treatment, remaining high until 16 h ( Figure 6C). This increase was more evident in cells pre-treated with Compound C, in which the release of mTOR inhibition by AMPK resulted in a higher pS6K phosphorylation, both at time 0 and in response to high methionine ( Figure 6C). High methionine also induced activation of the Akt pathway, involved in cell proliferation and survival [45], in both cell lines, although in HepG2 cells, it was less persistent over time when AMPK was inhibited ( Figure 6C). High Methionine Reduces Cancer Associated Phenotypes We previously showed that, in yeast cells, methionine induced a slow-down of growth rate, especially in cells lacking Snf1/AMPK activity [33]. The increase of methionine concentration in the medium (up to 1.5 g/L, versus 15 mg/L in regular medium) induced a slight slow-down of growth rate both in HepG2 and in Huh7 cell lines ( Figure 7A,B). Inhibition of AMPK with Compound C slightly reduced growth rate in both cell lines, but drastically impaired growth when combined with high methionine in the medium ( Figure 7A,B). These results suggest that the effect of methionine on cellular proliferation is a conserved feature that deserves further investigation. In addition to a higher proliferation, one of the most relevant features of cancer cells is their ability to migrate and to form colonies from single cells, to give metastasis [46]. To analyze the effect of methionine on cell migration, we used Boyden chambers and migration of serum starved cells was assessed in the presence or absence of high methionine concentration and/or Compound C. Cell migration through the membrane of the transwell was significantly impaired in high methionine in both cell lines, even more when AMPK was inhibited ( Figure 7C,D). We then tested the ability of single cells to form colonies in the presence of high methionine and/or inhibition of AMPK. As shown in Figure 7E,F, colony formation was reduced by high methionine supplementation, especially in the presence of Compound C ( Figure 7E,F). Altogether, our results suggest that high methionine supplementation inhibits cancer associated phenotypes, especially in combination with AMPK inhibition. The Effect of High Methionine and Compound C is Specific for Liver Cancer Cells Although Compound C has been extensively used to inhibit AMPK activity, it may also inhibit other kinases. To discern whether the effect of Compound C on growth inhibition in high methionine condition was specifically due to AMPK inhibition, we silenced the expression of AMPKα/α' subunits in HepG2 and Huh7 cells and tested their growth in the presence of high methionine in the medium ( Figure 8A,B). We found that cells with siAMPKα/α' showed a reduced growth at 72 h in the presence of high methionine, thus, confirming that high methionine had a negative effect on growth when AMPK activity was low (either due to chemical inhibition or to reduction of the catalytic subunit). These data confirm that the effect of Compound C on growth rate was mediated by AMPK inhibition, and not by non-specific effects of the compound. Finally, to analyze whether the effect of high methionine and AMPK inhibition was specific for liver cancer cells or could be observed also on other cancer cell types, we performed the growth assay and the clonogenic assay on three cell lines deriving from different tumors: SW480 colorectal cancer cells, A549 lung cancer cells and MCF7 breast cancer cells. High methionine and/or Compound C had no effect on the growth of SW480 and A549 cells, while high methionine slightly reduced the growth of breast MCF7 cells also in combination with AMPK inhibition ( Figure 8C). However, the ability of forming colonies from single cells was not impaired in none of the cell lines tested ( Figure 8D), although in MCF7 cells high methionine induced the formation of smaller colonies (not shown). Thus, we can assume that the effect of this treatment on cancer associated phenotypes is specific for liver cancer cells. Discussion The role of methionine has been long investigated in many different fields [13]. Indeed, it is well known that methionine restriction extends lifespan in different model systems, from budding yeast to Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells [47][48][49][50]. Methionine restriction also affects the cardiovascular system [51] and bone development [52]. On the contrary, the relationship between methionine and human cancers progression is still very ambiguous, most probably because it is cancer specific. Different studies showed that methionine restriction delays cancer progression. This was reported for instance in colon and prostate cancer animal models [53,54], as well as in breast cancer in vitro and in vivo [55,56]. On the contrary, other reports indicate that methionine supplementation induces cell-cycle arrest and transcriptional alterations in breast and prostate cancer cells [57,58]. It was reported that S-adenosyl-methionine (SAM) supplementation inhibits liver cancer cell invasion in vitro [19], by inducing changes in the methylation state of DNA, that lead to downregulation of genes involved in growth and metastasis, already known to be upregulated in liver cancer cells. In addition, in a rat model of hepatocarcinogenesis [59], as well as in a mouse model for inflammation-mediated HCC [20], SAM administration exerted a chemopreventive effect on HCC development. However, although a short-term treatment with SAM showed positive effects in the mouse model, a long-term administration did not affect tumor growth and hepatocyte proliferation [20]. Here, we explored the combination of methionine administration (the precursor of SAM) and AMPK inhibition in vitro. AMPK is a dual role kinase, being either anti-or pro-tumorigenic depending on the context, on the stage of tumor development and on the cancer type [6]. We showed that, in liver cancer cells, high methionine concentration in the medium reduces cell growth inhibits colony formation and cell migration in two different liver cancer cell lines ( Figure 7) and, remarkably, these phenotypes were increased when high methionine was combined with AMPK inhibition (Figures 7 and 8). This is perfectly in line with what we observed in budding yeast, where growth rate reduction due to methionine in the medium was largely dependent on the presence of an active Snf1/AMPK pathway [33]. Moreover, methionine induces an activation of Snf1/AMPK in S. cerevisiae, as we observe in liver cancer cells (Figure 6), highlighting that AMPK involvement in the response to methionine is a conserved feature in eukaryotic systems. An intriguing aspect of methionine response is the activation of the mTOR pathway ( Figure 6), which is coherent with the reported effect of SAM (the first metabolite of methionine) on mTOR activation through SAM-sensor upstream of mTOR1 (SAMTOR) [44]. However, the intracellular level of most amino acids was downregulated in cells grown in high methionine (Figure 4), and this probably leads to the observed downregulation of proteins involved in translation and tRNA synthesis (Figures 1, 2, 4 and 5) and to the reduction of growth rate (Figure 7). This condition-active mTOR with reduced translational capacity-reminds the condition of cycloheximide treatment, in which mTOR phosphorylates pS6K [60] although growth is impaired, thus, producing the effect of a counter circuit in the cell. High methionine has also a strong effect on metabolome and proteome remodeling, as also reported in yeast cells [33], especially when combined with Compound C. In fact, reduction of intracellular amino acid levels and alterations in metabolites of the TCA cycle were found in both yeast and liver cancer cells. Interestingly, the observed increase of proteins and metabolites of the TCA cycle could be a direct consequence of methionine metabolism to homocysteine, which can be then converted to α-keto-butyrate and enter the mitochondria. However, in yeast cells, the effect of methionine on mitochondrial functionality was much more evident, probably due to the fact that yeast cells have a fermentative metabolism in the presence of glucose. On the contrary, in human cells, which have a mixed respirative and fermentative metabolism, the effect on mitochondria better emerges by integrating metabolomics and proteomics data. These results, together with the reduction of polyamine levels (which are associated to growth rate), could explain at least in part the observed reduction of cancer phenotypes. Why does methionine metabolism have this anti-tumor role on liver cancer cell lines, contrary to other cancer cells? It should be noted that methionine metabolism in the liver is very peculiar, since the liver is the organ where most of the methionine is converted to S-adenosyl-methionine and where only the gene MAT1A is expressed. Therefore, most of the observed effects could be due to this liver-specific metabolism, although we cannot exclude the possibility that methionine could carry out also other functions more related to protein synthesis. In fact, Mato and Lu suggest that liver cancer cells, in contrast to normal non-proliferating, differentiated hepatocytes, tend to utilize methionine mainly for protein synthesis [17]. An interesting translational application of our results could be to directly increase methionine uptake through the diet, both in animal models and in human patients. Methionine should easily reach the liver, since it is the physiological organ where it is metabolized. It should have no side effect on normal hepatocytes, since SAM was shown to have negligible effects on primary untransformed liver cells [19]. Therefore, further investigations should explore the possibility that alterations in methionine dietary consumption, in combination with pharmacological treatments, could have clinically relevant outcomes in liver cancer patients. Figure S1: Hierarchical clustering heatmap from One-way ANOVA analysis of the entire set of metabolites differentially expressed.	v3-fos-license
2021-05-10T00:04:09.321Z	2021-01-31T00:00:00.000	234094807	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://academicjournals.org/journal/AJMR/article-full-text-pdf/CD327C665871.pdf", "pdf_hash": "66f88293836e1ec2d98a23ecd4823c8f75392663", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:29", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "sha1": "561e628a0ca806817f414a04c5e84edb9af68db3", "year": 2021 }	pes2o/s2orc	Isolation, identification and growth conditions of calcite producing bacteria from urea-rich soil Bacterial chemical reactions, such as urea hydrolysis can induce calcium carbonate precipitation. The induced production of calcium carbonate formed by microorganisms has been widely used in environmental and engineering applications. The present study aimed to isolate, identify and optimize growth conditions of urease positive bacteria from urea rich soil in Gaza Strip. Bacterial isolates, which tolerated ≥10% urea concentration, were selected for the investigation. Eight isolates recovered and identified to be spore forming, urease positive, alkaliphile, halotolerant, and presumptively belonged to Bacillus species. All isolates showed best growth at temperature 37°C, and pH 9-9.5. After exposure to UV irradiation, most isolates showed improved tolerance to urea concentration, however, other strains showed a decline in their adaption to urea concentrations. The mutant form of isolate in soil sample #3 showed the highest tolerance to urea concentrations at all exposure intervals, when compared with wild type. Moreover, all isolates precipitated calcium carbonate. The locally recovered isolates are promising contributors in the process of calcite Biomineralizaion and may be utilized in the remediation of concrete cracks, increase of compressive strength of concrete, decrease water permeability, and solve the problems of soil erosions. INTRODUCTION Biological precipitation of minerals (Bio-mineralization) is a widespread phenomenon in the microorganism's world, and is mediated by bacteria, fungi, protists, and even by plants. Calcium carbonate (Calcite) is one of those minerals that naturally precipitate as a by-product of microbial metabolic activities (Seifan and Berenjian, 2019). Microbial metabolic activities facilitate calcium carbonate (calcite) precipitation, in a well-studied process called microbial induced calcium carbonate precipitation (MICP) (Zambare et al., 2019). MICP usually occurs due to the chemical alteration of the environment induced by the microbial activity (Sarikaya, 1999;Stocks-Fischer et al., 1999;Warren et al., 2001;De Muynck et al., 2010a). Bacteria can be invested as a major player in the MICP phenomenon through various mechanisms. The most significant mechanism is the bacterial ureolytic activity (Stocks-Fischer et al., 1999;Warren et al., 2001;Krajewska, 2018). Urea hydrolysis can be facilitated by *Corresponding author. E-mail: [email protected]. Tel: 00970594157573. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License bacteria that can produce urease (urea amidohydrolase) enzyme and thus are able to induce CaCO 3 precipitation (Stocks-Fischer et al., 1999;Hammes and Verstraete, 2002;Phillips et al., 2013;Bhaduri et al., 2016). Calcium carbonate precipitation is a chemical process controlled mainly by four key factors: (1) calcium ions concentration, (2) dissolved inorganic carbon (DIC) levels, (3) the pH, and (4) the availability of nucleation sites (Hammes and Verstraete, 2002;Seifan and Berenjian, 2018). Over recent years, MICP has received considerable attention and has been proposed as a potent solution to address many environmental and engineering issues (Seifan and Berenjian, 2019). It has been intensely investigated in bulk systems, sand columns (Dhami et al., 2013a;Seifan et al., 2016;Tziviloglou et al., 2016), and bio-cementation processes (Seifan et al., 2016). It has been found that MICP may drive many potential applications in civil engineering such as enhancing stability of slopes and dams, reducing the liquefaction potential of soil, road construction, prevention of soil erosion, increase durability and compressive strength of concrete, as well as the repair of the cracks in concrete (De Muynck et al., 2010a;Stabnikov et al., 2011, Shashank et al., 2016. Many bacterial species have been studied to exploit their abilities in the biomineralizing of calcite (MICP). One of the most robust ureolytic bacteria is Sporosarcina pasteurii (formerly known as Bacillus pasteurii). S. pasteurii is facultative anaerobes, spore forming, bacilli bacteria. It utilizes urea as an energy source and eliminates ammonia which increases the pH in the environment and generates carbonate, causing Ca 2+ and CO 3 2to be precipitated as CaCO 3 (Clive, 1990, Stocks-Fischer et al., 1999Chahal et al., 2011). Other studies showed the role of bacteria that are mostly related to Bacillus spp. in the process of MICP (Stocks-Fischer et al., 1999;Elmanama and Alhour, 2013;Ali et al., 2020). The aim of this study is to isolate, identify, and optimize growth conditions of locally isolated urease-producing bacteria that are able to produce calcite crystals. Sample sources and characteristics This study utilized soil samples varying in urea content from Gaza Strip. About 100 g of each sample were collected during June 2020 from the following sources: (1) Sea coastal sand from Rafah city beach, (2) outlet sewage water (treated sewage), (3) inlet sewage water (untreated sewage), (4) soil sample with cat`s urine from Gaza city, (5) coastal sand with dog`s urine from Rafah city beach, (6) sand with dog`s urine from Rafah city, (7) agricultural soil with dog`s urine from Gaza city, (8) agricultural soil with dog`s urine from Rafah city, (9) urea rich soil from greenhouse in Gaza city, and (10) ammonia rich soil from a greenhouse in Gaza city. Sample processing and bacterial isolation Five grams of soil samples were mixed with 20 ml sterile saline (stock suspension) and dilutions 10 -1 and 10 -2 were made. A volume of 0.1 ml of the stock suspension as well as the two dilutions were cultured onto 2 and 3% urea containing Nutrient Agar (NA) plates (HiMedia, India). The media were prepared according to HiMedia manufacturer recommendations. Extra pure urea suspensions (Honeywell Riedel-de Haen, Germany) were filtered, then added to media after autoclaving and cooling to 50°C. Cultures were incubated at 37°C, and plates were examined after 24 and again after 48 h. Bacterial tolerance to high urea concentration Bacterial isolates were obtained as a pure culture and then cultured on 5, 8, 10, 12, and 15% urea enriched NA media, incubated at 37°C for 48-72 h, and after the incubation period bacteria were harvested to be cultivated in nutrient broth and agar plates. Bacterial isolates tolerated ≥ 10% urea concentration were selected for further testing. pH profile The isolates were inoculated into 3 ml of Nutrient Broth tubes with pH scale of 1 to 14. A bacterial suspension was made and the turbidity was adjusted to 0.5% McFarland standard, incubated for 24 h at 37°C, and growth has been measured as turbidity at O.D 600 nm using CT-2200 spectrophotometer (Chrom Tech, Taiwan). Results were recorded against a blank of bacterial suspension. 1N HCl (HiMedia, India) and 1N NaOH (Frutarom, Palestine) were used to adjust the pH. An additional nutrient broth tubes at pH 7 were inoculated with the bacterial isolates, incubated at 37°C, and the change in pH was monitored during growth, using a pH meter (Jenway pH Meter 3510 /mV, USA ) results were recorded after 30 min, 1 , 2, 4, 8, 24, and 32 h of inoculation. Effect of temperature on growth Bacterial isolates were inoculated into nutrient broth tubes (HiMedia, India), adjusted to 0.5% McFarland standard, incubated for 24 h at 0, 4, 25, 37, 45 and 60°C, and the turbidity was measured using spectrophotometer at O.D 600 nm. Ultraviolet (UV) induced mutagenesis for bacterial isolates The selected isolates were grown overnight in NB + 2% urea in a shaking incubator (Boeco, Germany) at 37°C. The isolates were washed three times with sterile phosphate-buffered saline, resuspended in urea free and sterile NB. The turbidity of cell suspensions was adjusted to a 0.5% McFarland reagent and exposed to UV light using a Philips 20 W germicidal lamp for 2-20 min with 2 min intervals. From each exposure interval, a loopful of the exposed bacteria was cultured onto urea-based agar (HiMedia, India). After incubation of 24 h at 37°C, a single, well-defined colony was chosen, cultivated on NA plates, and then inoculated onto NA with varying urea concentrations; 5, 8, 10, 12 and 15% respectively. After incubation, bacterial growth was observed and compared to wild type growth on the different urea concentrations. Mini-scale of calcium carbonate precipitation Bacterial isolates were subjected to calcium carbonate production test as described previously (Ghosh et al., 2019). Alive bacterial isolates were inoculated into nutrient broth (NB) containing both urea and calcium chloride (NBUC), NB with only urea (NBU), and NB with only calcium chloride (NBC). The same procedures were repeated with autoclave killed bacterial suspension (pellet and supernatant filtrate). To all tubes, a concentration of 0.012 g/L phenol red was used as a pH indicator. NBUC and NBU were prepared to contain 2% of urea. NBUC and NBC were prepared to contain 2.8 g/L of calcium chloride. Urea and calcium chloride solutions were filter sterilized and separately added to phenol red containing NB before bacterial inoculation. A urease enzyme reagent obtained from Blood urea nitrogen kit (Biosystems, Spain) was used as a positive control, while Escherichia coli ATCC 25922 (urease negative) was used as a negative control. Non-inoculated tubes were used as a validity control. All tubes were incubated at 37°C for 24 h. The trial was performed in triplicate. Bacterial isolates characteristics After the incubation of soil samples cultures, one to four colonies of isolated bacteria were selected from those plates containing few and well isolated colonies. Colonies were creamy white or pale yellow to bright orange colored and slightly convex with an entire margin. Selection of the suitable urease producing isolates By culturing all isolates on 5, 8, 10, 12, and 15% urea agar media, those that tolerated ≥ 10% urea concentration has been chosen to proceed with. Bacterial Identification and biochemical characterization All selected isolates were spore-forming, Gram-positive bacilli, catalase and urease positive. Table 2 shows the phenotypic characteristics of the eight isolates and indicates the biochemical tests that have been used in the identification process. ABIS online Software has been used in bacterial identification (Costin and Lonut, 2017). Table 3 shows the presumptive identification of the selected isolates according to the ABIS online Software. Growth conditions The optimal pH at which all selected isolates showed the highest turbidity and rapid growth ranged from 7 to 10, with a preference to the pH 9 (Figure 1), thus all tested isolates are moderate alkaliphiles. For most isolates, the pH of media has increased during growth to reach the maximum of 9 to 9.5. All isolates showed significant growth at temperature 37°C (Table 4). Most isolates showed halophilic characteristic as they grew at NaCl concentration up to 5%. Urea tolerance for bacterial isolates after UV exposure In general, most isolates showed improved tolerance to urea concentration after exposure to UV light when difference of tolerance to urea between wild type and mutant form. Mini-scale calcium carbonate precipitation experiment NBUC tubes for all live isolates, and the pure urease enzyme showed change in pH from neutral to alkaline (yellow to pink), and a precipitate of calcium carbonate were noticed at the bottom of the tubes. NBU tubes for all live isolates and urease enzyme showed only change in pH. NBC tubes for all live isolates and urease enzyme showed neither a change in pH nor calcium carbonate precipitation. Changes in color and pH indicate ureolytic activity (Table 6). A comparison of NBUC of live bacteria versus killed isolates (both killed cells and supernatant) and E. coli, all of them were unable to change pH, so there was no urea hydrolytic activity due to the absence of the enzyme. Consequently, there was no calcium carbonate precipitation. Unchanged non-inoculated tubes suggests that results obtained are reproducible and representative (Table 6). Precipitate containing and non-containing tubes were examined under light microscope to confirm the presence of calcium carbonate crystal (Figure 2). DISCUSSION The present study was conducted to isolate, characterize, and optimize locally adapted urease-releasing bacteria that inhabits urea rich soils. Microbial activity that involves the cleavage of urea into ammonia and carbon dioxide by the urease enzyme, leading to the precipitation of carbonate ions as calcium carbonates. This potentially useful application explains the need to enhance urease production by various methods among candidate microorganisms (Vempada et al., 2011). The biochemical profile of the selected isolates showed that all isolates belong to the genus Bacillus (Table 3). This is similar to a previous study that isolated and characterized urease positive bacteria from urea rich soils, in which several isolates were mostly related to the Bacillus group (Ali et al., 2020). Despite the differences in their characteristics, the obtained isolates showed similar behavior in their ureolytic capability. This is in agreement with the findings of Stocks-Fischer et al. (1999); Hammes et al. (2003) and Stabnikov et al. (2011) that reported the same ureolytic Bacillus strains that can be isolated and cultivated using the same followed protocols of isolation and cultivation. Phenotypic and biochemical profiles of the isolates were matched to those Bacillus species reported previously that proved active in MICP process (Stocks-Fischer et al., 1999;Elmanama and Alhour, 2013). Ureolysis-driven MICP is a phenomenon that has many applications for biochemical and engineering purposes (Omoregie et al., 2020). It has been widely investigated for soil stabilization, healing of concrete cracks, restoration of limestone surfaces, preventing soil erosions, and treatment of industrial wastewater and removing heavy metals (Whiffin et al., 2007;Sarda et al., 2009;Van paassen, 2009;De Muynck et al., 2010a;De Muynck et al., 2010b;Wu et al., 2019). All obtained isolates showed ureolytic activity, tolerance to high urea concentrations, as well as calcium carbonate production. This suggests that isolates are potential candidates for the applications of MICP. Isolates 10.1 that was identified as B. mycoides, has been previously isolated and showed an efficient role of increased sand consolidation and compressive strength of cement (Elmanama and Alhour, 2013). Isolate 8.3 has been identified as B. licheniformis, has been reported in a previous study that it was able to precipitate calcium carbonate by ureolysis (Helmi et al., 2016). Bacteria are previously known to breakdown urea in order to: (1) elevate the ambient pH (Burne and Marquis, 2000), (2) consume it as a nitrogen source (Burne and Chen, 2001), and (3) use it as a source of energy (Mobley and Hausinger, 1989). The amount and rate of urea that can be cleaved were influenced by the urea and calcium source (Wang et al., 2017). In this system, urea is the source of the carbonate. The more urea is supplied, the more CaCO 3 can be produced, if a sufficient amount of calcium ions is available (Wu et al., 2019). In this study, isolates that were selected tolerated and grew in the presence of 10 -15% urea concentration. This because urease activity, as well as, calcium carbonate production rate depend on urea concentration. A previous study utilized S. pasteurii and emphasized the role of urea containing cultural medium in the proliferation of bacteria. Moreover, it reported that bacteria cultivated with urea displayed a healthier cell surface and more negative surface charge for calcium ion binding than the bacteria have been cultivated without urea (Ma et al., 2020). Increasingly, it has been reported that the bacterial concentration and ureolytic activity are important contributors in the efficiency of MICP process. The urea hydrolysis is an extremely slow process, whereas the presence of urease enzyme can substantially increase the hydrolysis of urea (De Belie et al., 2018). Therefore, the selection of the bacterial isolates with higher ureolytic activity is desirable for the higher production of calcium carbonate. However, it has been shown that when the content of urea is excessive, bacterial growth and ureolytic activity are inhibited. For instance, when the urea concentration was greater than 0.75 mol/L, the amount of urea breakdown was decreased and thus appears as an inhibitory component. The reason could be due to too high urea molecule transportation over the cell A B membrane into the cell, at elevated urea concentrations, inhibiting other cellular processes. Therefore, a certain amount of bacteria can only metabolize a certain amount of urea hydrolysis (Wu et al., 2019). In our study, the local isolates were halo-tolerant, and corroborate with the findings of previous studies (Stabnikov et al., 2013). The observation of the pH tolerance profile of bacterial isolates showed a common moderate alkaliphile property. The best growth was at pH range 7-10 with a preference to pH 9. This is in agreement with a previous study that showed the good alkali tolerance of B. cereus which was successfully used to heal concrete cracks (Stabnikov et al., 2013;Wu et al., 2019). Generally, the optimal pH range for bacterial growth is 7 to 8. Under higher alkaline conditions (pH 9 -12) bacteria can still grow but at a much-declined rate. Although the pH is relatively high in fresh concrete, the pH at cracks may drop to 8-11 due to carbonation, exposure, and humidity (De Muynck et al., 2010a). Above pH 11, the bacteria have a limited capacity to precipitate CaCO 3 , thus limited ability to heal cracks. This implies that bacterial spores will keep dormant after being embedded in the concrete matrix (pH > 12), and only start to become active after cracks appear and crack surface pH drops (Wang et al., 2017;Wu et al., 2019). Therefore, alkaline pH is the primary factor by which bacteria promote calcite precipitation (Castanier et al., 2000;Fujita et al., 2000). Another study showed that the calcium carbonate yield (mg calcium carbonate/CFU) in the presence of Bacillus species increases when bacteria grown at a relatively high pH in compared with those bacteria that grown at uncontrolled pH solution (Seifan et al., 2017). Another study investigated the factors affecting the S. pasteurii induced biomineralization process, reported that the rise in medium pH to 9.5 accelerate bacterial growth (Ma et al., 2020). This may be promising that Bacillus isolates in this study can be used to heal concrete cracks. Especially, in the pH range of 7-11, bacteria will have a remarkable ureolytic activity, which ensures the decomposition of urea and the precipitation of CaCO 3 . This meets also with (Phang et al., 2018) findings, It has been reported that some bacterial ureases exhibited high activity in alkaline conditions at pH of 9. In the present study, the effect of temperature on isolates growth showed a temperatures range from 25 to 40°C. Bacterial mediated urea hydrolysis is an enzymatic reaction controlled by many factors including temperature. It has been reported in the literature that temperature affects bacterial activity, urease activity, and therefore reaction rate. Hence, the rate of formation of biogenic CaCO 3 and crack healing efficiency will be affected as well. Urease activity is stable between 15 and 25°C, and an increase in temperature (until 60°C) results in increased urease activity (Whiffin, 2004;Peng and Liu, 2019). Isolates that were exposed to UV irradiation were compared with their corresponding wild type isolates for the ability to tolerate higher urea concentrations. Most isolates showed improved tolerance to urea concentration. However, other strains showed a decline in their adaption to urea concentrations. This suggests that the mutagenesis process is random and did not correlate to the time of exposure to UV light. This is similar to the findings of a previous study, which used UV irradiation on S. pasteurii in order to improve urease activity (Wu et al., 2019). The established calcium carbonate precipitation process showed that all NBUC tubes containing the viable isolates showed accompanied ureolytic and calcite precipitation activity. On the other hand, NBUC tubes containing the autoclave-killed isolates (pellet or supernatant) showed neither ureolytic nor calcite precipitation activity. This suggests that bacteria activity and urease positivity is a principal contributing to pH change due to urea cleavage, as well as calcium carbonate precipitation. In all NBC tubes inoculated with the viable isolates there was no calcium carbonate precipitation observed. This suggests that calcium carbonate production is enhanced by the change of pH. In NBC tubes (without urea), there was no difference in color change or calcium carbonate precipitation between live bacteria, killed bacteria, or E. coli. All NBU tubes inoculated with the viable isolates showed a change in pH as a proof for the ureolytic activity they possess. Negative control (E. coli) showed no change in pH or calcite production. These findings matched a previous study that reported the ability of urease producing bacteria S. pasteurii to produce calcium carbonate crystals under the same conditions (Ghosh et al., 2019). This is in agreement with the previous studies that reported that Bacillus sp. is with high respect in compared with other genus and that this might be due to their physiological ability to adapt to stressed conditions (Helmi et al., 2016). In conclusion, this study successfully and easily isolated several Bacillus species from locally collected soil samples. These strains are alkaliphile, grow well at pH 7-10, and tolerate high urea concentrations. They showed calcite biomineralizing properties and may be employed in bacterial remediation of concrete cracks, increasing the compressive strength of concrete, decreasing water permeability, and solve the problems of soil erosions. Further studies on a larger scale are recommended to confirm the findings.	v3-fos-license
2018-08-13T13:04:43.119Z	2018-08-13T00:00:00.000	51965916	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fpls.2018.01081/pdf", "pdf_hash": "e2888e7267437fd8ade61a59ce2491e96947745b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:34", "s2fieldsofstudy": [ "Medicine", "Chemistry", "Environmental Science" ], "sha1": "e2888e7267437fd8ade61a59ce2491e96947745b", "year": 2018 }	pes2o/s2orc	AromaDb: A Database of Medicinal and Aromatic Plant’s Aroma Molecules With Phytochemistry and Therapeutic Potentials In traditional, herbal medicine, and aromatherapy, use of essential oils and their aroma compounds have been known since long, for the management of various human diseases. The essential oil is a mixture of highly complex, naturally occurring volatile aroma compounds synthesized by medicinal and aromatic plants as secondary metabolites. Essential oils widely used in pharmaceutical, cosmetic, sanitary, food industry and agriculture for their antibacterial, antiviral, antifungal, antiparasitic, insecticidal, anticancer, neuroprotective, psychophysiological, and anti-aging activities. Moreover, volatile aroma compounds comprise a chemically diverse class of low molecular weight organic compounds with significant vapor pressure. However, aroma compounds produced by plants, mainly attract pollinators, seed dispersers and provide defense against pests or pathogens. However, in humans, about 300 active olfactory receptor genes are involved to detect thousands of different aroma compounds and modulates expression of different metabolic genes regulating human psychophysiological activity, brain function, pharmacological signaling, and therapeutic potential. Keeping in mind this importance, present database, namely, AromaDb (http://bioinfo.cimap.res.in/aromadb/) covers information of plant varieties/chemotypes, essential oils, chemical constituents, GC-MS profile, yield variations due to agro-morphological parameters, trade data, aroma compounds, fragrance type, and bioactivity details. The database includes 1,321 aroma chemical structures, bioactivities of essential oil/aroma compounds, 357 fragrance type, 166 commercially used plants, and their high yielding 148 varieties/chemotypes. Also includes calculated cheminformatics properties related to identification, physico-chemical properties, pharmacokinetics, toxicological, and ecological information. Also comprises interacted human genes affecting various diseases related cell signaling pathways correlating the use of aromatherapy. This database could be a useful resource to the plant’s growers/producers, an aroma/fragrance industrialist, health professionals, and researchers exploring the potential of essential oils and aroma compounds in the development of novel formulations against human diseases. In traditional, herbal medicine, and aromatherapy, use of essential oils and their aroma compounds have been known since long, for the management of various human diseases. The essential oil is a mixture of highly complex, naturally occurring volatile aroma compounds synthesized by medicinal and aromatic plants as secondary metabolites. Essential oils widely used in pharmaceutical, cosmetic, sanitary, food industry and agriculture for their antibacterial, antiviral, antifungal, antiparasitic, insecticidal, anticancer, neuroprotective, psychophysiological, and anti-aging activities. Moreover, volatile aroma compounds comprise a chemically diverse class of low molecular weight organic compounds with significant vapor pressure. However, aroma compounds produced by plants, mainly attract pollinators, seed dispersers and provide defense against pests or pathogens. However, in humans, about 300 active olfactory receptor genes are involved to detect thousands of different aroma compounds and modulates expression of different metabolic genes regulating human psychophysiological activity, brain function, pharmacological signaling, and therapeutic potential. Keeping in mind this importance, present database, namely, AromaDb (http://bioinfo.cimap.res.in/aromadb/) covers information of plant varieties/chemotypes, essential oils, chemical constituents, GC-MS profile, yield variations due to agromorphological parameters, trade data, aroma compounds, fragrance type, and bioactivity details. The database includes 1,321 aroma chemical structures, bioactivities of essential oil/aroma compounds, 357 fragrance type, 166 commercially used plants, and their high yielding 148 varieties/chemotypes. Also includes calculated cheminformatics properties related to identification, physico-chemical properties, pharmacokinetics, toxicological, and ecological information. Also comprises interacted human genes affecting various diseases related cell signaling pathways correlating the INTRODUCTION Both flowers and leaves of plants emit aroma compound that moved through the air and is detected by the olfactory system of animals. The complexity of aroma is still challenging because smell of any flower or plant is not due to a single chemical compound. Although plants and flowers consist of many chemical compounds, all of them do not contribute in the aroma. As the rose scent majorly influenced by major constituent compound (-) -cis-rose oxide and minor constituent compounds, namely, beta-damascenone (family -rose ketones) and betaionone of the plant's essential oil, while other compounds like geraniol, nerol, (-) -citronellol, farnesol, and linalool contributions as minor. Aroma is a mixture of volatile compounds with a molecular weight <300 and high vapor pressure, but the complete group of volatile compounds comprises thousands of inorganic and organic compounds stemming from major pathways of secondary metabolism (Frauendorfer and Schieberle, 2006). There are three significant pathways which involved in the biosynthesis process of the main aroma components in plants, such as the shikimic acid pathway by which eugenol (cloves) biosynthesized, degradation of lipids for the formation of short-chain alcohols and aldehydes and terpenoid pathway by which geraniol (Rose) and menthol (Peppermint) are synthesized. These aroma molecules act as semiochemical (mixture or chemical that carries a message for the purpose of communication), pheromones, defense mechanism, allow animals to recognize and detect individuals. The pheromone aroma molecules are essential for mating choices, sexual behavior, fertilization, and nursing and to warn kin in situations of danger (alarm pheromones) and to defend against predators. Aroma molecules are also involved in communication and interaction like plant-plant interaction (Das et al., 2013) and plant-animal interactions (Herrera and Pellmyr, 2002). These interactions and communications were performed through pollination (Schaefer et al., 2004), while another property of aroma molecule is a plant's defense response against herbivores (Glaum and Kessler, 2017). Bacteria also emit a wealth of aroma molecules with an influence on plants, fungi, animals, and bacteria (Vet and Dicke, 1992;Heil and Silva Bueno, 2007). Aroma compounds can also be found in food, spices, perfumes, wine, fragrance oils, and essential oils. These molecules form biochemically during ripening of fruits and other crops. Aroma fragrance molecules are of great commercial interest, resulting in many applications of volatile aroma molecules in research, health, food, cosmetic, and health industries. A comprehensive information of plants based aroma molecules (2D and 3D structures), aroma or fragrance type, essential oils, respective commercial plants varieties available for cultivation in India and abroad, therapeutic potential in terms of biological activities, physicochemical, stereo, and pharmacokinetics (ADMET) properties of aroma molecules, interacted human genes and corresponding essential oil's export/import trade data trend information are not publicly available for both scientific research and industrial use. It was, therefore, our goal to develop a database AromaDb which cover basic and advance information of aroma molecules and provide a platform for comparative analysis and quantitative structure aroma relationship studies (QSAR). The AromaDb database would be helpful to answers the queries of researchers, industries, and growers related to aroma compounds and essential oils. However, a variety of database resources of, essential oils, scent, fragrance and flavor (synthetic) components are already reported but still limited at some extent focused on certain subgroups, for example, SuperScent (Dunkel et al., 2009), OdorDB, Pherobase, EssOilDB (Kumari et al., 2014); ScentBase 1 , AroChemBase 2 , and Flavornet 3 ( Table 1). Consequently, these databases are good but useful for special purposes, and therefor there is a need for a comprehensive listing of commercially important volatile aroma molecules found in plant's essential oils from different local and globally grown aromatic plants with information of chemotype-specific varieties, type of fragrance (aroma), physicochemical properties of aroma molecule, chemical identification, chemical structures (2D & 3D) for free downloads, Pharmacokinetic properties (ADMET), effect of aroma on human genes, their vapor pressure and logP, " and export and import trade data trends in terms of global demand and business turnover in India and abroad, so that to guide the future research directions for researchers, growers, farmers, and industries. Data Selection and Resources Data were retrieved from the national and international literature and various web database resources or papers published in national and international journals. The database includes records from more than 100 scientific journals related to aroma (fragrance) and flavor. Following web resources were used to select the required data, such as selection of floral (Baldwin et al., 2006) ( Table 2). The abstracts were screened searched against chemical names and synonyms of chemical compounds on given literature search engines. The information regarding adverse effect or allergic responses (skin irritation toxicity) by some aroma molecules is covered in the AromaDb database under fields "physical and ADMET properties" which was calculated through TOPKAT module of Discovery Studio v3.5 software (Accelrys, San Diego, CA, United States). Compounds human genes interaction data was retrieved from the EPA (United States Environmental Protection Agency) 4 and ACToR 5 information portal by searching all compounds using CAS ID. Database Development Backend Information AromaDb database is developed on Apache HTTP server, which is platform independent and available as open-source software. The database is developed on MySQL for storing the information in the backend. The database website front end is developed in PHP, HTML, CSS, and JavaScript. AromaDb comprises basic and advances molecular information retrieved from different resources as shown in Figures 1A,B. Structure Similarity Search Tool Application and Structure Editor Structure editor and structure similarity search tools in AromaDb for structural similarity within the database and from the extended database, i.e., ChemAxon SMILES based search option was used, for displaying 3D structures, JSmol was used which is available as open-source JavaScript viewer for chemical structures 4 https://www.epa.gov/ 5 https://actor.epa.gov/actor/home.xhtml in 3D 6 . JSmol is the extension of the Java-based molecular visualization applet Jmol 7 as an HTML5 JavaScript-only web app. Similarly, JSME (JavaScript Molecular Editor) was applied for the built-in molecule editor, which allows the user to screen with self-edited molecules. JSME is a free molecule editor written in JavaScript 8 . Database Content The major parameters in the database were plants, variety, essential oils, chemical molecules, chemical group, trade data, structural data, and biological activity of essential oil, compounds and their interacting genes, etc. For better performance, all the data is kept distributed in several interrelated tables logically. For wide complex searching search engine was used to check each link of the database website and accordingly showed the matched results ( Figure 1B). All the information in AromaDb database can be categorized into three categories: primary, secondary, and tertiary information. The database information was manually curated and selected from various sources such as published international literature, CSIR-CIMAP, Lucknow 9 essential oils monograph, web link annual reports, journal (JMAPS; Journal of Medicinal and Aromatic Plant Sciences; Baldwin et al., 2006), newsletters, and books. The primary information has been retrieved from the literature, these information's consists of the following major fields namely: (i) plant details (ii) essential oil name, and (iii) plant variety. The secondary information, which was derived from the plant essential oil (primary information) includes the essential oil description details, chemical constituents, major minor compound details, content percentage, and export-import essential oil trade data. The tertiary information is further derived from secondary information which comprises detailed information about the essential oil compounds as followed: IUPAC name, Chemical class biochemical classes (e.g., terpene hydrocarbons and oxygenated compounds: phenols, alcohols, aldehydes, ketones, esters, lactones, coumarins, ethers, and oxides), Fragrance type, Physical and chemical properties, Absorption and Metabolism information, Toxicological information, Ecological information, Hazards information, and compound bioactivity data for therapeutic use or drug formulation development. This information provides the user a comprehensive aroma molecules database, together with substantial options, such as chemical compounds search based on structural similarity using compound Canonical SMILES within databases. Figure 2 summarizes snapshots of database home page (Figure 2A), plant and varieties details ( Figure 2B), and essential oil details ( Figure 2C). Results of Structure Editor and Structure Similarity Search Tools The AromaDb database provides the diverse and commercially important aroma molecules, fragrance types, essential oils, aromatic plants, trade data, and other industrially important information as required while formulating any herbal product formulation for human uses. We have incorporated various tools for making the database easy and more convenient for users. The database contents could be accessible with different search tools, options, e.g., simple and advance search tools. The simple search tool is powered by Google search engine, which search data from within the AromaDb database and also from public databases, where another mining tool search the query within the database, the Advance search tool option further divided into two types: (i) search based on structural similarity (within AromaDb database) and (ii) search based on structural physicochemical and pharmacokinetics (ADMET) properties. The database search fields enable the user to look for compounds using physicochemical properties, plants, varieties, chemotype, essential oil, aroma molecules, chemical classification, biological activity, etc. Database allows the user to choose certain functional groups, species or range of molecular weights, which search whole entries and retrieve the user required results. To mine the database user require a 2D molecular structure or structure canonical SMILES code (Simplified Molecular Input Line Entry System) or a MOLfile of interested aroma compounds. Structure drawing/editing option is provided in the AromaDb database with the help of JSME (JavaScript Molecular Editor; JSME Homepage 10 ). With the help of JSME user can draw either full structure or part of it. The most similar database entries are listed in the order of structural similarity. For each compound, details of plant, plant name, variety, essential oil constituents, chemical class, ADMET properties, 2D/3D chemical structures, trade data, and the SMILES based similarity search percentage are presented. Furthermore, a similarity search to find the most similar compounds analogs are provided. Additionally, similar information can be viewed separately by accessing database header fields or the menu. Information About Putative Therapeutic Free Human Targets or Genes in AromaDb The other useful feature of the AromaDb database is the information of aroma molecules and related interacting human therapeutic genes or proteins involved to modulate the biological system biological pathways and for unraveling the underlying mechanism of action and therapeutic potential of these aroma molecules along with supporting references. Also enlisted the therapeutic important aroma molecules along with the option to see the related aromatic plants, essential oils, export and import trade data, and other information. Display of Essential Oil Trade Data Information The graphical analysis of trade data suggests the consumption and demand data trend in India and the world or own country (India) as a guideline or forecasting tool to the aroma or essential oil industries and growers (farmers or producers) to streamline their resources, economy, and time according to expected demand of respective essential oils in the world and therefore indirectly helps to improve the socioeconomical condition of farmers and producers. Here, the database entries have been clustered according to the quality of their aroma properties. A manually verified upload option through email allows the scientific community to contribute to the database. Here, the user can import a MOL-file together with corresponding information of the compound. The AromaDb database will be updated on a quarterly basis. Comparative Display of Physical Properties and Safety Prediction Data The database features the properties related to aroma molecule, hazard identification, exposure controls and personal protection, physical and chemical properties, toxicological and ecological information. Compound identification properties include chemical name, IUPAC name and chemical class. Hazards identification includes properties related to physical hazards, health hazards such as skin corrosion and ocular irritancy property. Toxicological properties such as irritation, absorption level, aqueous solubility level, LogP (octanol/water partition coefficient), polar surface area (PSA), and blood-brain barrier level indicate the precautionary properties related to exposure controls and personal protection (eye/face protection, skin protection, respiratory protection, and thermal hazards). Physical and chemical properties indicated by molecular weight, molecular formula, fragrance type, LogP (n-octanol/water), H-bond donor and H-bond acceptor, rotatable bond, topological polar surface area (TPSA), IUPAC name, InChIKey, aqueous solubility level, vapor pressure, PubChem (NCBI, United States) (Wang et al., 2009) database ID and SMILES information. Toxicological information includes properties related to information on likely routes of exposure such as inhalation, skin contact, eye contact, ingestion, symptoms related to the physical, chemical and toxicological characteristics, information on toxicological effects, e.g., acute toxicity (LD 50 -dermal/oral, rabbit/rat, mg/kg), skin corrosion/irritation, serious eye damage/eye irritation, respiratory or skin sensitization, germ cell mutagenicity (genotoxic), carcinogenicity, reproductive toxicity, specific target organ toxicity (single exposure/repeated exposure), and aspiration hazard. However, the ecological information includes properties related to eco-toxicity (environmentally hazardous), persistence, and degradability. Since these aroma molecules used in aroma based natural therapy (aromatherapy) in traditional herbal medicine system of India (Ayurveda and Unani) and other East Asian countries, therefore, database covered properties related to pharmacokinetics such as absorption, distribution, metabolism, excretion, and toxicity (ADMET). These properties indicate drug-likeness properties, ADME compliance, and toxicity risk assessment data. Moreover, the database also includes information related to the molecular interaction of aroma molecules/essential oils with human proteins (genes) directly or indirectly affecting the metabolic processes and therefore causing their useful biological activity or responses. This information is supported with cross-references or proper evidence based on reported publications and therefore offer a possibility for biological interpretation of these aroma molecules. Moreover, an investigation of medical effects is also possible by browsing the "biological activity" field for each essential oils/aroma molecules. The comparative trends or pattern plots shows the emitted aroma compounds of the chosen species compared with all other plants species, which emit these compounds. The comparative data analysis through graph plots shows the unique pattern of aroma molecules (fingerprints), essential oil, yield, major constituent percentage (chemotype), trade analysis trend, and others based on changes in different agromorphological parameters, e.g., soil type, stress conditions, temperature, weather type, months wise oil yield, etc., which are important feature for distinguishing between more or less useful features/parameters during agriculture practices and/or cultivation by farmers, or industrial growers. As an example, a snapshot represents these calculated properties of aroma showing molecule, chemical identification, description and 3D structure for download snapshot represented in the given figure ( Figure 3C). DISCUSSION It was difficult to explore such information about plants, its variety, essential oil constituents, and essential oil trade data, on a single platform in addition to the aroma molecules information about its physicochemical properties, absorption and metabolism information, toxicological information, ecological information, Hazards identification, and compound biological activity with its fragrance and interacting human genes information. Browsing and Searching Database Contents Database contents can be seen in two ways: (i) browsing different data fields available on the header menu on the home page such as, plants, essential oils, aroma molecules, chemical classes of aroma molecules, biological activity, and trade data, and (ii) searching database contents through advanced search option and wild search through Google's custom search option. Users can search any text within the database based on text similarity concept through Google's custom search option (wild search or complex search) available in the header of the database. A similar analysis with the help of representing snapshots of the database showing database home page search parameters (Figure 2A), plant and varieties details (Figure 2B), and essential oil details ( Figure 2C). Properties Search by Entering Values Besides, users can also limit searches using ranges of physiochemical data, e.g., maximum and minimum values of TPSA (Topological Polar Surface Area) (Figures 3A-C). Database snapshots of these properties and parameters showing chemical constituents of essential oil, GC-MS data and months wise variations in oil yield (Figure 3A), essential oil trade data and comparative graphical plots (Figure 3B), and aroma molecule description and 3D structure for downloads ( Figure 3C). Similar Structures Search and Downloading of Aroma Molecules Users can search and download interested aroma molecules (2D and 3D chemical structures) through structure search option. In this option, users can draw or edit any structure of their interest and convert it to SMILES and MOL file format and subsequently search whole AromaDb database aroma molecules and resulted most similar matched structures available in the database and can easily see details of these matched small molecules or free downloads the structures. For example, if the user draws the phenyl ring in the JME editor and enter the option either "get SMILES" or get MOL file' resulted in SMILES or MOL file data would be shown on the other side of JME editor and subsequently enter the key similarity search within database, results of matched structures showed in the new web page, with no exact match aroma molecule found, and enlisted the similar matched compounds, e.g., (E)-cinnamyl acetate, 1,1-diisobutoxy-2-phenylethane, 1,1-dimethoxy-2benzylideneheptane, etc. All these matched structures have phenyl ring in common. Moreover, the user can see further details of each matched compound by entering key on "details" button. Details of aroma compound include calculated properties related to aroma molecule identification, hazards, physical and chemical properties, pharmacokinetic properties, toxicological information, and ecological information. The database also represents the 2D structure, 3D structure visualization in 3D structure viewer window with spin-on/off option so that to see the structural conformations quickly with free download option. The compounds search outputs are oriented to explore the matched compounds by following the "details" hyperlink to the table of physical, ADMET and safety properties described earlier. Figure 4 showing snapshots of advanced search options based on different properties, fragrance type and toxicological parameters of aroma molecule (Figure 4A), drawing tool for structure-based search option as well as SMILES or MOL file basis (Figure 4B), and results of structure-based search based on SMILES basis structural similarity (exact match and similar hits; Figure 4C). Search by Selecting Data Fields Similarly, naive users can directly see and select the interesting data through search menu buttons provided at the end of the home page of aroma database and the further user may browse more information without wasting time on thinking about aroma (fragrance), aroma molecules name, essential oils, plant varieties, and plants. This type of search display option will help the fresh researchers, scholars, and students. For example, a new user can directly browse the home page header menu field "Plants, " which will display the list of all aromatic plants with their common and scientific names and a number of their available plant varieties or highly yielding chemotypes. Users can see more details of these plants by clicking on the button "View Detail." For example, if the user wishes to search for Menthol mint plant (Mentha arvensis), which included at present 11 mint plant varieties or chemotypes based on different aroma molecules constituent's ratio variations. Users can see more details such as brief introduction, family, localization, uses, essential oils types, and the name of 11 mint varieties used by Indian growers or farmers. Users have the option to print or save this data. In this page, users have two options to see further details; (i) essential oil types and (ii) variety details. For example, if the user searches for M. arvensis MAS-1 essential oil details, database will display brief introduction, major constituent Menthol with 84% with multiple minor components such as Menthone (5.8%), etc., comparative graph plot showing percentage ratio of different chemical constituents of M. arvensis MAS-1 essential oil (Figure 2C), and the Indian scientists or researchers contribution, if any, showed in the references. Users have the option to print (or save) this data and graph plot. Also, the user can directly move to see details of major or minor compounds and yield percentage by simply enter on the name of compounds. Moreover, if the user enters on M. arvensis variety Gomti button, the database will display a tabular text data showing brief details of plant, variety name, major constituents of given variety such as Menthol 74% in ratio, menthone 12.6%, isomenthone 3.7%, and methyl acetate 2.9%. Beside this database show details of essential oil, GC-MS graph (if available) of essential oil, major compounds peak in GC data, compound property, year of plant variety release and complete reference (Figures 3A-C). Agronomic Parameters Based Gaphical Data Analysis At the same time, the database also covers data related to different agronomic parameters based and provide an option to the user to see either in tabular or graphical forms. For example, in the case of M. arvensis Gomti variety, the database covers five observation readings revealing variations in Z-Linalool oxide compound percentage ranges from 2.2 to 2.7% from January to May 2015. This data suggest that the highest yield of Z-Linalool oxide was 2.7 February. Likewise, the user can see other parameter based graph plot analysis and get benefited from future cultivation, for example, if the user sees the Himalaya variety of M. arvensis, there are five readings based on the age of the plant (in days) and menthol compound yield percentage. This data showed that the study was performed for 30 days old plants to 150 days old plant (menthol mint) and menthol yield ranges from 70.66 to 82.18%. Graphical analysis revealed that highest yield obtained at 120 days old plant (Figure 2). A similar analysis with the help of representing snapshots of the database showing database home page search parameters (Figure 2A), plant and varieties details (Figure 2B), and essential oil details ( Figure 2C). CONCLUSION The AromaDb database is a useful tool to retrieve information about aroma molecules, aroma or fragrance types, essential oils, plants varieties, bioactivity of essential oils or aroma molecules, toxicological and ecological data, and trade data. The database provides, 3D structures of aroma compounds for free downloads and option to see the essential oil yields or constituents percentage variation trends at different agromorphological conditions during plant growth. The included data on aroma molecules along with a focus on associated plants and their essential oils chemotype (varieties) will enable systematic experimental approaches on the relation between structural similarities, essential oils, and aroma (fragrance) type and aroma chemical classes. Besides, last 18 years global export and import trade data of plants essential oils will educate the growers or farmers to prioritize the cultivation of aromatic plants based on expected global demand. Furthermore, structure comparisons of self-edited molecules with the database aroma molecules as well as the external database may allow a first rough estimation of the potential aroma of new chemicals. The AromaDb database is a free resource with embedded screening functions for aroma molecule based on molecular weight, plants, varieties, essential oils, fragrance or aroma type, toxicological and ecological information (allergic or toxic responses). AUTHOR CONTRIBUTIONS YK carried out the database entry, data searching and retrieval, download, designed 2D and 3D structures, participated in the data analysis, and manuscript writing. OP developed the preliminary database offline using PHP and MySQL for initial data entry. HT add the trade data of essential oil based plants commodities and economic profile in Indian currency. ST provided 25 aroma molecules to the database and other properties. MG also provided small molecules and corrected chemical classes of aroma molecules and corrected the chemistry part. L-UR provided data related to Indian plants varieties, essential oils details, constituents, and contributed in the design of the database. RL guided to add published in-house data related to aroma molecules, essential oils, aroma plants, and bioactivity. MS coordinated in database web hosting and IT support. FK conceived the study, contributed to its design, ER relationship, and drafted the manuscript. MD Addition of updated bioactivity (in vitro) data for essential oil/aroma molecules with their cros references. All authors read and approved the final manuscript. ACKNOWLEDGMENTS We are thankful to the Director, CSIR-CIMAP, Lucknow, India for rendering essential research facilities and support. YK, OP,	v3-fos-license
2018-04-03T04:27:43.190Z	2002-09-13T00:00:00.000	39043069	{ "extfieldsofstudy": [ "Chemistry", "Biology", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/277/37/34287.full.pdf", "pdf_hash": "1c9b4ef8f9b35ff6d4dc17f32b10e6a399a97e9d", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:35", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "2dc3746e1c9fa93b12a978063bc88142f68d2cf3", "year": 2002 }	pes2o/s2orc	An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner. The caspase protease family plays a central role in the implementation of apoptosis in vertebrates (4,5). Caspases are constitutively expressed in healthy cells, where they are synthesized as precursor proteins (procaspases). Caspases are activated upon processing of procaspases into ϳ20-kDa (p20) and 10-kDa (p10) mature fragments, in addition to the N-terminal prodomain. The caspase family is broadly divided into two groups: initiator caspases (caspase-8, -9, and -12) and effector caspases (caspase-3, -6, and -7). Initiator caspases undergo autoprocessing for activation in response to apoptotic stimuli. Active initiator caspases in turn process precursors of the effector caspases responsible for dismantling cellular structures. Recent studies have suggested the existence of a novel apoptotic pathway in which caspase-12 functions as the initiator caspase in response to a toxic insult to the ER, 1 such as by treatment with tunicamycin (an inhibitor of glycosylation), thapsigargin (an inhibitor of the ER-specific calcium ATPase), or calcium ionophores (1). Caspase-12 is specifically activated in cells subjected to ER stress. Furthermore, caspase-12-deficient cells are resistant to inducers of ER stress, suggesting that caspase-12 is significant in ER stress-induced apoptosis (1). ER stress has received growing attention because it is considered a cause of pathologically relevant apoptosis, and it is particularly implicated in neurodegenerative disorders (6). However, the mechanism of caspase-12-mediated apoptosis has been unknown, mainly due to the lack of identification of caspase-12 substrates. In this study, we have examined the susceptibility of procaspases to active caspase-12 and have shown that procaspase-9 can specifically be cleaved by caspase-12 in vitro. Recent studies show that multiple death signals converge on the mitochondrion (7). Damaged mitochondria release cytochrome c, which facilitates conformational changes in Apaf-1, the specific activator of procaspase-9 (2, 3). The cytochrome c⅐Apaf-1 complex called an apoptosome (8,9) is thought to recruit procaspase-9 through interaction between Apaf-1 and procaspase-9 and facilitate autoactivation of caspase-9. Active caspase-9 then activates caspase-3, the major effector caspase that is responsible for destruction of various substrates (4,5). Cytochrome c release from mitochondria has also been observed in ER stress-induced apoptosis of several cell lines, including mouse embryonic fibroblast cells (10,11). The in vitro cleavage of procaspase-9 by caspase-12 described above can be achieved in the absence of cytochrome c, suggesting the presence of the ER stress-specific caspase cascade, which comprises caspase-12, -9, and -3 in this order. For examination of the role of caspase-12 in activation of the caspase cascade in vivo, however, it would be desirable to use conditions in which cytochrome c is not released from mitochondria; otherwise, caspase-9 could be activated by the cytochrome c⅐Apaf-1 mechanism, independent of caspase-12. We thus used a murine myoblast cell line, C2C12, to study caspase-12, because our preliminary data showed that ER stress induces the activation of caspase-12 and apoptosis in the cell line without the release of cytochrome c from mitochondria. This result suggests that cytochrome c release is not essential for ER stress-induced apoptosis. We took advantage of the fact that cytochrome c is not released to examine the mechanism of caspase cascade activation in the absence of mitochondrial damage, focusing on events that occur downstream of caspase-12 activation. Examination of Mitochondrial Transmembrane Potential-Apoptosis was induced in C2C12 cells, and then the cells were stained with the MitoSensor reagent (CLONTECH) according to the manufacturer's protocol. Preparation of S-100 from C2C12 Cells-The C2C12 cell 100,000 ϫ g supernatant was prepared according to the method described in Liu and Wang (12). Briefly, cells were disrupted in buffer containing 250 mM sucrose by a Dounce homogenizer. The supernatant was centrifuged in a microcentrifuge for 10 min, and subsequently at 100,000 ϫ g for 30 min in a tabletop ultracentrifuge (Beckman Coulter, Inc.). Yeast Two-hybrid Screening-The split LexA protein system was used for two-hybrid screening according to the method of Brent as described by Gyuris et al. (16). The caspase-12 p10 fragment (Thr 319 -Asn 419 ) was used as the bait for the screening of a HeLa cell cDNA library. From ϳ2 ϫ 10 7 transformants, we obtained 15 positive clones, all of which contained sequences derived from the MAGE-3 mRNA. The 5Ј ends of the cDNAs were located between codons 81 and 94. We cloned the full-length coding region of MAGE-3 (314 amino acids) for further analysis by polymerase chain reaction amplification of a human testis cDNA library (CLONTECH). GST Fusion Protein Pull-down Assay-GST-MAGE-3 protein (1 g) and histidine-tagged caspase-12 (0.1 g) were incubated with 10 l of glutathione Sepharose-4B beads (Amersham Biosciences) for 1 h at room temperature in 150 l of 20 mM phosphate buffer, pH 7.0, containing 200 mM NaCl and 0.02% Triton X-100. Anti-hexahistidine monoclonal antibody (CLONTECH) was used for the detection of caspase-12 p20. Proteins that bound glutathione resin were analyzed by Western blotting. In Vitro Cleavage of Radiolabeled Procaspases-In vitro synthesis of 35 S-labeled proteins and their detection by autoradiography were achieved as described previously (15). For mutant analysis, mutations at specific aspartic acid residues in procaspases were introduced by the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Introduction of mutation was confirmed by DNA sequencing. 35 S-labeled procaspase (0.2 l of the labeled protein solution) was incubated for a cleavage assay at 37°C with caspase-12 (0.28 g) for 4 h. Resistance of procaspase-12 to cleavage by active caspase-12 was examined by addition of recombinant MAGE-3 to the procaspase-12 cleavage assay solution. Procaspase-12 whose active site Cys residue had been replaced with Ser was synthesized in vitro in the presence of [ 35 S]methionine. 35 S-Labeled mutant procaspase-12 (0.2 l of the labeled protein solution) was incubated with active caspase-12 (0.28 g) for 45 min in the presence or absence of MAGE-3. In Vitro Activation of Caspase-9 in S-100 by Caspase-12-Cytochrome c-free cytosol from C2C12 cells (10 g of proteins) was treated with recombinant caspase-12 p30 (0.8 g) at 37°C for 4 h. Activation of caspase-9 and -3 was examined by Western blot analysis. Five micrograms of proteins were loaded on each lane. As a positive control for caspase-9 activation, the cytochrome c-free cytosol was incubated with 10 M bovine cytochrome c (Sigma-Aldrich) and 1 mM dATP for 60 min at 37°C. For inhibition of caspase-9 activity, LEHD-fluoromethylketone (BioVision, Palo Alto, CA) was added to the cytosol before the addition of caspase-12. Stable Cell Lines-MAGE-3 stable cell lines of C2C12 were generated as follows. MAGE-3 cDNA was cloned into pcDNA3.1(Ϫ) vector (Invitrogen). The plasmid DNA was linearized by ScaI digestion before transfection. Transfection was performed with a Superfect transfection reagent (QIAGEN) according to the manufacturer's protocol. MAGE-3 cDNA cloned into the pcDNA3.1(Ϫ) vector (Invitrogen) was used for stable transfection. Stable transfectants were grown in medium containing 600 g/ml G418 (Invitrogen) for 2 weeks before cloning. RESULTS Procaspase-9 Is a Substrate of Caspase-12-We examined how caspase-12 processing is linked to the activation of other caspases. For an in vitro cleavage assay, we produced recombinant caspase-12 (p30) whose N-terminal prodomain had been removed and replaced with a hexahistidine tag. The p30 protein undergoes efficient autoprocessing into p20 and p10 peptides when overexpressed in E. coli (p30* in Fig. 1A). A mutant p30 (p30C/S), whose active site Cys is substituted with Ser, is not processed in E. coli (Fig. 1A). The mature caspase-12 (p30) exhibits proteolytic activity and cleaves procaspase-12 into 35and 12-kDa fragments (Fig. 1B, lane 16). The cleavage site was located at Asp318, because a procaspase-12 mutant in which Asp318 was replaced with Ser was resistant to caspase-12 digestion (data not shown). Asp318 is also the cleavage site for autoprocessing in E. coli (Fig. 1A), as revealed by amino acid sequencing of p10 by the Edman degradation method (data not shown). p30 cleaves caspase-9 ( Fig. 1, B and C) but not other caspase precursors (murine caspase-1 and -2, and human caspase-3, -6, and -8) under the experimental conditions. Note that processing site sequences between p20 and p10 are highly conserved between murine and human caspase-3, -6, -7, and -8. Mutation analysis of caspase-9 ( Fig. 1C) indicates that caspase-12 cleaves at specific Asp residues in the linker region between p20 and p10 in the procaspase-9 polypeptide (LDSD349 and SEPD353 in the murine caspase-9, PEPD315 in the human caspase-9). Asp 353 of the murine caspase-9 and Asp 315 of human caspase-9 have been reported to be the cleavage sites for the activation of procaspase-9 (17,18). The caspase-9 cleavage observed in vitro thus suggests the possibility that caspase-9 can be activated by caspase-12 during ER stress-induced apoptosis. Under the experimental conditions used, murine caspase-9 contains another cleavage site(s) for caspase-12 in vitro, cleavage at which generates 27-and 20-kDa fragments. We did not further analyze these additional cleavage site(s) because we could detect neither 27-nor 20-kDa caspase-9 fragments in apoptotic cells (described below; data not shown). Procaspase-7 seems to be only slightly processed by active caspase-12 (Fig. 1B, lane 10). The in vitro cleavage of procaspase-7 was not studied further because processing of procaspase-7 is undetectable in C2C12 cells subjected to ER stress (Fig. 1D). Cytochrome c Is Not Essential for ER Stress-induced Caspase Activation-Several reports have demonstrated that ER stress causes mitochondrial damage, which results in cytochrome c release from mitochondria (e.g., Refs. 10,11). Cytochrome c in cytosol and Apaf-1 can induce activation of caspase-9 (2, 3). To examine whether there is an ER stress-specific caspase cascade that is initiated by caspase-12, we used a murine myoblast cell line, C2C12, because this cell line undergoes ER stress-induced apoptosis without cytochrome c release from mitochondria. Cytosolic extracts (S-100) of tunicamycin-or thapsigargintreated C2C12 cells contain cytochrome c at the same level as that detected in S-100 fractions prepared from untreated cells ( Fig. 2A). Nevertheless, more than 50% of the cells undergo apoptosis (see below). Cytochrome c release per se, however, is functional in C2C12 cells, because treatment of C2C12 cells with etoposide or serum deprivation induces apoptosis at a similar level of lethality and with a significant release of cytochrome c. After apoptosis was induced by ER stress inducers, the mitochondrial transmembrane potential was maintained in apoptotic cells (small cells with condensed nuclei), as in the case of untreated cells, which was exhibited by mitochondrial accumulation of fluorochromes and their conversion to emit the orange color (Fig. 2B). Etoposide-treatment of C2C12 cells resulted in decrease in mitochondrial transmembrane potential, which was monitored by the green color of the fluorochromes in the cytosol (Fig. 2B). These results suggest that mitochondria in C2C12 cells do not suffer severe damages from ER stress, thus releasing little cytochrome c into cytosol. Treatment of C2C12 cells with ER stress inducers, either tunicamycin or thapsigargin, results in the processing of procaspase-12 (48 kDa, Fig. 2C) and apoptosis. A 35-kDa fragment was detected by antibodies specific to the p20 region (1). Caspase-9 and caspase-3 are also activated in C2C12 cells treated with ER stress inducers (Fig. 2C). The activation of caspase-3, one of the most downstream caspases, suggests that the ER stress-specific caspase cascade comprises caspase-12, -9, and -3. It has been suggested that calpain is involved in activation of caspases in cultured glial cells after deprivation of oxygen and glucose (19). In the apoptotic C2C12 cells, however, cleavage of a calpain substrate, Bcl-XL, was not detected (Fig. 2C), suggesting that caspase activation in C2C12 cells treated with ER stress inducers is independent of calpain. Direct Activation of Caspase-9 by Caspase-12-We then examined whether caspase-9 activation occurs by the cleavage of procaspase-9 by active caspase-12 without the release of cytochrome c in cell extracts. Incubation of the S-100 fraction of untreated C2C12 cells with active caspase-12 results in a pattern of cleavage of procaspase-9 that is similar to that observed in S-100 of apoptotic C2C12 cells, the cleavage products being a doublet of 35-kDa fragments (Fig. 3A, lanes 2 and 4). A FIG. 1. Procaspase-9 is a substrate of caspase-12. A, purification of the caspase-12 p30 protein overexpressed in E. coli. Either wild-type p30* or the inactive mutant (C/S) protein was tagged with hexahistidine at the N terminus and purified by Ni-column affinity chromatography. Proteins were detected by Coomassie Brilliant Blue staining. B, procaspase-9 and procaspase-12 are specifically cleaved by active caspase-12. 35 S-Labeled procaspases were incubated with (ϩ) or without (Ϫ) active caspase-12 at 37°C for 4 h and analyzed by SDS-polyacrylamide gel electrophoresis as described previously (15). Arrowheads indicate cleavage products. C, cleavage sites within procaspase-9 are processing sites for activation. Mutation of specific Asp residues (Asp-349 and Asp-353 in murine procaspase-9 and Asp-315 in human procaspase-9, respectively) significantly reduces cleavage by caspase-12 (ϩ). Arrowheads indicate cleavage fragments. D, caspase-7 is not activated in C2C12 cells under ER stress (TG, thapsigargin; TUN, tunicamycin). The Western blot was probed with an anti-caspase-7 monoclonal antibody. control experiment showed that addition of cytochrome c and dATP to S-100 of untreated cells also caused processing of procaspase-9 into 35-kDa fragments that appeared as a doublet on the blot (Fig. 3B, lane 3). The lower band was less intense than the upper band in the case of caspase-12-induced processing (Fig. 3B, lane 2) and the apoptotic S-100 fractions (lane 4). The ratio of these 35-kDa fragments was different from that observed in the cytochrome c-treated S-100 (Fig. 3B, lane 3). It remains to be revealed whether the difference in the ratio of these fragments reflects a difference in mechanism of processing. Suppression of Procaspase-12 Processing by Its Binding Protein-We have recently isolated by yeast two-hybrid screening from a HeLa cell cDNA library a human cancer antigen, MAGE-3, as a protein that specifically binds the caspase-12 p10 fragment (see "Experimental Procedures"). Because MAGE-3 can suppress the activity of procaspase-12, as described below, we used the protein to examine the significance of caspase-12 activation in ER stress-induced apoptosis in C2C12 cells. MAGE-3 is a member of the MAGE gene family and is expressed in various types of tumor but not in normal tissues except for the testis (24). Although the specific interac- tion between caspase-12 and MAGE-3 is intriguing, it remains unclear whether MAGE-3 plays any role in caspase regulation in human cells (see "Discussion"). MAGE-3 does not bind to other caspases, such as caspase-9 (of either murine or human origin), as tested by the two-hybrid assay (results of murine caspase-1, -9, and -11 and human caspase-3, -6, and -7 are shown in Fig. 4A). The MAGE-3 protein can also bind both the caspase-12 p10 fragment and procaspase-12 in mammalian cells. When MAGE-3 is expressed in COS-1 cells by transient transfection it can be co-precipitated with FLAG-tagged p10 (Fig. 4B, lane 3) or FLAG-tagged procaspase-12 (lane 7) using an anti-FLAG antibody. MAGE-3 was not co-precipitated with FLAG-tagged p10 fragments of murine caspase-2 and human caspase-8, whose binding ability could not be examined by the two-hybrid assay because of significant background activity (data not shown). Fig. 4C shows that p30C/S (unprocessed p30) co-precipitates with GST-tagged MAGE-3 (lanes 3 and 4). Under the same conditions, however, p30* (processed) is not efficiently co-precipitated by GST-MAGE-3 (Fig. 4C, lanes 1 and 2), suggesting that MAGE-3 does not efficiently bind the p10 fragment in active caspase-12. It is possible that the p10 fragment within mature caspase-12 is not fully accessible to MAGE-3 because of steric hindrance by the p20 portion. X-ray crystallographic analyses of caspase-1 and caspase-3 have suggested that they undergo a conformational change upon maturation (25)(26)(27). This conformational change may occur in caspase-12 and result in the p10 fragment being less exposed for binding to MAGE-3. Consistent with the binding of MAGE-3 to unprocessed caspase-12, MAGE-3 protects procaspase-12 from cleavage by active p30* in a dose-dependent manner (Fig. 4D, lanes 2-7). Substitution of MAGE-3 with bovine serum albumin fails to inhibit cleavage (Fig. 4D, lane 9). It is less likely that MAGE-3 blocks active caspase-12 by acting as a competitive inhibitor. In Fig. 4D, lane 7, small amounts of the 35-and 12-kDa fragments can be detected, indicating the presence of caspase-12 activity. Under such conditions, excessive levels of active caspase-12 are expected to be protected from inhibition by MAGE-3. However, an enhancement of cleavage was not detected in the presence of 4-fold higher levels of caspase-12 (lane 8). In contrast, when twice as much substrate is added to the reaction mixture in the presence of MAGE-3, both p35 and p12 cleavage products are produced at the same levels as in the absence of MAGE-3 (Fig. 4E, lane 2). It is more likely that MAGE-3 protects procaspase-12 from processing by specifically binding the p10 portion of the precursor. This result is consistent with our observation that the affinity of MAGE-3 for p30C/S is much higher than that for active caspase-12 (Fig. 4C). Suppression of Caspase-12 Activation Resulted in Suppression of Caspase-9 Activation and Apoptosis in Vivo-To examine the involvement of caspase-12 in the activation of the caspase cascade, we established stable transfectants (C2C12/ MA21) of C2C12 cells that overexpress MAGE-3 (Fig. 5A). Colocalization of MAGE-3 with endogenous caspase-12, an ERassociated protein (1), in C2C12/MA21 was observed by double immunostaining (Fig. 5B), although signals of free MAGE-3 proteins (red color) were still evident in the merged image. This observation was supported by a cell fractionation experiment, where MAGE-3 was detected in the microsomal fraction as well as in S-100 (Fig. 5C). Treatment of either parental C2C12 cells FIG. 4. A caspase-12 binding protein suppresses processing of procaspase-9. A, MAGE-3 binds to caspase-12 p10 but not to other caspases (p10) in the yeast two-hybrid system. Positive control (P), the active Gal4 transcription factor; negative control (N), empty vector. B, MAGE-3 binds to caspase-12 p10 in cells. Cell lysates were prepared from COS-1 cells transfected with plasmids bearing MAGE-3 (lanes 1-8) or FLAG-tagged caspase-12 (procaspase-12, lanes 1 and 3; caspase-12 p10, lanes 5 and 7), and proteins were precipitated using the anti-FLAG affinity gel. Lanes 2, 4, 6, and 8, a vector control for FLAG-caspase-12. MAGE-3 proteins were detected by Western blot analysis with an anti-MAGE-3 rabbit polyclonal antibody. C, coprecipitation of the GST-MAGE-3 fusion protein with an unprocessed form of caspase-12 (p30C/S). Lanes 1 and 2, p30*; lanes 3 and 4, p30C/S. Lanes 1 and 3, GST control; lanes 2 and 4, GST-MAGE-3. D, procaspase-12 bound to MAGE-3 is resistant to cleavage by active caspase-12. 35 S-Labeled procaspase-12 was prepared by in vitro transcription and translation (15). Lane 1, intact procaspase-12; lanes 2-7, procaspase-12 incubated with active caspase-12 (0. or a vector control line (C2C12/vec2) with ER stress inducers leads to morphological changes typical of apoptosis. Over 50% of C2C12/vec2 cells exhibit apoptotic morphology after 24 h treatment with tunicamycin or thapsigargin, as indicated by the small round shape of the cells (Fig. 5D). The nuclei of these round cells are fully condensed, as visualized by staining with Hoechst 33342 (data not shown). However, C2C12/MA21 cells undergo apoptosis at the same low background level (Ͻ 5%) observed in untreated cells under the same conditions (Fig. 5D). Activation of caspase-12 is almost completely suppressed in C2C12/MA21 cells treated with ER stress inducers (Fig. 5E). Processing of caspase-9 and caspase-3 also does not take place in MAGE-3 overexpressing cells. Both C2C12/MA21 cells and C2C12/vec2 cells respond to ER stress and elicit the unfolded protein response (reviewed in Ref. 28), as demonstrated by the induction of BiP, an ER-specific heat shock protein (Fig. 5F). These data indicate that MAGE-3 overexpression renders cells resistant to ER stress by suppressing the activation of caspase-12. Therefore, caspase-12 is a critical component of the apoptotic machinery that responds to ER stress, confirming the previous observation (1) that caspase-12 null mice are resistant to the toxic effects of ER stress (e.g., intraperitoneal injection of tunicamycin). Furthermore, concomitant inhibition of the activation of other caspases (caspase-9 and -3) in stably transfected C2C12/MA21 cells strongly suggests that caspase-9 and -3 are located downstream of caspase-12 in the ER stress-specific caspase cascade. These results suggest that procaspse-9 is a substrate of caspase-12 in vivo as well as in vitro. Both C2C12/ MA21 and C2C12/vec2 cells undergo apoptosis when treated with staurosporine, a protein kinase inhibitor (data not shown), indicating that the apoptotic machinery per se is functional. This result supports the idea that the suppressive effect of MAGE-3 is specific for the ER stress-induced apoptotic pathway mediated by caspase-12. DISCUSSION Our data suggest the following: 1) caspase-12 activation triggers the caspase cascade in response to ER stress; 2) pro-caspase-9 is a substrate of caspase-12 and caspase-9 activation can be achieved in cells without the release of cytochrome c from mitochondria; and 3) proteolytic signals in the cascade are transmitted from caspase-12 to an effector caspase (caspase-3) via caspase-9 (Fig. 6). An Apaf-1/cytochrome c-independent mechanism of caspase-9 activation has recently been reported for dexamethasone-induced apoptosis of multiple myeloma cells (29). Because recombinant caspase-9 prepared from E. coli exhibits protease activity (30), it is obvious that Apaf-1 (and cytochrome c) is not essential for the activation of caspase-9. However, the lack of cytochrome c release in C2C12 cells does not exclude the possibility that Apaf-1/cytochrome c is involved in other cell lines. Cytochrome c release has been observed in both mouse and rat embryonic fibroblast cells subjected to ER stress (10,11). It is likely that caspase-9 activation can be achieved by caspase-12-dependent cleavage, by an Apaf-1/cytochrome c mechanism, or by both means (Fig. 6). A similarly complex mechanism by which apoptosis is triggered has been described previously for the death receptor mediated pathway (31). Stimulation of death receptors (e.g., Fas) results in the activation of caspase-8, which in turn activates effector caspases in a direct manner. Alternatively, caspase-8 may cleave Bid, a pro-apoptotic member of the Bcl-2 family, and the cleaved Bid may in turn induce cytochrome c release through mitochondrial damage (32,33). Our studies present another example of redundancy in the mechanisms by which apoptosis is executed. It is unclear, then, how cytochrome c release is induced by ER stress in cell lines other than C2C12 cells. ER stress induces cytochrome c release in rat fibroblast cells in a caspase-8-and Bid-independent manner (11). Possible mediators linking the ER to mitochondria, as suggested by recent studies, include the c-Abl tyrosine kinase (10) and calcium (34). The present study reveals that C2C12 cells are useful for the study of the ER stress-specific caspase cascade because a simpler mechanism probably operates in these cells. Comparison of C2C12 cells with other cell lines would contribute to the dissection of the mechanism of ER stress-induced apoptosis. To conclude that caspase-12 initiates the ER-specific caspase cascade in a direct manner, it should be critical to show that caspase-12 cleaves procaspase-9 at the processing site for activation, and the cleavage product (caspase-9) is active. We have demonstrated the specific cleavage and activation of procaspase-9 by purified caspase-12. Furthermore, we have shown direct correlation between suppression of caspase-12 activation and suppression of caspase-9 activation (and apoptosis) in vivo using the caspase-12 binding protein. These data strongly suggest that caspase-12, activated in response to ER stress, cleaves procaspase-9 to initiate the ER stress specific caspase cascade. During preparation of this article, Ellerby's group reported that Apaf-1 Ϫ/Ϫ knockout cells undergo ER stressinduced apoptosis (35). This result indicates that the cytochrome c⅐Apaf-1 complex is not essential for apoptosis induced by ER stress. They also showed that transient overexpression of a catalytic mutant of caspase-12 results in partial resistance of the knockout cells to ER stress and demonstrated that procaspase-9 can be cleaved by microsomal fractions, although the cleavage site has not been determined. These data are consistent with our findings described above in terms of the dependence of caspase-9 activation on caspase-12. In this study, we also identify MAGE-3 as a protein that specifically binds procaspase-12. MAGE-3 has been detected in tumor cell lines, including melanoma cell lines (24). Because the precise human ortholog of murine caspase-12 is not yet known (36), it remains unclear whether MAGE-3 plays any role in caspase regulation in human cells. Our preliminary data show that endogenous MAGE-3 is detected in the microsomal fraction as well as in the S-100 fraction in several human tumor cell lines so far examined (e.g., Jurkat, HeLa), although the abundance in the microsomal fraction depends on cell lines (data not shown). Our results also show that overexpression of antisense MAGE-3 rendered Jurkat cells less resistant to ER stress induced by A23187, whereas the sense construct did not affect the resistance of Jurkat cells (data not shown). These results support the theory that specific expression of MAGE-3 in tumor cells may be involved in resistance of tumor cells to ER stress. It is interesting to note that MAGE-3 is more often expressed by metastatic melanomas than primary tumors (24). Although the involvement of MAGE-3 in resistance to ER stress has not been studied in detail, a correlation between malignancy and resistance to thapsigargin is evident in human melanoma cells (37,38). These observations together suggest the possibility that MAGE-3 may regulate the human caspase-12 ortholog. We have shown that murine caspase-12 can cleave procaspse-9 of both murine and human origins, although their cleavage site sequences are not identical (Fig. 1C). It is interesting to note that the processing sites within procaspase-9 of both origins are functionally conserved so that they can be cleaved by caspase-12, implying the presence of a functional homolog of caspase-12 in human cells. Prolonged ER stress contributes to cell death and is linked to the pathogenesis of several different neurodegenerative disorders (39). It is possible that suppression of caspase-12 activation per se generates little toxicity in mammalian bodies, because caspase-12 null mutant mice do not show abnormalities during either development or adulthood (1). Therefore, a study of the specific interactions between MAGE-3 and procaspase-12 may provide a basis for the development of therapeutic reagents against unwanted activation of caspases caused by ER stress.	v3-fos-license
2014-10-01T00:00:00.000Z	2008-09-24T00:00:00.000	38636532	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://journals.iucr.org/e/issues/2008/10/00/is2330/is2330.pdf", "pdf_hash": "e0ba656b8bc3236e2aa1c0e27694b6be474defe5", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:52", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "debd403f694e9b562d2c23d5ca138cdda67bf17b", "year": 2008 }	pes2o/s2orc	1,1′-Dimethyl-4,4′-(propane-1,3-diyl)dipyridinium tetrabromidocadmate(II) In the cation of the title compound, (C15H20N2)[CdBr4], the dihedral angle between the two pyridine rings is 70.85 (5)°. An intermolecular π–π interaction between the pyridine rings [centroid–centroid distance = 3.900 (4) Å] is observed. The CdII atom has a distorted tetrahedral coordination. In the cation of the title compound, (C 15 H 20 N 2 )[CdBr 4 ], the dihedral angle between the two pyridine rings is 70.85 (5) . An intermolecularinteraction between the pyridine rings [centroid-centroid distance = 3.900 (4) Å ] is observed. The Cd II atom has a distorted tetrahedral coordination. Compound (I), as shown in Fig. 1, consists of a 1,3-bis(1-methyl-4-pyridinium)propane cation and a tetrabormocadmate anion. As result of the flexible propane chain, the two pyridine rings have seriously torsion with the dihedral angle of 70.85 (5)°. The Cd II atom is coordinated by four Br atoms to a tetrahedral divalent anion. The mixture was stirred for 20 min at room temperature and then sealed in a Teflon-lined stainless steel autoclave with a 23 ml capacity at 428 K for 72 h. After cooling to room temperature, the filtered solution was slowly evaporates and 7 days later colourless block-shaped crystals were obtained; these were washed with deionized water, filtered, and dried in air (yield 46% based on Cd). S3. Refinement H atoms were placed geometrically (C-H = 0.93-0.97 Å) and refined as riding, with U iso (H) = 1.2U eq (C) or 1.5U eq (methyl C) . The highest residual electron density peak is located at 1.223 (3) Å from Cd atom. The molecular structure of (I), with the atom-labeling scheme and 30% probability displacement ellipsoids. Figure 2 A partial packing view of (I) along the c axis. For the sake of clarity, H atoms have been omitted. Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.	v3-fos-license
2018-04-03T01:01:57.413Z	2017-01-01T00:00:00.000	27832178	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/14756366.2017.1344236?needAccess=true", "pdf_hash": "17ab2c25bb72c6f1af56b60d393719f7e33e6cd6", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:76", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "sha1": "d1a4243fcf85870ee00a703c32de12da029b8189", "year": 2017 }	pes2o/s2orc	Primary mono- and bis-sulfonamides obtained via regiospecific sulfochlorination of N-arylpyrazoles: inhibition profile against a panel of human carbonic anhydrases Abstract A diverse set of mono- and bis-sulfonamide was obtained via a direct, chemoselective sulfochlorination of readily available yet hitherto unexplored N-arylpyrazole template. Biochemical profiling of compounds thus obtained against a panel of human carbonic anhydrases (hCA I, hCA II, hCA IV and hCA VII) revealed a number of leads that are promising from the isoform selectivity prospective and exhibit potent inhibition profile (from nanomolar to micromolar range). The observed SAR trends have been rationalized by in silico docking of selected compounds into the active site of all four isoforms. The results reported in this paper clearly attest to the power of direct sulfochlorination as the means to create carbonic anhydrase focused sets in order to identify isoform selective inhibitors of closely related enzymes. Introduction Spiking a carbo-or heterocyclic compound with a primary sulfonamide group has manifested itself a remarkably efficient strategy to render the molecule somewhat inhibitory toward human carbonic anhydrase (hCA) due to prosthetic Zinc binding by that group (with numerous limitations currently known to that approach)and, ultimately, determine which of the resulting molecules will have (or at least show tendencies to have) favourable isoforminhibitory profiles 1 . These much-needed, early leads can subsequently scrutinized from a structural viewpoint, advanced into clinical status (such as SCL-0111 2 ) or gain a more evolutionary look (such as compound 1 3 developed for treatment of cancer metastases). It's been a long-standing dogma that the currently available landscape of drugs acting via pan-isoform inhibition mechanism (e.g. acetazolamide, methazolmide, dorzolamide and brinzolamide) are all efficacious but suboptimal in terms of inhibiting several isoforms at the same time ( Figure 1 They are poor research tools on the one hand (i.e. any attempt to link the biology perturbed by them to reality would be a dicey undertaking). On the other hand, cleaner isoform selectivity of a therapeutic agent has always been a holy grail of pharma companies: such drugs are considered to have fewer off-targets, which usually means less side-effects 5 . There is one more aspect as to the isoform hCA selectivity worth mentioning here, perhaps even more puzzling. The localization of various hCA isoforms within the cell is uneven and some are more or become more important than others, especially when the disease strikes ( Figure 2). Take hCA IX that can is expressed on a cell membrane and became the main defenders of cells in tumors 6 . Clearly, we need tools to tackle hCA isoform selectivity. One such tool to use would be chemical diversity and, indeed, numerous chemically diverse series of carbonic anhydrase inhibitors (CAIs) have been profiled today 4 . The power of multicomponent chemistry to deliver CAIs has been relatively underutilized today as was recently reviewed 7 and we are currently working to fill this void. Herein, we report a somewhat intermittent approach, namely, a systematic conversion of a set of N-arylpyrazines 2a-t into tractable and SAR-informative set of primary mono-and bis-sulfomamides. The substrates are relevant to, among other pharmacologically sound molecules, known blockbuster antipyretic Celebrex as well as tricyclic congeners 3a-e earlier reported by Marini (Figure 3) 8 . Moreover, through some less systematic approach, the direct sulfochlorination of (hetero)aromatics has very recently given rise to: (i) 5-thienyl-1,3-oxazolecarboxamides 4 (where a remarkable potency toward hCA II with K i ¼ 0.01 nM was achieved) 9 and (ii) a series of isoxazole bis-sulfonamides, exemplified by 5 clearly offering an alternative ZBG binging mode and a remarkable K i of 9.4 nM against hCA IV, an extremely rare hCA to target with such a potency and selectivity (Figure 4) 10 . These isolated and nonetheless successful results, prompt us to undertake a direct sulfochlorination approach to produce compounds which would not only provide a wealthy entry into the realm of CAI but also provide the reader with an easy-to-read compendium of methods on direct shofochlorination of Celebrexlike N-arylpyrazoles. Chemical synthesesgeneral All reactions were carried out in oven-dried glassware in atmosphere of nitrogen. Melting points were measured with a Buchi ffl-520 melting point apparatus and are uncorrected. Thin-layer chromatography was carried out on Silufol UV-254 silica gel plates using an appropriate mixture of ethyl acetate and hexane. Compounds were visualized with short-wavelength UV light. 1 H NMR and 13 C NMR spectra were recorded on Bruker MSL-300 spectrometers in DMSO-d 6 using TMS as an internal standard. Elemental analyses were obtained at Research Institute for Chemical Crop Protection (Moscow, Russia) using Carlo Erba Strumentazione 1106 analyser. Mass spectra were recorded using Shimadzu LCMS-2020 system with electron impact (EI) ionization. All and reagents and solvents were obtained from commercial sources and used without purification. General procedure 1 (GP1): regiochemically unambiguous preparation of monosulfonamides 7-8, not requiring chromatographic separation regioisomers of sulfonyl chlorides 10-11 To a well-stirred ice-cold mixture of 6.76 g (58.1 mmol) or of chlorosulfonic acid and 0.76 g (6.4 mmol) thionyl chloride was added, in small portions, an appropriate precursor 6 (5.8 mmol). The mixture was heated at the temperature and for the period of time indicated in Tables 1-3. The reaction mixture was cooled to ambient temperature and poured over ice (250 g). The resulting mixture was extracted with chloroform (100 ml). The organic layer was separated, washed with water (200 ml), 5% aqueous K 2 CO 3 , dried over anhydrous CaCl 2 and filtered through a short plug of silica. The volatiles were removed in vacuo and the residue dissolved in acetone (15 ml) and the resulting clear solution was treated with 25% aqueous ammonia solution (29.0 mmol). The resulting mixture was heated at 50 C for 30 min, concentrated in vacuo and the residue was dispersed in water (50 ml) the resulting fine precipitate was separated by filtration, washed with more water (100 ml) and air dried. Crystallization from isopropyl alcohol provided analytically pure mono-(7-8) and bis-sulfamides (9) in yields indicated. 1-(4-Sulfamoylphenyl The procedure is analogous to GP1 except for after evaporation of chloroform, the mixture of regioisomeric mono-sulfonyl chlorides was fractionated on silica gel using an appropriate gradient of ethyl acetate in hexanes as eluent, fractions containing different isomers of mono-sulfonylchlorides were pooled separately, concentrated in vacuo and then, also separately, converted to respective mono-sulfonamides (by treatment with 25% aqueous ammonia) which were characterized. Docking studies The crystal structure of hCA I (pdb code 1AZM 11 ), hCA II (pdb code 2AW1 12 ), hCA IV (pdb code 1ZNC 13 ), and hCA VII (pdb code 3ML5 14 ) was taken from the Protein Data Bank 15 . After adding hydrogen atoms and removing complexed ligands, the four proteins were minimized using Amber 14 software 16 and parm03 force field at 300 K. The four proteins were placed in a rectangular parallelepiped water box, an explicit solvent model for water, TIP3P, was used and the complexes were solvated with a 20 Å water cap. Sodium ions were added as counter ions to neutralize the system. Two steps of minimization were then carried out; in the first stage, we kept the protein fixed with a position restraint of 500 kcal/mol Å 2 and we solely minimized the positions of the water molecules. In the second stage, we minimized the entire system through 5000 steps of steepest descent followed by conjugate gradient (CG) until a convergence of 0.05 kcal/Å·mol. Automated docking was carried out by means of the AUTODOCK 4.2 program 17 using the improved force field 18 . Autodock Tools was used in order to identify the torsion angles in the ligand, add the solvent model and assign the Kollman atomic charges to the protein. The ligand charge was calculated using the Gasteiger method. The sulfonamide group involved in the interaction with the Zinc ion was considered as deprotonated, as reported in literature 19,20 . A grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant were used for the energetic map calculations. Using the Lamarckian Genetic Algorithm, the docked compounds were subjected to 100 runs of the Autodock search, using 500,000 steps of energy evaluation and the default values of the other parameters. Cluster analysis was performed on the results using an RMS tolerance of 2.0 Å and the best docked conformations were taken into account. Carbonic anhydrase inhibition assay An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity 21 . Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.005 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear leastsquares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house [22][23][24][25] . Compound synthesis In order to create and investigate a set of the required compounds for this study, we began by performing synthesis of mono-sulfonamides 7a-n or 8a-i as well as of bis-sulfonamides 9a-s by direct sulfochlorination of a large set of N-arylpyrazoles 6a-t (all of which are known compounds and/or can be prepared according to straightforward technique 26 ), followed by conversion of the respective mono-and bis-sulfochlorites 9, 10 and 11 on treatment with aqueous ammonia (Scheme 1). The regiospecificity of the sulfochlorination was unequivocally established for every substrate 6a-t by means for correlational NOESY spectroscopy (ESI) and is depicted in Figure 4 in a straightforward fashion. On close observation, it is the relative stereoelectronic character of the phenyl vs. the pyrazole unit that governed the direction of the first sulfochlorination. With some exceptions, dimethylpyrazole unit was the first affected with electron-neutral or moderately electron-rich aryls. There are a few mixed situations (6f and 6h). Clearly, introduction of an anisyl group swayed the sulfochlorination completely to that group and made it impossible to produce bis-sulfonamides from those compounds (leading to only tar formation when attempting (6i-k, 6m). Some compounds (6f, 6h, 6q) allowed making mono-sulfonamides at both the phenyl and the pyrazoles portions of the molecule, thereby contributing even more to the diversity of this hCA-probing set compounds. Altogether, to the best of our knowledge, the one presented in Figure 4 is the most comprehensive mono-and bis-sulfochlorination match presented to-date. Of course, some "arrows" require rather mild conditions to realize; othersa lot more forcing conditions, particularly when it comes to achieving the second sulfochlorination. For clarity of the presentation in Figure 4, the reader is referred to Tables 1-3 and the ESI for specific reaction times and temperatures. Compound 7o which constitutes an important SAR point could not be prepared as described in Scheme 1, was prepared by a direct route 26 shown in Scheme 2. Biological activity The inhibitory profile obtained for mono-sulfonamides 7a-o in a stopped-flow kinetics assay against human CA I, II, IV and VII is shown in Table 1. Several observations emerge from the data in Table 1. Clearly, some respectable hCA II levels are achievable. Compounds 7c and 7o are distinctly acetazolamide-like. The difficult-to-inhibit hCA IV is not giving high inhibition results throughout, considering the "detrimental methoxy" phenomenon present in 7g-7l (previously noted by us 10 and tentatively justified). When it comes to hCA IV isoform, some striking restoration of potency is observed in 7b (on top of a high selectivity) and, particularly, in 7n (where overall selectivity is not that good). The hCA VII selectivity of compound 7j is also quite notable (and was not ablated, in this case, by the "detrimental methoxy" phenomenon). Altogether, from this set alone, compounds 7b and 7j can be developed as selective probes for hCA IV and VII, respectively. The set of compounds presented in Table 2 is marked by a virtual absence of hCA IV activity. The compounds can be regarded as weaker, nonselective analogues of acetazolamide and are primarily of interest as a reference set to compare with the bissufonamide set discussed below. Among bis-sulfonamides 9a-r, several instances of restoring specific inhibitory potencies (compared to the respective monosulfonamide parts 7 or 8) can be noted, which is suggestive of a possibility of alternative binding mode compared to either 7 or 8. Most notable example is provided by compound 9l whose analogue 7k was inactive throughout the panel (most likely, due to the "detrimental methoxy" effect noted earlier). However, the potency is restored against three isoforms in 9l, which is indicative of the inhibitor's binding to the target at the pyrazole sulfonamide portion. Notable examples of isoform selectivity identified within 9a-r set include: 9d (selective hCA IV inhibitor); 9h (selective inhibitor of hCA I) which, cf. 7f, demonstrates the power of an additional sulfonamide in ablating activity against all other isoforms; 9q (selective hCA II inhibitor). In silico modelling In order to identify the possible binding mode of the new monoand bis-sulfonamides disclosed herein and also rationalize the SAR trends observed, representative compounds were docked into the hCA I, II, IV and VII X-ray structures. Figure 5(A) shows the docking of compound 7b into the active site of hCA IV. The sulfonamide group acts as a zinc binding group (ZBG) and forms hydrogen bonds with the protein backbone and the hydroxy group of T225; the phenyl ring does not show important lipophilic interactions whereas the methyl substituent in inserted into a lipophilic cleft mainly delineated by V142, I163, L224 and V233. With regards to the pyrazole ring, it points towards H88 and shows an H-bond with T226. The docking analysis of this compound into hCA I, II and VII highlights a completely different binding mode for these three enzymes. As shown in Figure 6, in all three cases the pyrazole ring points towards the entrance of the binding site and the phenyl ring shows lipophilic interactions with V121, V142 and L197 (hCA II numbering). The sulfonamide group acts as a ZBG with an uncommon coordination, with one of the two oxygens that coordinates the zinc ion and the nitrogen that forms an Hbond T198. This different binding disposition is in agreement with the selectivity profile of this compound (potent inhibition of hCA IV with virtually no activity against the other three isoforms) and could be due to small differences in the lipophilic cleft in which the methyl group interacts in hCA IV, as this enzyme exhibits the non-conserved I163 that is substituted in the other three CA subtypes by a Leucine residue. The substitution of the methyl with a methoxy substituent in the benzene ring (as in compound 7h) triggers the loss of hCA IV inhibition activity. As shown in Figure 5(B), the methoxy group is not able to interact into the lipophilic cleft mainly delineated by V142, I163, L224 and V233 and for this reason this compound shows a binding disposition very similar to that observed for compound 7b into hCA I, II and VII ( Figure 6). The pyrazole ring points towards the entrance of the binding site, the phenyl ring shows lipophilic interactions with V142, I163, V165 and L224 whereas one of the two oxygens of the sulfonamide group coordinates the zinc ion and the amide nitrogen forms an H-bond T225. Compound 8h highlights a good hCA I, II and VII inhibition activity with selectivity against hCA IV. Docking studies suggests that this compound interacts with a similar binding disposition into the four different CA subtypes. The sulfonamide group acts as the ZBG and forms hydrogen bonds with the protein backbone and the hydroxy group of T198 (hCA II numbering), the pyrazole ring shows a lipophilic interaction with the conserved L197 whereas the chlorophenyl group shows lipophilic interactions with the conserved V121, L140 and L197. Furthermore, this aromatic ring shows a lipophilic interaction with F130 (L132 for hCA I and F133 for hCA VII) that partially occludes the binding site cavity (see Figure 7). In hCA IV this residue is substituted by an asparagine residue and corresponds to a region that in hCA IV is far away from the binding site, thus leaving the chlorophenyl ring more exposed to the solvent (Figure 7). Finally, the analysis of the docking results for compound 9r suggests that in the four CA subtypes the sulfonamide group attached to the o-chlorophenyl fragment act as the ZBG. The phenylpyrazole portion shows lipophilic interactions with F130, L140, L197, P201 (hCA II numbering), and the sulfonamide group attached to the pyrazole ring forms an H-bond with the oxygen backbone of P200 ( Figure 8). Conclusions In this work, we systematically harnessed the power of direct sulfochlorination of a series of known, diversely substituted N-arylpyrazole to arrive at three distinct series of compounds. In each series, SAR generalizations have been made and a number of selective compounds (working against only one target in the panel of four or having high selectivity indices) have been identified. The observed selectivity patterns have been rationalized by modelling. The compounds thus identified can serve as isoformselective tool inhibitors to probe for cellular processes and their linkage to particular hCA isoforms.	v3-fos-license
2019-11-14T17:13:53.344Z	2019-11-04T00:00:00.000	209725024	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2019/ra/c9ra06991g", "pdf_hash": "b5d54c9a199bb36d3a0e0a52cbaba60e38fde32b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:95", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "263b34035b1fef539013fd6a368c4ea2c69e6ef4", "year": 2019 }	pes2o/s2orc	Iron oxide encapsulated by copper-apatite: an efficient magnetic nanocatalyst for N-arylation of imidazole with boronic acid N-Arylation of imidazole was carried out with various arylboronic acids on iron oxide encapsulated by copper-apatite (Fe3O4@Cu-apatite), producing excellent yields. Firstly, the iron nanoparticles were prepared using a solvothermal method, and then they were encapsulated by copper-apatite to obtain magnetic Fe3O4@Cu-apatite nanocatalysts. Several physico-chemical analysis techniques were used to characterize the prepared nanostructured Fe3O4@Cu-apatite catalyst. The prepared Fe3O4@Cu-apatite was used as a nanocatalyst for N-arylation of imidazole with a series of arylboronic acids with different substituents to reaffirm the effectiveness of this magnetic nanocatalyst. The Fe3O4@Cu-apatite nanocatalyst can also be easily separated from the reaction mixture using an external magnet. More importantly, the as-prepared Fe3O4@Cu-apatite exhibited good reusability and stability properties in successive cycles. However, there was a notable loss of its catalytic activity after multiple cycles. Introduction Over the years, N-arylheterocycles have gained prominence due to their presence in a wide variety of natural products and bioactive compounds. They have also played an important role as building blocks in organic synthesis. 1,2 There is a special emphasis on N-aryl imidazole because it exhibits antipsychotic, antiallergic, and herbicidal 3,4 properties, as well as others. The development of a mild and highly efficient method for the synthesis of N-arylheterocycles over classical Ullman type, 5,6 nucleophilic aromatic substitution reactions, 7 or coupling with organometallic reagents has recently gained considerable attention in synthetic chemistry. 8,9 But the harsh conditions of these reactions, such as very high temperature and strong bases have restricted and limited their applications. At the turn of the 21 st century, recent development of Narylation with boronic acids unleashed the power of Cheteroatom bond formation reaction due to the mild reaction conditions (room temperature, weak base and ambient atmosphere). [10][11][12][13] However, the synthetic scope of this reaction is strongly limited due to some disadvantages, such as product contaminationtoxic waste produced aer separation of the catalyst, which cannot be easily recovered aer the reaction. 14 These disadvantages can be overcome by anchoring the metal on suitable supports, which can be easily recovered, and then potentially be reusable with a minimal amount of product contamination. Recently, diverse forms of heterogeneous catalysts have been developed for N-arylation, such as cellulose supported copper(0), 15 polymer supported Cu(II), 16 MCM-41-immobilized bidentate nitrogen copper(I), 17 CuO nanoparticles 18 and Cu-exchanged uorapatite. 19 Magnetic separation has also received a lot of attention as a solid catalyst separation technology, since it can be very efficient and fast. Numerous studies have focused on the immobilization of copper catalytic systems on a magnetic medium based on iron in order to separate them by the simple application of a magnet. 20,21 In the same way, Alper and co-workers have immobilized Cu(I) catalyzed on the surface of Fe 3 O 4 magnetic nanoparticle-supported L-proline as a recyclable and recoverable catalyst for N-arylation of heterocycles. Calcium phosphate, such as hydroxyapatite and its derivates appear very attractive due to their ion-exchange capability, adsorption capacity, and acid-base properties. 22 Hydroxyapatite based materials have been used in the biomedical eld 23,24 lately and in some important chemical transformations. [25][26][27][28] The latter applications are related to its well-known ability to immobilize divalent and trivalent metal ions, by partial cationic exchange with calcium. [29][30][31][32][33] Recently, the possibility to combine the properties of apatite with other inorganic phases within coreshell nanostructures has attracted great attention. For instance, hydroxyapatite/TiO 2 , 34 hydroxyapatite/carbon nanotubes, 35 and hydroxyapatite/SiO 2 (ref. 36) core-shell nanoparticles have been studied for different applications, such as photocatalytic processes, uorescence imaging and drug delivery. Indeed, the combination of hydroxyapatite with iron oxide particles could be useful to design sorbents that combine large activity and easy recovery via magnetic separation. 37,38 Hence, several iron oxidehydroxyapatite nanocomposites have been described in the literature. [39][40][41][42][43][44][45] However, the Fe 3 O 4 @Cu-apatite has only rarely been explored, and to the best of our knowledge, the use of Fe 3 O 4 @Cu-apatite as a heterogeneous catalyst for the N-arylation of imidazole has not been reported in literature. Fe 3 O 4 is one of the cheaper magnetic oxide, which act in this study as a high surface area framework that is coated by the growth of Cu-apatite shell. The Fe 3 O 4 core has to facilitate the recycle of nanocatalysts through magnetic separation. This present work was achieved within our progressive program to develop an ecofriendly and efficient approach for the synthesis of various products. [46][47][48][49][50] This is the background upon which this work is situated upon, and the objective is based on the synthesis and characterization of Fe 3 O 4 @Cu-apatite nanoparticles, and then their use as magnetic catalysts for the N-arylation of imidazole with arylboronic acids (Scheme 1). Materials and apparatus Copper(II) nitrate (Cu(NO 3 ) 2 $3H 2 O, $98.0%), ferric chloride (FeCl 3 $6H 2 O), calcium nitrate (Ca(NO 3 ) 2 $4H 2 O, $99.0%), ammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), sodium acetate (CH 3 COONa), ethylene glycol (EG), sodium hydroxide (NaOH), ammonia solution (NH 3 $H 2 O, 30%), poly (ethylene glycol) (PEG, 1000) were purchased from Aldrich Chemical Company and were used as received without any further purication. Deionized water was used in all experiments. Xray diffraction (XRD) patterns of the catalysts were obtained at room temperature on a Bruker AXS D-8 diffractometer using Cu-Ka radiation in Bragg-Brentano geometry (q-2q). SEM and STEM micrographs were obtained on a Tecnai G2 microscope at 120 kV. The average diameter of sample was quantied from the SEM image using ImageJ soware. The gas adsorption data was collected using a Micromeritics 3Flex Surface characterization analyzer, using nitrogen. Prior to nitrogen sorption, all samples were degassed at 150 C overnight. The specic surface areas were determined from the nitrogen adsorption/desorption isotherms (at À196 C), using the BET (Brunauer-Emmett-Teller) method. Pore size distributions were calculated from the N 2 adsorption isotherms with the "classic theory model" of Barret, Joyney and Halenda (BJH). Fourier transformation infrared (FT-IR) spectra of samples in KBr pellets were measured on a Bruker Vector 22 spectrometer. Magnetic properties of Fe 3 O 4 and Fe 3 O 4 @Cu-apatite were investigated in a MPMS-XL-7AC superconducting quantum interference device (SQUID) magnetometer. The magnetic measurements were performed from À15 000 to 15 000 Oe at room temperature. The element content of Fe 3 O 4 @Cu-apatite materials was determined by an Elemental analysis was realized using inductively coupled plasma atomic emission spectrometry (ICP AES; Ultima 2 -Jobin Yvon). The 1 H NMR spectra were recorded in CDCl 3 or DMSO d6, using a Bruker Avance 600 spectrometer. Catalyst preparation 2.2.1. Preparation of Fe 3 O 4 nanoparticles. The magnetic iron oxide nanoparticles used in this study were synthesized using a solvothermal method, adapting methodologies described in the literature. 51 In a typical procedure, 5.0 mmol of the FeCl 3 $6H 2 O was dissolved in 20 mL of ethylene glycol, and then 20 mmol of NaOH was added to the resultant mixture with vigorous stirring. Then, the obtained EG solution was transferred to a Teon-lined stainless-steel autoclave and sealed. Aer reacting at 180 C for 6 h, the autoclave was cooled down to ambient temperature and the mixture was withdrawn from the reactor with a magnet bar to get the Fe 3 O 4 magnetic NPs, which were then washed with water and ethanol and dried at 40 C for 24 h. 2.2.2. Fe 3 O 4 @Cu-apatite nanocomposite. The Fe 3 O 4 @Cuapatite nanocomposite was prepared along a hydrothermal route. 52 Firstly, 0.30 g of Fe 3 O 4 NPs was dispersed in 100 mL distilled water, and then 12 g of urea was added. Then, 10 mL of a solution produced from a mixture of Ca(NO 3 ) 2 $4H 2 O (0.5904 g) and Cu(NO 3 ) 2 $4H 2 O (0.604 g) was added into the above solution in drops (molar ratio of Ca : Cu 1 : 1). Aer heating at 90 C for 2 h, 10 mL of (NH 4 ) 2 HPO 4 (0.1981 g) solution was also added in drops and the pH value of the solution was tuned to 10 with ammonia water (molar ratio of Ca : P 10 : 6). Aer 0.5 h, the solution was transferred into a Teon-lined stainless-steel autoclave, where it was sealed and heated at 165 C for 12 h. Finally, the Fe 3 O 4 @Cu-apatite NPs were collected using a magnet bar. General procedure of imidazol's N-arylation with arylboronic acid In a 50 mL round-bottomed ask, imidazole (1 mmol), phenylboronic acid (1.2 mmol), K 2 CO 3 (1.5 mmol) and Fe 3 O 4 @Cuapatite (15 mol%) were added and stirred in MeOH under air at 60 C for the required time, monitoring by TLC. Aer completion, the mixture was diluted with H 2 O and the product was extracted with EtOAc (3 times). The combined extracts were washed with brine (3 times) and dried over Na 2 SO 4 . The product was puried using column chromatography (60-120 mesh silica gel, eluting with EtOAc-hexane). The structures of the prepared products were conrmed by 1 H NMR and assigned on the basis of their spectral data in comparison with those reported in the literature. Catalyst characterization XRD was used to analyze the crystalline phases of the asprepared Fe 3 O 4 and Fe 3 O 4 @Cu-apatite samples (Fig. 1). The Table 1. The calculation of the BET surface area for Fe 3 O 4 is 22 m 2 g À1 and the pore volume is 0.0305 cm 3 g À1 . The surface area and pore volume of the Fe 3 O 4 @Cu-apatite are 84 m 2 g À1 and 0.1350 cm 3 g À1 , respectively. This change in surface properties was assumed to be due to the coating of Cu-apatite on the surface of Fe 3 O 4 nanoparticles. The rough surface provides more pores for nitrogen adsorption. The nitrogen sorption isotherm of Fe 3 O 4 is type IV, displaying a hysteresis loops type H3, indicating the mesoporous nature of the material (Fig. 3a). For the Fe 3 O 4 @Cuapatite, the nitrogen sorption isotherm is type (IV) with a hysteresis loop type H3, which proves the existence of mesopores according to the IUPAC manifests (Fig. 3b). The corresponding pore size distribution curve indicates that Fe 3 O 4 and Fe 3 O 4 @Cu-apatite have a centralized pore size distribution around 4 and 11 nm, respectively, which corresponding to mesoporous materials (inset Fig. 3a and b). SEM was used to study the surface morphology of Fe 3 O 4 and Fe 3 O 4 @Cu-apatite (Fig. 4) Fig. 5a and b, it can be observed that Fe 3 O 4 NPS show a very good dispersion of spherical particles. These Fe 3 O 4 spheres were further used as cores for the growth of Cu-apatite shells to obtain the Fe 3 O 4 @Cu-apatite core-shell nanostructures. Compared to the Fe 3 O 4 cores, the outside surfaces became coarse aer the growth process, as shown in Fig. 5c and d. These results indicate that the Cu-apatite nanoparticles were successfully loaded onto the Fe 3 O 4 sphere surfaces, which clearly demonstrates the formation of core-shell nanostructures. We have also noticed that Cu-apatite was still tightly anchored on the surface of Fe 3 O 4 even aer the severe conditions under which the sample preparation for STEM analysis were conducted (a long time of mechanical stirring and sonication), suggesting the existence of strong interactions between Cu-apatite and Fe 3 O 4 . The magnetic properties of Fe 3 O 4 and Fe 3 O 4 @Cu-apatite were investigated using SQUID from À15 000 to 15 000 Oe at room temperature. As shown in Fig. 6 This superparamagnetism can make the magnetic nanoparticles dispersible in the solution with negligible magnetic interactions between each other, which avoids magnetic clustering. As shown in the Fig. 7, Fe 3 O 4 @Cu-apatite can be dispersed in deionized water to form a stable brown suspension before magnetic separation. However, when a magnet was placed close to the reaction vessel for a while, it could be observed that the synthesized samples were rapidly attracted to the magnet side, and a nearly colorless solution was obtained. N-Arylation of imidazole over Fe 3 O 4 @Cu-apatite catalyst Firstly, the activities of some samples were screened, such as: Fe 3 O 4 , Cu-apatite, Fe 3 O 4 @Cu-apatite and Cu-apatite@Fe 3 O 4 catalyzing imidazole N-arylation using phenyl boronic acid as a model reaction. The resultant obtained results are summarized in Table 2. First of all, the reaction doesn't occur without the addition of a catalyst ( Table 2, entry 1), and the iron oxide is also inactive in this reaction (Table 2, entry 2). However, the reaction of N-arylation of imidazole was occurring when Cuapatite was used as catalyst with a yield of 98% (Table 2, entry 3). These results can be explained by the presence of the copper, which play an important role as catalytic element for enhancing the cross-coupling reaction. Additionally, coating iron oxide by copper-apatite (Fe 3 O 4 @Cu-apatite) in order to separate them by the simple application of a magnet has no signicant effect on its catalytic activity ( Table 2, entry 4) with a yield of 97%. On the other hand, coating Cu-apatite by Fe 3 O 4 decrease their catalytic activity ( Table 2, entry 5), this can be explain by the decrease of the contact surface of the catalytic element and the reactants. This result shows the importance of the catalytic system developed in this work (Fe 3 O 4 @Cu-apatite) for the N-arylation of imidazole. The inuence of various reaction parameters, such as type of solvent, nature of the base, reaction temperature, and catalyst Table 3. Initially, with 20 mol% of the catalyst and K 2 CO 3 as a base, the screening of the effect of different solvents showed that H 2 O may not be the optimal solvent, when the reaction was carried out in EtOH or MeOH, the yield of the product 1-phenyl-1H-imidazole was isolated in 87 and 97%, respectively (Table 3, entry 1 and 3). Aer optimizing the solvent, the effect of the base was studied (Table 3, entries 4-6), and it was found that the best yields were obtained by using K 2 CO 3 as base. Otherwise, it was noted that the presence of the base was necessary for the achievement of the reaction as reported in the literature. This is explained by the role of the base to activate the boronic acid in the mechanism of the reaction. The study of the inuence of temperature, in the case of using MeOH, indicated that this reaction is very sensitive to changes in temperature (Table 3, entries 7-10). According to these studies we can conclude that temperature is a key parameter in this type of reaction, and the optimum yield was obtained at 60 C. Furthermore, the decreasing the catalyst loading to half results in a yield of 84% (Table 3, entry 12), which demonstrate the inuence of the catalyst amount in this type of reactions. Thus, the optimum conditions for this reaction are at 60 C in MeOH in the presence of 15 mol% of nanocatalyst with respect to imidazole and K 2 CO 3 (1 mmol) as the base (Table 3, entry 11). With the optimized reaction conditions in hand, a variety of arylboronic acids were chosen as the substrates in this Nimidazole cross-coupling reaction and the results are shown in Table 4. The substitution effects of arylboronic acid were investigated. It was found that the reaction with phenylboronic acids with an electron donating group afforded better yields ( Table 4, entries 1-5) than with electron withdrawing groups ( Table 4, entries 6). Similar observation was made when indole was used in place of imidazoles to obtain the corresponding Narylindole (Table 4, entries [8][9][10]. Another benet of this nanocatalyst system consists of its ease of recyclability. The coupling of imidazole with arylboronic acid was chosen as a model reaction for the reusability study (Fig. 8). Aer the cross-coupling reaction, the recovered catalyst was washed twice by dichloromethane and water, dried at room temperature before being reused under similar conditions for the next run. It has to be noted that the catalytic behavior of the Fe 3 O 4 @Cu-apatite catalyst remains nearly the same for the ve successive runs. Aer the 5th cycle, a notable loss of the catalytic activity was observed. The N-arylation of N-arylheterocycles over Fe 3 O 4 @Cu-apatite was compared with other catalysts reported in the literature as tabulated in Table 5. From this, we can show clearly that our Fe 3 O 4 @Cu-apatite catalyst exhibited a best catalytic activity of N-arylation of imidazole comparing with other catalytic systems. In order to investigate the behavior of the Fe 3 O 4 @Cu-apatite catalyst during recycling experiments, XRD and STEM analysis were performed (Fig. 9). It was observed from the XRD pattern of the Fe 3 O 4 @Cu-apatite catalyst that, aer one catalytic cycle, the crystalline composition of the catalyst remained unchanged. Also, from STEM analysis, Cu-apatite was still tightly anchored on the surface of Fe 3 O 4 aer one catalytic cycle. The catalytic effect of copper ions leached from the Fe 3 O 4 @Cu-apatite was studied in the N-arylation reaction, which was carried out under the optimum conditions. Aer 2 hours of heating, the catalyst was removed and the remaining reaction mixture was reheated. It was observed that the concentration of the desired product didn't increase even aer heating for an additional 8 h. To further evaluate the catalytic effect of copper ions leached from the Fe 3 O 4 @Cu-apatite, ICP analysis was used against the catalyst before and aer reaction. The copper concentration of the catalyst was found to be 1.83 wt% for the fresh catalyst and 1.82 wt% aer one catalytic cycle in N-arylation of imidazole, which conrms negligible copper leaching. Conclusion In summary, magnetic Fe 3 O 4 @Cu-apatite core-shell nanocatalysts have been successfully prepared using a hydrothermal method. The physico-chemical properties of these materials were evaluated by FT-IR, XRD, SEM, STEM as well as the adsorptiondesorption of nitrogen. The prepared catalysts showed potential capability for the preparation of N-arylimidazoles through the Narylation reaction under mild conditions. Thereaer, the optimal reaction conditions were explored, and methanol was identied as the optimum solvent of the reaction, which is environmentally friendly. Furthermore, the Fe 3 O 4 @Cu-apatite can also be easily separated by an external magnet and reused for ve runs with only a slight decrease in its catalytic activity. Conflicts of interest The authors declare that they have no competing interests.	v3-fos-license
2020-02-12T14:04:21.041Z	2020-02-01T00:00:00.000	211079385	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2304-8158/9/2/156/pdf", "pdf_hash": "1f51f4e710ea0126de585ebbce037159279030f0", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:121", "s2fieldsofstudy": [ "Environmental Science", "Chemistry" ], "sha1": "4bb6de7754312a32a78a72662af1ab2db7f7007c", "year": 2020 }	pes2o/s2orc	HPTLC-DESI-HRMS-Based Profiling of Anthraquinones in Complex Mixtures—A Proof-of-Concept Study Using Crude Extracts of Chilean Mushrooms High-performance thin-layer chromatography (HPTLC) coupled with negative ion desorption electrospray ionization high-resolution mass spectrometry (DESI-HRMS) was used for the analysis of anthraquinones in complex crude extracts of Chilean dermocyboid Cortinarii. For this proof-of-concept study, the known anthraquinones emodin, physcion, endocrocin, dermolutein, hypericin, and skyrin were identified by their elemental composition. HRMS also allowed the differentiation of the investigated anthraquinones from accompanying compounds with the same nominal mass in the crude extracts. An investigation of the characteristic fragmentation pattern of skyrin in comparison with a reference compound showed, exemplarily, the feasibility of the method for the determination of these coloring, bioactive and chemotaxonomically important marker compounds. Accordingly, we demonstrate that the coupling of HPTLC with DESI-HRMS represents an advanced and efficient technique for the detection of anthraquinones in complex matrices. This analytical approach may be applied in the field of anthraquinone-containing food and plants such as Rheum spp. (rhubarb), Aloe spp., Morinda spp., Cassia spp. and others. Furthermore, the described method can be suitable for the analysis of anthraquinone-based colorants and dyes, which are used in the food, cosmetic, and pharmaceutical industry. Introduction Anthraquinones represent a large family of naturally occurring pigments, which are produced by plants, microbes, lichens, insects, and fungi [1]. Besides their coloring properties, these natural products exhibit a broad range of bioactivities such as antibacterial, antiparasitic, anti-inflammatory, fungicidal, insecticidal, laxative, antiviral, and anticancer but also DNA intercalating properties [2][3][4][5][6][7]. The chemical structure of anthraquinones is based on an anthracene skeleton with two keto groups in position 9 and 10. The basic core unit can be further substituted at various positions and connected with sugar molecules, forming the corresponding glycosides [8,9]. In the literature, about 700 anthraquinone derivatives are described, in which emodin, physcion, catenarin, and rhein are the most frequently reported [9][10][11]. Two hundred of these are described for flowering plants, which also occur in edible plants and vegetables such as Rheum, Aloe and Cassia species, while the remaining ones are produced by lichens and fungi [7,8,12]. The analysis of anthraquinones is of interest due to their wide range of application. A continuous improvement of the analytical techniques is needed to overcome difficulties with respect to interference with various types of matrices and low abundance of the analytes within complex mixtures [7]. Desorption electrospray ionization mass spectrometry (DESI-MS) represents a powerful ambient ionization mass spectrometric technique, which enables a direct ionization of analytes from surfaces with subsequent mass spectrometric detection [30][31][32]. The coupling of DESI-MS with high-performance thin-layer chromatography (HPTLC) provides a robust methodological approach for the separation and highly sensitive detection of secondary metabolites in plants and fungi [33][34][35]. Furthermore, this method is suitable for the fingerprint analysis of crude extracts in natural product research [36,37]. Recently, the detection of excreted polyhydroxyanthraquinones from the surface of fungal culture agar plates using DESI-MS in negative ion mode was reported [38]. In the present paper, we report the development of a rapid profiling method of anthraquinones, exemplified with the analysis of different crude extracts from Chilean dermocyboid Cortinarii concerning their anthraquinone pattern based on the combination of HPTLC with negative ion DESI-HRMS. For this proof-of-concept study, extracts from fruiting bodies of six dermocyboid Cortinarii were investigated for the occurrence of the known anthraquinones emodin, physcion, endocrocin, dermolutein, hypericin, and skyrin. Furthermore, the possibility of performing MS/MS experiments on the desorbed analytes directly from the HPTLC plate was exemplarily shown for the bisanthraquinone skyrin in comparison with data obtained from direct-infusion MS experiments. Reagents and Chemicals The authentic reference compounds endocrocin (3), hypericin (5) and skyrin (6) were available from the in-house compound library of the Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany. Methanol and toluene were used at analytical grade. Ethyl formate was purchased from Merck (Darmstadt, Germany) and formic acid from Roth (Karlsruhe, Germany). LC-MS grade methanol was obtained from Merck (Darmstadt, Germany), and purified water was prepared by Merck Millipore Milli-Q equipment (Darmstadt, Germany). Table S1). Voucher specimens are deposited in the Fungarium of Concepción University (CONC-F). A duplicate is deposited at the Leibniz Institute of Plant Biochemistry. Sampling Sites and Extraction Air-dried fruiting bodies (2 g) were homogenized using 15 mL of acetone in a blender followed by an ultrasonic extraction for 15 min to remove interfering compounds such as fatty acids from the material. After vacuum-supported filtration, the fungal material residue was further extracted twice with 15 mL methanol each. The resulting extracts were filtrated and dried under reduced pressure using a rotary evaporator. The crude methanolic extracts were redissolved in methanol and directly spotted on the HPTLC plate for chromatographic separation. DESI-Orbitrap-MS and MS 2 All experiments were performed using a 2D-DESI source (Omnispray System OS-3201, Prosolia, Indianapolis, IN, USA) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) operated in the negative ion mode. The DESI source settings were as follows: spray voltage, 3 kV; solvent flow rate, 2 µL/min; nebulizing gas (nitrogen), pressure, 7 bar; tip-to-surface distance, 2-2.5 mm; tip-to-inlet distance, 3.5 mm; incident angle (relative to the surface plane), 55 • . The DESI spray solvent was 50:50 (v/v) methanol/water. MS experiments were performed by continuously scanning every HPTLC band in the y-direction (from Rf 0 to 1.0) at a surface velocity of 200 µm/s while acquiring mass spectra in full scan mode (m/z 150-1500; resolution 30,000) and 150 µm/s in MS 2 mode. Collision-induced dissociation was performed using normalized collision energies (NCE) of 35 and 50 (arbitrary units) and an isolation width of ± 2 Da. The data were evaluated using the software Xcalibur 2.2 SP1 (Thermo Fisher Scientific). Method Development The normal-phase HPTLC plates were developed for 55 mm in one dimension to enable the separation of anthraquinones according to their polarity ( Figure 1). The geometry of the source, the composition of the spray solvent, the flow rate as well as the scanning rate were optimized for the analysis. To enhance the ionization and desorption efficiency, different mixtures of methanol and water (with and without formic acid as additive) were tested as spray solvents. A mixture of methanol and water of 1:1 (v/v) yielded the best results. During optimization, a flow rate of 2 µL/min showed good results to obtain adequate signal intensities. On the other hand, higher flow rates led to a partial detachment of silica gel particles. Additionally, different velocities of the DESI spray head were tested to obtain an efficient number of precursor ions for the MS 2 experiments. Lower scan rates led to better signal intensities due to the better desorption of the analytes from the surface of the HPTLC plates. Therefore, we used a lower velocity for the MS 2 experiments in the final measurements than in the full scan runs. Each band was recorded by scanning the surface in the y-direction (Rf 0 to 1.0) with an automated DESI source coupled to an Orbitrap Elite mass spectrometer within a total run time of 4.6 min. Before starting the experiment, the spray head was positioned on the application line of the HPTLC followed by the manual start of the MS measurement. Profiling of Anthraquinones in Crude Extracts The pigment pattern of the methanolic extracts of dermocyboid Cortinarii Cortinarius (Figure 1) was analyzed by high-performance thin-layer chromatography (HPTLC) coupled to desorption electrospray ionization (DESI) mass spectrometry in the negative ion mode. An unspotted HPTLC band was scanned to assign background related peaks ( Figure S1) and to ensure the absence of the target compounds before applying the crude extracts on the HPTLC plate. No anthraquinone-related peaks could be detected by scanning the empty band on the HPTLC plate after running with the solvent system. This is demonstrated by the extracted ion chromatograms based on the theoretical calculated m/z value of the [M-H] − ions using an 25 ppm window (four decimals) ( Figure S2).The established analytical method was applied to identify anthraquinones 1-6 ( Figure 2). These anthraquinones were chosen for this proof-of-concept study because their occurrence in different Cortinarius and Dermoybe species is described in the literature [15]. The assignment of the structures is based on their elemental composition determined by high-resolution mass spectrometry (HRMS) ( Table 1 and Table S2). Table 1. Detected anthraquinones (1-6) using HPTLC-desorption electrospray ionization (DESI)-high-resolution mass spectroscopy (HRMS). Table 1. Due to the resolving power of the orbitrap detector, a differentiation of isobaric ions was possible as shown in the EIC of dermolutein (4, Figure 3B). The anthraquinone peak m/z 327.0505 is clearly separated from other accompanying ions at the same nominal mass using a resolution of 30,000. Comparing the pigment patterns of the different fungal extracts (Table 1) Based on the retention time and the velocity of scanning the HPTLC bands (see Equation (1)), Rf values can be calculated and compared with the Rf values determined directly from the HPTLC plate ( Table 2). The results of the developed HPTLC plates of the extracts and the reference compounds (see Figures S7-S10) were reproducible and comparable, exemplified based on the extracted ion chromatograms of endocrocin ( Figure S11). Therefore, the determination of Rf values based on the retention time of the HPTLC-DESI-MS measurements of UV/VIS inactive analytes could be possible. Rf = t R (min) × velocity (mm/s) × 60 × 1/distance from application line to solvent front (mm) Rf = t R × 0.200 mm/s × 60 × 1/55 mm (1) Structural Characterization Using MS 2 Experiments As an example, the fragmentation behavior of the bisanthraquinone skyrin (6) was investigated by a MS 2 measurement compared with the results obtained directly from the extract, data of a reference compound measured by HPTLC-DESI-HRMS and with direct infusion DESI-HRMS ( Figure 4A-C, Table S3). Skyrin (6), in its MS 2 spectrum, shows a base peak ion at m/z 493.0923 ([M-H-CO 2 ] − , calcd for C 29 H 17 O 8 − 493.0929, Figure 4A, Table S3). Furthermore, a loss of carbon suboxide (C 3 O 2 ) is observed at m/z 469.0926 (calcd for C 27 H 17 O 8 − 469.0929), indicating a 1,3-dihydroxybenzene feature, which is also described for flavones and other polyphenols [39,40]. The obtained data are in good agreement with the reported MS 2 data of skyrin [41]. Conclusions Crude extracts of six Chilean dermocyboid Cortinarii were investigated by HPTLC-negative ion DESI-HRMS concerning the occurrence of the anthraquinones physcion (1), emodin (2), endocrocin (3), dermolutein (4), hypericin (5), and skyrin (6). The compounds were identified by their elemental composition. It should be pointed out that the high-resolution mass spectrometry (HRMS) approach also allows a mass spectral distinction of isobaric ions as demonstrated for the detection of dermolutein (4) whose nominal mass is accompanied by other compounds in the crude extract. Furthermore, the implementation of fragmentation experiments (MS 2 ) for anthraquinones on HPTLC surfaces is possible, as exemplarily shown for the detection of skyrin (6) in the extract of C. (D) austronanceiensis, and could be a valuable tool for the presence of these compound classes. The corresponding results are in good agreement with the data obtained by direct infusion and in comparison with the LC-MS data reported in literature. HPTLC provides good separation efficiencies and can be performed in an automated and controlled way with respect to the sample application and the development of the plate. In classical approaches, a derivatization of the HPTLC plate is needed; however, combined with DESI-MS, this step is not required. Although the separation power of HPTLC is lower than in (U)HPLC, several analyses can performed with one plate and within a short analysis time. In case of the presented approach a HPTLC plate (total length 100 mm) with a developing length of 55 mm, and a total scanning time of only 4.6 min was necessary to obtain the presented results. After the extraction of the material, no further sample preparation steps are necessary, and the crude extracts can be directly applied to the plate, representing an advantage compared with other analytical techniques. In summary, the obtained results illustrate the feasibility and capacity of HPTLC-DESI-HRMS to provide a rapid first screening method for the analysis of anthraquinones in complex mixtures, which may be used in the analysis of anthraquinones in food, plants, fungi, dyes, and cosmetic and pharmaceutical products.	v3-fos-license
2016-03-22T00:56:01.885Z	2014-07-01T00:00:00.000	11790460	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.mdpi.com/1422-0067/15/7/11799/pdf", "pdf_hash": "8d7f3d685b697c92c1f3d07843cf3c462bb11bce", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:149", "s2fieldsofstudy": [ "Chemistry", "Biology" ], "sha1": "8d7f3d685b697c92c1f3d07843cf3c462bb11bce", "year": 2014 }	pes2o/s2orc	Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition. Introduction The genomic integrity of the cell is constantly threatened by DNA damage, nucleotide changes, deletions or recombinations, or epigenetic modifications, leading to mutations. A complex machinery of interacting DNA processing repair enzymes protects the cell from these distortions. Typical targets of such repair enzymes are abasic sites, damaged or alkylated nucleotides or non-native bases, such as uracil. Glycosylase enzymes recognize mismatches and damage and specifically remove the wrong base. The resulting abasic site is then processed further by other DNA repair enzymes. Preferred sites are CpG sites [1], which are the target for human methyltransferase. From the many structures of glycosylases complexed to damaged DNA [2,3], it is known that damaged, mispaired or wrong bases are flipped out of the helical DNA duplex into the enzyme's active site. In the debate on how glycosylase enzymes recognize a damaged or mispaired base, two mechanisms are discussed. One is a passive mechanism in which the enzyme detects extra-helically-exposed, already, at least partially, flipped-out bases. This mechanism implies that base pair opening up to several degrees of flipping is more likely for damaged/mispaired bases than for intact canonical ones. The alternative mechanism involves flipping of the base, while the enzyme travels along the DNA, relying on the enzyme specifically enhancing the flip-out of its target base [4,5]. In addition to the deamination products of 5-methyl-cytosine (mCyt), thymine DNA glycosylase (TDG) has been reported to be involved in active DNA demethylation through the removal of the oxidized derivatives of 5-methyl-cytosine: 5-formyl-cytosine (5fC) and 5-carboxyl-cytosine (5caC) will be recognized and expelled by TDG, following the base excision repair pathway [6][7][8], whereas 5-hydroxymethyl-cytosine (5hmC) and 5-methyl-cytosine (5mC) are not processed by the glycosylase enzyme. Human TDG has been crystallized in complex with DNA containing various mismatches, including G:5caC and G:5hmU (5-hydroxymethyl-uracil). 5caC is also flipped out of the double-stranded DNA into the enzyme's active site, but exhibits a conformation different from those reported for TDG complexed to substrate analogues [8,9]. The post-reactive complex of TDG with caC did not show any interactions of protein side-chains with the DNA major groove where the methyl groups are located, but, instead, interactions with the phosphate group of the flipped nucleotide [8]. This finding led the authors to suggest that the discrimination between different 5-methyl groups is achieved or at least facilitated by other means of recognition. It has been speculated that, e.g., G:5fC and G:5caC form mismatch-like wobble hydrogen bonding pattern via an amino-imino protonation equilibrium [10,11] that is shifted towards the imino site for formyl and carboxyl cytosine, stabilized by the possibility to form an intra-molecular hydrogen bond to the carbonyl oxygen atom. NMR studies of 5-formyl-2'-deoxycytidine in DMSO and calculations of isolated 5fC and 5caC bases in implicit water, however, suggest that the amino form is energetically more stable and, thus, predominant [12]. Glycosylase enzymes are discussed as following a multi-step interrogation pathway to discriminate their target base from non-cognate ones. Partial distortion of the DNA helix, intra-helical interrogation to detect a lesion and base flipping in varying degrees are thought to ensure that only the substrate-base is processed. Biochemical DNA binding data show binding to C, 5mC and 5hmC to be significantly weaker than binding of substrate bases. This has been interpreted as a discrimination step before base flip and reactive complex formation [7,13]. However, among the possible base substrates analyzed, only thymine and uracil variants would have a non-Watson-Crick hydrogen bonding pattern that is likely to stabilize distorted, wobble pairs or even partially-flipped conformations [14] that are easy to recognize by the repair enzyme. Molecular simulations have proven to be a powerful tool for obtaining information on the structure and dynamics at the atomic level and have been used successfully to analyze interactions between proteins and DNA. There is a vast literature on molecular dynamics studies of DNA analyzing the structural and dynamical differences of various damages, lesion or mispairs [15][16][17][18][19][20]. Simulations of base flip have been successfully conducted on free DNA [21][22][23][24][25][26] and in complex with different DNA repair enzymes [26][27][28][29][30][31][32], applying various flavors of enhanced molecular dynamics. The recognition and base flip of cytosine has been studied in a series of molecular dynamics studies [26,30,31]. Huang et al. investigated the spontaneous base flip in free DNA in aqueous solution, in a binary complex with HhaI methyltransferase, and in a ternary complex containing protein, DNA and the cofactor, S-adenosylhomocysteine (SAH). They observed the free energy barrier for the cytosine base flip in uncomplexed DNA to be independent of the flanking sequence. In contrast, when complexed to the methyltransferase and cofactor, the barriers for base flipping have been found to be significantly higher in non-cognate sequences than in cognate DNA. We have previously shown that DNA containing a single T:G mispair exhibits local dynamics significantly different from DNA without such a mispair. The T:G wobble pair shows a distorted conformation compared to T:A or C:G pairs. Our free energy calculations show that thymine is much more probable to be flipped than cytosine in a C:G pair or thymine in a T:A pair, a fact that can be exploited by the repair enzymes. Moreover, a partially open state of the T:G mispair, which we observe to be transiently occupied also in the unbiased simulations, is supposedly easy to be recognized by the searching repair enzyme [14]. These results suggest that DNA repair enzymes, such as glycosylases, can first recognize local distortions in the base steps and base-pair geometries, which deviate from normal B-form DNA. Those distorted sites are then likely to be further examined by the repair enzyme, including the attempt to flip out the putative mispaired or damaged base into the active site of the repair enzyme. In the present paper, we analyze the dynamics of DNA containing oxidized and intact cytosine, so as to reveal which structural and dynamical differences might facilitate the discrimination of the target bases for removal (5fC and 5caC) over the very similar C, 5mC and 5hmC bases. DNA Conformation We have examined the conformation of the DNA double helix carrying the different forms of oxidized and intact methyl cytosine, analyzing the local conformation at the central G:Cox pair. Figures 1 and 2 show the free energy profiles of the intra-and inter-base pair parameters involving the methyl cytosine. The free energy minima for all rotational parameters are the same. The only difference between the models is the slightly larger range of tilt and roll angle explored by formyl-cytosine and a similarly slightly larger range of buckle angle sampled by 5-hydroxymethyl-cytosine. Furthermore, for the translational base-pair and base-step parameters, only marginal differences can be observed between the different forms of methyl cytosine. The free energy profile for the shear parameter is less smooth in the case of native and 5-hydroxymethyl-cytosine compared to the higher oxidized forms. However, both the free energy minimum and range of the shear translation are similar for all types. Figure 3 shows the free energy profile of the flip angle around its equilibrium values, computed from the unbiased simulations. Except for the 5-carboxyl-cytosine (minimum at˜48°), all models exhibit a similar free energy minimum of the flip angle, between˜42°and˜46°. Base Flip The free energy computed for the rotation (flip) of a single base out of the DNA double helix is plotted in Figure 4. A complete rotation of the base, including the passage of the minor groove, requires very high forces and leads to a deformation of the DNA. We therefore restricted the flip to a rotation through the major groove. Free energy profiles of the pseudo-dihedral flip angle evaluated from the unbiased MD simulations of 5-methyl-cytosine (green), 5-hydroxymethyl-cytosine (maroon), 5-formyl-cytosine (blue) and 5-carboxyl-cytosine (orange), respectively. Free energy profile of the base flip for 5-methyl-cytosine (green), 5-hydroxymethyl-cytosine (maroon), 5-formyl-cytosine (blue) and 5-carboxyl-cytosine (orange). The pseudo dihedral coordinate is illustrated in Section 4.2.2. The positions of the free energy minima computed from the biased simulations at˜48°are virtually the same for 5fC and 5caC. The less oxidized 5-hydroxymethyl-cytosine exhibits a free energy minimum of the flip angle at a slightly smaller value of˜45°, and the unoxidized methyl-cytosine shows a most probable flip angle at˜38°(see Figures 3 and 4). This is a shift towards a smaller flip angle by˜5°c ompared to the unbiased simulation. For 5hmC, we also observed a small difference in the biased compared to the unbiased simulation, however, this time, towards a larger flip angle. Table 1 lists the occupancies of the hydrogen bonds in the base pair between the different forms of methyl-cytosine and guanine, as well as hydrogen bonds between the methyl-cytosine base and bulk water. The inter-base pair hydrogen bonds are of similar strength in all models investigated here with a somewhat larger standard deviation in the case of 5-hydroxymethyl-cytosine. Furthermore, the hydrogen-bond interaction between the O 2 oxygen atom and bulk water is comparable in all four models. A significant difference, however, can be observed for the hydrogen bonds formed between the N4 nitrogen atom and bulk water: the higher oxidized forms, 5-formyl-and 5-carboxyl-cytosine, show only little occupancy for that hydrogen bond. This can be attributed to the fact that the N4 amino group forms an intra-molecular hydrogen bond with the formyl or carboxyl oxygen atom, respectively. Although an intra-molecular hydrogen bond can, in principle, also be formed between N4 and the hydroxyl oxygen atom, this is not the case, and hence, the interaction between N4 and bulk water appears not to be influenced by the extra hydroxyl group. Table 1. Occupancies of hydrogen bonds between DNA base pairs computed from the simulation of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC) and 5-carboxyl-cytosine (5caC). The hydrogen-bonds between the oxygen atom and bulk water in the two forms carrying one oxygen atom (5hmC and 5fC) are comparably probable, whereas 5-carboxyl-cytosine not only forms hydrogen bonds to bulk water with both oxygen atoms, but also these hydrogen bonds are significantly stronger than those of the other oxidized methyl-cytosine forms. Hydrogen Bonds and Solvent Accessibility We have furthermore computed the solvent accessible surface area and the radial distribution function of bulk water around the methyl-cytosine bases and the (oxidized) methyl groups. Whereas the solvent accessibility of the entire base is almost the same for all forms of methyl-cytosine investigated, there is a small effect of the methyl group itself. With increasing oxidation of the methyl group, and, hence, also increasing size, the solvent accessible surface area also increases ( Table 2). Only in the case of carboxyl-cytosine, also the base exhibits a somewhat larger solvent accessible surface area than the less oxidized forms, which is perfectly explained by the carboxyl group extending to the solvent ( Figure 5). Table 2. Solvent accessible surface area (Sasa) of the base and the oxidized methyl group in 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC) and 5-carboxyl-cytosine (5caC). The water accessibility computed for the differently oxidized methyl cytosine bases as quantified by the radial distribution of water molecules surrounding the base (Figure 6) is again very similar for all forms of methyl cytosine, and again, the carboxylated form is the exception, which shows a higher probability for water molecules to be in the first solvation shell, i.e., at a distance of˜1.8Å. At the larger distances, the probability of finding a water molecule agrees in all cases studied, showing that the second, third or higher solvation shells are not affected by the differences in the oxidation level. When analyzing the distribution of water molecules around the methyl groups only, the difference between the carboxylated form and the other ones becomes even more pronounced. Here, a rather large peak can be observed at a distance of˜1.8Å from the methyl group, indicating a well-ordered first solvation shell. The significantly larger height of that peak can be attributed to two oxygen atoms as opposed to only one in the other forms that are likely to form hydrogen bonds to bulk water molecules (cf., also, Table 1). Furthermore, 5-formyl-cytosine also shows a peak at that distance, albeit smaller than that of 5-carboxyl-cytosine. 5-hydroxymethyl-cytosine shows a somewhat lower probability for water molecules at that distance from the methyl group and appears to lack any structural order that can be interpreted as a first solvation shell. For 5-methyl-cytosine only at the distance of a second or even third solvation shell, the water distribution becomes significant. This is at a distance at which all three uncharged models are rather similar. Radial distribution functions g(r), with r = Radius, of water surrounding (a) the base and (b) the (oxidized) methyl groups in the central 5-methyl-cytosine (green), 5-hydroxymethyl-cytosine (maroon), 5-formyl-cytosine (blue) and 5-carboxyl-cytosine (orange). Discussion Our molecular dynamics simulations indicate that the conformations and flexibility of DNA carrying oxidized forms of 5-methyl-cytosine (5-hydroxymethyl, 5-formyl and 5-carboxyl) are essentially the same as those of DNA with native 5-methyl-cytosine. All base-pair and base-step parameters investigated differ only marginally for the four methyl-cytosine models. The pseudo-dihedral angle defining the coordinate for the base flip suggests that all four methyl-cytosine forms are rather unlikely to flip-out spontaneously. The free energy barrier computed for the flip out of the DNA through the major groove is between 9 and 12 kcal/mol, comparable to the free energy barrier calculated for unmethylated cytosine [14,21,30] and significantly higher than the free energy barrier for the base flip of a mispaired thymine, both computed in a previous study [14]. Our data suggest that the intrinsic probability of the target base for wobble conformations and displacement towards base-pair opening or flip that has been found for thymine is not present in the methyl-cytosine forms investigated here and, therefore, cannot be exploited by the enzyme. However, we cannot rule out the idea that the different equilibria between the amino-imino tautomeric variants (see, e.g., [8], Figure 7) of the oxidized methyl cytosines do play a role in target site recognition [8]. Whereas the NMR studies and calculations of individual methyl cytosines in [12] very convincingly conclude that the amino form is prevailing, the formation of imino-tautomers can still be different in solvated DNA and, therefore, also be more likely (or less unlikely) for formyl and carboxyl-cytosine than for native and 5-hydroxymethyl-cytosine. Following that assumption, formyl and carboxyl-cytosine are also more probable to form "wobble pairs" with guanine that are similar to mismatches in their displaced conformations, as well as in a reduced energy requirement for base flip compared to intact (amino) Watson-Crick base pairs. Simulations of imino forms and calculations of amino-imino equilibria in solvated DNA are subject of an ongoing study. Another possibility to recognize oxidized methyl-cytosine in their amino forms is by direct contacts between protein and base. In contrast to the rather indirect recognition of distorted wobble pairs whose specific hydrogen bonds are buried in the DNA helix to a large extent, even for partially-flipped conformations, the (oxidized) methyl group is comparably easy to be accessed and probed by protein residues. Our simulation data show that the oxidized forms that are processed by the glycosylase enzyme, formyl and carboxyl, indeed show more hydrogen-bond interactions with bulk water, a higher probability of forming a (structured) first solvation shell and also have a larger solvent accessible surface area. Hence, it is conceivable that the protein can directly access the oxidized site and form specific contacts that might destabilize the Watson-Crick state. That way, discrimination would be achieved by energetically favoring the flip of the target bases only. For the cytosine-specific methyltransferase, M.HhaI, such an "energetic recognition mechanism" has been reported in which the enzyme's specificity depends on the ability to exclusively facilitate flipping of the target base: a lowering of the free energy barrier for base flip is only observed upon the formation of specific protein-DNA interactions, such as hydrogen bonds to the target cytosine [30,31]. An additional discrimination possibility is via the very low probability of the amino group forming hydrogen bonds to bulk water in the 5-formyl and 5-carboxyl forms, as opposed to 5-methyl and 5-hydroxymethyl cytosine. Similarly, hydrogen bonds that can (or cannot) be formed with water can be envisaged to be formed (or not) between the enzyme and the base. As a result, the protein would no longer interrogate a site that allows hydrogen bonds to be formed with its amino group N4 atom. This amino group is not present in thymine, and hence, contacts as formed between protein and the amino group are lacking here, too. This suggests that direct readout via hydrogen bonds that can be formed in the non-cognate bases as opposed to all bases known to be processed by the repair enzymes is an essential element of target base recognition. The systems were solvated with explicit water, using the TIP3Pmodel [34], extending to at least 10Å beyond the DNA in each direction in a tetragonal box for the unbiased simulations of size (x = 90Å, y = z = 60Å) and in a larger cubic box (x = y = z = 90Å) to allow for flipping of the central base. Twenty four Na + counter-ions were added to neutralize the system and an excess of Na + and Cl − ions to obtain a physiological concentration of 150 mM NaCl. The addition of the ions was carried out by the random substitution of water oxygen atoms. Simulations were performed using periodic boundary conditions, and the long-range electrostatic interactions were treated using the particle mesh Ewald method [35] on a 96 × 60 × 60 charge grid for the unbiased and on a 96 × 96 × 96 charge grid for the biased simulations, respectively. A non-bonded cut-off of 12Å was applied. The short range electrostatics and van der Waals interactions were truncated at 12Å using a switch function starting at 10Å. The solvated structures were minimized using 5000 steps of steepest descent, followed by minimization with the conjugate gradient algorithm, with solute atoms harmonically constrained until an energy gradient of 0.01 kcal/(mol·Å) was reached. The system was then gradually heated for 30 ps to 300 K with 1 K temperature steps with harmonic restraints on the solute atoms. The systems were equilibrated in three different stages with the numbers of particles, pressure (1 bar) and temperature kept constant (NPT ensemble) during 75 ps. In the first 25 ps, velocities were rescaled every 0.1 ps, and in the second 25 ps, Langevin dynamics were used to maintain a constant temperature. Pressure control was introduced in the third 25 ps and in the production run using the Nosé-Hoover Langevin piston with a decay period of 500 fs. The harmonic restraints were gradually lifted (to 0.5, 0.25 and 0.05 kcal/(mol·Å 2 )) in the three equilibration stages. Unbiased MD Simulations After equilibration, unbiased NPT production runs were performed for 60 ns. The integration time step was 2 fs, and coordinates were saved with a sampling interval of 2 ps. All covalent bond lengths involving hydrogen atoms were fixed using the "SHAKE"algorithm [36]. Three independent MD simulations were carried out by assigning different initial distributions of starting velocities to the minimized systems. Adaptive Biasing Force (ABF) MD Simulations For the simulation of the base flip, we applied the adaptive biasing force (ABF) method [37][38][39]. In ABF, the reaction coordinate is discretized into small bins. Sampling is carried out along the reaction coordinate in a continuous fashion. In each bin, samples of the instantaneous force acting along the reaction coordinate are accrued up to a certain threshold. If this threshold is reached, the adaptive biasing force is applied, so as to "drive" the system into the next bin. The reaction coordinate for the base flip has been defined as a pseudo-dihedral angle between the flipping base, the sugar moiety of the same nucleotide, the sugar of the next nucleotide and the base of the next nucleotide, plus the base and sugar of the opposing nucleotide downstream (see Figure 8). This definition of the flipping coordinate is the same as we had used in an earlier study [14] and is similar to the one proposed and applied in [21,40,41]. The potential of mean force (free energy profile) was obtained by discretizing the reaction coordinate between 10°and 180°into windows of a 2°width, and in each window, 2000 samples were collected before the bias was applied. For all systems, we carried out three ABF simulations of 60 ns in length, starting with different initial velocities. Figure 8. Definition of the reaction coordinate for the base flip simulations: the flip angle is a pseudo-dihedral between the centers of mass of the flipping base (red shade), the sugar moiety of the same nucleotide (yellow shade), the sugar moiety of the next nucleotide (green shade) and the base of the next nucleotide, plus the complementary base in the other DNA strand (blue shade). Analysis For all analyzes (unbiased and ABF simulations), properties were evaluated for each run individually. Then, the averages and standard errors over the respective individual runs were calculated. In the analyses of the unbiased MD simulations, the first 10 ns of each trajectory were not included. The conformations of the G:oxC pairs in the DNA were characterized by calculating twelve helical parameters, six inter-base pair parameters (the three rotational parameters: roll, tilt and twist; and the three translational parameters: slide, rise and shift) and six intra-base pair parameters (rotation: buckle, propeller, opening; and translation: stagger, shear, stretch) that define the local DNA geometry. These parameters were measured using the Curve+ [42] suite of programs. Hydrogen-bond occupancies were calculated as the ratio of the time when the hydrogen bond is formed to the total time of the trajectory. Two atoms are considered here to form a hydrogen bond if the acceptor-donor distance is <3.0Å and the acceptor-hydrogen-donor angle is >135 • . Water accessibility of the (oxidized) methyl group was analyzed by calculating the solvent accessible surface area of that group. Solvent accessible surface areas have been computed by placing a probe sphere of radius r vdW + 1.4Å in contact with the atomic van der Waals sphere, both centered at the atom. The parts of the surface spheres where the center of the spherical probe can be placed without penetrating other atoms add up to the solvent accessible surface area [43]. Force Field Parameter Development Bonded parameters were obtained from the ParamChem program [44][45][46][47][48]. For deriving the atomic charges of the oxidized methyl cytosine, we followed the procedure recommended in [45]. We have first geometry-optimized the oxidized bases (without sugar or phosphate groups) in vacuum at the Hartree-Fock/6-31G(d)level to a convergence criterion of 10 −6 a.u. using Gaussian G09 [49]. Then, a water molecule was added to the optimized structures at several different positions that allow for hydrogen bonds to be formed, and the relative orientation of the two molecules was optimized. The geometries and interaction energies were then used as a reference for fitting the charges of the oxidized bases. Charges were fit applying a Monte Carlo procedure to minimize the error of water-base distances and interaction energies. As starting values of the atomic charges, we used the Mulliken charges obtained from the Hartree-Fock calculations. Only charges of the oxidized methyl group and the C5 host atom were adapted, so as to keep the new residues compliant with the existing CHARMM force field [45]. The Monte Carlo runs were repeated several times for 1000 steps each. The final charges are listed in the Supplementary (Tables S1 and S2). Programs All molecular images were generated with VMD (visual molecular dynamics) [50]. Structural analysis was performed using standard programs; Curve+ [51], Gromacs [52] tools and home-made scripts. The molecular dynamics simulations have been carried out using the program NAMD2.9 and applying the CHARMM27 force field. Simulations have been performed on the local Linux cluster of the physics department, on the ZEDATuniversity cluster (soroban), and using resources of the North-German Supercomputing Alliance (HLRN). Conclusions The different oxidized forms of methyl-cytosine investigated in this study show no intrinsic difference regarding their preference for a certain base-pair or base-step conformation. Moreover, the energy required to flip the methyl-cytosine base out of the DNA helix is similar in all four cases, indicating that the target base cannot be easily discriminated by the probability for base flip. Differences in the solvent accessibility and, in particular, different hydrogen bond patterns of the amino group N4 observed for the different forms of methyl-cytosine suggest a recognition mechanism in which the glycosylase enzymes attempt to form direct contacts. To what extent the imino forms of the cytosine bases that can form mismatch-like conformations and wobble-pair hydrogen bonding patterns contribute to recognition remains to be investigated.	v3-fos-license
2018-04-03T03:49:46.580Z	2009-09-08T00:00:00.000	36555909	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/284/46/32089.full.pdf", "pdf_hash": "60a602168dc876103d4f8f633d68ef9dd73fb181", "pdf_src": "Highwire", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:163", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "40fd5cfeefb789ae5181c9f93533ef4d8eefd00e", "year": 2009 }	pes2o/s2orc	X-ray Crystal Structure of Michaelis Complex of Aldoxime Dehydratase*♦ Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R–CH=N–OH) to their corresponding nitrile (R–CN). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe3+ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE. Nitrile compounds are important intermediates in some industrial processes to produce nylon and acrylic fibers, insecticides, and pharmaceuticals. Although one of the most useful methods for nitrile production is dehydration of aldoxime, the chemical dehydration of aldoxime used in the industrial process requires harsh conditions. Therefore, a more environmentally benign process of aldoxime dehydration is needed, for which a biological dehydration of aldoxime is a possible candidate. In nature, some microbes have an "aldoxime-nitrile pathway" (supplemental Fig. 1), where aldoximes are metabolized to the corresponding carboxylic acids through nitriles formed by dehydration of aldoximes with aldoxime dehydratase (Oxd; 3 EC 4.99.1.5). There are two pathways for the conversion of nitriles to carboxylic acids. One is hydrolysis of nitriles by nitrilase, and the other is the combination of the reactions catalyzed by nitrile hydratase and amidase (1)(2)(3)(4). Nitriles are the important intermediate not only in some industrial processes but also in this biological system. The detailed characterization of such a biological process to produce nitriles will give some useful information to develop an environmentally benign process for the production of nitriles in the industrial field. Oxd is a new heme-containing enzyme that works as a hydrolyase (2,5,6). The enzymatic activity of Oxd is dependent on the oxidation state of the heme iron, although the reaction catalyzed by Oxd is not a redox reaction. Ferrous Oxd containing a Fe 2ϩ heme shows the enzymatic activity, but ferric Oxd containing a Fe 3ϩ heme does not. Previous spectroscopic analyses reveal a novel mechanism, where the change in the coordination mode of the substrate plays a crucial role for the regulation of the enzymatic activity (7) (supplemental Fig. 1). Although the oxygen atom of aldoxime is coordinated to the ferric heme, the nitrogen atom of aldoxime is coordinated to the ferrous heme (7). The dehydration reaction proceeds only via N-coordinated substrate in the ferrous heme. The organic substrate is directly coordinated to the heme iron in dehydration of aldoxime, which is a unique example among heme enzymes, although the coordination of O 2 or H 2 O 2 to the heme is well known in the heme-containing oxygenases, catalases, and peroxidases. It is proposed that the dehydration reaction of the heme-bound aldoxime proceeds in a general acid-base catalysis with a histidine working as a catalytic residue in the distal heme pocket (7)(8)(9)(10). However, the detailed reaction mechanisms remain to be elucidated mainly because the structural information of Oxd was lacking. In this study, we determined the crystal structures of Oxd from Rhodococcus sp. N-771 (OxdRE) in the substrate-free and substrate-bound forms. We could determine the crystal structure of the Michaelis complex of OxdRE by means of the unique property of OxdRE for the substrate binding. As the reaction spontaneously proceeds when mixing ferrous Oxd and the substrate, the crystallization of the substrate-bound OxdRE in the ferrous form (the Michaelis complex of OxdRE) is not possible by the usual methods. However, we could prepare the crystal of the Michaelis complex of OxdRE by the reduction of the crystal of the substrate-ferric Oxd complex using x-ray radiation under cryogenic temperature. The structures of the resting state and the Michaelis complex provide structural insights into the mechanisms of substrate recognition and the catalysis of OxdRE. EXPERIMENTAL PROCEDURES Protein Expression, Purification, and Enzymatic Activity Assay-The expression, purification, and enzymatic activity assay of OxdRE were performed according to reported methods (7), with a slight modification as described below. The OxdRE used in this work has an additional 20 N-terminal residues encoded in the plasmid, including a His 6 tag. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene). The deoxyoligonucleotide sequences used to introduce the desired mutations are shown in supplemental Table S1. Introduced mutations were confirmed by DNA sequencing with an ABI PRISM 310 genetic analyzer (Applied Biosystems). Cultures of Escherichia coli stains BL21(DE3) (for wild type, E148Q, and F306A), HMS174(DE3) (for R178Q), and BL21CodonPlus(DE3)RIL-X (for S219A and H320A) carrying each expression plasmid were grown under agitation at 140 rpm in 1 liter of LB medium containing 50 g/ml ampicillin and 0.5 mM ␦-aminolevulinic acid for heme synthesis at 37°C. OxdRE expression was induced by adding isopropyl-␤-D-thiogalactopyranoside to a final concentration of 1 mM after 5 h of growth. The cells were incubated under shaking with 100 rpm at 20°C for 18 h for BL21(DE3) and HMS174(DE3) or 36 h for BL21CodonPlus(DE3)RIL-X, after which the cells were harvested by centrifugation. Harvested cells were resuspended in lysis buffer (0.1 M MOPS buffer (pH 6.8) containing 0.3 M NaCl and 10 mM imidazole). The suspension was treated with 0.2 mg ml Ϫ1 lysozyme at 4°C for 1 h and then with DNase mixture (10 g ml Ϫ1 DNase I, 5 mM MgCl 2 , and 0.5 mM MnCl 2 ) for 30 min. The resulting solution was centrifuged at 100,000 ϫ g for 1 h, and the cellular debris was discarded. The supernatant was loaded onto a nickel-nitrilotriacetic acid agarose (Qiagen) column. The column was washed with 3 bed volumes of lysis buffer followed by 10 bed volumes of lysis buffer containing 50 mM imidazole. The adsorbed proteins were eluted with lysis buffer containing 250 mM imidazole. The OxdRE-containing fractions were com-bined and dialyzed against 40 mM MOPS buffer (pH 6.8). The dialyzed sample was loaded on a RESOURCE Q column (GE Healthcare), and the adsorbed proteins were eluted under a R work is calculated for 95% of reflections used for structure refinement. R free is calculated for the remaining 5% of reflections randomly selected and excluded from refinement. d r.m.s.d., root mean square deviation. linear gradient of NaCl from 0 to 0.75 M. The OxdRE-containing fractions were combined, concentrated, and subsequently purified using a HiLoad 26/60 Superdex 200 pg column (GE Healthcare). OxdRE was concentrated by centrifugation with Amicon Ultra (Millipore). OxdRE was oxidized to the ferric form by adding excess potassium ferricyanide. Potassium ferricyanide was removed by a prepacked PD-10 column (GE Healthcare), which was previously equilibrated with 20 mM HEPES buffer (pH 7.4) (Hampton Research). The purity of the sample used in the crystallization experiments was ϳ98%, as estimated by SDS-PAGE. The OxdRE activity was assayed at 30°C under anaerobic conditions by measuring the amount of phenylacetonitrile formed from Z-phenylacetaldoxime. The assay reaction was started by the addition of Z-phenylacetaldoxime (5 mM in the final concentration) dissolved in N,N-dimethylformamide into a 500 mM phosphate buffer (pH 7.0) solution containing 5 mM sodium dithionite and 1 M purified OxdRE, in a final volume of 0.5 ml. High pressure liquid chromatography analyses were performed as described previously (7). The activity was measured at least three times for each sample. The average values are shown in Table 2. Crystallization-Proteins were crystallized with hanging drops of 3 l of protein mixed with an equal volume of precipitant solution in a 24-well crystallization VDX M TM plate (Hampton Research) at 20°C. OxdRE was concentrated to 30 mg ml Ϫ1 in 10 mM HEPES buffer (pH 7.4). Tris-(2-carboxyethyl)phosphine was added to the concentrated sample to a final concentration of 2 mM, and the mixture was centrifuged at 50,000 ϫ g for 20 min before crystallization. Initial screening using Natrix screening kit (Hampton Research) revealed substrate-free OxdRE crystals. The final crystallization condition of the substrate-free OxdRE was 18% (w/v) polyethylene glycol 4000, 75 mM sodium cacodylate (pH 7.4), and 100 mM magnesium acetate. For cryoprotection, the crystal was soaked in a reservoir solution containing 16% (w/v) sucrose and 12% (w/v) trehalose. To prepare substrate-bound crystals by the soaking method, substrate-free crystal was soaked for a few minutes in a reservoir solution supplemented with 16% (w/v) sucrose, 12% (w/v) trehalose, and 2% (v/v) substrate (BOx or PrOx). For co-crystallization of OxdRE and BOx, the concentrated OxdRE solution (10 mg ml Ϫ1 ) was mixed with BOx at a final concentration of 2% (v/v) and incubated for 1 h at 20°C before starting crystallization. The final crystallization condition was 17% (w/v) polyethylene glycol 2000 monomethylether, 100 mM Trizma (Tris base)/HCl (pH 7.0), and 200 mM calcium acetate. The crystal was soaked in reservoir solution containing 22% (w/v) xylitol and 2% (v/v) BOx for cryoprotection. Data Collection, Structure Determination, and Refinement-The optical absorption spectra of the crystals were measured by a single-crystal microspectrophotometry system (11) on the beam line BL44B2 (SPring-8, Hyogo, Japan). Diffraction data were collected on beam lines BL41XU and BL44B2 (SPring-8). All image data were collected at 90 K. The HKL2000 (12) or XDS (13) program was used for integration of diffraction intensity and scaling. Initial phases of substrate-free crystal were calculated by the SHELX-97 (14) and HKL2MAP (15) programs from multiwavelength anomalous dispersion (16) data measured at the absorption edge of the iron atom (supplemental Table S2). Phases were further improved and extended to 1.8 Å resolution data. Most parts of the atomic model of OxdRE were built automatically using the ARP/warp (17) program. The heme and remaining residues were manually built using the graphics program Coot (18). The model was refined using REFMAC5 (19). Non-crystallographic symmetry restrains were applied to the core region of the protein molecule throughout all refinement cycles. The model geometry was checked using the program WHAT IF (20). Crystal structures of the substratebound form were determined by the molecular replacement method using the MOLREP program (21). The substrate-free OxdRE structure was used as a search model. The open and closed states were defined based on the conformations of the A-and C-chains in PrOxsoaked crystal and of the co-crystallized BOx-bound OxdRE, respectively. The ␣10 and following 3 10 showed the positive density for the close state. The occupancies of alternative conformations were not refined by the program but estimated from manual adjustment in 0.05 steps, which is followed by the refinement and the check of B-factor of each atom and F o Ϫ F c map. The additional N-terminal 20 residues derived from the vector, including the His tag, were disordered in density for most of the polypeptide chains. Coordinates-The coordinates for OxdRE and BOxbound OxdRE prepared by co-crystallization and for BOxbound OxdRE and PrOx-bound OxdRE prepared by the soaking method were deposited in the Protein Data Bank (PDB) with accession codes of 3A15, 3A17, 3A18, and 3A16, respectively. RESULTS AND DISCUSSION Overall Structure and Protein Fold-Statistics of the data collection and refinement of OxdRE are summarized in Table 1. The overall crystal structure of substrate-free OxdRE was solved by multiwavelength anomalous dispersion (16) and refined at a 1.8 Å resolution (supplemental Table 2). OxdRE formed a homodimer with non-crystallographic two-fold symmetry (Fig. 1A), consistent with previous gel filtration analysis results (22). Each monomer contained one heme molecule. The ␣10 helix of one monomer interacted with the ␣10 helix of the other to create the dimer interface, which was stabilized by hydrogen bonds and electrostatic interactions. Each monomer of OxdRE has a ␣ϩ␤ structure consisting of an elliptic ␤-barrel flanked on both sides by ␣-helices (Fig. 1B). The barrel was composed of eight ␤-strands, ␤32 ␤41 ␤22 ␤5-5Ј1 ␤11-11Ј2 ␤8-8Ј1 ␤92 ␤101, with all strands exhibiting an antiparallel pattern. Some regions of ␤-strands failed to form hydrogen bonds with the neighboring strand because of the insertion of a short turn or loop, which deformed the ␤-barrel. The interior of the ␤-barrel primarily consisted of hydro-phobic residues. A hydrogen-bonding network involving the side chains of Arg-218, Tyr-98, Tyr-278, Tyr-319, and Asp-80 was also found (supplemental Fig. 2). There was no space to create a cavity or channel inside the ␤-barrel. Structural data base searches using DALI (23) and MATRAS (24) indicated that the eight-stranded elliptic barrel structure and secondary structure arrangement of OxdRE show a low similarity (z-score in DALI Ͻ8) to known proteins with a ␣ϩ␤ ferredoxin-like fold consisting of four-stranded antiparallel ␤-sheets packed as a ␤-barrel against two kinked ␣-helices. OxdRE displayed a unique topology of ␤-strands in the barrel (22 31 12 41 82 51 62 71) (supplemental Fig. 3), which was not identical to two repeats of (22 31 12 41) that are seen in other ferredoxin-like folds (25). Although a hypothetical protein TT1485 that shows ␣ϩ␤ ferredoxin-like fold is assumed to have heme binding ability (26), the topology and the location of proposed heme-binding pocket of TT1485 are distinct from those of OxdRE (supplemental Fig. 4). Thus, the structure of OxdRE was evaluated as a hemoprotein containing a novel ␤-barrel topology. Environment of the Heme in the Substrate-free OxdRE-The crystal of the substrate-free OxdRE was obtained in ferric form, in which the heme iron was in the Fe 3ϩ state. However, the heme was reduced by x-ray radiation, as confirmed from the visible crystal spectra obtained with the single-crystal microspectrophotometry system. As data collections for the structural analyses were begun after heme reduction was complete, the structure of the substrate-free OxdRE reported here is the same as that of ferrous OxdRE. The heme exists between the ␤-barrel and the helix in the hydrophobic pocket composed of non-polar side chains of Trp-172, Ile-217, Ile-238, Leu-242, Leu-249, Leu-289, Trp-292, Ile-302, Phe-303, Phe-306, and Phe-307 (supplemental Fig. 5). His-299 in the ␣10 helix was the proximal ligand of the heme iron. The orientation of the His-299 imidazole ring was stabilized by a hydrogen bond between the N ␦1 atom of His-299 and the O atom of the main chain at position 293 (Fig. 2). A water molecule (w1) coordinated to the distal coordination site of the heme in the substrate-free form (Fig. 2). The heme 6-propionate group was directed to the proximal side, and its conformation was stabilized by interactions with two waters (w2 and w3) and the side chain of Ser-174 (supplemental Fig. 5B). The 7-propionate was directed to the distal side and interacted with the side chain of His-169 and the main chain amide of Gly-170 (supplemental Fig. 5B). A water molecule (w4) was shared by two propionates for hydrogen bonding (supplemental Fig. 5B). One of the ␤-strands in the barrel provided His-320, whose N ␦1 atom is located within a distance capable of a hydrogen bonding with w1. The side chain of Ser-219 also formed a hydrogen bond to w1 (Fig. 2). Another water molecule (w5) interacted with the side chains of Ser-219 and Gln-221 near w1. In the distal heme pocket, a hydrogenbond network existed among His-320, Glu-143, and Arg-178 (Fig. 2). Preparation of the Crystal of the Michaelis Complex for OxdRE- The structure of substrate-free OxdRE was determined, as described above. Determination of the structures of the resting state and the Michaelis complex of OxdRE should provide useful information for the elucidation of the molecular mechanisms of OxdRE catalysis. In principle, crystallization of the Michaelis complex of a native enzyme is not possible because the complex is a transiently formed intermediate on enzymatic catalysis that is formed only at very low concentrations. We obtained the crystal of the Michaelis complex by using the unique properties of OxdRE. The substrate-bound ferric form of the OxdRE crystal was obtained by crystallization of ferric OxdRE in the presence of the substrate or by soaking the substrate-free crystal of ferric OxdRE in a substrate-containing solution. Ferric OxdRE is an inactive form that forms a dead-end complex with the substrate. The oxidation state of the heme iron and the coordination mode of the substrate were different between this dead-end complex and the Michaelis complex (supplemental Fig. 1). We postulated that the crystal of the OxdRE Michaelis complex could be obtained by reduction of the heme iron in the crystal of the dead-end complex using x-ray radiation under cryogenic temperature. The optical absorption spectra of the substrate-free and substratebound OxdRE crystals were changed upon x-ray radiation (Fig. 3). The spectral change in the Q-band region indicates that the heme iron in the substrate-bound crystal was also reduced by x-ray radiation at 90 K, as was also the case for substrate-free OxdRE. The heme iron began to be reduced within seconds by the x-ray beam of SPring-8 BL44B2, and reduction was complete within 60 s (Fig. 3C). We collected x-ray diffraction images for structural analyses after confirming the reduction of the heme iron by microspectrophotometry. The structures of the BOx-bound OxdRE and PrOx-bound OxdRE were determined at 1.8 and 1.6 Å resolutions, respectively (supplemental Fig. 6). The nitrogen atom of aldoxime was coordinated to the heme in these complexes, indicating that reduction by x-ray radiation has successfully reconstructed the coordination structure of the aldoxime-heme complex from the inactive to the active form, even in the crystal. The overall structure of the substrate-bound OxdRE was almost the same as that of the substrate-free OxdRE, but there were some local conformational changes upon substrate binding, as discussed below. Environment of the Heme in the Michaelis Complex of OxdRE-The structures around the heme of PrOx-and BOx-bound OxdRE are shown in Figs. 4 and 5, which clearly reveal that PrOx and BOx bound to the heme iron via the N atom. The structures around the heme were similar in PrOx-and BOxbound OxdREs. The substrate alkyl group was surrounded by hydrophobic residues (Met-29, Leu-145, Phe-306, and Leu-318) in the distal side of the heme, but there was no specific arrangement of hydrophobic residues to discriminate the specific alkyl group (Fig. 5). A large cavity existed on the distal side of the heme (Fig. 6A). OxdRE has a broad substrate specificity capable of converting various aryl-and alkyl-aldoximes to their corresponding nitriles (22). This is because the large cavity does not prevent accommodation of various substrates. The OH group of the heme-bound substrate formed two hydrogen bonds with Ser-219 and His-320. In the distal heme pocket, the hydrogen-bond network was retained among Glu-143, Arg-178, and His-320 (Fig. 5), as was the case of the substrate-free form. Conformational differences between the substrate-free and substrate-bound forms were not observed in these distal residues. Although no conformational differences were observed at the distal side of the heme among the determined structures, conformational variations were observed at the proximal side of the heme (Fig. 6B). The region 294 -315, including the proximal ␣-helix (␣10) and the following 3 10 helix (4), displayed a conformational variation in each monomer in the asymmetric unit of the crystal. In the dimer of the substrate-free form, two conformations were present: one subunit with an open form and another with a closed form. In the open form, the substratebinding cavity in the distal side of the heme was connected to the protein surface through a channel formed between the 3 10 helix (309 -312) and the loop (24 -29). This cavity will serve as the substrate access and product release channel (Fig. 6A). However, the entrance of the channel on the protein surface was closed in the closed form (Fig. 6C), in which the proximal ␣-helix was rotated ϳ15°(as viewed from the heme) and the 3 10 helix deviated ϳ5 Å to cover the channel entrance. The conformational change from the open to the closed form resulted from the sliding of Phe-306 by 3 Å vertically to the heme plane, accompanied by rotation of the proximal ␣-helix. This movement of Phe-306 disconnected the substrate-binding cavity from the substrate access/product release channel (Fig. 6A). Thus, the 3 10 helix and side chain of Phe-306 act as a gate for the channel, modulating substrate access and product release. Substrate-bound OxdRE displayed different populations of two conformations, depending on crystal preparation method. PrOx-bound OxdRE prepared by soaking the OxdRE crystal in PrOx for 30 s contained two dimers (AB and CD) in an asymmetric unit. Although the A-and C-chains had open conformation, the B-and D-chains had mixtures of open and closed forms, with estimated occupancies of 75 and 25% for the open and closed forms, respectively (supplemental Fig. 7A). BOxbound OxdRE prepared by the soaking method had a similar population of the two forms as the PrOx-bound OxdRE (supplemental Fig. 7B). However, BOx-bound OxdRE prepared by co-crystallization had mainly the closed conformation (occupancy Ͼ90%) for all chains in the asymmetric unit. These results suggest that the open and closed forms are in equilibrium in solution and that the substrate binding can shift the equilibrium from the open to the closed form. As complete equilibration may not be achieved in the crystal prepared by soaking in PrOx or BOx, the substrate-bound OxdREs prepared by the soaking method will have a mixed conformation. Reaction Mechanism of OxdRE-The apparent role of the heme in OxdRE is to tether the substrate in the catalysis by aldoxime dehydratase. The crystal structures determined in this study reveal that hydrogen bonding between the OH group of aldoxime and the side chains of Ser-219 and His-320 controls the specific orientation of the heme-bound substrate suitable for the elimination of the OH group of aldoxime and that these residues and the heme create a prefixed site for substrate recognition and binding. An inducedfit type conformational change was not observed upon substrate binding at the distal heme pocket, the substrate-binding site in OxdRE. His-320 also serves as a catalytic residue for the elimination of the substrate OH group to form H 2 O, consistent with previous mutagenesis studies (7)(8)(9)(10). The H320A mutant lacked enzymatic activity ( Table 2). A hydrogen-bond network existed among Glu-143, Arg- 178, and His-320 that fixed the proper orientation of His-320 toward the heme-bound substrate. Based on analyses of the resonance Raman spectra of CO-bound aldoxime dehydratase from Pseudomonas chlororaphis B23 (OxdA), Konishi et al. (9) have proposed that the catalytic His, which corresponds to His-320 in OxdRE, is in imidazolium form in the resting state, in which both of the nitrogen atoms of the imidazole ring are protonated. The hydrogen bond between Glu-143 and His-320 will play an important role not only for control of the proper orientation of His-320 but also for stabilization of the imidazolium form of His-320. Taken together, we propose the reaction scheme as shown in Fig. 7. Previous studies using a cavity mutant of OxdRE revealed that electron donation from the proximal ligand to the heme iron accelerates the dehydration reaction (27). The hydrogen bond between His-299 and the oxygen atom of the main chain will strengthen the imidazolate character and electron donation ability of the proximal His, partially regulating the enzymatic activity of OxdRE. These results suggest another functional role for the heme in which the substrate coordination polarizes the NϭC bond, assisting in product formation. Mutagenesis studies confirmed the functional role of Glu-143, Arg-178, Ser-219, and His-320 deduced from the structural analyses. Mutation of Glu-143, Arg-178, Ser-219, His-320, or Phe-306 resulted in a 60 -90% loss in enzymatic activity ( Table 2). E143Q OxdRE displayed similar activity to that of H320A OxdRE, which lacked the catalytic residue. This result indicates that the proper orientation of His-320, which is mainly achieved by a hydrogen bond with Glu-143, is crucial for the enzymatic activity of OxdRE. The effect of the mutation of Phe-306 suggests that disconnection of the substrate-binding cavity from the substrate access channel and bulk solution, which is induced by substrate binding to the heme, controls the hydrophobicity/hydrophilicity of the distal heme pocket for the reaction to proceed efficiently.	v3-fos-license
2016-05-04T20:20:58.661Z	2012-11-27T00:00:00.000	16338095	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0050299&type=printable", "pdf_hash": "08ed31994644acbee44aab7031a2303bb6b72a6b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:219", "s2fieldsofstudy": [ "Biology" ], "sha1": "08ed31994644acbee44aab7031a2303bb6b72a6b", "year": 2012 }	pes2o/s2orc	Electroendocytosis Is Driven by the Binding of Electrochemically Produced Protons to the Cell’s Surface Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method to electroporation for the incorporation of macromolecules into cells. The present study explores the underlying mechanism of electroendocytosis and its dependence on electrochemical byproducts formed at the electrode interface. Cell suspensions were exposed to pulsed low electric field in a partitioned device where cells are spatially restricted relative to the electrodes. The cellular uptake of dextran-FITC was analyzed by flow cytometery and visualized by confocal microscopy. We first show that uptake occurs only in cells adjacent to the anode. The enhanced uptake near the anode is found to depend on electric current density rather than on electric field strength, in the range of 5 to 65 V/cm. Electrochemically produced oxidative species that impose intracellular oxidative stress, do not play any role in the stimulated uptake. An inverse dependence is found between electrically induced uptake and the solution’s buffer capacity. Electroendocytosis can be mimicked by chemically acidifying the extracellular solution which promotes the enhanced uptake of dextran polymers and the uptake of plasmid DNA. Electrochemical production of protons at the anode interface is responsible for inducing uptake of macromolecules into cells exposed to a pulsed low electric field. Expanding the understanding of the mechanism involved in electric fields induced drug-delivery into cells, is expected to contribute to clinical therapy applications in the future. Introduction Electropermeabilization of the cell membrane by pulsed high electric fields has been used in the last three decades to induce the uptake of molecules, in particular DNA, into the intracellular compartment of the cell [1]. The excepted paradigm in this research field has been that electropermeabilization occurs via short-lived pores in the plasma membrane that are formed when the cross-membrane potential difference reaches a threshold value of ,200 mV [2,3,4]. However, the mechanism for uptake of large molecules, DNA in particular, has not been fully resolved [5]. Since the 1990s, numerous reports have shown that in addition to electropermeabilization, the application of high electric fields can induce endocytic-like processes [6,7,8,9,10,11]. The fact that both plasma membrane permeabilization and membrane vesiculation can occur during and following cell exposure to the high electric fields, has hindered the identification of the mechanism(s) underlying electroendocytosis. A significant advance in exploring this phenomenon was the discovery that exposure of cells to nonpermeabilizing pulsed train of low electric fields (LEF), leads to a stimulated uptake of different fluid phase and adsorptive fluorescent probes of low and high molecular weight via endocytic-like pathway [12,13]. The exposure to LEF was reported to generate an alteration of cell surface, leading to elevated adsorption of macromolecules such as bovine serum albumin (BSA), dextran and DNA, as well as to an enhanced uptake [14]. This surface alteration, attributed to the electropho-retic segregation of charged mobile lipid and protein entities in the cell plasma membrane, was suggested to be responsible both for enhanced adsorption and stimulated uptake, via change of the plasma membrane curvature that enhances budding processes [14]. Recently, an important development in the understanding of the mechanism that underlies endocytic-like uptake was reported, revealing that high concentration of hydrogen ions at the cells' surface can induce inward membrane vesiculation and uptake of macromolecules [15]. Since the exposure of cells to high or low electric fields, which leads to electropermabilization and electroendocytosis, involves direct contact between the electrodes and the cells' medium, the cells are expected to be exposed to electrochemical byproducts created at the electrode-solution interface. In the present study we examined the involvement of radical oxygen species (ROS) and elevation in hydrogen-ion concentrations in the uptake of macromolecules induced by low pulsed train of electric fields. Our finding reveals that uptake only occurs at the proximity of the anode and that electrochemical acidification of the extracellular media, is sufficient to enhance the uptake of macromolecules, including DNA, by cells via endocytic-like pathway. Cell Culture COS-7 cells (ATCC No. CRL-1651, monkey kidney fibroblastlike cells) and HaCaT cells (human keratinocyte [16]) were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with L-glutamine (2 mM), 10% FCS and 0.05% PSN solution. All cells were grown in 75 cm 2 tissue culture flasks (Corning) at 37uC, in a humid atmosphere of 5% CO 2 in air. Cells were harvested before reaching ,80% confluence by using 0.25% trypsin solution (with 0.05% EDTA) for 5 min at 37uC. The cells were centrifuged (1 min at 400 g), their solution aspirated and then re-suspended in growth medium. All culture media, antibiotics, trypsin and serum products were purchased from Biological Industries (Beit Haemek, Israel). Exposure Set-up for Low Electric Fields Exposure of cells to low-intensity trains of unipolar rectangular voltage pulses was carried out by employing an electric pulse generator (Grass S44 Stimulator, West Warwick, USA). Exposure of cells in a three-compartment exposure set-up [17] consisted of a rectangular chamber made from polystyrene, 15 mm long, 10 mm wide, with 0.5 cm 2 area planar platinum electrodes positioned on the extreme sides (Fig. 1). The chamber is partitioned by two porous membranes (polyethersulfone, 0.8 mm pores and 200 mm thickness) into three distinct and equal compartments: anode, cathode and a central one. The passage of charged low MW fluorescent probes from the anode or cathode compartments into the central one was monitored using tryphan-blue (TB) or Luciferyellow (LY) by measuring their fluorescence (480/590 nm or 430/ 530 nm, respectively). The passage of hydrogen ions and oxidative intermediates into the central compartment was monitored by a pH electrode or by TMB color conversion, respectively. The electric field parameters applied were monitored on-line by recording the voltage and the current (by means of a wide band current probe, Pearson) using an oscilloscope. Typically, a cell suspension is exposed to a train of low electric fields (LEF) consisting of unipolar rectangular pulses with duration of 180 ms, frequency of 500 Hz for the total time of exposure of one minute. The temperature of the solution during exposure was measured using fiberoptic temperature sensors (FISO Technologies, Quebec, Canada). The measured mean transient temperature rise at the end of a 1 min exposure of cells to LEF of 20V/cm at 24uC was 1.460.6uC (mean 6 SD). Uptake Studies Cells were harvested, re-suspended in DMEM (glucose 1 mg/ ml, FCS 5%), and incubated (37uC, 5% CO 2 ) as designated by the experimental setup. When no pre-treatments were required, the cells were kept in the DMEM for the same duration. Following incubation period, the cells were centrifuged at 400 g for 1 min, the solution aspirated, and cells were re-suspended in HBSS. Cell suspensions (1610 6 cells/ml, 0.5 ml per compartment) were exposed to LEF in the three-compartment device in the presence of dextran-FITC for 60 s at room temperature (,24uC). The samples were then immediately diluted with 5 fold larger volume of ice-cold DMEM-H (25 mM HEPES supplemented DMEM without phenol red) and kept on ice. Exposure of cells to pulses of low pH was done through titration of HBSS with HCl for 60 s, terminated by the addition of 3x volume of cold DMEM-H. Cells in the control group were incubated with dextran-FITC for 60 s at room temperature, after which they were moved to ice as described above. Each experiment was repeated independently at least 9 times spread over three different days. Removal of the Dextran-FITC fraction Adsorbed to Cell Surface For determining the fraction of the fluorescent dextran adsorbed to the cells' membrane, cell suspensions were pre-cooled to 4uC and exposed to LEF for 60 s in the presence of dextran, thereby eliminating the contribution of endocytosis. For measuring the cell auto-fluorescence, LEF exposure was carried out in the absence of the external fluorescent probe. In preparing for analysis, the cells were washed twice in DMEM-H medium by centrifugation (400 g for 1 min, Sorvall RT6000D, Du-Pont USA). To further remove remaining dextran adsorbed to the cell membrane, the cell suspensions were centrifuged again, supernatant aspirated and the pellet re-suspended with 0.5 ml DMEM-H supplemented with 2 unit/ml of dextranase for 10 min at 24uC. Cells were washed again and the cell pellet was re-suspended with 0.2 ml DMEM-H. Shortly before FACS analysis, PI (25 mg/ml) as a label for membrane-permeable cells and TB (0.01% w/v) as a fluorescence quenching agent for extracellular FITC [18] were added to each sample. Microscopic examination of the cells verifies the absence of detectable fluorescent corona around the cell membrane (Fig. 2). Flow Cytometry (FACS) Flow cytometry analysis was carried out by FACSort (Becton @ Dickson, San Jose, CA), employing a 488 nm argon laser excitation. The green fluorescence of FITC was measured via 530/30 nm band filter, the red fluorescence of PI was measured via a 585/42 nm band filter. To eliminate signals due to cellular fragments, only those events with forward scatter and side scatter comparable to whole cells were analyzed. Ten thousand cells were examined for each sample and data were collected in the list mode. The analysis of flow cytometry data was performed using WINMDI 2.9 software (Joe Trotter, The Scripps Research Institute). For each sample the geometrical mean was calculated without including PI stained cells. The efficiency of probe loading was characterized by cell labeling intensity using fold of induction, (FI) that was calculated as the ratio of the geometric mean of fluorescence in experimental samples to that of control nonexposed ones. Determination of Oxidative Stress in Solution Extracellular media analysis was carried by employing TMB, a colorless solution that is transformed into blue when oxidized. TMB was added to the solution before or after its exposure to LEF and the solution collected and stabilized with 1.25 M HCl, which transforms its color to yellow. Optical density of the stabilized oxidation product was measured at 450 nm employing a microplate reader (GENius, Tecan, USA). Determination of Intracellular Oxidative Stress Preloading cells with 10 mM of Carboxy-H 2 DCFDA was performed by incubating the cells (at 37uC in humid atmosphere with 5% CO 2 ) for 90 min in serum-free DMEM. H 2 DCF-DA is essentially a cell-permeable, non-fluorescent molecule that can be transformed by the action of intracellular esterases to a charged, cell non-permeable H 2 DCF molecule. In this form the molecule can be oxidized, mainly by super oxides and radical hydroxyls to the green fluorescent dichlorofluorescein (DCF) [19]. Harvested cells were incubated and loaded with carboxy-H 2 DCF-DA. Shortly after the loading process the cells were washed once with HBSS before being exposed to the electric field under the designated experimental setup. Following the exposure, cell suspensions were washed and prepared for FACS analysis as described. All washing steps were performed with ice cold solutions. Quantitative and Qualitative Evaluation of Solution's pH pH analysis in the medium shortly after LEF exposure was determined by pH electrode (SevenEasy, Mettler). For qualitative evaluation of the transient pH formed at the anode surface during the application of electric current, we used irreversible pH sensitive paper indicators (PANPEHA, Sigma-aldrich) prepared by cutting 5 mm by 7 mm rectangular section from the original paper strips. The pH indicator paper was placed perpendicularly to the electrode surface plane and in one quick movement dipped in and pulled out from the anodic compartment solution followed by gentle placement on a blotting paper. Osmolarity Solutions were analyzed employing an osmometer (Vapro 5520, Wescor), based on the evaporation point method. Medium Conductivity Conductivity was lowered by replacing some of the soluble salt ions with sucrose. 300 mM sucrose solution in water was used for diluting HBSS at several ratios to final sucrose concentrations from 200 mM down to 50 mM. Plasmid DNA A 4.7 kb pAcGFP1-C1 plasmid, (Clontech, Takara), which encodes a GFP reporter gene, was used for the DNA transfection . Cell images following LEF induced uptake of dextran-FITC. COS-7 cells were exposed to LEF in the anode compartment for 60 s in the presence of dextran-FITC (38 kD). The cells were washed as described in section 2.5 and mounted on glass slide in PBS containing 0.01% TB. Images of cells were acquired by SCLM in the DIC (images A and C. arrows point to the cell nucleus) and fluorescence (485/530 nm, images B and D) modes. Images A and B depict cells exposed to LEF at ,4uC. Images C and D depict cells exposed to LEF at room temperature. doi:10.1371/journal.pone.0050299.g002 and uptake studies. The DNA was prepared using PureYield TM Plasmid Midiprep System (Promega), according to manufacturer's protocol and dissolved in nuclease free water. The quantity and quality of the plasmid was assessed using NanoDrop ND-1000 spectrophotometer (Thermo Scientific) by light absorption at 260/ 280 nm ratio and by 1% agarose gel electrophoresis. For uptake studies the plasmids were labeled with Cy3 using the Label ITH Nucleic Acid Labeling Kit, Cy3 (Mirus), according to the manufacturer's protocol. Positive control transfections were performed using TurboFect TM (Fermentas) transfection reagent according to the manufacturer's protocol. Microscopy Scanning Confocal Laser Microscope (SCLM; LSM 410, Zeiss, Germany) was used for acquiring microscopic cell images. In SCLM, computer-generated images of 0.5 mm optical sections were obtained at the approximate geometric center of the cell as determined by repeated optical sections. The images were processed by axiovision software (Zeiss, Germany) and image annotation was made using Illustrator CS software (Abode, USA). Statistical Analysis Statistical analyses were performed by student's t-test and one way ANOVA, using Microsoft Excel. Electrically Induced Uptake is not Uniform in between the Electrodes In order to differentiate between the contributions of the electric field itself and its electrochemical byproducts to the extent of uptake, we constructed an exposure system where the cells could be maintained in three compartments; anodal, central and cathodal (Fig. 1). These compartments are separated by two highly porous (0.8 mm) and tortuous membranes. These membranes possess very low electric resistance when placed in physiological solutions (device resistance is 47.6 V without the membranes and 50 V with both of them), yet are able to delay the diffusion time between the compartments due to internal tortuous structure which provides a longer travel path for the passing molecules. This was experimentally verified using two low molecular weight dyes (LY and TB) which showed no traceable penetration across the membrane barriers in the three compartment exposure system, following one minute of exposure to LEF. COS-7 cell suspensions were placed in the three compartment exposure device employing Pt electrodes. Uptake was induced by exposure to LEF (20 V/cm, 200 mA/cm 2 , 180 ms pulse width, 500 Hz repetition rate) for 60 s at 24uC, in the presence of dextran-FITC (38 kD, 0.1 mM). Following their exposure, the cells were washed by PBS and analyzed by FACS. Cells exposed to LEF in the anodal compartment showed an ,3 fold increase in cellular uptake of dextran-FITC relative to the constitutive uptake in the absence of LEF (Table 1, P,0.05 by t-test, n = 9). However, no enhanced uptake was found in cells present in the other two compartments, i.e. the central and cathodal ones. Electrically Induced Uptake Depends on Current Density more then on Electric Field Strength The relative contribution of the electric field and the electric current to the elevated uptake of dextran-FITC was further examined by exposing COS-7 cells to LEF in dextran solutions possessing different electrical conductivities. In order to improve assay sensitivity, we employed a 71 kD dextran (0.1 mM) possessing a higher number of FITC conjugates per molecule. Conductivity was altered by substituting NaCl with sucrose while maintaining iso-osmotic conditions. Data is presented only for the cells exposed in the anode compartment, since cells in the other compartments (i.e. cathodal and central) showed no enhanced uptake over the constitutive level. The results reveal that variation in electric field strength has minor impact on cellular uptake of dextran-FITC at a given current density (Fig. 3A, B and C). Explicitly, Fig. 3D describes the uptake as function of electric current density (at 20 V/cm) possessing a second order polynomial dependency. Electrically Induced Uptake is Independent of Electrochemically Produced ROS Electric current is known to promote oxidation at the anode interface through the production of reactive oxidative intermediates. The term oxidative stress (OS) refers to an imbalance between cellular production of reactive oxidative species (ROS) and their disintegration, leading to elevation of intracellular oxidative activity and consequent damage. Such OS, either extracellular or intracellular, could play a role in the electrically induced uptake. In order to study this possibility we first examined the formation of ROS in the three-compartment exposure set-up. ROS was monitored by measuring the colorimetric oxidation of TMB present in solution during its exposure to LEF. TMB in the anodic compartment solution changed its color during electric current application, but addition of TMB immediately after LEF termination failed to produce any color conversion. Supplementing the solution with 2 mM sodium ascorbic acid (SAA), prior to its exposure to LEF in the anodic compartment was sufficient to prevent TMB oxidation during the exposure ( Table 2). The intracellular OS was monitored by the fluorescence intensity of DCF of cells preloaded with H 2 DCF. Only cells exposed to LEF in the anode compartment developed enhanced fluorescence intensity (Table 1). COS-7 cells exposed to LEF in the presence of an antioxidant (2 mM SAA), possessed 66% less DCF fluorescence intensity ( Table 2). Cells were loaded with the intracellular antioxidant DHA by incubation in glucose free DMEM with 5% FCS and 1 mM DHA for 90 min. Once in the cytosol, DHA, the stable reduced form of ascorbic acid, is converted to ascorbic acid by intracellular enzymes [20,21]. Pre-loading the cells with DHA before their exposure to LEF was sufficient to completely abolish the DCF fluorescence increase ( Table 2). In order to determine the role played by OS in LEF induced uptake, COS-7 cells exposure to LEF was carried out in the presence of dextran-FITC (71 kD, 0.1 mM). Dextran uptake was compared between cells in standard HBSS, cells in HBSS Uptake of Dextran-FITC in terms of exposed/constitutive uptake. b ROS determined by DCF relative fluorescence in terms of exposed/unexposed cells. Dependence of Electrically Induced Uptake on Acidification in the Anode Compartment Electrolysis of water is known to be responsible for acidification near the anode and alkalization near the cathode. However, the exposure of HBSS supplemented with 100 mM HEPES to LEF in the anode compartment, produced no steady-state changes in the solution's pH (pH 7.4) or osmolarity (290 mOs), relative to its original values, when measured after LEF termination. The exposure of HBSS without additional buffering shows that the solution's pH is shifted with dependence on the electrode, i.e. alkaline near the cathode and acidic near the anode (Table 1). In order to monitor the transient pH change occurring at the anode's surface during the exposure to LEF, an irreversible pH-sensitive paper indicator was dipped-in perpendicularly to the electrode surface and pulled out immediately. This method verified the existence of a transient drop in spatial pH profile near the anode interface down to the range of 1.0-2.0 units. In order to validate the role of the transient acidification during exposure in the enhanced uptake by LEF we varied the concentration of HEPES buffer (60 mM to 150 mM) in the exposure solution (HBSS), in the presence of dextran-FITC (71 kD, 0.1 mM). Solution's iso-osmolarity was maintained by replacing NaCl with HEPES and current density of 200 mA/cm 2 was kept constant for all experimental instances. Fig. 4A reveals that reducing the buffer concentration in the exposure solution strongly increased the extent of dextran-FITC uptake by COS-7 cells in a sigmoid-like plot, having the steepest slope occurring between 80 mM and 70 mM HEPES. For simulating the effect of electrochemical acidification on the cellular uptake of dextran-FITC, COS-7 cells were suspended in 10 mM MES buffered HBSS, and the solution's pH was lowered by titration with hydrochloric acid. Following 60 s exposure to external low pH in the presence of dextran-FITC (71 kD, 0.1 mM), the cell suspensions were immediately diluted with excess volume of cold DMEM to restore the normal pH 7.4. Fig. 4B reveals the dependence of cellular uptake on external pH level. This dependence is characterized by a gradual linear increase from pH 7.0 to pH 4.0, with a steep sigmoid-like elevation in uptake in the range of pH 4 to pH 3. Low pH Induces the Entry of Naked DNA Plasmid into Cells Cultures of HaCaT cells were incubated in either HBSS (pH 7.4) or HBSS with 20 mM MES (tittered to pH 5) in the presence of 0.6 nM (0.1 mg/well) plasmid tagged by Cy3 for a period of 45 min at 24uC. Following treatment, the cells were prepared for SCLM analysis by fixation with karnovsky's solution, followed by 3 washes with PBS and further staining of the cell's surface and nucleus with Cholera toxin subunit-B (0.2 mg/ml, Alexa 488 conjugated) and 1 mg/ml DAPI, respectively. Confocal fluorescence imaging was used to demonstrate the uptake of the Cy3 tagged DNA plasmid at pH 5, compared to uptake at pH 7.4 (Fig. 5). For the study of DNA transfection efficiency, cultures of HaCaT cells were incubated for 1 hr in either HBSS (pH 7.4) or HBSS with 20 mM MES buffer (tittered to pH 5) in the presence of 4.8 nM (3 mg/well) plasmid. The duration of the incubation was limited by the tendency of the cells to detach from the culture surface in response to prolonged exposure to low pH. Following their incubation with the DNA plasmid, the cultures were washed with HBSS and incubated in a growth medium for 48 hours. The extent of transfection by the plasmid was measured by employing FACS for counting the fraction of cells that express the GFP fluorescent protein. The potency of the plasmid was verified by inducing its transfection in the cells by standard TurboFect TM (Fermentas) protocol. Despite the apparent entry of plasmids into the cells under low pH conditions, only a very small fraction (,0.1%) of the cells expressed the encoded fluorescent protein, similar to the transfection level found in the control cells, exposed to the plasmid at pH 7.4. Discussion Electroendocytosis, an electric-field induced endocytic-like process, was previously reported to enable the uptake of macromolecules by cells [12,13,14]. In the present study we examine the possibility that electroendocytosis is driven by the formation of electrolytic byproducts. The impact of the cells' location relative to the electrodes on the extent of uptake was examined in an exposure device where the cells are restricted to one of three partitions -near the anode, near the cathode or at 5-10 mm from the electrodes. When employing this exposure set-up, essentially the same electrical field prevails in all three compartments. However, no increase in cellular uptake was observed in the central and the cathodal compartments, whereas the cells in the anodal one demonstrate an impressive increase of uptake (See Table 1). This finding does not comply with an effect driven by an electric field. Furthermore, it is shown that the enhanced uptake depends on the electric current density rather than on the electric field strength (Fig. 3). All together, these findings imply that the electrically induced uptake is more likely to be associated with the electrochemical byproducts formed in the anodic compartment than with the electric field per-se. This conclusion points out a clear mechanistic difference from that of electroporation, which depends on electric field strength. A differentiation between LEF induced uptake and an electroporation driven one was already previously discussed [12]. The main difference between electroporation and inward vesiculation is the fact that the later phenomenon does not involve membrane permeability change. One way to demonstrate the existence of permeability change of the cell membrane for small molecules is to load the cells with the small molecules and to examine whether they undergo enhanced efflux following exposure to electric field. Such studies have been carried out previously [12] and no enhanced efflux was detected. Electrolysis at the anode-solution interface produces radical oxidative species as well as increased concentration of hydrogen ions. We show that electrochemical oxidative intermediates which are formed in the anodal compartment are instrumental in elevating the intracellular oxidative stress. However, while the presence of extracellular and intracellular antioxidants can attenuate this cellular oxidative stress, it conveys no significant impact on the extent of electrically induced uptake. Electrohydrolysis is the decomposition of water due to an electric potential applied across a pair of electrodes. If electrohydrolysis occurs in low water conductivity, H + cations and OH 2 anions will remain at the interface of their respective electrodes. This will lead to electrode polarization and unless a very large electric potential is applied to cause an increase in water ionization, the electrolysis will be slowed down. If the conductivity of the water is raised through a dissociated electrolyte (e.g. NaCl), the electrolyte anions and cations neutralize the buildup of charges at the electrodes interface, allowing for the flow of electricity and the continued hydrolysis. Thus while the electrode potential drives the water decomposition, its rate depends on the current that pass through the electrodes [22]. Anodic hydrolysis generates a marked decrease in the local pH at the anode interface. Due to the high buffer capacity of the medium, this local elevation of hydrogen ion concentration has a transient nature, diminishing both temporally and spatially [23]. Thus, cells located away from the anode interface are expected to be less susceptible to the effect of electrochemically produced acidity. The dependence of LEF induced uptake on buffer capacity, presented in Fig. 4A, leads us to propose that an elevated concentration of hydrogen ions is the major contributor to the phenomenon of electrically induced uptake. This dependence of uptake on extracellular proton concentration is in-line with our finding that electroendocytosis shows higher response to anodal current density than to electric field. Protons are produced by anodal electrolysis of water. In our experimental setup, higher current densities are associated with higher solution Results are given as folds of fluorescence geometrical mean 6 SD of induced uptake relative to constitutive uptake. (A) Cells were exposed to LEF in the anode compartment for 60 s at different concentrations of HEPES buffered solutions. There is a statistically significant difference between all adjacent points (P,0.05 by t-test), except between the 60 mM and the 70 mM (P.0.05 by t test). (B) Cells exposed for 60 s to solutions tittered to different pH, in the presence of 71 kD dextran-FITC. The insert depicts a better detailed view in the pH range between 7.4 and 4.0. A statistically significant difference from the constitutive cell uptake begins at pH 5.8 (2.6 fold, P,0.01 by t-test, n = 9) down to pH 1.6. Cells' uptake did not change significantly between pH 3.0 and pH 1.6 (P.0.05, one way ANOVA, n = 9). doi:10.1371/journal.pone.0050299.g004 conductivities and these electrical parameters are responsible for the translocation of hydrogen ions away from the anode interface into the bulk solution and their rate of production at the anode. Therefore higher current densities would be expected to raise both protons production and their accessibility to the cells. We simulated the electrochemical low pH environment by chemically exposing the cell suspension to a low pH in the range of 7.4 to 1.7 (Fig. 4B). The extent by which the cells internalize dextran-FITC as a function of buffer capacity or pH possess a sigmoid-like curve. These findings are in line with our recent report [15] where we show (by electron microscopy) that the exposure of the cell's surface to extracellular low pH promotes the formation of endocytic-like membrane invaginations into the cytoplasm and detail the kinetics of the enhanced uptake of macromolecules into the cells. This conclusion is also supported by a previous study [12], showing that the endocytic routes, consisting of clathrin-dependent pathways and caveolin-dependent pathway, contribute very little to the LEF-induced uptake. Low pH has been shown before to induce inward membrane tubules in lipid vesicles [24] and to induce the formation of plasma membrane vesicle and uptake of macromolecules in cells [15]. The mechanism that enforces a membrane to invaginate was suggested to rely on elevated concentration of extracellular hydrogen ions which bind to the anionic charged sites on the external membrane leaflet. This would result in local reduction of charge density on the external membrane surface, with consequent increase in local cross-membrane charge asymmetry. Such a state would yield a negative value for the spontaneous curvature of the plasma membrane and impose upon it an inward bending [25,26]. An inward bending of the plasma membrane is the initiating event of endocytic-like processes [27] which consequently lead to the formation of a membrane connected vesicle and finally the scission of the bud neck and separation of the vesicle from the mother membrane. Two studies of electroendocytosis have suggested a dependence of cellular uptake on the strength of the applied electric field [7,28], seemingly contradicting our results. As we pointed earlier, the electric potential is responsible for the decomposition of water and the production of hydrogen ions at the anode. Therefore, under experimental conditions of constant current density in a conductive medium (such as biological solutions), electric field would be correlated with enhanced hydrolysis. However, as the distribution of ions into the solution is less efficient at low current densities, the effect of protons on cells' membrane (and consequent uptake) is restrained (note the trendline in Figs. 3B and 3C). In the study reported by Mahrour et al. [28], a bipolar signal was chosen to avoid electrophoresis and to minimize electrochemical reactions at the electrodes. However the extent of this ''minimized'' contribution and the transient pH changes in the proximity of the electrodes were not reported. Furthermore, the role of the medium's conductivity and buffer capacity were not studied with relation to the cell's uptake. In fact, it is reported that under mild alkaline pH, the stimulatory effect of the electric field on uptake was lost [28]. In a recent study by Lin et al. [7], electroendocytosis was shown to constitute endocytis-like processes, but its dependence on the medium's conductivity, buffer capacity or transient pH changes was not examined. The possible involvement of endocytic-like uptake in electroporation has been reported before. In one important example, DNA was efficiently taken up by large unilamellar vesicles exposed to a short pulse of electric field as a result of the electro-stimulated formation of endosome-like vesicles rather than via field-induced membrane pores [29]. Other studies have elucidated the involvement of endocytosis in the uptake of plasmid DNA, as a complementary process to the absorption of DNA to the plasma membrane by the applied electric field. Translocation of these DNA complexes through the membrane is implied to occur after, rather than during, electric pulse application [6,7,8,9,10,11]. It should be pointed that under our experimental protocol, the cells' suspension was cooled immediately after the termination of electric pulses, leaving very little time for endocytosis to commence. As electroporation is associated with lowered pH at the anodic front [23], the consequent cells' membrane protonation may play part in the insertion of DNA into cells. This assumption was examined in the present study by exposing the cells to pH 5 in the presence of a naked plasmid DNA. The results, shown in Fig. 5, demonstrate that the plasmids have entered the cells and could indeed be found in their cytoplasm following exposure to low pH. However, very few cells were able to successfully undergo transfection by this method. This outcome is in line with previous reports showing that the mere entry of naked DNA into the cytoplasm is not sufficient for the induction of transfection, mainly due to the nuclear barriers that need to be crossed [30]. Conclusions The present study reveals that uptake induced by pulsed low electric field (electroendocytosis) is restricted to the vicinity of the anode. The extent of the uptake depends on anodic current density more than on electric fields possessing values ,65 V/cm. Our experimental findings show that the major component to induce such uptake is the production of surplus hydrogen ions by anodic hydrolysis. This type of uptake is possibly an inherent companion of electroporotion, the practice of which involves acidification of the solution near the anode.	v3-fos-license
2020-01-23T09:09:39.382Z	2020-01-21T00:00:00.000	213695085	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1996-1073/13/3/521/pdf?version=1580729096", "pdf_hash": "e8dbc630e25cf155c6043ff0bb52f57d0539d6b0", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:223", "s2fieldsofstudy": [ "Chemistry", "Environmental Science" ], "sha1": "c6072f19313ab5eac9182fa09182dc409381e0c0", "year": 2020 }	pes2o/s2orc	Comparison of Catalytic Activity of ZIF-8 and Zr / ZIF-8 for Greener Synthesis of Chloromethyl Ethylene Carbonate by CO 2 Utilization : The catalytic activity of both ZIF-8 and Zr / ZIF-8 has been investigated for the synthesis of chloromethyl ethylene carbonate (CMEC) using carbon dioxide (CO 2 ) and epichlorohydrin (ECH) under solvent-free conditions. Published results from literature have highlighted the weak thermal, chemical, and mechanical stability of ZIF-8 catalyst, which has limited its large-scale industrial applications. The synthesis of novel Zr / ZIF-8 catalyst for cycloaddition reaction of ECH and CO 2 to produce CMEC has provided a remarkable reinforcement to this weak functionality, which is a signiﬁcant contribution to knowledge in the ﬁeld of green and sustainable engineering. The enhancement in the catalytic activity of Zr in Zr / ZIF-8 can be attributed to the acidity / basicity characteristics of the catalyst. The comparison of the catalytic performance of the two catalysts has been drawn based on the e ﬀ ect of di ﬀ erent reaction conditions such as temperature, CO 2 pressure, catalyst loading, reaction time, stirring speed, and catalyst reusability studies. Zr / ZIF-8 has been assessed as a suitable heterogeneous catalyst outperforming the catalytic activities of ZIF-8 catalyst with respect to conversion of ECH, selectivity and yield of CMEC. At optimum conditions, the experimental results for direct synthesis of CMEC agree well with similar literature on Zr / MOF catalytic performance, where the conversion of ECH, selectivity and the yield of CMEC are 93%, 86%, and 76%, respectively. Introduction The effective transformation and utilization of anthropogenic carbon dioxide (CO 2 ) is a subject of political and environmental debates in recent years, which have been actively pursued by the academia and energy industries in order to promote a sustainable environment [1]. The current level and accumulation of CO 2 in the atmosphere is high and requires urgent attention [2]. However, regardless of environmental regulations and discharge limits placed on greenhouse gases emitted into the atmosphere, CO 2 is believed to be environmentally benign, abundant, nontoxic, non-flammable, and a readily available C1 source for the synthesis of organic carbonate [3]. Therefore, the synthesis of cyclic organic carbonates via the cycloaddition of CO 2 and epoxides is one of the most promising reaction schemes because of its 100% atom efficiency [4]. Cyclic organic carbonates such as chloromethyl ethylene carbonate (CMEC), propylene carbonate (PC), styrene carbonate (SC), and ethylene carbonate (EC) are widely used as polar aprotic solvents, electrolytes for lithium-ion batteries, automobile, cosmetic, fuel additives materials, alkylating and carbonylating reagents, and fine chemicals for pharmaceuticals [5,6]. In the past two decades, several attempts have been made to develop greener and sustainable catalytic systems for chemical fixation of CO 2 . This includes conventional solid catalysts such as zeolites, salen Cr(III) complexes, metal oxides, quaternary ammonium salts, polymer-supported catalysts, ionic liquids (ILs), etc. However, these attempts have failed to yield satisfactory results as most of these catalysts require high temperature and/or pressure (usually around 453 K and pressure higher than 8 atm), further separation and purification steps, and low product yield [7]. This is uneconomical from a commercial point of view and hence, the research has been directed to employ a novel catalyst that provides solutions to all these shortfalls i.e., metal organic framework (MOF). Although, microporous materials such as zeolites, crystalline aluminosilicate, activated carbon, etc. have been known for their high surface area and high porosity, however, their applications have been limited especially in the field of heterogeneous catalysis due to difficulty in pore modification [7]. Metal organic framework (MOF) catalysts are identified as multidimensional porous polymetric crystalline organic-inorganic hybrid materials with exceptional characteristics including an ultrahigh specific surface area, enormous pore spaces, and ordered crystalline structure [8]. MOFs have emerged as a suitable candidate for the synthesis of organic carbonates from CO 2 and epoxide due to their unique heterogeneity and reusability requirements [9]. MOF-based catalysts often display higher catalytic activity than their corresponding homogenous catalysts as evidenced in many catalytic reactions such as ring opening, addition reactions, oxidation reactions, hydrogenation and isomerization [10]. Zeolitic imidazolate frameworks, (ZIFs), is one of the subclasses of MOFs with a similar structure to zeolites. It has attractive structural properties and intrinsically lower density. Many experiments involving ZIF-8 have shown great applications in multidisciplinary fields such as catalysis, drug deliveries, purification and gas storage [11]. Recently, the stability of MOFs for large-scale industrial applications has been questioned in many published papers [11][12][13][14][15]. This is due to their weak thermal, chemical, and mechanical stability due to the structure of inorganic bricks and the nature of the chemical bonds they form with the linker [15]. In order to improve this weak thermal functionality and gain in-depth knowledge of their catalytic activities, Cavka et al. [16] was the first group to synthesize Zr-based MOFs designated as zirconium 1,4-dicarboxybenzene, UiO-66 for photocatalysis [17]. The test conducted by the group found that the increased stability of the Zr-based MOFs is owing to the Zr-O bonds formed between the cluster and carboxylate ligands [18]. Several other groups have thereafter explored this opportunity, which has seen the increased application of Zr-based MOFs in many research activities. Demir et al. [19] utilized UiO (University of Oslo) type zirconium metal-organic frameworks in a solvent-free coupling reaction of carbon dioxide (CO 2 ) and epichlorohydrin (ECH) for the synthesis of epichlorohydrin carbonate (ECHC). The results of their experiments have increased the use of zirconium-based (Zr-based) MOFs for the catalytic synthesis of organic carbonates from CO 2 and epoxides. From our experiments, the synthesis of Zr-doped MOF (Zr/ZIF-8) for the cycloaddition reaction of CO 2 and ECH in the synthesis of chloromethyl ethylene carbonate (CMEC) has demonstrated reasonable thermal stability under relatively mild reaction conditions without using any solvent or co-catalyst. Although, the synthesis of several Zr-based MOFs have been reported in recent times (albeit in early stages), only a few were employed for catalytic studies even more rarely for the synthesis of organic carbonates from CO 2 and epoxides. Zr-based MOFs have exhibited increased structural tailorability as a result of the organic linkers in the catalyst frameworks [19]. Zirconium-based MOFs have demonstrated proof-of-concept applications in several areas such as toxic analyte, catalysis, gas storage, vivo drug delivery, and bio-sensing [20]. In this paper, a novel Zr/ZIF-8 has been successfully synthesized using the conventional solvothermal method. The prepared catalyst has been assessed as an innovative greener and sustainable heterogeneous catalyst for the direct synthesis of CMEC from CO 2 and ECH. The effect of various reaction parameters has been investigated and critically analyzed. These include the effect of reaction time, catalyst loading, temperature, CO 2 pressure, and stirring speed. Catalyst reusability studies of Zr/ZIF-8 was also investigated to establish its stability and reusability for the synthesis of CMEC. Catalysts Preparation Preparation of ZIF-8 and zirconium-doped ZIF-8 (Zr/ZIF-8) were synthesized according to a method, which was previously described elsewhere [20,21]. Briefly, 8 mmol of zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O 99.99%) and zirconium (IV) oxynitrate hydrate (ZrO(NO 3 ) 2 ·6H 2 O, 99.99%) solutions in a stoichiometric ratio of Zn:Zr = 10:0 and Zr:Zn = 9:1 (to synthesis ZIF-8 and Zr/ZIF-8 respectively) were dissolved in 6.2 mmol of methanol. A separate solution of 14.2 mmol of 2-methylimidazole and 600 mml of methanol was prepared in another flask, which was added by dropwise addition to the Zr-Zn-based solution. The mixture conducted in an ambient temperature under nitrogen flow was vigorously stirred for 6 h. The Zr-doped ZIF-8 crystals were collected and separated by centrifugation at 300 rpm for 30 min. The solution was washed thoroughly with methanol three times and then dried at room temperature conditions. The crystals were left to dry overnight at 373 K. The greyish-white powders of Zr-ZIF-8 samples were further washed with DMF for 24 h in order to remove any excess of an unreacted organic linker. The solution was then heated at a temperature of 373 K in order to activate it. The samples were allowed to cool to room temperature naturally before being capped in a vial and refrigerated, ready for use in catalytic reactions. The obtained samples were identified with a stoichiometric ratio of Zr:Zn = 10:0 and Zr:Zn = 1:9 for ZIF-8 and Zr/ZIF-8 respectively. Experimental Procedure for the Synthesis of Chloromethyl Ethylene Carbonate (CMEC) In a typical cycloaddition reaction, a 25 mL stainless steel high-pressure reactor was initially charged with a specific amount of Zr/ZIF-8 catalyst and the limiting reactant, epichlorohydrin. A desired temperature was set on the reactor's panel controller; the reactor was then sealed and stirred continuously at a known stirring speed. At the desired temperature, a specific amount of liquid CO 2 was charged through a supercritical fluid (SCF) pump into the reactor. The reaction was left for the desired reaction time. After the reaction was completed, the reactor was cooled down to room temperature and the mixture was collected and filtered. The catalyst was separated, washed with acetone, and dried in a vacuum oven. A known amount of methanol (used as internal standard) was added to the product and analyzed using a gas chromatograph (GC). The effect of different reaction parameters was investigated. These include catalyst loading, stirring speed, CO 2 pressure, temperature, and reaction time. Reusability studies of both catalysts were also carried out in order to investigate the stability of the catalysts for the synthesis of chloromethyl ethylene carbonate. Method of Analysis A specific quantity of internal standard, methanol added to a known sample of the product was analyzed using a gas chromatography (GC) (Model: Shimadzu GC-2014). The stationary phase was a capillary column with dimensions (30 m length, 320 µm inner diameter, and 0.25 µm film thickness). Oxygen (99.9%) and hydrogen (99.9%) were used as ignition gases. The carrier gas used for the mobile phase was a high purity helium (99.9%) with a flow rate maintained at 1 mL min −1 . A temperature program was developed for the system where both the injector port and detector temperatures were kept isothermally at 523 K. The other selected program includes split ratio of 50:1 and injection volume of 0.5 µL. The column temperature was initially maintained at 323 K for 5 min, then followed by a Energies 2020, 13, 521 4 of 25 temperature ramp at a flow rate of 50 K min −1 to a temperature of 523 K with a 12 min run for each subsequent samples. The chromatogram shows that ECH peak at~3.5 min, methanol at~3.8 min, CMEC at~11 min. Proposed Reaction Mechanism The proposed reaction mechanism involves two steps: The ring-opening of epoxides by a catalyst; Incorporation of carbon dioxide into the opening to form the cyclic carbonate. The coupling reaction of CO 2 with epoxides can be initiated by activating either the epoxide or CO 2 or both at the same time [22]. This reaction, using a suitable heterogeneous catalyst, produces desired organic carbonates along with other side products. Figure 1 shows reaction pathways 1, 2, and 3 with corresponding products being chloromethyl ethylene carbonate, 3-chloropropane 1,2-diol, and 2,5-bis (chloromethyl)-1,4-dioxane respectively. The epoxide is activated when the oxygen atom interacts with the Lewis acid, this is then followed by a nucleophilic attack that provokes the opening of the epoxide ring [23] as shown in Figure 2. The activation of CO 2 can occur both through a nucleophilic attack with the oxygen atom as a nucleophile or an electrophilic attack with the carbon atom as an electrophile [24]. Figure 2 shows a proposed reaction mechanism for the synthesis of CMEC, where R is an alkyl group, A is a metal atom with a Lewis acid site, while B is an oxygen atom with a Lewis basic site. Zr/ZIF-8 is a dual-functional catalyst, which contains both the acidic and basic sites that are associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. The by-products identified with the coupling reaction of CO 2 and ECH as identified by the GC analysis are 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane (see Figure 1). Figure 3 shows the schematic representation of the reaction of CO 2 and ECH to produce CMEC. Energies 2020, 13, x FOR PEER REVIEW 4 of 26 program was developed for the system where both the injector port and detector temperatures were kept isothermally at 523 K. The other selected program includes split ratio of 50:1 and injection volume of 0.5 μL. The column temperature was initially maintained at 323 K for 5 min, then followed by a temperature ramp at a flow rate of 50 K min -1 to a temperature of 523 K with a 12 min run for each subsequent samples. The chromatogram shows that ECH peak at ~3.5 min, methanol at ~3.8 min, CMEC at ~11 min. Proposed Reaction Mechanism The proposed reaction mechanism involves two steps:  The ring-opening of epoxides by a catalyst;  Incorporation of carbon dioxide into the opening to form the cyclic carbonate. The coupling reaction of CO2 with epoxides can be initiated by activating either the epoxide or CO2 or both at the same time [22]. This reaction, using a suitable heterogeneous catalyst, produces desired organic carbonates along with other side products. Figure 1 shows reaction pathways 1, 2, and 3 with corresponding products being chloromethyl ethylene carbonate, 3-chloropropane 1,2-diol, and 2,5-bis (chloromethyl)-1,4-dioxane respectively. The epoxide is activated when the oxygen atom interacts with the Lewis acid, this is then followed by a nucleophilic attack that provokes the opening of the epoxide ring [23] as shown in Figure 2. The activation of CO2 can occur both through a nucleophilic attack with the oxygen atom as a nucleophile or an electrophilic attack with the carbon atom as an electrophile [24]. Figure 2 shows a proposed reaction mechanism for the synthesis of CMEC, where R is an alkyl group, A is a metal atom with a Lewis acid site, while B is an oxygen atom with a Lewis basic site. Zr/ZIF-8 is a dual-functional catalyst, which contains both the acidic and basic sites that are associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. The by-products identified with the coupling reaction of CO2 and ECH as identified by the GC analysis are 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane (see Figure 1). Figure 3 shows the schematic representation of the reaction of CO2 and ECH to produce CMEC. Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5° min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuK radiation ( = 1.5406° A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM)). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron Figure 2. Proposed reaction mechanism for the cycloaddition reaction of CO 2 to ECH over an acid-base pairs. R is an alkyl group, M is a metal atom (acidic site), and O is oxygen atom (basic site). Energies 2020, 13, x FOR PEER REVIEW 5 of 26 Figure 2. Proposed reaction mechanism for the cycloaddition reaction of CO2 to ECH over an acid-base pairs. R is an alkyl group, M is a metal atom (acidic site), and O is oxygen atom (basic site). Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5° min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuK radiation ( = 1.5406° A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM)). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5 • min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuKα radiation (λ = 1.5406 • A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron spectroscopy (XPS) of the samples was recorded on the krato axis ultra DLD photoelectron spectrometer, a surface Energies 2020, 13, 521 6 of 25 science instrument SSx-100 using a monochromatic Al KR X-ray source operating at 144 W. Raman spectroscopy measurements of the specimen were taken at room temperature with the Horiba Jobin Yvon LabRAM spectrometer equipped with an aHeNe laser operating at a wavelength of 633 nm (E ex = 1.96 eV) and Coherent Innova 70 ion laser at a wavelength of 458 nm, 488 nm, and 514 nm. Catalyst Characterization The X-ray diffraction patterns of ZIF-8, Zr/ZIF-8 and recycled Zr/ZIF-8 are shown in Figure 4, confirming that Zr/ZIF-8 has high crystal stability under the normal reaction conditions. These results are in agreement with simulated patterns reported in other literature [25][26][27][28]. The decrease in peak intensity of these diffractions was also observed at (2θ = 28-35 • ) indicating the effect of excess doping of Zr into the ZIF-8 framework. The XRD pattern of Zr/ZIF-8 also show a characteristic peak of ZIF-8 with no diffraction peak of zirconium nitrate. temperature with the Horiba Jobin Yvon LabRAM spectrometer equipped with an aHeNe laser operating at a wavelength of 633 nm (Eex = 1.96 eV) and Coherent Innova 70 ion laser at a wavelength of 458 nm, 488 nm, and 514 nm. Catalyst Characterization The X-ray diffraction patterns of ZIF-8, Zr/ZIF-8 and recycled Zr/ZIF-8 are shown in Figure 4. Figure 4, confirming that Zr/ZIF-8 has high crystal stability under the normal reaction conditions. These results are in agreement with simulated patterns reported in other literature [25][26][27][28]. The decrease in peak intensity of these diffractions was also observed at (2θ = 28-35°) indicating the effect of excess doping of Zr into the ZIF-8 framework. The XRD pattern of Zr/ZIF-8 also show a characteristic peak of ZIF-8 with no diffraction peak of zirconium nitrate. Although, the peak intensity of Zr/ZIF-8 may be slightly lower when compared to commercial Basolite Z1200, purchased from Sigma Aldrich. Nevertheless, the experiments of Nordin et al. [29] They establishes that guest molecules (such as zirconium) occupying MOF pore spaces may cause pattern destructive and subsequently, a retarded gas uptake capacity in the MOF. A further and indepth examination of the XRD patterns of the specimen beyond this study could reveal some surprising details as doping of zirconium into ZIF-8 could enlarge its pore spaces [30], thereby inducing a crystallographic defect in the Zr/ZIF-8 catalyst. The nitrogen adsorption-desorption isotherms of ZIF-8, Zr/ZIF-8, and the recycled Zr/ZIF-8 catalysts are shown in Figure 5. The samples were measured at liquid temperature of 77k at 373 K for 24 h. The three isotherms showed an attribute of a microporous framework with a sharp hysteresis loop of P/P0 between 0.8 and 1.0. However, the pristine ZIF-8 catalyst demonstrates a typical type-I isotherm behavior [31] while Zr/ZIF-8 and the recycled Zr/ZIF-8 catalysts both shows typical type-IV Although, the peak intensity of Zr/ZIF-8 may be slightly lower when compared to commercial Basolite Z1200, purchased from Sigma Aldrich. Nevertheless, the experiments of Nordin et al. [29] They establishes that guest molecules (such as zirconium) occupying MOF pore spaces may cause pattern destructive and subsequently, a retarded gas uptake capacity in the MOF. A further and in-depth examination of the XRD patterns of the specimen beyond this study could reveal some surprising details as doping of zirconium into ZIF-8 could enlarge its pore spaces [30], thereby inducing a crystallographic defect in the Zr/ZIF-8 catalyst. The nitrogen adsorption-desorption isotherms of ZIF-8, Zr/ZIF-8, and the recycled Zr/ZIF-8 catalysts are shown in Figure 5. The samples were measured at liquid temperature of 77k at 373 K for 24 h. The three isotherms showed an attribute of a microporous framework with a sharp hysteresis loop of P/P0 between 0.8 and 1.0. However, the pristine ZIF-8 catalyst demonstrates a typical type-I isotherm behavior [31] while Zr/ZIF-8 and the recycled Zr/ZIF-8 catalysts both shows typical type-IV isotherms with a type H 4 hysteresis loop in the range of P/P0 = 0.4-0.8 indicating the presence of mesopores [32]. Meanwhile, an increase in the volume adsorbed at low relative pressure is consistent with interparticle voids, which is indicative of dual macro-mesoporosity of the Zr/ZIF-8 lattice according to International Union of Pure and Applied Chemistry (IUPAC) classification [33,34]. Figure 6 shows the morphologies and microstructures of ZIF-8, fresh and recycled Zr/ZIF-8 catalysts using the scanning electron microscope (SEM) with an average particle size diameter of 100 μm. Figure 6a shows an evolution of ZIF-8 crystal from cubes with 6 faces (100) to intermediates shapes, and finally to a more stable equilibrium rhombic dodecahedral shape with edges exposing 12 faces (110) [41]. The specific BET surface area (S BET ) of the catalysts has been calculated using the BET equation. The pore size distribution was derived from the nonlinear density functional theory (DFT) model (calculated using computer software). The surface area and micropore volume of Zr/ZIF-8 was generally lower than ZIF-8 as shown in Table 1. The lower BET surface area and pore volume of Zr/ZIF-8 may be caused by the blockage of the pore cavities of the host molecule as a result of deposition of zirconium particles in the ZIF-8 shell, a phenomenon that has been previously reported by Na et al. [35]. Surprisingly, the total pore volume and the BET specific surface area of recycled Zr/ZIF-8 catalyst both decreased after the reaction. This observation may be attributed to agglomeration of coke deposits in the pore spaces, resulting in the blockage of some micropores and mesopores. [36]. These results reflect a good pore size distribution of the samples microporous network [37,38]. Although, variation may exist in particles BET surface area and pore volume from one literature to another, this may be attributed to post-synthesis work-up procedures such as further purification processes and activation of MOF samples [39]. The BET surface area as shown in Figure 5 is in agreement with the previous literature [40]. Figure 6 shows the morphologies and microstructures of ZIF-8, fresh and recycled Zr/ZIF-8 catalysts using the scanning electron microscope (SEM) with an average particle size diameter of 100 µm. Figure 6a shows an evolution of ZIF-8 crystal from cubes with 6 faces (100) to intermediates shapes, and finally to a more stable equilibrium rhombic dodecahedral shape with edges exposing 12 faces (110) [41]. Figure 6c showed a very small change after the cycloaddition reaction. A close examination of the SEM images of Figure 6a,b shows no significant effect of attrition on the overall particle aggregation between the two structures. The SEM image of recycled Zr/ZIF-8 in Figure 6c showed rather small isolated monodispersed particles with a well-defined truncated rhombic dodecahedron structure caused by the presence of dopant in the host molecule. Essentially, the SEM images of the samples are consistent with the XRD results and the thermal stability of Zr/ZIF-8 as shown in the catalyst reusability studies. It is worth mentioning that, the increased average crystal size of the recycled Zr/ZIF-8 catalyst in the range of~100-170 nm (Figure 6c) may be attributed to Ostwald ripening and/or recrystallization effect [42], a phenomenon which explains a possible increase in the average crystal size of the reused catalyst during cycloaddition reaction, especially at a higher temperature (reaction temperature 353 K). Energies 2020, 13, x FOR PEER REVIEW 8 of 26 6a (ZIF-8) framework. The slight alterations are a genuine indication of a stable Zr/ZIF-8 catalyst comparing to the report of Pang et al. [41]. Furthermore, the hexagonal shape of the recycled catalyst in Figure 6c showed a very small change after the cycloaddition reaction. A close examination of the SEM images of Figures 6a and 6b shows no significant effect of attrition on the overall particle aggregation between the two structures. The SEM image of recycled Zr/ZIF-8 in Figure 6c showed rather small isolated monodispersed particles with a well-defined truncated rhombic dodecahedron structure caused by the presence of dopant in the host molecule. Essentially, the SEM images of the samples are consistent with the XRD results and the thermal stability of Zr/ZIF-8 as shown in the catalyst reusability studies. It is worth mentioning that, the increased average crystal size of the recycled Zr/ZIF-8 catalyst in the range of ~100-170 nm (Figure 6c) may be attributed to Ostwald ripening and/or recrystallization effect [42], a phenomenon which explains a possible increase in the average crystal size of the reused catalyst during cycloaddition reaction, especially at a higher temperature (reaction temperature 353 K). Low-magnification TEM images of the samples were carried out in order to examine the structural changes taking place on the surface of the samples. Figures 7a and 7b showed well-shaped high-quality homogenous crystals with a remarkable rhombic dodecahedral shape and average crystal size of about 100 μm which conforms to earlier literature [42]. It can be observed from the image in Figure 7b that there are no obvious aggregations or changes in particle size and morphology Low-magnification TEM images of the samples were carried out in order to examine the structural changes taking place on the surface of the samples. Figure 7a,b showed well-shaped high-quality homogenous crystals with a remarkable rhombic dodecahedral shape and average crystal size of about 100 µm which conforms to earlier literature [42]. It can be observed from the image in Figure 7b that there are no obvious aggregations or changes in particle size and morphology from Figure 7a. The TEM image of the recycled catalyst (Figure 7c) shows that the catalyst crystals were highly stable during the cycloaddition reaction of CO 2 and ECH. Figure 8 shows the Fourier-transform infrared spectroscopy (FTIR) spectra of ZIF-8, Zr/ZIF-8, and the recycled Zr/ZIF-8 with an absorption region of 500-4000 cm −1 . The three samples show several bands with no substantial difference in the spectra. For example, a typical adsorption band at 423 cm −1 is attributed to the Zn-N bond vibrations indicating that zinc molecules of the imidazole ring are well-knitted during the reaction to nitrogen atoms in 2-methylimidazolate (2-Hmim) linkers to form the ZIF frameworks [43]. The absorption spectra at 2926 cm −1 can be ascribed to the aromatic moieties, while the spectra at 3133 cm −1 can be attributed to the aliphatic imidazole ring due to C-H stretching [44]. The missing adsorption spectra in the region of 3400 to 2200 cm −1 is a strong indication of a fully deprotonated imidazole ring during the formation of the ZIF-8 frameworks [44]. The strong sharp peak at 1449 cm −1 can be assigned to the C-C bonding in the benzene ring. The peak at 1579 cm −1 can be attributed to C=N vibrations mode [45], while the spectra in the band range between 1100 and 400 cm −1 can be assigned to C-N stretching vibrations. The small peaks at 1245 and 1255 can be assigned to C-N and C≡N groups respectively indicating the presence of imidazole molecules in the samples frameworks. The Zr-N bonding vibration located between 550 and 620 cm −1 in Zr/ZIF-8 catalyst [46]. All characteristic peaks of ZIF-8 can be observed both in Zr/ZIF-8 and the recycled Zr/ZIF-8, indicating a successful combination and interaction between Zr and ZIF-8. This observation Figure 8 shows the Fourier-transform infrared spectroscopy (FTIR) spectra of ZIF-8, Zr/ZIF-8, and the recycled Zr/ZIF-8 with an absorption region of 500-4000 cm −1 . The three samples show several bands with no substantial difference in the spectra. For example, a typical adsorption band at 423 cm −1 is attributed to the Zn-N bond vibrations indicating that zinc molecules of the imidazole ring are well-knitted during the reaction to nitrogen atoms in 2-methylimidazolate (2-Hmim) linkers to form the ZIF frameworks [43]. The absorption spectra at 2926 cm −1 can be ascribed to the aromatic moieties, while the spectra at 3133 cm −1 can be attributed to the aliphatic imidazole ring due to C-H stretching [44]. The missing adsorption spectra in the region of 3400 to 2200 cm −1 is a strong indication of a fully deprotonated imidazole ring during the formation of the ZIF-8 frameworks [44]. The strong sharp peak at 1449 cm −1 can be assigned to the C-C bonding in the benzene ring. The peak at 1579 cm −1 can be attributed to C=N vibrations mode [45], while the spectra in the band range between 1100 and 400 cm −1 can be assigned to C-N stretching vibrations. The small peaks at 1245 and 1255 can be assigned to C-N and C≡N groups respectively indicating the presence of imidazole molecules in the samples frameworks. The Zr-N bonding vibration located between 550 and 620 cm −1 in Zr/ZIF-8 catalyst [46]. All characteristic peaks of ZIF-8 can be observed both in Zr/ZIF-8 and the recycled Zr/ZIF-8, indicating a successful combination and interaction between Zr and ZIF-8. This observation is a strong indication that the frameworks of ZIF-8 have not been affected after the incorporation of Zr. This results in agreement with the report of Giraldo et al. [47] experiments. The X-ray photoelectron spectroscopy (XPS) spectra in Figure 9 clearly shows the chemical state of the element present in pristine ZIF-8 frameworks (Zn, C, N) and O, while those elements present in Zr/ZIF-8 sample include Zn, C, N, O, and Zr species. Figure 9a exhibits high resolution XPS spectra showing two strong peaks with binding energy of 1044.3 and 1021.1 eV which can be assigned to Zn 2p ½ and Zn 2p 3/2 components, respectively, confirming the presence of Zn (II) ions attached with nitrogen in the imidazole ring [48]. This result is consistent with the XRD results. With the incorporation of Zr into ZIF-8, the binding energy of Zn 2p 1/2 and 2p 3/2 have slightly increased, this could be as a result of the chemical environment of zinc and the interaction between zinc and zirconium. All spectra have been normalized to the magnitude of the Zn 2p 3/2 and Zn 2p 1/2 peaks, so that changes in intensity are relative to the amount of Zn in the surface region. Similarly, Figure 9b shows high-resolution N1s spectra of all samples. The N1s spectra can be deconvoluted into three characteristic peaks found at 399.0 and 399.8 and 398 eV which can be assigned to the pyridinic, pyrrolic, and graphitic, respectively. These can be related to the N species of the 2-methyl imidazole ring [48]. C1s spectra shows four different characteristic peaks corresponding to C-C at 284.1 eV, C-N at 285.8 eV, C-O at 286.4 eV, all assigned to the 2-methyl imidazole ring [49]. The low peak found at 283.4 eV could be as a result of Zr doping into ZIF-8 frameworks [49]. Figure 9d shows high resolution O1 spectra that has been deconvoluted into two characteristic peaks with binding energy 532.3 and 531.8 eV corresponding to O 2− found in Zn-O bonding and carboxylate species, respectively [50]. The relatively low peak intensity of Zr-O in O1s, C1s, and N1s is a strong indication that the ZIF-8 frameworks are not affected by the presence of dopant, which perfectly agreed with the result of Mao et al. [51]. and Zn 2p 3/2 components, respectively, confirming the presence of Zn (II) ions attached with nitrogen in the imidazole ring [48]. This result is consistent with the XRD results. With the incorporation of Zr into ZIF-8, the binding energy of Zn 2p 1/2 and 2p 3/2 have slightly increased, this could be as a result of the chemical environment of zinc and the interaction between zinc and zirconium. All spectra have been normalized to the magnitude of the Zn 2p 3/2 and Zn 2p 1/2 peaks, so that changes in intensity are relative to the amount of Zn in the surface region. Similarly, Figure 9b shows high-resolution N1s spectra of all samples. The N1s spectra can be deconvoluted into three characteristic peaks found at 399.0 and 399.8 and 398 eV which can be assigned to the pyridinic, pyrrolic, and graphitic, respectively. These can be related to the N species of the 2-methyl imidazole ring [48]. C1s spectra shows four different characteristic peaks corresponding to C-C at 284.1 eV, C-N at 285.8 eV, C-O at 286.4 eV, all assigned to the 2-methyl imidazole ring [48]. The low peak found at 283.4 eV could be as a result of Zr doping into ZIF-8 frameworks [48]. Figure 9d shows high resolution O1 spectra that has been deconvoluted into two characteristic peaks with binding energy 532.3 and 531.8 eV corresponding to O 2− found in Zn-O bonding and carboxylate species, respectively [49]. The relatively low peak intensity of Zr-O in O1s, C1s, and N1s is a strong indication that the ZIF-8 frameworks are not affected by the presence of dopant, which perfectly agreed with the result of Mao et al. [50]. Raman spectra of ZIF-8, Zr/ZIF-8 and the recycled Zr/ZIF-8 were observed using a Renishaw Ramascope 1000 (model: 52699). Figure 10 shows that Zr/ZIF-8 exhibited several Raman spectra at the following peaks 687, 892, 1149, 1186, 1462, 1568, 2931, 3114, and 3131 cm −1 similar to ZIF-8 spectra. The spectra at 1116 and 1484 cm −1 corresponding to bands D and G, respectively, found in the Raman spectrum of ZIF-8, have not been observed in the Zr/ZIF-8 and the recycled Zr/ZIF-8 spectra. This may be as a result of a split of the main bands at 1143 and 1508 cm −1 as previously reported by Biswal et al. [51]. The spectra found at 278 cm −1 may be attributed to Zn-N stretching, while the spectra at 683, 1143, 1456, and 1508 cm −1 are attributed to imidazole ring puckering, C5-N vibrations, methyl bending, and C4=C5 stretching, respectively, which are similar to the observation of Tanaka et al. [52]. The remaining spectra can be assigned to stretching and bending on the imidazole ring [53]. With doping of Zr into the ZIF-8 frameworks, the peaks at 1116 and 1484 disappeared with no significant change in main peaks on spectra [54]. The spectra of three samples shows similar vibration modes, which confirms structural equality in the frameworks. Raman spectra of ZIF-8, Zr/ZIF-8 and the recycled Zr/ZIF-8 were observed using a Renishaw Ramascope 1000 (model: 52699). Figure 10 shows that Zr/ZIF-8 exhibited several Raman spectra at the following peaks 687, 892, 1149, 1186, 1462, 1568, 2931, 3114, and 3131 cm −1 similar to ZIF-8 spectra. The spectra at 1116 and 1484 cm −1 corresponding to bands D and G, respectively, found in the Raman spectrum of ZIF-8, have not been observed in the Zr/ZIF-8 and the recycled Zr/ZIF-8 spectra. This may be as a result of a split of the main bands at 1143 and 1508 cm −1 as previously reported by Biswal et al. [52]. The spectra found at 278 cm −1 may be attributed to Zn-N stretching, while the spectra at 683, 1143, 1456, and 1508 cm −1 are attributed to imidazole ring puckering, C5-N vibrations, methyl bending, and C4=C5 stretching, respectively, which are similar to the observation of Tanaka et al. [53]. The remaining spectra can be assigned to stretching and bending on the imidazole ring [54]. With doping of Zr into the ZIF-8 frameworks, the peaks at 1116 and 1484 disappeared with no significant change in main peaks on spectra [54]. The spectra of three samples shows similar vibration modes, which confirms structural equality in the frameworks. There are three distinct phases of weight loss experienced by both samples as indicated in Figure 11. It can be observed from the thermogram that, both catalysts experienced a very small initial weight loss of about 3% in the region from 298 to 373 K in the first phase. This can be attributed to loss of water and some guest molecules (e.g., methanol) and possibly some unreacted species trapped in the pore cavities of the framework. As the temperature was further increased through the second phase, Zr/ZIF-8 experienced a gradual and steady weight loss up to 723 K and then remained stable thereafter until 973 K. Conversely, ZIF-8 experienced a rapid and significant weight loss of around 54% up to 823 K, attributing the decomposition of some absorbed organic ligand and the final weight loss phase experienced the collapse of the ZIF-8 structure at high temperature [53]. It is worth noting that materials stability of the ZIF-8 framework can be attributed to the incorporation of zirconium in ZIF-8. A similar observation was reported by Wang et al. [54] in the doping of lanthanum into ZIF-8. After the decomposition, approximately 39% of the starting weight remained. From this observation, it can be concluded that the Zr/ZIF-8 catalyst frameworks have remained structurally stable and this is consistent with XRD and SEM. There are three distinct phases of weight loss experienced by both samples as indicated in Figure 11. It can be observed from the thermogram that, both catalysts experienced a very small initial weight loss of about 3% in the region from 298 to 373 K in the first phase. This can be attributed to loss of water and some guest molecules (e.g., methanol) and possibly some unreacted species trapped in the pore cavities of the framework. As the temperature was further increased through the second phase, Zr/ZIF-8 experienced a gradual and steady weight loss up to 723 K and then remained stable thereafter until 973 K. Conversely, ZIF-8 experienced a rapid and significant weight loss of around 54% up to 823 K, attributing the decomposition of some absorbed organic ligand and the final weight loss phase experienced the collapse of the ZIF-8 structure at high temperature [54]. It is worth noting that materials stability of the ZIF-8 framework can be attributed to the incorporation of zirconium in ZIF-8. A similar observation was reported by Wang et al. [55] in the doping of lanthanum into ZIF-8. After the decomposition, approximately 39% of the starting weight remained. From this observation, it can be concluded that the Zr/ZIF-8 catalyst frameworks have remained structurally stable and this is consistent with XRD and SEM. Catalytic Activity After catalyst characterization, the catalytic activity of the novel materials was compared with ZIF-8 for the synthesis of chloromethyl ethylene carbonate from CO2 and epichlorohydrin under solvent-free conditions. It is interesting to note that the combination of acid and basic sites (Lewis and Brönsted site) existing in the MOF catalyst may improve the catalytic activity of both samples. The reactions were carried out under the same conditions of 353 K reaction temperature, 8 bar CO2 pressure, 10% (w/w) catalyst loading, 8 h reaction time, and 350 rpm of stirring speed. From the Table 2, it follows that at optimum CO2 pressure of 8 bar, reaction time of 8 h, catalyst loading of 10 % w/w, and variable temperature, Zr/ZIF-8 exhibits a higher catalytic activity than ZIF- Catalytic Activity After catalyst characterization, the catalytic activity of the novel materials was compared with ZIF-8 for the synthesis of chloromethyl ethylene carbonate from CO 2 and epichlorohydrin under solvent-free conditions. It is interesting to note that the combination of acid and basic sites (Lewis and Brönsted site) existing in the MOF catalyst may improve the catalytic activity of both samples. The reactions were carried out under the same conditions of 353 K reaction temperature, 8 bar CO 2 pressure, 10% (w/w) catalyst loading, 8 h reaction time, and 350 rpm of stirring speed. From the Table 2, it follows that at optimum CO 2 pressure of 8 bar, reaction time of 8 h, catalyst loading of 10 % w/w, and variable temperature, Zr/ZIF-8 exhibits a higher catalytic activity than ZIF-8 (Zr/ZIF-8: 93%, 86%, 76%; and ZIF-8: 77%, 74%, 52%) for conversion, selectivity, and yield respectively at the same reaction conditions. The presence of acid and/or basic site in heterogeneous catalyst has significantly catalyzed the reaction of CO 2 and ECH to produce CMEC [42]. Effect of Different Heterogeneous Catalysts Catalysts are very important parts of any chemical reaction; they contain active sites, which are able to speed up the kinetics of chemical reaction by reducing the activation energy. Different types of homogenous and heterogeneous catalysts have been synthesized to catalyze the reaction of CO 2 and epoxide to produce corresponding organic carbonates. In order to assess the stability and effectiveness of the samples, the catalytic activity of both ZIF-8 and Zr/ZIF-8 was investigated in the synthesis of chloromethyl ethylene carbonate from CO 2 and epichlorohydrin. Table 2 shows the effects of the two catalysts for the conversion of epichlorohydrin, selectivity and yield of chloromethyl ethylene carbonate. The catalysts were synthesized using solvothermal method as per standard procedures. The samples were heat-treated at about 373 K in order to enhance an improved catalytic activity and were labelled as ZIF-8 and Zr/ZIF-8 for pure and doped samples, respectively. The reaction of CO 2 and ECH to produce CMEC was carried out in a 25 mL high-pressure reactor at 353 K reaction temperature, 8 bar CO 2 pressure, 10% catalyst loading, and 8 h reaction time. It can be seen from Table 2 that when ZIF-8 was used to catalyze the reaction of CO 2 and ECH, the conversion of ECH, selectivity, and the yield of CMEC were 77%, 74%, and 52% respectively. However, incorporating zirconium into ZIF-8 has significantly increased catalytic performance of Zr/ZIF-8 with the conversion of ECH, selectivity and the yield of CMEC being 93%, 86%, and 76% respectively, although, the presence of side products were reported in both reactions by GC analysis. These side products include 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane. With similar pore spaces and same embedded Lewis acid metal sites in both ZIF-8 and Zr/ZIF-8 catalysts, the increase in the catalytic activity of Zr/ZIF-8 as shown in Figure 12, may be ascribed to high CO 2 affinity via the introduction of zirconium into MOF, which has significantly increased those pore spaces of ZIF-8 [55]. A fine balance of proximity between pure and Zr-doped MOF was critically examined by Demir and research group. Their experimental results in the solvent-free coupling reaction of ECH and CO 2 to produce epichlorohydrin carbonate (ECHC) concluded that 79.6% yield of ECHC and 97.3% selectivity were achieved after 2 h using Zr-MOF catalyst (Zr/MOF-53). It is however Effect of Temperature The cycloaddition reaction of CO2 and epoxide can be referred to as exothermic in nature. The influence of temperature on the cycloaddition of CO2 to ECH to produce CMEC was investigated between temperature ranges of 323 to 373 K. All experiments were conducted with optimized reaction conditions, which were determined during our previous studies with a 10% catalyst loading and 8 bar CO2 pressure for 8 h and a stirring speed of 350 rpm. Table 2 shows the catalytic performance of Zr/ZIF-8 and ZIF-8 as a function of temperature, CO2 pressure, reaction time, and catalyst loading. It can be depicted from Figure 13 that the conversion of epichlorohydrin, selectivity and yield of CMEC were temperature-dependent. Generally speaking, variation in temperature has similar trends in the catalytic activity of both frameworks; the conversion of epichlorohydrin, the selectivity and yield of CMEC increases as temperature increases from 323 to 353 K. However, incorporating zirconium into ZIF-8 has significantly improved the performance of Zr/ZIF-8 with the conversion of ECH, selectivity and yield of CMEC as 93%, 86%, and 76% respectively, while ZIF-8 gave a conversion of ECH, selectivity and yield of CMEC as 77%, 74%, and 52%, respectively, under the same optimum reaction temperature. Further increase in reaction temperature beyond 353 K was unfavorable to selectivity and yield of CMEC in both systems. A slight decrease of the CMEC yield (from 76% to 75%; Zr/ZIF-8 and 52% to 51%; ZIF-8) was observed upon an increase in temperature. This may be due to the formation of diols and dimers of epichlorohydrin [57] and a small amount of by-products such as polymerized CMEC could also affect the yield. Adeleye et al. [58] reported that the increase in the reaction temperature caused a decrease in carbonate yield, due to the decomposition of the catalyst at a higher temperature. Kim et al. [59] also concluded that the reaction temperature for optimal performance is dependent on the nature of the catalyst employed. Therefore, for this set of experiments, the optimized reaction temperature for both frameworks in the synthesis of chloromethyl ethylene carbonate was 353 K. All the subsequent experiments for the chloromethyl ethylene carbonate were conducted at 353 K. To affirm the superior catalytic performance of Zr/ZIF-8 over ZIF-8, nitrogen adsorption and desorption isotherms of the two frameworks were collected and presented in Table 1. Zr/ZIF-8 showed higher CO 2 adsorption capacity which explains in part the improved catalytic performance. Effect of Temperature The cycloaddition reaction of CO 2 and epoxide can be referred to as exothermic in nature. The influence of temperature on the cycloaddition of CO 2 to ECH to produce CMEC was investigated between temperature ranges of 323 to 373 K. All experiments were conducted with optimized reaction conditions, which were determined during our previous studies with a 10% catalyst loading and 8 bar CO 2 pressure for 8 h and a stirring speed of 350 rpm. Table 2 shows the catalytic performance of Zr/ZIF-8 and ZIF-8 as a function of temperature, CO 2 pressure, reaction time, and catalyst loading. It can be depicted from Figure 13 that the conversion of epichlorohydrin, selectivity and yield of CMEC were temperature-dependent. Generally speaking, variation in temperature has similar trends in the catalytic activity of both frameworks; the conversion of epichlorohydrin, the selectivity and yield of CMEC increases as temperature increases from 323 to 353 K. However, incorporating zirconium into ZIF-8 has significantly improved the performance of Zr/ZIF-8 with the conversion of ECH, selectivity and yield of CMEC as 93%, 86%, and 76% respectively, while ZIF-8 gave a conversion of ECH, selectivity and yield of CMEC as 77%, 74%, and 52%, respectively, under the same optimum reaction temperature. Further increase in reaction temperature beyond 353 K was unfavorable to selectivity and yield of CMEC in both systems. A slight decrease of the CMEC yield (from 76% to 75%; Zr/ZIF-8 and 52% to 51%; ZIF-8) was observed upon an increase in temperature. This may be due to the formation of diols and dimers of epichlorohydrin [56] and a small amount of by-products such as polymerized CMEC could also affect the yield. Adeleye et al. [57] reported that the increase in the reaction temperature caused a decrease in carbonate yield, due to the decomposition of the catalyst at a higher temperature. Kim et al. [58] also concluded that the reaction temperature for optimal performance is dependent on the nature of the catalyst employed. Therefore, for this set of experiments, the optimized reaction temperature for both frameworks in the synthesis of chloromethyl ethylene carbonate was 353 K. All the subsequent experiments for the chloromethyl ethylene carbonate were conducted at 353 K. Effect of CO 2 Pressure CO 2 pressure is another important factor influencing the cycloaddition of CO 2 to epoxides. The pressure of carbon dioxide has been established as one of the most crucial factors affecting the conversion, yield, and selectivity of cyclic carbonate [59]. The reaction of epichlorohydrin and CO 2 to produce chloromethyl ethylene carbonate was examined by varying the CO 2 pressures. For this study, the experiments were carried out at 353 K, 10% catalyst loading, and 350 rpm for 8 h. The selectivity and yield of CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO 2 pressure increases from 2 to 8 bar. These results indicate that the catalytic performance of the Zr/ZIF-8 depends on the concentration of available CO 2 at the reactive sites. Similar variation was observed in the catalytic activity of the two frameworks with changing CO 2 pressure where the selectivity and yield of CMEC increased from 57% and 37% to 77% and 52%, respectively, at the same pressure of 8 bar of CO 2 as in the case of Zr/ZIF-8. Figure 14 demonstrates the dependence of CO 2 pressure on the yield of CMEC. It can be observed from the graph that the CMEC yield increased with increasing pressure, the maximum of the CMEC yield was reached at 8 bar. By increasing the CO 2 pressure more than 8 bar, a negative effect was observed on both reaction systems, where both yield and conversion experience a slight drop. Wang et al. [60] observed that the introduction of too much CO 2 dissolves in epoxide may result in the formation of CO 2 -epoxide complex, and retards the interaction resulting in a lower conversion. Similar results were also reported by Onyenkeadi et al. [61], where the introduction of higher pressure of CO 2 dissolved in the epoxide and becomes an unfavorable factor due to the difficulty of separating CO 2 and ECH. This condition inhibits the reaction between ECH and catalyst, thus resulting in lower yield [62]. Liang et al. [63] also reported that many diols and dimers of epichlorohydrin were produced as side products at high pressure. Based on the experimental results and theoretical study, it can be concluded that 8 bar CO 2 pressure was the optimum and all subsequent experiments for the CMEC synthesis were carried out at a CO 2 pressure of 8 bar. Effect of CO2 Pressure CO2 pressure is another important factor influencing the cycloaddition of CO2 to epoxides. The pressure of carbon dioxide has been established as one of the most crucial factors affecting the conversion, yield, and selectivity of cyclic carbonate [60]. The reaction of epichlorohydrin and CO2 to produce chloromethyl ethylene carbonate was examined by varying the CO2 pressures. For this study, the experiments were carried out at 353 K, 10% catalyst loading, and 350 rpm for 8 h. The selectivity and yield of CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO2 pressure increases from 2 to 8 bar. These results indicate that the catalytic performance of the Zr/ZIF-8 depends on the concentration of available CO2 at the reactive sites. Similar variation was observed in the catalytic activity of the two frameworks with changing CO2 pressure where the selectivity and yield of CMEC increased from 57% and 37% to 77% and 52%, respectively, at the same pressure of 8 bar of CO2 as in the case of Zr/ZIF-8. Figure 14 demonstrates the dependence of CO2 pressure on the yield of CMEC. It can be observed from the graph that the CMEC yield increased with increasing pressure, the maximum of the CMEC yield was reached at 8 bar. By increasing the CO2 pressure more than 8 bar, a negative effect was observed on both reaction systems, where both yield and conversion experience a slight drop. Wang et al. [61] observed that the introduction of too much CO2 dissolves in epoxide may result in the formation of CO2-epoxide complex, and retards the interaction resulting in a lower conversion. Similar results were also reported by Onyenkeadi et al. [62], where the introduction of higher pressure of CO2 dissolved in the epoxide and becomes an unfavorable factor due to the difficulty of separating CO2 and ECH. This condition inhibits the reaction between ECH and catalyst, thus resulting in lower yield [63]. Liang et al. [64] also reported that many diols and dimers of epichlorohydrin were produced as side products at high pressure. Based on the experimental results and theoretical study, it can be concluded that 8 bar CO2 pressure was the optimum and all subsequent experiments for the CMEC synthesis were carried out at a CO2 pressure of 8 bar. Influence of Reaction Time The effect of varying the reaction time on the yield of CMEC was investigated by carrying out a set of coupling reaction of CO2 and epichlorohydrin using both ZIF-8 and Zr/ZIF-8 catalysts. For this study, all experiments were conducted at 353 K and 8 bar CO2 pressure with 10% (w/w) catalyst loading of ZIF-8 and Zr/ZIF-8. Figure 15 demonstrates the influence of reaction time on CMEC yield and selectivity. The results shown on the graph illustrates that the yield increased continuously at the beginning and reached 76% and 52% within 8 h for Zr/ZIF-8 and ZIF-8, then decreased to 75% and 51% respectively, indicating that a slight change in the reaction condition can influence the product formation in a reaction. Similarly, the conversion of ECH was observed to increase from 353 to 366 K when the reaction time was increased from 2 to 8 h. However, when the reaction time was increased further to 10 h and above, a progressive decrease in conversion of ECH was recorded. A similar observation was previously reported in the conversion of ECH to chloropropene carbonate with Zn-ZIF-67 by Adeleye et al. [65]. According to him, conversion of epoxides reaches an equilibrium plateau at optimum reaction time. This phenomenon is referred to as induction period. The induction period is attained when the CO2 and epoxides sufficiently diffuse into the catalytic frameworks of the ZIF-material to reach the active sites of the catalyst and then be converted to the organic carbonate. Beyond the induction period, low conversion of epoxides as well organic carbonates may be observed. From Figure 15, it can be concluded that prolonged reaction time produces lesser ECH conversion and consequently lesser CMEC yield and selectivity. Based on the experimental results and theoretical study, the reaction time of 8 h was considered the optimum for ZIF-8 and Zr/ZIF-8. Influence of Reaction Time The effect of varying the reaction time on the yield of CMEC was investigated by carrying out a set of coupling reaction of CO 2 and epichlorohydrin using both ZIF-8 and Zr/ZIF-8 catalysts. For this study, all experiments were conducted at 353 K and 8 bar CO 2 pressure with 10% (w/w) catalyst loading of ZIF-8 and Zr/ZIF-8. Figure 15 demonstrates the influence of reaction time on CMEC yield and selectivity. The results shown on the graph illustrates that the yield increased continuously at the beginning and reached 76% and 52% within 8 h for Zr/ZIF-8 and ZIF-8, then decreased to 75% and 51% respectively, indicating that a slight change in the reaction condition can influence the product formation in a reaction. Similarly, the conversion of ECH was observed to increase from 353 to 366 K when the reaction time was increased from 2 to 8 h. However, when the reaction time was increased further to 10 h and above, a progressive decrease in conversion of ECH was recorded. A similar observation was previously reported in the conversion of ECH to chloropropene carbonate with Zn-ZIF-67 by Adeleye et al. [64]. According to him, conversion of epoxides reaches an equilibrium plateau at optimum reaction time. This phenomenon is referred to as induction period. The induction period is attained when the CO 2 and epoxides sufficiently diffuse into the catalytic frameworks of the ZIF-material to reach the active sites of the catalyst and then be converted to the organic carbonate. Beyond the induction period, low conversion of epoxides as well organic carbonates may be observed. From Figure 15, it can be concluded that prolonged reaction time produces lesser ECH conversion and consequently lesser CMEC yield and selectivity. Based on the experimental results and theoretical study, the reaction time of 8 h was considered the optimum for ZIF-8 and Zr/ZIF-8. Effect of External Mass Transfer in Heterogeneous Catalytic Processes Mass transfer limitations play significant roles in chemical reactions by controlling the rate of reaction towards the desired product. In homogenous catalytic reaction, the effect of mass transfer between the phases is mostly negligible. However, in a heterogeneous catalytic reaction, the reaction rate significantly relies on the mass or diffusion between these phases. Mass transfer is typically higher in porous solid or fine particles of nanoscale than large nonporous catalyst [66], transfer of material from the exterior to the interior of a particle happens through pores that open to the external surface, which provides access to the interior of the crystallite material [66]. A typical example is zeolitic imidazolate framework (ZIF-8). In the heterogeneous catalytic conversion of CO2 and epoxide, the internal and external gradient of transport materials between system phases lowers the activity and selectivity of the catalyst towards the desired product [67]. It is important to know that when designing a new catalyst and directing such a catalyst to be selective towards a particular desired product mass transfer resistance and the kinetics are key functions [67]. In cycloaddition reaction of CO2 with ECH, the physicochemical properties of the catalyst and the operating conditions all have a direct effect on the activity of the catalyst as well as the quality of CMEC formed [68]. When a chemical reaction occurs on an active surface, intraparticle diffusion takes place through the pores and the film surrounding the solid catalyst [68]. The coupling reaction of ECH with CO2 to produce chloromethyl ethylene carbonate is an exothermic reaction. In order to reduce or eliminate the effects of mass transfer resistance, it is recommended to employ a highly porous heterogeneous catalyst [68]. The influence of mass transfer on the reaction of ECH and CO2 to synthesize CMEC at 353 K reaction temperature for 8 h with a Effect of External Mass Transfer in Heterogeneous Catalytic Processes Mass transfer limitations play significant roles in chemical reactions by controlling the rate of reaction towards the desired product. In homogenous catalytic reaction, the effect of mass transfer between the phases is mostly negligible. However, in a heterogeneous catalytic reaction, the reaction rate significantly relies on the mass or diffusion between these phases. Mass transfer is typically higher in porous solid or fine particles of nanoscale than large nonporous catalyst [65], transfer of material from the exterior to the interior of a particle happens through pores that open to the external surface, which provides access to the interior of the crystallite material [65]. A typical example is zeolitic imidazolate framework (ZIF-8). In the heterogeneous catalytic conversion of CO 2 and epoxide, the internal and external gradient of transport materials between system phases lowers the activity and selectivity of the catalyst towards the desired product [66]. It is important to know that when designing a new catalyst and directing such a catalyst to be selective towards a particular desired product mass transfer resistance and the kinetics are key functions [66]. In cycloaddition reaction of CO 2 with ECH, the physicochemical properties of the catalyst and the operating conditions all have a direct effect on the activity of the catalyst as well as the quality of CMEC formed [67]. When a chemical reaction occurs on an active surface, intraparticle diffusion takes place through the pores and the film surrounding the solid catalyst [67]. The coupling reaction of ECH with CO 2 to produce chloromethyl ethylene carbonate is an exothermic reaction. In order to reduce or eliminate the effects of mass transfer resistance, it is recommended to employ a highly porous heterogeneous catalyst [68]. The influence of mass transfer on the reaction of ECH and CO 2 to synthesize CMEC at 353 K reaction temperature for 8 h with a range of stirring speed between 320 and 550 rpm in an autoclave reactor. It was observed that there was no significant change in the conversion of ECH (~93), selectivity (~86), and the yield of CMEC (~76) when the stirrer speed was maintained above 330 rpm. Therefore, it was concluded that there was no effect of external mass transfer resistance on the experimental conditions. Effect of Catalyst Loading To investigate the influence of catalyst loading on the CMEC synthesis, several number of experiments were performed by varying the molar ratio of both ZIF-8 and Zr/ZIF-8 catalyst to ECH. For this study, all experiments were conducted at 353 K and 8 bar CO 2 pressure for 8 h. The results of varying catalyst loading are presented in Figure 16. It can be observed from the graph that by increasing the catalyst loading, there was a corresponding increase in ECH conversion, yield, and selectivity of CMEC. For example, for the experiments conducted with catalyst loadings from 2.5%-7.5%, there was a significant increase in ECH conversion, yield, and selectivity of CMEC. Also, for the experiment conducted at 10% (w/w) of catalyst loading, there was a sharp increase of ECH conversion, yield, and selectivity of CMEC from 90%-96%, 45%-56%, and 73%-79%, respectively. According to Maeda et al. [68], the decrease in epoxide conversion may be ascribed to a decrease in the substrate concentration around the pore cavities of the catalyst at higher catalyst loading. This effect neutralizes the Brönsted acid centers of the catalyst, thereby preventing the interaction between the acidic sites of the catalyst and the oxygen atom of epoxide from the ring opening. This consequently reduces the epoxides conversion to organic carbonates. Considering the percentage error of ±2%, it can be concluded that the number of active sites for ECH and CO 2 to react and produce CMEC was large enough at 10% (w/w) catalyst loading. From the results obtained with respect to catalyst loading, 10% (w/w) was the optimum. From the experimental results for both ZIF-8 and Zr/ZIF-8 catalysts, it is satisfactory to conclude that 10% (w/w) catalyst loading was considered the optimum and further experiments were carried out at 10% (w/w) catalyst loading. Energies 2020, 13, x FOR PEER REVIEW 20 of 26 range of stirring speed between 320 and 550 rpm in an autoclave reactor. It was observed that there was no significant change in the conversion of ECH (~93), selectivity (~86), and the yield of CMEC (~76) when the stirrer speed was maintained above 330 rpm. Therefore, it was concluded that there was no effect of external mass transfer resistance on the experimental conditions. Effect of Catalyst Loading To investigate the influence of catalyst loading on the CMEC synthesis, several number of experiments were performed by varying the molar ratio of both ZIF-8 and Zr/ZIF-8 catalyst to ECH. For this study, all experiments were conducted at 353 K and 8 bar CO2 pressure for 8 h. The results of varying catalyst loading are presented in Figure 16. It can be observed from the graph that by increasing the catalyst loading, there was a corresponding increase in ECH conversion, yield, and selectivity of CMEC. For example, for the experiments conducted with catalyst loadings from 2.5%-7.5%, there was a significant increase in ECH conversion, yield, and selectivity of CMEC. Also, for the experiment conducted at 10% (w/w) of catalyst loading, there was a sharp increase of ECH conversion, yield, and selectivity of CMEC from 90%-96%, 45%-56%, and 73%-79%, respectively. According to Maeda et al. [69], the decrease in epoxide conversion may be ascribed to a decrease in the substrate concentration around the pore cavities of the catalyst at higher catalyst loading. This effect neutralizes the Brönsted acid centers of the catalyst, thereby preventing the interaction between the acidic sites of the catalyst and the oxygen atom of epoxide from the ring opening. This consequently reduces the epoxides conversion to organic carbonates. Considering the percentage error of ±2%, it can be concluded that the number of active sites for ECH and CO2 to react and produce CMEC was large enough at 10% (w/w) catalyst loading. From the results obtained with respect to catalyst loading, 10% (w/w) was the optimum. From the experimental results for both ZIF-8 and Zr/ZIF-8 catalysts, it is satisfactory to conclude that 10% (w/w) catalyst loading was considered the optimum and further experiments were carried out at 10% (w/w) catalyst loading. Figure 13a,b shows the effect of varying reaction temperature on catalysts' selectivities towards CMEC. For example, it can be observed that when the temperature was increased from 50 to 80 • C, both catalysts show a corresponding increase in selectivities from 68% and 50% to 86% and 74%, respectively. However, when the temperature was increased beyond the 353 K, a marginal decrease in selectivities was observed in both frameworks, demonstrating that the 353 K was the optimum temperature for the reaction. Meanwhile, the gas chromatography-mass spectroscopy (GC-MS) analysis of the samples shows that 17.3% of 2,5-bis (chloromethyl)-1,4-dioxane (by-product) formed at 353 K, this may explain in part why a drop in catalysts' selectivities was recorded for both samples. Similar results and by-product have been previously reported with ZIF-8 by Carron et al. [69]. They also agree that almost 100% selectivity of ZIF-8 to chloropropene carbonate was achieved at a temperature of 393 K, but decreased to 78.6% when the temperature was increased to 403 K. Effect of Reaction Conditions on Catalysts Selectivity to Chloromethyl Ethylene Carbonate In addition to the effect of temperature on catalysts' selectivities, the influence of varying CO 2 pressure on catalysts' selectivities was also investigated. According to Figure 14a,b, the selectivity of the catalysts towards CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO 2 pressure was increased from 2 to 8 bar. These results indicates that the activity and selectivity of both catalysts were influenced by the concentration of available CO 2 at the reactive sites. Although, similar effect was observed in the responses of both catalysts to variation in CO 2 pressure, however, the results show that Zr/ZIF-8 has higher selectivity than the ZIF-8 catalyst, where the selectivity of both catalysts increased from 69% and 60% to 87% and 77%, respectively, for Zr/ZIF-8 and ZIF-8catalysts. Concersely, both samples experienced decline in selectivities from 87% and 77% to 85% and 70% for ZIF-8 and Zr/ZIF-8, respectively, when the CO 2 pressure was increased beyond the optimum level of 8 Bar. Miralda et al. [70], further argues that ZIF-8 is a dual-functional catalyst with both acidic and basic sites that have been associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. This bifunctional characteristic enhances the catalyst selectivity for cycloaddition reaction. In a separate report, Miralda et al. [70], also ascertained that it is likely that Lewis acid sites associated with Zn 2+ ions in the ZIF-8 framework play the vital role in catalyzing the reaction of epichlorohydrin and CO 2 to chloropropene carbonate. They further explained that the presence of basic nitrogen atoms of the imidazole ligand, probably, favours the adsorption and binding of CO 2 as well as activation of the carbon-oxygen bonds in CO 2 . In agreement with other similar doped ZIF-8, the open metal centers in the Zr/ZIF-8 has the potential to easily activate the epoxides and the basic sites present in the frameworks. This could be the reason for the higher selectivity that were observed in the solvent-free ECH-CO 2 cycloaddition reactions under mild conditions. Comparatively, the higher selectivity of Zr/ZIF-8 than ZIF-8 towards CMEC may be attributed to the presence of zirconium (Zr). According to a 2019 publication by de Caro et al. [71], the effect of Zr doping on Mg-Al hydrotalcite, the catalyst has significantly increased its selectivity from 90% to >99% towards glycerol carbonate (GC). Reusability of ZIF-8 Catalysts Reusability is an important and essential feature of any heterogeneous catalyst in order to be considered useful in industrial applications [71]. The influence of catalyst reusability on the catalytic properties of ZIF-8 and Zr/ZIF-8 in the cycloaddition reaction was investigated. The experiments were carried out in a high-pressure reactor at optimum reaction conditions, i.e., at 353 K, 8 bar with fresh 10% (w/w) ZIF-8 catalyst loading, for 8 h, and at a stirring speed of 350 rpm. The catalysts after Run 1 in the cycloaddition reaction were washed with ethanol and acetone, centrifuged, and oven-dried at 343 K for 12 h before reuse. The recovered catalysts were reused for up to 7 subsequent experiments following the same experimental procedure. ZIF-8 showed a progressive loss in catalytic activity after each run as shown in Figure 17, while Zr/ZIF-8 exhibited no loss of activity indicating the catalyst stability for cycloaddition reaction of CO 2 epichlorohydrin. Yuan et al. [72] stated that the presence of dopant in ZIF-8 show that zirconium is more stable and resilient during the reaction. There was no significant change in the conversion of ECH, selectivity, and yield of CMEC using Zr/ZIF-8. Although, a very slight decrease in the yield of CMEC from 70% (fresh) to 69% (recycled) was observed. The low catalytic activity of the recycled Zr/ZIF-8 catalyst may be ascribed to formation of carbonaceous materials during the cycloaddition reaction as previously reported by Yuan et al. [72]. Furthermore, the XRD and FT-IR analyses results confirmed that Zr/ZIF-8 maintained its crystallinity throughout the reaction process. Zr/ZIF-8. Although, a very slight decrease in the yield of CMEC from 70% (fresh) to 69% (recycled) was observed. The low catalytic activity of the recycled Zr/ZIF-8 catalyst may be ascribed to formation of carbonaceous materials during the cycloaddition reaction as previously reported by Yuan et al. [73]. Furthermore, the XRD and FT-IR analyses results confirmed that Zr/ZIF-8 maintained its crystallinity throughout the reaction process. Conclusions Zr/ZIF-8 has been successfully designed and assessed as a greener and highly efficient CO 2 -reduction catalyst for the synthesis of CMEC. Although ZIF-8 is criticized by many researchers as thermally unstable for the synthesis of organic carbonates from CO 2 and epoxide, however, our experiments have confirmed that the introduction of zirconium into ZIF-8 could strengthen the weak functionality, making it tenable for large-scale industrial applications. Several authors have utilized zirconium to reinforce different kinds of MOF experiments in order to achieve optimum results. However, their attempts have been unsatisfactory, partly because a firm balance between the required percentage of zirconium dopant and their host molecules was not established for those particular experiments. It may also be worth mentioning that this work has utilized a 10% dopant of zirconium for such a tremendous catalytic activity of Zr/ZIF-8. The stability tests carried out on both samples show that Zr/ZIF-8 demonstrates higher stability compared with single metal ZIF-8. It has been concluded from the experimental results that there is a direct relationship between variation in the reaction conditions and ECH conversion, CMEC yield, and selectivity. From the experimental results, it can be observed that Zr/ZIF-8 catalyst displayed high epoxide conversions and high selectivity to chloromethyl ethylene carbonate at 353 K, without using any solvent or co-catalyst. Lewis acid copper (II) sites in the ZIF-8 frameworks promote adsorption of CO 2 on the solid surface and its further conversion to CMEC. The activity of reused Zr/ZIF-8 catalyst showed consistent stability over seven subsequent runs. The optimum reaction condition for the experiments was found at 353 K, 8 bar CO 2 pressure, and 8 h using fresh 10% (w/w) Zr/ZIF-8 catalyst loading for this reaction. Therefore, the development of a novel Zr/ZIF-8 catalyst for the synthesis of CMEC from CO 2 and ECH provided an efficient and promising greener route for CO 2 utilization.	v3-fos-license
2019-05-07T13:28:51.753Z	2019-01-01T00:00:00.000	145969556	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": null, "oa_url": null, "pdf_hash": "e5eb5df4f1817e3eb2b44d6d873a8635bb653290", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:252", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "e5eb5df4f1817e3eb2b44d6d873a8635bb653290", "year": 2019 }	pes2o/s2orc	Antitubercular Activity of the Fungus Gliocladium sp. MR41 Strain Tuberculosis (TB) is a leading cause of death worldwide from infectious diseases and its inadequate treatment has led to emergence of resistant strains. The emergence of these strains renders the search for new drugs for the treatment of TB. The aim of this study was the evaluation of the anti-TB activity of the extract from fungus Gliocladium sp. MR41, and bioassay-guided fractionation and identification of majority compounds was carried out. Fungal strain culture was lyophilized and extracted by maceration in Ethyl Acetate (EtOAc). This extract was fractionated by liquid-liquid partitioning and chromatographic techniques, and the compounds were identified by their spectroscopic data. Furthermore, the EtOAc extract, fractions, and pure compounds were tested on Mycobacterium tuberculosis using the Microplate Alamar Blue Assay. From the bioactive AcetoNitrile Fraction (AcNF; MIC = 3.13 µg/mL) of the EtOAc extract, four compounds were isolated: ergosterol (1), ergosterol-5, 8-peroxide (2), 1, 6-di-O-acetyl-2,3,4,5-tetrahydroxy-hexane (3), and allitol (4). Only 2 exhibited potent activities against M. tuberculosis (MIC = 0.78 µg/mL). Additionally, this is the first report, to our knowledge, of polyols 3 and 4 from this fungus. Introduction Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis, which typically affects the lungs (pulmonary TB), but it can also affect other sites (extrapulmonary TB) (1). TB gives rise to poor health in millions of people each year, and in 2016, it was the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. In 2016, there were an estimated 10.4 million new cases of TB and 1.3 million deaths in worldwide. Treatment lasts about 6 months with five firstline drugs (Streptomycin, Isoniazid, Rifampin, Ethambutol, and Pyrazinamide). Misuse of these drugs, in addition to inconsistent or partial treatment, has led to the development of multiDrug-Resistant (MDR) TB and eXtensively Drug-Resistant (XDR) TB (2). There is an urgent need for new anti-TB drugs. The genus Gliocladium belongs to the Moniliaceae family and its species are saprophytic fungi that generally live on the ground, although they have also been found in association with plants and animals of aquatic environments (10). In recent times, species of this genus have received attention concerning their biological and chemical properties. Some compounds isolated from species of the genus Gliocladium have exhibited important biological activities, such as secalonic and heptelidic acids with cytotoxic and antibacterial activities, respectively (11, 12). From Gliocladium sp. FO-1513 were isolated the polyisoprene compounds glisoprenins A and B that inhibited acyl-CoA: cholesterol acyltransferase activity (13). In addition, from G. roseum KF-1040 were isolated some roselipins -structures of highly methylated C20 fatty acid, mannose, and arabinitol-that inhibitied diaciylglycerol acyltransferase activity (14). Recently, a diketopiperazine, alkaloid cyclo-(glycyl-L-tyrosyl)-4,4-dimethylallyl ether, was isolated from a strain of Gliocladium sp., and this compound exhibited strong antimicrobial activity against Micrococcus luteus (15). Sterol derivatives and anthraquinones were isolated from G. catenulatum, and these compounds inhibited the proliferation and growth of a myelogenous leukemia line (K562) (16). Our research groups reported the anti-TB activity of the Ethyl Acetate (EtOAc) extracts of three strains of the Gliocladium genus from Yucatán, México (17). In addition, another strain of Gliocladium sp. MR41, was isolated from leaf litter in Veracruz, México (18). In our present contribution, the extract from this fungal strain was tested in-vitro against M. tuberculosis, and bioassay-guided fractionation and the identification of majority compounds were carried out. General chemical experimental procedures Silica gel 60 (Merck, Darmstadt, Germany) or Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for Column Chromatography (CC). Detection in Thin-Layer Chromatography (TLC) was achieved under Ultra Violet (UV) light and by spraying with the phosphomolybdic acid reagent, followed by heating for 5 min at 105 ºC. Melting point was determined on a Mel-Temp II. InfraRed (IR) spectra were recorded on KBr discs on a Nicolet Protégé 460. 1 H-, while 13 C-Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bucker Avance 400 Ultra Shield spectrometer in CDCl 3 or D 2 O, with Tetra Methyl Silane (TMS) as internal standard. EIMS were obtained on an Agilent Technologies 6890N gas chromatograph coupled with an Agilent Technologies 5975B intermass selective detector. Fungal culture The strain of Gliocladium sp. MR41 was obtained from the culture collection of the Centro de Investigación Científica de Yucatán, previously identified by molecular taxonomy (18). This strain was reactivated on Petri dishes with corn agar and incubated during a 12/12-h light/dark photoperiod at 25 °C for about 7-8 days. Finally, the hyphae/spores of this fungus was obtained and inoculated onto fermented rice as substrate, as described previously (18), Anti-TB activity of the fungus Gliocladium sp. MR41 strain and this was incubated for 40 days at 25 °C with a 12/12-h light/dark photoperiod. At the end of the growth, the fungal cultures were frozen, lyophilized, and powdered. Extraction and Purification Several batches of the ground fungal material were extracted by maceration in EtOAc (3×) at room temperature. The solvent was filtered and evaporated in vacuo to yield a dry EtOAc extract (8.0 g). This extract was fractionated by liquid-liquid partition, with n-hexane and acetonitrile (3×, 2:1, 1:1, and 1:1, v/v), and both layers were evaporated under reduced pressure to produce the Hexanic (HexF) and AcetoNitrile Fractions (AcNF). During the evaporation of AcNF, a precipitate was obtained and purified by crystallization with acetone to yield compound 1 (230 mg). The AcNF (510 mg) was subjected to CC on Sephadex LH-20 and eluted with MeOH to yield six fractions (F1-F6). The bioactive F6 (47.7 mg) was chromatographed by vacuum liquid chromatography on silica gel and eluted with n-hexane, n-hexane: CH 2 Cl 2 , CH 2 Cl 2 : MeOH, and MeOH as ascending polarity gradient to yield 18 subfractions (SF1-SF18) according to TLC analysis. The SF1 was purified by flash CC on silica gel using mixtures of n-hexane:EtOAc with gradient elution to yield compound 2 (4.0 mg). The SF9 was partitioned with EtOAc and water (1:3, v/v), and the aqueous layer was lyophilized to yield compound 3 (11.5 mg). The SF15 was purified by successive crystallizations with acetone to yield compound 4 (10.1 mg). Antimycobacterial assay The in-vitro assay was assessed on the M. tuberculosis H37Rv strain (ATCC 27294) susceptible to all five first-line anti-TB drugs (Streptomycin, Isoniazid, Rifampin, Ethambutol, and Pyrazinamide). The microorganism was inoculated in 13 × 100-mm screw-capped tubes containing 3 mL of sterile Middlebrook 7H9 broth (Difco, Detroit MI, USA), supplemented with 0.2% glycerol and enriched with 10% Aleic acid, Albumin, Dextrose, and Catalase (OADC) (Difco) incubated at 37 °C in 5% CO 2 atmosphere. Anti-TB activity was determined by the Microplate Alamar Blue Assay (MABA) described previously by . The crude extract, fractions, or pure compounds were dissolved with DiMethyl SulfOxide (DMSO). All samples were tested using a concentration range of 100-0.39 µg/ mL, and the maximal concentration of DMSO in the assays was 1.2% (v/v). MIC was defined as the lowest concentration of each sample that prevented the color change from blue to pink. In each microplate, 1.00-0.031 µg/mL of Rifampin was included as positive control. In addition, a blank (extract of fermented rice) and DMSO were included as negative and solvent controls, respectively. All evaluations were carried out in triplicate. Extraction and Purification The fungus Gliocladium sp. MR41 was massively cultured and the EtAcO crude extract was obtained. This extract was solventpartitioned to obtain HexF and AcNF. The bioassay-guided fractionation of AcNF led to the obtaining of four pure compounds: 1-4 ( Figure 1). Structural Identification of compounds Compounds 1 and 2 were identified as ergosterol and its derivative, ergosterol-5,8peroxide, respectively, according to their spectral data and by comparison with standard samples. In addition, two compounds were purified, which presented similar spectral data. Compound 3 presented an intense band of alcohol (3328 cm -1 ) and ester (1743 cm -1 ) groups. Compound 4 showed no carbonyl group and, and its 1 H-NMR spectra showed signals typical of the oxygenated protons (3.62-3.81 ppm). These spectral data indicated a polyol structure; then, its melting point (154−156 °C) 9 The fungus Gliocladium sp. MR41 was massively cultured and the EtAcO crude extract was obtained. This extract was solvent-partitioned to obtain HexF and AcNF. The bioassayguided fractionation of AcNF led to the obtaining of four pure compounds: 1-4 (Figure 1). matched with that reported for a hexytol named allitol (4). On the other hand, the ester group of compound 3 was confirmed by their 1 H and 13 C NMR spectrum. These corresponded to two acetate groups (20.8 and 173.2 ppm), since it is a symmetrical structure and it correlated in all proton-spectra integrations. Therefore, compound 3 was identified as an ester derivative denominated 1,6-di-O-acetyl-allitol. Relative stereochemistry at positions C-4 and C-5 was established by the interaction observed on Two-Dimensional-Nuclear Overhauser Enhancement SpectroscopY (2D-NOESY), and this established a trans relationship between H-4 and H-5. Discussion In the search for novel alternative natural products in the discovery and development of new active drugs against M. tuberculosis, our research group has been screening fungi isolated from several states of México. Among these, a non-previously tested Gliocladium sp. MR41 strain revealed a remarkable anti-TB property (MIC = 6.25 μg/mL), justifying the bioassayguided purification process of its extract that led to the identification of ergosterol-5,8-peroxide 2 (MIC = 0.78 μg/mL) as responsible for its bioactivity. Previously, this compound 2 has been reported as deriving from the medicinal plant Ajuga remota (MIC = 1 μg/mL) (20). There are no reports, to our knowledge, of compounds isolated from the Gliocladium genus with antitubercular activity; however, the fungi of other genera belonging to the Hypocreales order of the Moniliaceae family such as Paecilomyces tenuipes produced beauvericin (3) 100 Allitol (4) 100 Rifampin 0.062 EtOAc: Ethyl AcetateHexF: n-Hexane Fraction; AcNF: AcetoNitrile Fraction; F: Fraction. (MIC = 12.5 μg/mL) (21). This is the first report, to our knowledge, of polyols 3 and 4 from this fungus. Allitol (4) is an acyclic and rare sugar possessing a low abundance in nature and with an exorbitant cost (22). It was previously isolated from the fungus Tylopilus plumbeoviolaceus (23). No reports exist, to our knowledge, for allitol (4) with respect to its biological activity in the scientific literature; however, its isomeric forms, such as mannitol, have been reported to possess activity against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus (24). Our research contributes to the chemical composition and antitubercular activity of the fungus Gliocladium sp. MR41.	v3-fos-license
2016-05-04T20:20:58.661Z	2013-09-19T00:00:00.000	14449565	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CC0", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0072131&type=printable", "pdf_hash": "a43a65905bf54a25c0b541851a6891e07833ff00", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:258", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "sha1": "a43a65905bf54a25c0b541851a6891e07833ff00", "year": 2013 }	pes2o/s2orc	Inhibition of HIV-1 Reverse Transcriptase-Catalyzed Synthesis by Intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide Adducts To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N 6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT. Introduction DNA polymerase interactions with their nucleic acid substrates have gained considerable interest in recent years with the determination of the crystal structures of several polymerase-DNA complexes, including but not limited to human immunodeficiency virus type-1 (HIV-1 1 ) reverse transcriptase (RT) [1,2], A-family DNA polymerases (Klenow fragment of Escherichia coli DNA polymerase I [3], Thermus aquaticus DNA polymerase [4,5], Bacillus stearothermophilus DNA polymerase I [6], and T7 DNA polymerase [7]), and the Xfamily DNA polymerase β (pol β) [8,9,10]. Although the general shape of the polymerase domain is likened to a partially opened right hand with fingers, palm, and thumb subdomains, there is significant structural diversity in the composition of the residues that comprise these subdomains. The general features revealed by the structural characterization of these polymerase-DNA complexes have shown that polymerases interact with the duplex region of their nucleic acid substrates primarily through sugar-phosphate backbone and minor groove interactions. Further, it has been recognized that protein-DNA interactions occur primarily through sequence-specific DNA hydrogen bond donors and acceptors in the DNA major groove, while the minor groove offers very little hydrogen bond selectivity [11]. Additionally for the lentiviral reverse transcriptases, these polymerases have a series of amino acids in α-helix H that serve as a sensor of the DNA along its minor groove, the "Minor Groove Binding Track" (MGBT) [12]. The position of this helix places it in contact with newly synthesized duplex DNÃ 2-6 nucleotides from the 3´ end of the primer strand ( Figure 1). The structure of the DNA in this region is in the A-form near the polymerase active site and B-form near the RNase H domain, with a 40-45° bend at the junction. An α-helix in the thumb subdomain, helix H, interacts primarily with the primer strand in the region of the bend and contributes several key MGBT residues [12,13,14,15]. Previously, we have used site and stereospecific styrene oxide DNA adducts that were linked through either the N 6 exocyclic amino group of adenine or the N 2 exocyclic amino group of guanine, major and minor grooves, respectively as a probe of this MGBT interaction [16,17]. When the styrene oxide lesions were positioned in the minor groove, HIV-1 RT synthesis was terminated in the newly synthesized DNA, 4-7 nucleotides downstream of the lesions [16]. These data suggested that when α-helix H encountered the lesion, it became trapped with no further extension. When chemically identical adducts were positioned in the major groove, there was a compression of the corresponding minor groove and replication was blocked 1-3 nucleotides beyond the lesion [17]. These data are consistent with the interpretation that DNA adducts can indirectly terminate polymerization of HIV-1 RT by altering the flexibility of the DNA toward bending in the template-primer (T-P) stem and interfering with crucial enzyme-DNA contacts, especially those that involve α-helix H. While these earlier studies provided insights into the molecular interactions between the α-helix H and the minor groove of the newly synthesized DNA, these analyses were limited by the fact that the styrene oxide adducts were completely localized in either the minor or major groove. However, other types of DNA adducts are not only able to covalently attach to an exocyclic amino group, but also intercalate into the hydrophobic DNA core. To further an understanding of the mechanisms by which HIV-1 RTcatalyzed synthesis can be blocked, we adopted an experimental design in which the N 6 position of adenine was chosen as the site for adduction by the polycyclic aromatic hydrocarbon, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) that intercalates its aromatic rings into the helix [18,19,20,21]. Additionally, the design of the current experiments was expanded to position the lesion in either the template or the primer strand. This represents the first study to The crystallographic structure of a T-P/HIV-1 RT complex is shown (PDB ID 3V4I). The heterodimeric HIV-1 RT is shown in a ribbon representation (p66, blue; p51, gray). The molecular surface of the DNA duplex is dark gray with the 5 th base pair upstream of the 3´-primer terminus (3´) colored yellow. The major groove is indicated and α-helix H (solid blue ribbon) is situated in the minor groove. The residues of the MGBT are on one side of the α-helix that face the DNA (residues not shown). Enzymatic construction and purification of BPDEadducted and unadducted templates and primers Oligodeoxynucleotides (11-mers) containing the (+) or (-)anti-trans-BPDE lesion were synthesized and purified as previously described (Fig. 2) [22,23]. The sequence context of the lesion is the third position of codon 61 of human N-ras gene. All unadducted control oligodeoxynucleotides were synthesized by Midland Research Inc. The 73-mer oligodeoxynucleotide strands (Fig. 3B) were constructed as follows. Using a 46-mer scaffolding DNA, the phosphorylated adducted or unadducted control 11-mers were ligated to both a 30-mer and 32-mer on the 5΄and 3΄ ends of the 11-mer, respectively. The 32-mer was also fully phosphorylated prior to ligation. The 25-mer oligodeoxynucleotide primers were created by ligation of a phosphorylated 14-mer to the adductcontaining or control 11-mer, respectively. All oligodeoxynucleotides were gel purified through 10% denaturing polyacrylamide gels. As illustrated in Figure 3, two different constructs were made and utilized for primer extension reactions: a) the 27t/11p* represents a 11-mer adducted primer (5΄-CGGACAAGAAG-3΄) (p) annealed to a 27-mer template (t) in which the A with an asterisk (Figure 3) represents either C 10 S or C 10 R configuration of the BPDE-dA lesion located 5 nucleotides from the 3΄ terminus. The unadducted T-P is designated 27t/11p; b) the 73t/11p represents an unadducted 11-mer primer annealed to a 73-mer adducted template containing either the C 10 S or C 10 R configuration of the BPDE-dA lesion (indicated by ). The adduct was located 37 nucleotides from the 3΄ end of the template strand within the duplex. The unadducted T-P is designated 73t/11p. Thus, in all sequence contexts, the adduct always remains in the same position. Polymerase arrest assays Oligodeoxynucleotide primers (11-, 20-, and 25-mers) were 5΄ end-labelled with (γ 32 P) ATP (6000 Ci/mmol, DuPont-New England Nuclear, Boston, MA) using T4 DNA kinase (New England BioLabs) and purified from excess ATP by column chromatography. Extension reactions with the 11-mer primers were performed at 18 °C for 30 min, whereas the reactions using longer primers were conducted at 37 °C for 30 min. The template (250 fmol) to primer (50 fmol) ratio was 5 in all cases. Primer-template enzyme binding assay Solution conditions were similar to those described above for the polymerase arrest assay. HIV-1 RT (2.5, 5, or 10 fmol) was preincubated with adducted or unadducted 5´-32 P-labeled T-P (i.e., 27t/11p, 27t/11p; 73t/11p, 73/11p) at 18 °C for 30 min in the absence of dNTPs. The template (250 fmol) to primer (50 fmol) ratio was 5 in all cases. After the initial incubation with the 27t/11p or 11p, a competitor 33t/29p T-P was added in fivefold excess with dTTP, dCTP, and dGTP (330 µM each) and incubated at 18 °C. The competitor DNA binds free enzyme and enzyme that dissociates from the duplex used in the preincubation phase. DNA synthesis does not occur on the competitor T-P since the first two templating nucleotides are thymidines and dATP is omitted. The 29-mer primer of the competitor T-P was 32 P-labeled to monitor binding and extension. Aliquots were taken at 0.5, 3, 10, 20, and 30 min and analyzed as described above. When the 73t or 73t/11p was preincubated with HIV-1 RT, the competitor T-P was 27t/ 11p. In this situation, the only dNTP added was dATP along with the competitor DNA. The initial templating sequence of the 73t/11p is 3΄-GCG-5΄. The sequence of the 33-mer template and 29-mer primer were: 5΄-CGGACAAGAAGAATTCGTCGTGACTGGGAAAAC-3΄ and 5΄-GTTTTCCCAGTCACGACGAATTCTTCTTG-3΄, respectively. Results Primer extension analysis with unadducted (27t/11p) and C 10 S or C 10 R adducted 11-mer primers (27t/11p) Analyses of the co-crystal structures of HIV-1 RT with T-P DNA reveal that the enzyme makes a series of minor groove contacts in the newly synthesized duplex DNA. Additionally both major and minor groove lesions block the progression of HIV-1 RT that were located 1-3 and 4-7 nucleotides, respectively beyond the site of synthesis [16,17]. These data suggest that bulky DNA lesions can probe the interactions of the polymerase with newly synthesized DNA. However, the location of the previous adducts was limited to either residing in the major or minor groove and have not considered intercalation as an additional probe of the HIV-1 RT-DNA interaction. In order to investigate the replicative consequences of an intercalating DNA lesion, C 10 S-or C 10 R-BPDE adducts were positioned in the primer strand in the vicinity of the 45° DNA bend observed in the HIV-1 RT/DNA crystal structure [1]. The adducted and unadducted primers and full-length products exhibit differential electrophoretic mobilities due to the presence or absence of the BPDE adduct ( Figure 4). Comparative analyses of unadducted primer extensions (27t/ 11p) with the BPDE-adducted (27t/11p) revealed that HIV-1 RT was able to readily extend the unadducted (U) and C 10 S-BPDE (S) adducted 11-mer primers to form longer and fulllength products (Figure 4, RT, U and S, respectively). However in contrast, primer extension was severely restricted when HIV-1 RT utilized the C 10 R-BPDE (R) adducted 11-mer primer ( Figure 4, RT, R) with only a modest amount of single nucleotide extension and no replication detected beyond four incorporated nucleotides. These data demonstrate clear differences in the ability of HIV-1 RT to form productive polymerase complexes based on the BPDE stereochemistry within the primer strand. Additionally, these data are qualitatively similar to results obtained for HIV1-RT replication with templates containing N 2 G styrene oxide lesions [16]. In order to determine if this inhibition of extension using the C 10 R-BPDE-containing primer was unique, other DNA polymerases were also examined in order to compare their replication patterns relative to HIV-1 RT. In contrast to the data generated for HIV-1 RT, pol β ( Figure 4, pol β), Sequenase (Figure 4, Seq), and KF exo - (Figure 4, KF exo -) catalyzed efficient primer extensions on both control and adductcontaining primers. Specifically, for these other polymerases, there were no significant differences in the utilization of primers containing R or S adduct stereochemistries. These data therefore demonstrate that ongoing DNA synthesis by HIV-1 RT is strongly and specifically inhibited when the primer strand contains the C 10 R-BPDE intercalated major groove adduct 5 nucleotides upstream of the polymerase active site. Primer extension analyses with unadducted (73t/11p) and C 10 R or C 10 S-adducted 11-mer templates (73t * /11p) In order to determine if the same DNA lesions located in the complementary strand would similarly inhibit HIV-1 RT primer extension, long oligodeoxynucleotides (73-mers) were constructed such that the adducted nucleotides were centrally located, with the primer strand annealed opposite the base modification. As shown in Figure 5, on the control primer extension reaction using the unadducted (U) T-P duplex, HIV-1 RT catalyzed efficient synthesis, resulting in a majority of DNA products being full-length, with a very minor termination site observed following incorporation of 11 additional nucleotides. In contrast, unadducted primers bound to templates containing C 10 R-and C 10 S-BPDE adducts were very poorly extended and the minor population of extended primers were terminated with a pattern similar to that observed for the unadducted template ( Figure 5, lanes S and R). These data suggest that when either the C 10 R-or C 10 S-BPDE adducts was positioned within the portion of the newly synthesized DNA that interacts with α-helix H in RT, these templates cannot support replication. Binding affinity of HIV-1 RT on primer-template complexes containing BPDE adducts To determine whether the adducted 11-mer primer ( Figure 4) influenced the apparent DNA binding affinity of HIV-1 RT, a competition binding/dissociation and polymerization assay was used to estimate the HIV-1 RT dissociation rate constant from unadducted and adducted primers ( Figure 6). The dissociation rate constant is a sensitive monitor of overall DNA binding affinity, since the association rate constant, k on , is often diffusion-controlled [26]. HIV-1 RT was preincubated with an excess unlabeled unadducted T-P, containing either control unadducted or the C 10 R-or C 10 S-adducted t-p (27t/11p or 27t/11p * ) in the absence of dNTPs to form a polymerase/DNA complex. To measure relative rates of dissociation, a different 32 P-labelled T-P was added concomitantly with dTTP, dCTP, and dGTP. The experimental design severely restricted nucleotide incorporation on the original unadducted or adducted unlabeled complex, since the first two templating nucleotides were dT. Enzyme dissociations from each of the original complexes were monitored by elongation of the competitor DNA. Data shown in Figure 6 reveal a very similar rate of competitor primer extension, suggesting comparable dissociation/reassociation rates for the adducted and unadducted 11-mer primers. Discussion Insights into the molecular interactions between two macromolecules such as polymerases and DNA can be obtained either by altering specific resides in the enzyme by site-directed mutagenesis or by modifying the T-P complex by covalently attaching stereospecific adducts on either the template or primer strands. Deoxyadenosine adducted at N 6 with BPDE has been employed previously in single-stranded templates to examine translesion bypass synthesis with a variety of DNA polymerases [27,28,29]. In many instances, DNA polymerases terminate synthesis one nucleotide prior to, opposite, or beyond the lesion. The exact site of termination was dependent on the identity of the polymerase, the absolute configuration at C 10 (R versus S), and the local sequence context. As part of these previous studies, it was shown that HIV-1 RT generally terminated DNA synthesis one nucleotide prior to both the (-)-anti-trans-C 10 R and (+)-anti-trans-C 10 S BPDE adducts when DNA synthesis was limited to one polymerase encounter per T-P binding event. Under conditions that allowed multiple polymerase encounters, bypass DNA synthesis was observed with (+)-anti-trans-C 10 S BPDE, but termination occurs one nucleotide 5΄ to the (-)-anti-trans-C 10 R adduct. Blockage of DNA synthesis by the C 10 R adduct could not be overcome by annealing a primer in which the 3΄terminus was one nucleotide beyond the lesion [27]. These data demonstrate that under a variety of T-P conditions, the (-)anti-trans-C 10 R BPDE adduct was significantly more disruptive to polymerization than the corresponding (+)-anti-trans-C 10 S BPDE adduct. Stereochemical, structural and thermodynamic analyses of these two adducts in the same CAA sequence context provided significant insights into understanding the origins of these differences [19]. This study extended prior NMR analyses of duplex DNAs containing these lesions [18,20,21], in which molecular dynamics simulations revealed that the C 10 R-containing DNA was more stable by ~13 kcal/mol compared to the C 10 S-containing DNA. This destabilization is at least partially driven by syn-anti conformational heterogeneity around the glycosyl bond, resulting in diminished base stacking, helix unwinding and poor Watson-Crick base pairing. In the current study, to probe polymerase DNA-interactions in the duplex region of the T-P, two stereoisomers of BPDE were positioned 5 nucleotides upstream of the 3΄-OH of the primer strand or at a comparable position in the complementary template strand (Figure 3). Using these substrates, pol β, Sequenase, and KF exowere able to efficiently extend both the BPDE-adducted primers and unadducted primers that were annealed to a template containing the lesion. The crystal structures of these polymerases bound with DNA indicate that non-specific interactions occur with the DNA sugar-phosphate backbone and the minor groove [1,3,4,30]. These structures, as well as the high resolution structures of a pol I family polymerase bound to DNA [31], indicate that minor groove interactions can occur up to 5 base-pairs from the polymerase active site, and that the sugar-phosphate backbone interactions extend even further. The lack of a significant perturbation of primer extension by pol β, Sequenase, or KF exofor the C 10 Ror C 10 S-BPDE primers in which the adducted site was positioned 5 nucleotides into the duplex; suggest that this lesion does not significantly affect these polymerase sugarphosphate backbone or minor groove interactions with these polymerases. Since a dramatic bend in the DNA duplex 4-5 nucleotides upstream of the polymerase active site is only observed in crystallographic structures of HIV-1 RT (1,2), this bend must alter local dynamic properties of the surrounding nucleotides that are sensitive to DNA lesions and modulate events at the polymerase active site. Our prior investigations corroborate these conclusions such that minor groove DNA adducts severely block ongoing replication as the lesion passes through this helix-induced bend [16]. Similarly, adducts in the major groove that compress the width of the minor groove also restrict HIV-1 RT transit through this bend. These inhibitory effects are not seen with other DNA polymerases that do not induce this bend. In contrast, the present study focused on the influence of adducts placed either in the template or primer strands on HIV-1 RT catalyzed DNA polymerization. In contrast to the other polymerases examined, 11-mer primers containing the C 10 R-BPDE adduct or templates containing either the C 10 R-or C 10 S-BPDE adduct were strongly inhibitory to primer extension. Importantly, nucleic acid binding, processivity, and frameshift fidelity are influenced by hydrophobic and hydrogen bonding interactions within the DNA minor groove and a group of amino acid residues in the thumb subdomain, referred to as the MGBT that are highly conserved among lentiviral reverse transcriptases [12,14,15]. Similar to that observed with the other DNA polymerases examined in this investigation, HIV-1 RT readily extended the C 10 S-BPDE adducted 11-mer primer ( Figure 4). In contrast, HIV-1 RT Inhibition by PAHs PLOS ONE \| www.plosone.org under the same conditions, the C 10 R-BPDE adducted 11-mer primer was primarily extended by only a single nucleotide. As described above, these data are highly consistent with the findings by Yan et al [19],, who demonstrated major differences in the overall structures and stabilities of DNAs containing these adducts. The inability to extend the C 10 R-adducted primer was shown to be due to a diminished rate of nucleotide incorporation and not due to an increased dissociation rate constant for the T-P complex. The unadducted and adducted primers or templates had similar DNA binding affinities as judged by their apparent dissociation rate constants ( Figure 6). Since the adduct can be perturbing in either the primer or template strands when it is positioned 5 base pairs from the polymerase active site, DNA lesions adducted in the major groove must be able to structurally alter the minor groove that is monitored by the MGBT of HIV-1 RT. The MGBT interacts primarily with the primer strand in the minor groove two to six nucleotides upstream of the polymerase active site and alterations of these interactions decrease DNA binding and polymerase processivity [12]. We have previously noted that DNA lesions adducted in the major groove influence RT termination in the vicinity of the MGBT [17]. The solution structure of the C 10 R N 6 -deoxyadenosine-BPDE adduct in duplex DNA indicates that the pyrenyl moiety is intercalated in the helix from the major groove on the 3΄ side of the modified adenine [18,20,21]. This results in distortion of the adjacent base pairs altering the position of the purine and pyrimidine hydrogen bond acceptors in the DNA minor groove (N3 and O2, respectively). In contrast to the premature termination observed with the C 10 R-adduct, the C 10 S-adduct positioned in the primer strand did not perturb important polymerase-DNA interactions. The low T m of a duplex C 10 S N 6 -deoxyadenosine-BPDE opposite a complementary thymine base has precluded the determination of the solution structure of this stereoisomer [18,32]. However, the solution structure of the C 10 Sdeoxyadenosine adduct when positioned opposite a deoxyguanosine, indicates that the duplex exists in at least two conformers. The torsion angle of the glycosidic bond of the modified base adopts a syn-configuration in the major conformer [32] and an anti-configuration in the minor conformer [33]. The adduct was inserted into the helix on the 3΄-side of the modified adenine with both conformers [32,33]. The lack of an effect on HIV-1 RT termination suggests that the C 10 S-BPDE adduct does not significantly impact the DNA minor groove when positioned opposite thymidine. Furthermore, as shown in Figure 5, although synthesis from primers bound to both unadducted and adducted 73-mers showed minor pause sites, the unadducted template could be extended to full-length products. In contrast, only very modest replication could be initiated off of both of the BPDE-adducted templates. Importantly, this indicates that the major groove adducted templating adenine signifiicantly alters DNA minor groove interactions with RT in the vicinity of the 40-45° bend in the duplex DNA upstream of the primer terminus and highlights the strand specific nature of the influence of the C 10 S-BPDE adduct on RT replication.	v3-fos-license
2016-04-27T00:50:03.749Z	2014-10-15T00:00:00.000	16447389	{ "extfieldsofstudy": [ "Biology", "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0109824&type=printable", "pdf_hash": "e370e9a2130f9cdaae99d29ba8e458205660b754", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:275", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "e370e9a2130f9cdaae99d29ba8e458205660b754", "year": 2014 }	pes2o/s2orc	Protein Interactions of the Vesicular Glutamate Transporter VGLUT1 Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling. Introduction The high frequency of neurotransmitter release observed at many synapses requires mechanisms to recycle synaptic vesicle membrane, proteins, and transmitter locally at the nerve terminal. Several mechanisms have been proposed to underlie the efficient recycling of synaptic vesicle components: classical clathrinmediated endocytosis, budding from an endosomal intermediate, and rapid endocytosis after full fusion or kiss-and-run exocytosis [1,2,3,4]. Reformation of synaptic vesicles from the plasma membrane by classical clathrin-mediated endocytosis is very similar to endocytosis occurring in non-neural cells. It requires the recruitment of a clathrin coat by adaptor proteins (APs), the acquisition of curvature mediated by endophilin, epsin and other cytosolic proteins, scission of the nascent vesicle from the plasma membrane orchestrated by dynamin, followed by uncoating triggered by the phosphatidylinositol phosphatase synaptojanin [1,5,6]. Dynamin and syndapin are among the ''dephosphin'' proteins that are regulated by a cycle of calcium-dependent dephosphorylation and phosphorylation mediated by cdk5 and GSK-3 kinases [7,8,9]. Thus, synaptic vesicle recycling is driven by a sequence of protein interactions and enzymatic activities [10,11]. Models of the proposed mechanisms for synaptic vesicle recycling have assumed that the protein components of vesicles recycle together. Protein-protein interactions or retention of proteins in the cholesterol-rich synaptic vesicle membrane could cluster synaptic vesicle proteins upon exocytosis [12,13,14,15]. But synaptic vesicle proteins differ in their diffusion into the plasma membrane from the site of exocytosis. While synaptotagmin, synaptophysin and VGLUT1 maintain a synaptic localization after exocytosis [16,17,18,19], the v-SNARE VAMP2 rapidly diffuses away from the synapse [16,19]. VAMP2 and synaptotagmin may also exchange with a large cell surface reservoir of these proteins [16,20,21,22]. Despite differences in diffusion, some vesicle proteins appear to undergo endocytosis at the same rate [19]. In the case of VGLUT1, however, the rate of endocytosis depends on the intensity of the exocytotic stimulus and the endocytic pathway to which it is recruited, as directed by sorting signals in its protein sequence [18]. Although it is possible that synaptic vesicles retain their identity after exocytosis simply through the clustering of their components on the plasma membrane, the demonstration that synaptic vesicle proteins contain distinct sorting signals and are targeted to different endocytic pathways suggests that specific sorting of individual proteins to synaptic vesicles could be independently regulated [23,24,25,26]. Three distinct vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3) underlie the packaging of glutamate into synaptic vesicles [27][28][29][30][31][32][33][34][35][36][37][38]. VGLUT1 and 2, which are responsible for the majority of glutamatergic neurotransmission, exhibit similar transport activity in vitro, but are largely expressed in different cell populations [39]. Expression of VGLUT1 or 2 isoforms confers differences in membrane trafficking, which may underlie differences in glutamate release properties [25,30,40]. VGLUTs exhibit a high level of sequence homology in the transmembrane segments that mediate glutamate transport, but diverge considerably at their cytoplasmic termini. The C-terminal domain of VGLUT1 contains several consensus sequences for protein interaction and modification that suggest these regions play a primary role in differences in membrane trafficking between the isoforms. We previously found that VGLUT1 contains multiple dileucine-like trafficking motifs that direct trafficking by distinct pathways that use different clathrin adaptor proteins [18,25,26]. Further, interaction of a VGLUT1 polyproline domain with the Src homology 3 (SH3) domain-containing endocytic protein endophilin targets the transporter to a faster recycling pathway during prolonged stimulation. In addition to dileucine-like and polyproline motifs, VGLUT1 contains potential ubiquitination and phosphorylation sites, suggesting that posttranslational modifications may be involved in targeting and recycling of the transporter. In this work, we use VGLUT1 as a model synaptic vesicle protein to identify cis-acting sorting signals in the amino acid sequence and trans-acting factors that may direct protein sorting to specialized cellular membrane trafficking pathways involved in synaptic vesicle recycling. Reagents Cell culture reagents were from Life Technologies unless otherwise noted. All other chemicals were from Sigma-Aldrich. Antibodies, suppliers and dilutions used are listed in Table 1. Molecular biology, cell culture and transfection Overlap extension PCR mutagenesis and site-directed PCR mutagenesis (QuikChange, Agilent Technologies) were used to introduce epitope tags and mutations, which were verified by sequencing. For expression of bacterial glutathione S-transferase (GST) fusion proteins, cDNA fragments were inserted in frame into the multiple cloning site of the pGEX-5x vector (GE Healthcare). For expression of His-tagged fusion protein (His-PP1), cDNA fragments encoding amino acids 513-549 were inserted in frame into the multiple cloning site of the Ligand Expression Vector (Panomics/Affymetrix). GST fusions of the SH3 domains of human Lyn, Fyn and Src in pGEX vectors along with full-length mouse Lyn were obtained from Clifford Lowell (UCSF). Purified GST-Lyn-SH3 protein was purchased from Panomics. Myc-tagged Lyn was generated by amplifying fulllength mouse Lyn with primer encoding a myc tag followed by a four alanine linker immediately before the kinase. The resulting myc-Lyn was subcloned into the pcDNA1/Amp vector (Invitrogen) using standard molecular biological techniques. 3x-FLAG-tagged ubiquitin was obtained from Jeffrey Benovic (Thomas Jefferson). COS7 cells were obtained from UCSF Cell Culture Facility, grown in DME H-21 medium supplemented with 10% cosmic calf serum (Hyclone) and 1X pen/strep at 37uC in 5% CO 2 . Transient transfection by electroporation was performed as described [41]. Rat cortical neurons were isolated from embryonic day 18-20 Sprague Dawley rats (Charles River) of either sex. Prior to harvesting the embryos, pregnant female was placed in a 10.5 L acrylic chamber and euthanized with CO 2 asphyxiation at a flow rate of 1.05-3.15 L/min followed by bilateral thoracotomy. Embryos were quickly decapitated with sharp scissors and brains were removed from the skull in ice cold HBSS. Cortex was dissected in Hibernate E (BrainBits) followed by digestion with trypsin for 5-10 min at 37uC. Dissociated neurons were transfected using a SCN Nucleofector kit (Lonza), according to manufacturer's directions [18]. SH3 and WW domain arrays Purified His-PP1 was incubated with TranSignal WW (Cat #MA3030, MA3032) and TranSignal SH3 Domain Arrays (Cat. #MA3010, MA3011, MA3012, and MA3014) (Panomics/Affymetrix, now available from Gentaur, Belgium), and detected with anti-His antibody according to manufacturer's instructions [42]. The arrays were made by the manufacturer using the recombinant conserved binding sites of individual WW or SH3 domain proteins fused to GST. GST fusions are purified and immobilized onto a membrane. Each domain on the array is spotted in duplicate at 100 ng. WW domain arrays include 67 different human WW domains, whereas SH3 domain arrays include over 130 different domains. GST pull-down assays GST pull-downs were performed essentially as described [18]. 10 mg GST fusion proteins bound to 10 ml beads were rotated with 250 ml brain or COS7 cell lysates (,3 mg/ml) at room temperature (RT) for 60 min. Pelleted beads were washed with 1 ml lysis buffer and repelleted four times. Bound proteins were eluted by incubating with 10 ml reduced glutathione for 5 min at RT, then with 10 ml sample buffer for 5 min at RT. Eluted protein was subjected to SDS-PAGE and stained with Coomassie or silver, or subjected to immunoblotting. To prepare cell extracts, COS7 cells were washed, mechanically harvested and lysed in 10 mM Hepes-KOH, pH 7.4 and 150 mM NaCl containing protease inhibitors (PIs, 10 mg/ml phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, 2 mg/ml pepstatin A, 2 mg/ml E-64, 2 mg/ml aprotinin), and 2% Triton X-100 (TX-100) for 45 min on ice and the lysate cleared by centrifugation at 13,0006g for 15 min at 4uC. To prepare brain extracts used in the experiments of Figs. 3 and 4, rat brains were homogenized directly in 8 volumes 10 mM Hepes buffer, pH 7.4 with 0.32 M sucrose, pellet at 13,0006g, resuspended, solubilized in 8 volumes sucrose Hepes buffer with 2% TX-100, and pelleted at 50,0006g to remove cell debris. The concentration of soluble protein was assayed (Bradford reagent, BioRad) and equal amounts of protein incubated with GST fusions. Extracts from rat brain used in experiments shown in Fig. 6 were solubilized in 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, and 1% TX-100 containing PIs (1 mg/ml VGLUT1 Protein Interactions PLOS ONE \| www.plosone.org E64, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 2 mg/ml pepstatin, and 20 mg/ml PMSF), and sedimented at 20,0006g for 45 min at 4uC. The supernatant (,400 mg total protein) was incubated with 400 mg of GST fusion proteins immobilized on glutathione sepharose beads at 4uC for 2 h. After pelleting, beads were washed and bound protein was detected by immunoblot analysis with the appropriate antibodies (see below). Immunoprecipitation Cell and brain extracts were prepared as described above. For crosslinking experiments, cells were pretreated with 1 mM dithiobis (succinimidyl propionate) (DSP) for 2 h at 4uC, and quenched with 25 mM Tris [43]. Equal amounts of protein were incubated with anti-HA antibody for 1 h to overnight at 4uC, followed by incubation with Protein G sepharose beads (Roche) for 1 h. After washing 4 times with 10 volumes of lysis buffer, proteins were eluted by boiling in SDS-PAGE sample buffer, and subjected to immunoblotting. SDS-PAGE and immunoblotting Samples containing 20-50 mg of protein were mixed with Laemmli sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membrane. Membranes were blocked and immunoblotted with antibody in PBS containing 0.1% Tween and 5% nonfat dry milk, washed 3 times for 10 min, hybridized with appropriate horseradish peroxidase-coupled secondary antibodies (GE), followed by further washing, 3 times for 10 min. Detection of hybridization was performed by enhanced chemiluminescence (ECL, Thermo Scientific) and exposure of the membrane to X-ray film. Quantification of band intensities was performed using the lowest exposure that allowed detection of immunoreactive bands. ImageJ was used to determine the intensity of bands using the intensity of the respective fusion protein loaded on the same lane (revealed by Ponceau staining) to normalize the signal. Immunoblots shown are representative of at least three independent experiments. To determine statistical significance, two-tailed t-test or one-way ANOVA followed by Bonferroni's test was performed at p,0.05 as appropriate (GraphPad Prism). Quantification data are means 6 SEM of at least three independent experiments. P i metabolic labeling For metabolic labeling with 32 P i [44,45], cells were washed three times in medium lacking phosphate and then incubated for 2 h at 37uC in the presence of 0.5-1.0 mCi/ml 32 P i (Perkin Elmer). After labeling, cells were washed on ice with ice-cold HBSS containing PIs and phosphatase inhibitors (50 mM NaF, and harvested by scraping into the same buffer; pelleted by centrifugation at 50006g for 5 min at 4uC; and then resuspended by trituration in 1 ml of buffer with 2% TX-100 (homogenization buffer). After removal of the cell debris and nuclei by centrifugation at 14,0006g for 5 min at 4uC, SDS was added to the supernatant to a final concentration of 0.2%. For immunoprecipitation, the mixture was incubated overnight at 4uC with protein G sepharose prebound to monoclonal antibody to HA (Roche). Immune complexes were washed four times in homogenization buffer and resuspended in 2x sample buffer and the proteins separated by SDS-PAGE. Gels were fixed, dried and subjected to autoradiography. Ethics Statement All animal studies were conducted in accordance with the policies and approval from the Institutional Animal Care and Use Committee for the University of California, San Francisco (Institutional PHS Assurance #A3400-01; USDA Customer #9199, Registration #93-R-0440; AAALAC Accreditation #001084). VGLUT C-terminal sequence domains VGLUT1 and 2 exhibit a high degree of sequence homology, but diverge at their cytoplasmic termini, suggesting that these regions may mediate differences in trafficking between the two isoforms [18,25,30]. The C-termini of VGLUT1 and VGLUT2 both contain a potential dileucine-like internalization motif ( Fig. 1A underlined) consisting of two hydrophobic amino acids with acidic residues at -4 or -5 upstream, which are thought to mediate trafficking via clathrin adaptor proteins [46,47,48]. VGLUT1 and 2 also both contain two lysine residues on either side of a sequence rich in proline, glutamic acid, serine and threonine residues (PEST) (Fig. 1A, +). A web-based prediction program (PESTfind) identifies a second PEST domain in VGLUT1 (Fig. 1A). PEST domains can direct ubiquitination or calpain cleavage. VGLUT2 has been shown to undergo calpain cleavage under excitotoxic conditions [49]. The C-terminus of VGLUT1 also contains two polyproline domains not present in VGLUT2 (PP1 and PP2, Fig. 1A, bold). PP1 and PP2 each contain three sequences which fit the consensus for SH3 protein interaction domains (PXXP) [50]. PP1 also contains a consensus for a WW protein interaction domain (PPXY; Fig. 1B) [51]. We have previously shown that interaction of PP2 with endophilins accelerates VGLUT1 recycling, in a manner dependent on the dileucine-like trafficking motif also present in the C-terminus [18]. The proximal C-terminus of VGLUT1 also contains an acidic region with potential phosphorylation sites (SDESEMEDEVE, Fig. 1A, italics) that fits the consensus for casein kinase 2 (CK2) phosphorylation of serines 519 and 522, as identified by NetPhosK (CK2 consensus sequence S/T-D/E-X-D/E/pS). The serine residue immediately upstream of the VGLUT1 acidic dileucinelike motif (S504) is identified by NetPhosK as a potential substrate for CK1 and CK2. Although the sequence around S504 (EPEEMSEE) does not fit the canonical consensus sequence for CK1 or 2 (CK1 consensus sequence S/T(p)-X 2-3 -S/T), noncanonical substrates include sequences containing many negatively charged amino acids [52,53]. In addition, the sequence SYGAT is identical in all three VGLUT isoforms, and S540 is a predicted GSK-3 substrate, fitting the consensus sequence S/T-X-X-X-S/ T(p). The presence of these motifs suggests that the VGLUT1 Cterminus could organize protein interactions to drive trafficking. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of biochemical screening assays using the amino acid residues 513-549 of the rat VGLUT1 sequence. This region encompasses the first polyproline motif, the cluster of acidic amino acids containing consensus phosphorylation sites, and the PEST domains. The first polyproline domain contains consensus sequences for SH3 and WW domain interactions (Fig. 1B). Mutation of individual proline residues to alanine were used to selectively disrupt the consensus sequences of each of the three SH3 domain-binding motifs and the WW domain-binding motif independently (Fig. 1B, asterisks). Mutation P534A + P535A disrupts all three SH3 domain-binding motifs. Protein interaction arrays Our yeast two-hybrid screen using the entire VGLUT1 Cterminus had previously identified the SH3 domain-containing endophilins as interactors at the second PP domain, but did not identify any other interacting proteins [18]. To identify proteins Figure 1. Comparison of the C-termini of rat VGLUT1 and 2. (A) VGLUT1 and VGLUT2 both contain an acidic dileucine-like internalization motif (underlined) and two lysine residues on either side of a potential PEST ubiquitination domain (+). VGLUT1 contains two PP domains (bold) not found in VGLUT2. VGLUT1, but not VGLUT2, also contains a region of acidic amino acids with a CK2 phosphorylation consensus sequence, S/T-D/E-X-D/E/pS, (italics) containing two serine residues (shaded). In addition, the VGLUT1 acidic domain and PP1 together fit the consensus for a second PEST domain (+). (B) VGLUT1 PP1 contains three sequences that fit the consensus for SH3 protein interaction domains (PXXP) and one for a WW protein interaction domain (PPXY). Starred proline residues are mutated singly to alanine (P531A, P537A, P535A, P539A) to individually disrupt SH3 1, 2, or 3 (PXXP), or WW (PPXY) binding. The mutation P534A + P535A disrupts all three SH3 binding domains (see Fig. 4B interacting with VGLUT1 PP1, SH3 and WW domain arrays (Panomics/Affymetrix) were screened using a His-tagged VGLUT1 fusion protein encompassing amino acids 513-549. The arrays cover the majority of identified SH3 and WW domains found in the human genome. Membranes spotted in duplicate with GST fusions of SH3 and WW domains from more than 150 proteins were incubated with bacterial extract containing the tagged protein and washed extensively. Bound protein was detected using antibody to the His tag (Fig. 2). Several proteins that bound His-VGLUT1 PP1 fell into three general categoriestyrosine kinases, cytoskeletal adaptors, and ubiquitin ligases. The SH3 domain-containing proteins identified include multiple Src family tyrosine kinases (Fyn, Lyn, Src, Hck); and scaffolding/ adaptor proteins (e.g. Nck1, Nck2, intersectin, ArgBP2, sorting nexin 9), and endophilin ( Fig. 2A). WW domain-containing proteins identified in the screen include several E3 ubiquitin ligases (e.g. Nedd4, AIP4/Itch, WWP1, WWP2; Fig. 2B). Proteins expressed at low levels in brain and those with an established function unrelated to trafficking or neurotransmitter transport were excluded from further analysis. Biochemical analysis of SH3 domain-containing proteins To test for in vitro interaction of proteins identified in the SH3 array screen, we performed GST pull-down assays with candidate proteins that were detected above background in the array screen, and fit the criteria of a) at least modest brain expression and b) a subcellular localization or function consistent with interaction with VGLUT1. Detergent-solubilized rat brain extracts were incubated with GST fusions of SH3 domains (Panomics/Affymetrix) bound to glutathione sepharose beads. Proteins bound to the beads after washing were detected by immunoblotting with an antibody to VGLUT1 (Chemicon). Using this assay, we detect binding of VGLUT1 to distinct domains of the actin cytoskeletal adaptor Nck isoforms 1 and 2 (Fig. 3A). The three SH3 domains of the two isoforms of Nck (D1-3) were screened independently. Interaction with VGLUT1 is strongest in this assay for the second SH3 domain of Nck1 (Fig. 3A, Nck1 D2). We also detect interaction of VGLUT1 with the SH3 domain of Lyn, a protein tyrosine kinase (Fig. 3B). No binding of VGLUT1 to other proteins identified in the initial screen, EPS8, spectrin, ArgBP2 or SNX9 is detected by this method (Fig. 3B). To determine the site of interaction in VGLUT1, we used GST fusions of either the entire C-terminal cytoplasmic tail of VGLUT1 (VG1) and VGLUT1 lacking both PP domains (DPP1&2), the first PP (DPP1) or second (DPP2), to pull down candidate interactors. Detergent-solubilized rat brain extracts were incubated with GST fusions bound to glutathione sepharose, washed and detected by immunoblotting with antibodies to the SH3 domain-containing proteins Nck (BD Biosciences) and ponsin/c-Cbl interacting protein CAP (Upstate). Ponsin is a cytoskeletal adaptor of the sorbin homology (SoHo) family, which was reported to interact with VGLUT1 in a yeast two-hybrid screen [54]. Nck and ponsin from brain extracts bind to the GST fusion of the entire C-terminus of VGLUT1 or DPP1, but not to DPP1&2 or DPP2 (Fig. 3C,D). Similarly, the tyrosine kinase Lyn, from extracts of COS7 cells transfected with myc-tagged Lyn, binds preferentially to entire C-terminus of VGLUT1 and DPP1, but not to DPP1&2 or DPP2 (Fig. 3E). This suggests specific binding between PP2 and an SH3 domain of Nck, ponsin, and Lyn. To test the specificity of VGLUT1 binding to tyrosine kinases, we performed GST pull-downs using fusions of kinases that were detected in the array blots, and expressed in brain. VGLUT1 from brain extracts binds more strongly to GST-Lyn (0.876760.0644 a.u.) than GST-Fyn (0.362260.1034 a.u.) in vitro (Fig. 3F). No specific binding to GST-Src was detected (Fig. 3F). No binding of VGLUT1 to intersectin, CIN85, HIP55, osteoclast simulating factor (OSF) or vinexin was detected by GST pulldown assays followed by immunoblotting with specific antibodies (data not shown). Biochemical analysis of WW domain containing proteins GST fusions of the WW domains of several proteins identified on the arrays did not bind VGLUT1 from brain extracts (Fig. 4A). However, GST-VGLUT1 specifically pulls down the HECT domain E3 ubiquitin ligase Atrophin Interacting Protein AIP4/ Itch from brain extracts (Fig. 4B). This interaction is greatly decreased by deletion of PP1 (DPP1 and DPP1&2). Moreover, AIP4/Itch appears to specifically interact with the WW domain consensus binding site, PPXY, at the end of PP1. Disruption of the WW domain-binding consensus sequence (PPXY) by either mutating the proline residue at 538 and a tyrosine residue at 541 to alanine (DWW/PAXA) or the proline 538 residue alone (DWW/PAXY) reduces binding. In contrast, a GST fusion of VGLUT1 which disrupts all three SH3 consensus sequences by mutating proline residues 534 and 535 to alanine (DSH3/1,2,3), is able to bind AIP4/Itch in vitro (Fig. 4B). Since the closely related E3 Nedd4 is highly expressed in brain we also tested whether this protein interacts with VGLUT1. Indeed, Nedd4 is also pulled down by GST-VGLUT1 and GST-VGLUT1DPP2, but not GST, GST-VGLUT1DPP1 and GST-VGLUT1DPP1&2 (Fig. 4B). Two other HECT domain E3 ligases, WWP1 and 2, were not pulled down by GST-VGLUT1 (Fig. 4B). Taken together, this suggests both AIP4/Itch and Nedd4 specifically bind at the WW domainbinding consensus sequence in the first polyproline domain in the VGLUT1 C-terminus. To determine whether VGLUT1 interacts with AIP4/Itch in cells, we co-immunoprecipitated AIP4/Itch and HA-VGLUT1. Cultured rat cortical neurons were transfected with HA-VGLUT1 and AIP4/Itch and incubated with the cross-linking agent dithiobis(succinimidyl proprionate) (DSP) [43]. Detergent extracts were immunoprecipitated with HA or IgG control antibody, and immunoblotted with antibody to AIP4/Itch. AIP4/Itch was specifically co-immunoprecipitated with antibody to HA, but not control IgG (Fig. 4C). Therefore, the interaction of AIP4/Itch and VGLUT1 occurs in cells. To determine whether VGLUT1 is ubiquitinated in neurons, we transfected rat cortical neurons with HA-VGLUT1, AIP4/Itch, and 3x-FLAG-tagged ubiquitin and immunoprecipitated with HA antibody or control IgG. Immunoprecipitates were probed with FLAG antibody to detect ubiquitination. Two bands of approximately 58 and 74 kD were recognized by antibody to FLAG when immunoprecipitation was carried out with antibody to HA, but not IgG (Fig. 4D). Thus, HA-VGLUT1 is ubiquitinated under these conditions. Phosphorylation of VGLUT1 The C-terminus of VGLUT1 contains a cluster of acidic amino acids that includes a consensus sequence for serine phosphorylation (NetPhos 2.0) (Fig. 1). Like the PP domains, this motif is conserved in mammalian VGLUT1 homologs, but not in VGLUT2 or 3. This sequence is similar to acidic motifs found in several membrane proteins, including the vesicular monoamine transporter, VMAT2, the epithelial sodium transporter (ENaC), the endoprotease furin, vesicle associated membrane protein 4 (VAMP4), transient receptor potential polycystin-2 channel (TRPP2), and aquaporin 4 (AQ4). Trafficking of some of these proteins is influenced by CK2-mediated serine phosphorylation [44], [55][56][57][58][59][60]. In the case of aquaporin 4, CK2 phosphorylation regulates its sequential binding to AP-2 to mediate endocytosis, and then to AP-3 to mediate post-endosomal trafficking [57]. Additional phosphorylation motifs may be present in VGLUT1. Indeed, we have recently demonstrated that a negatively charged residue in the vesicular GABA transporter upstream of the dileucine-like motif can modulate trafficking [26]. The analogous residue in rat VGLUT1 (S504) also fits the consensus sequence for CK2 phosphorylation (NetPhos 2.0). In addition, the serine residue in the 540 SYGAT 544 sequence conserved in VGLUT1, -2, and -3 is also a potential phosphorylation site (NetPhos 2.0), although these were not tested here. To determine whether VGLUT1 is phosphorylated, we used 32 P i to metabolically label cultured rat cortical neurons transfected with HA-VGLUT1 at 37uC, followed by immunoprecipitation of VGLUT1 with immunoprecipitated with rat anti-HA antibodies. AIP4/Itch specifically co-immunoprecipitates with HA antibody. HA-VGLUT1 was detected with mouse anti-HA antibody. (D) Cultured rat cortical neurons transfected with HA-VGLUT1, 3x-FLAG-ubiquitin, and AIP4/Itch, were immunoprecipitated with rat anti-HA antibodies as in (C). Immunoprecipitates were probed with FLAG antibody to detect ubiquitination. Two bands of approximately 58 and 74 kD were specifically recognized by antibody to FLAG when immunoprecipitation was carried out with antibody to HA, but not IgG. Mouse anti-HA antibody was used to detect HA-VGLUT1. FT: flow through. doi:10.1371/journal.pone.0109824.g004 antibody to HA (Roche) in the presence of phosphatase inhibitors, and autoradiography [44,45]. A 32 P i labeled band approximately the predicted size of VGLUT1 is immunoprecipitated with antibody to HA, but not IgG (Fig. 5A). Substitution of serines 519 and 522 by alanine (SS/AA) within the acidic cluster decreases phosphorylation by ,60% (Fig. 5B). Alanine mutagenesis does not completely abrogate phosphorylation, consistent with possible additional phosphorylation sites in the VGLUT1 Cterminus. To gain more insight into possible downstream effects of VGLUT1 phosphorylation, we performed GST pull-down experiments utilizing VGLUT1 C-terminal mutants in which serines 519 and 522 were replaced with alanine (SS/AA) or aspartate (SS/ DD) to mimic the dephosphorylated and phosyphorylated states, respectively. GST fusions of wild type and mutant VGLUT1 Cterminus were bound to glutathione beads, incubated with rat brain homogenate, and analyzed by immunoblotting with antibodies to the proteins that interact at the polyproline domains. Binding to endophilins, Nedd4, AIP4/Itch, Nck, and ponsin was not affect by either of the serine mutations (Fig. 6A). We have recently shown that binding of the clathrin adaptor protein AP-2 at the dileucine-like motif is important for VGLUT1 recycling in neurons [25]. To determine whether phosphorylation could regulate interaction of the VGLUT1 C-terminus with AP-2, we investigated whether mimicking phosphorylation of serines 519 and 522 affects binding of AP-2 and VGLUT1. As expected, GST-VGLUT1 specifically pulls down AP-2 (Fig. 6B). Interestingly, mutation to alanine (SS/AA), which mimics a dephosphorylated state, reduces this interaction. Conversely, mimicking the phosphorylated state by substitution of aspartate for the same serines (SS/DD) increases this interaction (Fig. 6B). We also tested whether serine mutations affect binding to AP-3, which has a role in synaptic vesicle recycling under conditions that trigger activitydependent bulk endocytosis [18,61]. In contrast to AP-2, binding of AP-3 to VGLUT1 is not affected by mutation of serines 519 and 522 (Fig. 6C). Deletion of both polyproline domains (DPP1&2) prevents binding of the polyproline domain interacting proteins, but not AP-2, which binds at the upstream dileucine-like motif 504 SEEKCGFV 511 [25]. Thus, while binding of protein interactors at the polyproline domains is insensitive to phosphomimetic mutations of serines 519 and 522, binding of AP-2 is modulated by phosphomimetic mutations in VGLUT1. Discussion In this work, we investigated consensus sequences for protein interaction and post-translational modification contained in the cytoplasmic C-terminal tail of VGLUT1, paying particular attention to the domains that are conserved in mammals, but differentiate this transporter from the other VGLUT isoforms. Through a series of screening and binding assays we uncovered a remarkable network of interactors belonging to several classes of protein modulators of cellular function. The results show that VGLUT1 interacts in vitro with actin cytoskeletal adaptor proteins, a tyrosine kinase, and ubiquitin ligases. The results further show that VGLUT1 can undergo ubiquitination and phosphorylation. Moreover, phosphorylation may regulate protein interactions of VGLUT1. These findings can drive further investigation of how VGLUT1 interacts with specialized cell biological mechanisms to direct synaptic vesicle protein recycling. In protein arrays and GST pull-down assays, VGLUT1 PP2 interacts with an SH3 domain of Nck, an actin cytoskeletal adaptor containing one SH2 and three SH3 domains [62]. Through its SH3 domain, Nck can recruit proline-rich proteins to the plasma membrane or to multiprotein complexes found either in the cytoplasm or in association with the actin cytoskeleton. Nck activates actin polymerization [62,63]. Ponsin/CAP was also identified as a VGLUT1 interactor in this study, as well as in a previous yeast two-hybrid screen [54]. Ponsin contains a sorbin homology domain and three C-terminal SH3 domains. Ponsin, along with ArgBP2 and vinexin, belongs to the SoHo family of proteins that regulate actin-dependent processes [64,65]. Ponsin binds dynamin and promotes the formation of tubules decorated with actin [66]. The effects of actin disruption on synaptic vesicle recycling have been somewhat contradictory. However, there is evidence that actin is important in scaffolding of synaptic vesicles [67,68,69], their mobilization from synaptic vesicle pools [70][71][72][73][74][75], endocytosis after spontaneous release [76], ultrafast endocytosis milliseconds after exocytosis [3], and bulk endocytosis [77][78][79][80]. In addition, Nck could act as a scaffold to recruit other SH3 domaincontaining proteins. SH3 protein interacting with Nck, 90 kDa (SPIN90) is a Nck binding protein that also interacts with dynamin and syndapin, and regulates synaptic vesicle endocytosis [81]. Investigation of the functional consequences of VGLUT1 interaction with Nck or ponsin may help clarify the role of actin in synaptic vesicle recycling, or other aspects of VGLUT1 function. Here we also find that VGLUT1 PP2 specifically binds the tyrosine kinase Lyn. A role for Lyn in membrane protein trafficking remains unknown. The sequences around the two tyrosine residues in the VGLUT1 C-terminus are not identified as strong phosphorylation consensus motifs by a prediction program. It is possible that Lyn could exert an effect by phosphorylating other proteins involved in recycling. Tyrosine phosphorylation of synaptophysin and synapsin by Src may regulate some properties of synaptic strength [82,83]. Interestingly, Lyn has been shown to modulate dopamine release with effects on alcohol reward [84]. Notably, endophilin, Nck, ponsin, and Lyn all bind at PP2, an arginine-rich polyproline domain. It is possible that these proteins compete for binding with each other, perhaps modulated by the phosphorylation state of the transporter. Alternatively, different populations of the transporter may bind a different cohort of proteins. Further investigation will distinguish among these possibilities. Our screen did not uncover SH3 domain-containing proteins that bind to PP1. Instead, we discovered that VGLUT1 binds WW domain-containing ubiquitin ligases at a PPXY motif in PP1. Nedd4 and AIP4/Itch are HECT family E3 ubiquitin ligases each containing three or four WW domains, a Ca 2+ -dependent lipid binding C2 domain, and a HECT catalytic domain [85]. Nedd4mediated ubiquitination has been shown to regulate endocytosis of the sodium channel ENaC [86], and internalization and lysosomal trafficking of AMPARs and TrkA [87,88]. The closely related AIP4/Itch also interacts with PP1 in vitro. Deletion of AIP4/Itch in mice is associated with severe immune and inflammatory defects due to T cell receptor mistargeting [89]. However, the endosomally localized ubiquitin ligase AIP4/Itch is also highly expressed in neurons [90]. AIP4/Itch has been shown to interact with and ubiquitinate endophilin, which binds at PP2 of VGLUT1 [91]. Scaffolding of endophilin and ubiquitin ligase homologs signals endocytosis of several membrane proteins, including transporters, in mammals and yeast [92,93,94,95]. The C2 domain present in Nedd4 or AIP/Itch could serve to coordinate scaffolding at the membrane with changes in calcium levels. Two predicted PEST sequences in the cytoplasmic C-terminus of VGLUT1 could direct ubiquitination [49]. Immunoprecipitation experiments indicate that HA-VGLUT1 undergoes ubiquitination. Two sizes of ubiquitinated VGLUT1 bands could correspond to a mono-and a polyubiquitinated species. The conserved PEST sequence in VGLUT2 directs calpain cleavage of the transporter under excitotoxic conditions, but VGLUT1 is not cleaved by calpain [49]. The ubiquitination of VGLUT1 could suggest the potential for regulation of protein levels by degradation. Ubiquitination may also signal endocytosis of the transporter. These mechanisms could be similar to the post-endocytic sorting of receptors between recycling and degradative pathways [96]. Regulation of VGLUT1 degradation and trafficking has the potential to influence quantal size or the amount of transporter in different synaptic vesicle pools. In addition, phosphorylation of PEST sequences can influence ubiquitination and proteolysis [97][98][99][100][101]. In fact, we found evidence for phosphorylation of VGLUT1. Calcium-regulated cycles of protein dephosphorylation and rephosphorylation are important regulators of synaptic vesicle recycling and pool size at the presynaptic terminal [7,79,102,103,104]. Phosphorylation may also affect protein interactions [56,57,99,105,106,107]. To assess a potential role of phosphorylation on the interaction of VGLUT1 with other proteins, we used site-directed mutagenesis to replace identified residues with either alanine to mimic the unphosphorylated state of serines 519 and 522, or aspartate to mimic phosphorylation. We determined that these mutations affect the ability of GST-VGLUT1 to bind AP-2, but not AP-3. AP-2 is thought to be the main adaptor protein functioning at the plasma membrane to internalize synaptic vesicle protein cargoes. However, the alternate adaptors AP-1 and AP-3 have been shown to be involved in synaptic vesicle formation from endosome-like structures [61,108,109]. The difference in the modulation of AP-2 and AP-3 binding in vitro by serine mutation is consistent with distinct roles for the alternate adaptors for in VGLUT1 recycling. These serines are in a cluster of acidic amino acids in the C-terminus of VGLUT1 that, like the PP domains, is conserved in mammalian VGLUT1 homologs. This sequence is also similar to acidic motifs found in several related membrane proteins, including some whose trafficking are influenced by CK2-mediated serine phosphorylation [55][56][57][58][59][60]. The vesicular GABA transporter VGAT and the vesicular monoamine transporter VMAT2 are phosphorylated, but non-neuronal VMAT1 is not, suggesting phosphorylation as a specific regulatory mechanism for some vesicular transporters [44,110]. VGLUT1 contains unique domains that may reflect specialized mechanisms for regulation of its recycling, which could underlie the differences in physiological responses between neurons expressing VGLUT1 and the closely related VGLUT2. In addition to their important role in glutamate storage, VGLUTs serve as a model to understand how individual synaptic vesicle proteins recycle at the nerve terminal. In this work we investigated the VGLUT1 interactome. We identified several classes of interactors and post-translational modifications that suggest novel modes of regulation of synaptic vesicle protein recycling. Further studies will elucidate the physiological role of these modulators including the effects on neurotransmitter release. The data presented here provides a framework to understand how unique sorting sequences target individual synaptic vesicle proteins to pathways with different rates or destinations. Regulation of these mechanisms may in turn influence synaptic transmission.	v3-fos-license
2018-04-03T05:06:29.608Z	2016-09-09T00:00:00.000	11262843	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0161813&type=printable", "pdf_hash": "688d494959fddb45fa78acfb89a7e8824a45dbfd", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:312", "s2fieldsofstudy": [ "Biology" ], "sha1": "688d494959fddb45fa78acfb89a7e8824a45dbfd", "year": 2016 }	pes2o/s2orc	The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity Functional recovery from central neurotrauma, such as spinal cord injury, is limited by myelin-associated inhibitory proteins. The most prominent example, Nogo-A, imposes an inhibitory cue for nerve fibre growth via two independent domains: Nogo-A-Δ20 (residues 544–725 of the rat Nogo-A sequence) and Nogo-66 (residues 1026–1091). Inhibitory signalling from these domains causes a collapse of the neuronal growth cone via individual receptor complexes, centred around sphingosine 1-phosphate receptor 2 (S1PR2) for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) for Nogo-66. Whereas the helical conformation of Nogo-66 has been studied extensively, only little structural information is available for the Nogo-A-Δ20 region. We used nuclear magnetic resonance (NMR) spectroscopy to assess potential residual structural propensities of the intrinsically disordered Nogo-A-Δ20. Using triple resonance experiments, we were able to assign 94% of the non-proline backbone residues. While secondary structure analysis and relaxation measurements highlighted the intrinsically disordered character of Nogo-A-Δ20, three stretches comprising residues 561EAIQESL567, 639EAMNVALKALGT650, and 693SNYSEIAK700 form transient α-helical structures. Interestingly, 561EAIQESL567 is situated directly adjacent to one of the most conserved regions of Nogo-A-Δ20 that contains a binding motif for β1-integrin. Likewise, 639EAMNVALKALGT650 partially overlaps with the epitope recognized by 11C7, a Nogo-A-neutralizing antibody that promotes functional recovery from spinal cord injury. Diffusion measurements by pulse-field gradient NMR spectroscopy suggest concentration- and oxidation state-dependent dimerisation of Nogo-A-Δ20. Surprisingly, NMR and isothermal titration calorimetry (ITC) data could not validate previously shown binding of extracellular loops of S1PR2 to Nogo-A-Δ20. Introduction Neurons in the central nervous system (CNS) exhibit very limited capacity to regrow upon neurotrauma, preventing them from restoring disrupted networks after a spinal cord or brain injury. This is contrary to the situation in the peripheral nervous system (PNS), where regrowth of nerve fibres can occur to a much higher extent [1,2]. CNS-specific myelin-associated inhibitory molecules that actively prevent the outgrowth of neurons are an important factor accounting for this discrepancy [3]. One of the most prominent members of these inhibitory molecules is the 1163 residues (rat sequence) long membrane protein Nogo-A, also referred to as reticulon 4-A [4][5][6]. Nogo-A is expressed on the surface of oligodendrocytes where it exhibits an inhibitory signal for neurite growth [7,8]. Nogo-A acts as a stabilizer for the highly complex CNS wiring; it restricts synaptic plasticity and influences various intracellular processes such as shaping of the endoplasmic reticulum (ER), where particularly high Nogo-A levels are found [9][10][11]. Two domains of Nogo-A have been identified that impose inhibitory effects on neurite growth and cell migration: Nogo-A-Δ20 and Nogo-66 [7]. The Nogo-A-Δ20 domain, which contains 182 residues, is located in the middle of the 803 residues long Nogo-A-specific segment. In contrast, the 66 residues long Nogo-66 domain is situated between two long hydrophobic stretches at the C-terminus that Nogo-A shares with its much smaller isoforms Nogo-B and Nogo-C, as well as with other reticulon proteins. Neurons express distinct receptors for each of these inhibitory domains, i.e., sphingosine 1-phosphate receptor 2 (S1PR2) together with tetraspanin-3 for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) in association with co-receptors p75, Troy and Lingo-1 for Nogo-66 [12][13][14][15][16][17][18]. S1PR2 and NgR receptor complexes both lead to an activation of RhoA in the neuronal cytoplasm, which in turn causes destabilisation of the actin cytoskeleton and thus collapse of the neuronal growth cone as well as a general downregulation of the neuronal growth machinery [10]. Structural analysis at atomic resolution is a powerful approach to gain insight into the structure-activity relationship of proteins. To date, Nogo-66 is the only inhibitory domain of Nogo for which a structure has been determined [19]. For Nogo-A-Δ20, it is only known that it exhibits an overall unstructured conformation [20,21]. However, according to circular dichroism (CD) spectroscopy, this region seems to contain residual secondary structure [21]. This is supported by secondary structure prediction indicating the presence of residual conformations within the Nogo-A-Δ20 sequence [20,22]. Furthermore, the addition of zinc ions to Nogo-A-Δ20 induced a higher degree of α-helical content in circular dichroism [21]. However, the exact locations of putative structural elements within Nogo-A-Δ20 have not been known until now. The interaction between Nogo-A-Δ20 and the G-protein coupled receptor S1PR2 has been demonstrated biochemically and functionally [12]. Extracellular loops (ECLs) 2 and 3 of S1PR2 were concluded to be the primary binding sites for Nogo-A-Δ20, based on the nanomolar affinities of isolated ECL peptides in a microscale thermophoresis assay. However, only little is known about the exact binding mode and amino acid residues involved in this interaction. Here, we provide first structural data of Nogo-A-Δ20 with single-residue resolution using nuclear magnetic resonance (NMR) spectroscopy. The backbone of biologically active Nogo-A-Δ20 was assigned to a completeness of 94% using various triple resonance experiments, revealing three sites of marked α-helical propensity. A concentration-dependent dimerisation was found using diffusion NMR experiments. In addition, we investigated the interaction of S1PR2 with Nogo-A-Δ20 by titrating isolated ECL2 and ECL3 of S1PR2 to Nogo-A-Δ20. No conclusive binding data were obtained for these isolated fragments using NMR spectroscopy and isothermal calorimetry (ITC). Structural Propensities for Nogo-A-Δ20 Nogo-A-Δ20 was expressed as 13 C-and/or 15 N-labelled recombinant protein in E. coli to study its structural characteristics using CD and NMR spectroscopy. The CD spectrum of Nogo-A-Δ20 with its minimum at around 200 nm suggests a high proportion of unstructured regions, with some residual secondary structure (Fig 1). Addition of dodecylphosphocholine (FC12), which is required for structuring of Nogo-66 [19], only led to minor changes in the CD spectrum of Nogo-A-Δ20, indicating that FC12 induced no significant structural rearrangements. In accordance with the CD spectrum observed for Nogo-A-Δ20, a 2D-NMR [ 15 N, 1 H]-HSQC spectrum confirmed the intrinsically disordered character of Nogo-A-Δ20, as deduced from the low chemical shift dispersion in the 1 H dimension (Fig 2A). In order to obtain sequence-specific conformational and structural information, a backbone assignment was conducted. Standard pulse programs (HNCA, HNCACB) and non-standard experiments (HNN and HCAN) were recorded on [ 13 C, 15 N]-Nogo-A-Δ20. The low dispersion in the proton dimension, present in HNCA, HNCACB, and HNN spectra, together with many proline residues present in the sequence (13%, 23 prolines of 182 residues), interrupted the sequential assignment and posed a severe challenge. To overcome the discontinuity of the spectra along the backbone caused by proline residues, an HCAN spectrum was recorded. Here, the magnetisation is transferred from 1 H α to 13 C α and further on to N i and N i+1 , enabling a connection of a proline to its following residue [23] and allowing a sequential assignment through prolines. With this set of NMR experiments, 94% of the non-proline 13 C α and 13 C β and 83% of proline 13 C α -and 13 C β -frequencies in Nogo-A-Δ20 were assigned (Fig 2B). An unambiguous assignment was impossible for the residue stretches 575 PSFE 578 and 678 LIKETK 683 due to severe peak overlap (S1 Fig). The complete assignment can be found in Fig 2A and was deposited in the Biological Magnetic Resonance Bank (BMRB) with the ID 26653. The sequential assignment enables secondary structure analysis using secondary chemical shifts of Δδ 13 C α and Δδ 13 C β , which are the difference between the observed chemical shifts and corresponding random coil chemical shifts (S2 Fig) [24]. Positive Δδ 13 C α values and negative Δδ 13 C β values for several consecutive residues indicate an α-helical conformation. Conversely, negative Δδ 13 C α in combination with positive Δδ 13 C β indicate the formation of a β-strand. These two experimental values can be combined with the secondary structure propensity (SSP) algorithm resulting in a combined statistically more relevant value with positive values indicating α-helical and negative values suggesting β-strand conformations (Fig 3A) [25]. The SSP score for each residue was calculated from 13 C α and 13 C β chemical shifts and weighted over five residues. Thereby, many residues of Nogo-A-Δ20 showed values close to zero (Fig 3A), indicating a random coil-like structure with little secondary structure elements [26][27][28]. However, the three segments 561 EAIQESL 567 , 639 EAMNVALKALGT 650 , and 693 SNYSEIAK 700 contained positive SSP values above 0.1 for more than five consecutive residues indicating α-helical propensity. These three stretches relate well to three α-helices that were predicted in silico by PSIPRED v3.3 (residues 561-566, 637-648 and 696-700; S3 Fig) [22]. In order to corroborate the proposed helical propensity of the three segments, scalar couplings 3 J HNHα were measured. Secondary scalar couplings, Δ 3 J HNHα , were calculated by subtracting random-coil values [29] from the experimentally measured 3 J HNHα data. While positive Δ 3 J HNHα values show a tendency for β-sheets, negative values indicate turns or α- In the HNCACB, cross peaks belonging to C α , C α i-1 , C β , and C β i-1 are indicated, while in the HNN spectrum, the N i , N i-1 , and N i+1 are labelled. The HNCACB spectrum was recorded at a 600 MHz and the HNN spectrum was recorded at a 700 MHz NMR spectrometer at 6°C and pH 7.4. helical propensities [30]. All the three stretches proposed to be α-helical according to their secondary chemical shift values, i.e., 561 EAIQESL 567 , 639 EAMNVALKALGT 650 , and 693 SNY-SEIAK 700 , had negative Δ 3 J HNHα values supporting the presence of transient α-helices in these segments ( Fig 3B). An independent measure of both disorder and secondary structure can be obtained by 15 N { 1 H}-heteronuclear NOEs (HetNOE). While positive values close to 1 indicate structural rigidity of the backbone 15 N-1 H moieties, values close to 0 indicate dynamics in the range of~1 ns, and 15 N-1 H moieties with negative values are highly flexible with dynamics faster than~1 ns [31,32]. Most of the values of Nogo-A-Δ20 were slightly positive between 0.1 and 0.2, and extended runs of positive values were especially found at the locations of all three proposed αhelical stretches (Fig 3C). In addition, the N-and C-terminal regions of Nogo-A-Δ20 showed an elevated rigidity ( Fig 3C). Overall, the HetNOE data indicate a highly flexible state for Nogo-A-Δ20, as commonly found in IDPs [33,34]. Clustering of Nogo-A-Δ20 and of full-length Nogo-A is a highly discussed topic [7,14,35]. Pulse-field gradient NMR spectroscopy experiments were therefore recorded to determine the diffusion coefficient D of Nogo-A-Δ20 at two different concentrations. D 30 μM was found to be 2.18 ± 0.02×10 -11 m 2 /s (mean ± SD), and D 560 μM equalled 2.00 ± 0.01×10 -11 m 2 /s (Fig 4). Since two cysteines are present in Nogo-A-Δ20, we also tested the influence of their oxidation state Titration of S1PR2 Fragments to Nogo-A-Δ20 It has been shown that Nogo-A-Δ20 binds to isolated extracellular loops (ECL) 2 and 3 of sphingosine 1-phosphate receptor 2 (S1PR2) with affinities in the nanomolar range [12]. In order to identify the binding interface between ECL peptides and Nogo-A-Δ20 at atomic resolution, ligand titration studies were performed using NMR spectroscopy. First, ECL2 was titrated to 15 N-labeled Nogo-A-Δ20 at different molar ratios. A [ 15 N, 1 H]-HSQC spectrum with a resolution of 0.04 ppm in the 15 N and 0.028 ppm in the 1 H dimension was measured for each titration step at 6°C and pH 7.4 including a reference without addition of ECL2 (Fig 5 and S5 and S6 Figs). Even with a threefold excess of ECL2, no cross peak shifts were detected when compared to the spectrum without ECL2 (Fig 5B and S5C Fig). Normalized chemical shift changes of the combined 15 N and 1 H residues of Nogo-A-Δ20 were smaller than 0.005 ppm. These values were below the combined 15 N and 1 H detection resolution of 0.07 ppm, indicating the absence of conformational changes upon ECL2 addition. Since pronounced chemical shift changes were observed for several peaks of Nogo-A-Δ20 upon decreased pH (S7 Fig), it was hypothesised that a lower pH might be necessary for binding. However, a reduction of pH from 7.4 to 6.4 did not result in any peak shifts upon ECL2 titration (S6A and S5B Figs). Furthermore, a temperature increase from 6°C to 15°C to match the conditions of a previously published binding study more closely [12] did not result in any peak shifts upon ECL2 titration (S5D and S6B Figs). Subsequently, Nogo-A-Δ20 was investigated upon ECL3 titration. Similarly, no peak shifts could be detected ( Fig 5C and S8A Fig). Logarithmic intensities of Nogo-A-Δ20 at different conditions were plotted against its dependence on gradient strength. Nogo-A-Δ20 was measured with pulsed field gradient experiments at two concentrations (30 and 560 μM), shown as closed circles and squares, respectively. The diffusion coefficient was calculated according to [36], resulting in D 30 μM = 2.18 ± 0.02×10 -11 m 2 /s and D 560 μM = 2.00 ± 0.01×10 -11 m 2 /s (mean ± SD). Nogo-A-Δ20 was measured again in the presence of the reducing agent TCEP, shown as open circles (30 μM) and squares (560 μM). Here, the diffusion coefficients equalled D red 30 μM = 2.53 ± 0.01×10 -11 m 2 /s and D red 560 μM = 2.55 ± 0.02×10 -11 m 2 /s. ***, p < 0.0001; ns, not significant; red, reduced. doi:10.1371/journal.pone.0161813.g004 mM zinc ions were added to the sample. Again, no changes in the spectra could be detected upon ECL3 addition, indicating that zinc ions do not facilitate ECL binding (S6D and S8B Figs). Finally, as FC12 is required for folding of Nogo-66 [19], we explored the possibility that Nogo-A-Δ20 only binds to ECL2 in the presence of FC12. However, no changes in the Nogo-A-Δ20 CD spectrum were observed when ECL2 was added in the presence of FC12 (Fig 1). The missing shifts of [ 15 N, 1 H]-HSQC peaks might be explained by an intermediate exchange of the bound and unbound state. In this time regime, decreases of intensities of the amino acid residues participating in an interaction are anticipated. Therefore, the intensity ratios of Nogo-A-Δ20 in the presence vs. absence of ECL2 and ECL3 were calculated for each residue (Fig 6 and The intensity ratios at pH 7.4 at 6°C were found to have a random distribution near 1 for the Nogo-A-Δ20 to ECL2 ratios of 1 to 1 and 1 to 3, indicating no intermediate exchange. Intensity ratios at pH 6.4 at 6°C and at pH 7.4 at 15°C upon addition of ECL2 and the intensity ratio at pH 7.4 at 6°C upon addition of ECL3 have a larger deviation from the value 1, which might be rather attributed to an imperfect adjustment of pH and temperature than to ECL binding. Isothermal Titration Calorimetry (ITC) A third biophysical technique, ITC, was consulted to investigate the discrepancy between NMR spectroscopy and previous binding assay data [12]. Either ECL2 or ECL3 were titrated to 10 μM Nogo-A-Δ20. In both cases, only very small exothermic heat signals could be detected upon titration (S10 Fig). For ECL2, a small difference in the heat signals at low and high concentration could be observed. Furthermore, equally-sized heat signals were observed throughout the titration for ECL3. Cellular Activity Assay for Nogo-A-Δ20 In order to confirm that the obtained structural data correspond to a biologically active protein, and in order to exclude that the lack of peak shifts upon ECL titration was due to misfolding of Nogo-A-Δ20, we performed a 3T3 fibroblast spreading assay (Fig 7). Fibroblast spreading was markedly inhibited on isotopically labelled Nogo-A-Δ20 substrate, confirming intact inhibitory activity of the protein. Importantly, the IC 50 value was~40 pmol/cm 2 , which is a typical potency for Nogo-A-Δ20-induced inhibition of 3T3 fibroblast spreading [37]. In summary, Nogo-A-Δ20 is an intrinsically disordered domain as indicated by CD data, [ 15 N, 1 H]-HSQC peak dispersion, secondary chemical shift analysis and dynamic studies. Within the disordered region, three contiguous segments of α-helical propensity are found. All agree well with those indicated by a computational algorithm. Diffusion coefficients of Nogo-A-Δ20 suggest a dimerisation dependent on the concentration and oxidation state of the protein. While titration of ECL2 and ECL3 to Nogo-A-Δ20 did not induce pronounced peak shifts in [ 15 N, 1 H]-HSQC spectra or show marked thermal heat exchange in ITC measurements, the used batch of Nogo-A-Δ20 was found to be active in a 3T3 fibroblast spreading assay. Discussion We investigated Nogo-A-Δ20 using CD and NMR spectroscopy. We were able to obtain highquality NMR spectra for backbone assignment, which enabled us to derive structural data with atomic resolution. Our data show a high degree of disorder within the neurite growth and cell spreading inhibitory Nogo-A-Δ20 region. The largely random coil CD spectrum and narrow proton dispersion in [ 15 N, 1 H]-HSQC spectra confirm previous observations [20,21] and are extended by the experimentally obtained secondary structure analysis of secondary chemical shifts, 3 J HNHα scalar coupling, as well as the high degree of flexibility indicated by HetNOE measurements. Importantly, despite the lack of fully structured regions, isotopically labelled and thrombin-cleaved Nogo-A-Δ20 exerted its typical inhibitory activity in a 3T3 fibroblast spreading assay. Structural flexibility imposes a variety of advantages on proteins, ranging from an enlarged interaction surface and thus higher binding specificity to an elevated promiscuity towards binding partners [38][39][40]. As a consequence, IDPs are involved in a multitude of signalling pathways and appear in all three domains of life, i.e., Archaea, Bacteria, and Eukarya [41]. The intrinsically disordered Nogo-A-Δ20 has been shown to interact with various binding partners, such as S1PR2, tetraspanin-3, β1-integrins, cyclic nucleotide phosphodiesterase, and WWP1 [12,13,[42][43][44][45]. In addition, clustering of N-terminal Nogo-A fragments including Nogo-A-Δ20 has been described to enhance their inhibitory potency [14,42]. Our finding that the diffusion coefficient of Nogo-A-Δ20 is reduced at higher concentrations supports the notion of dimer formation. A high degree of flexibility might represent an important structural feature of this domain, increasing its surface area available for binding molecular target proteins and for homodimerisation. Additionally, Nogo-A is a multifaceted player implicated in neurite outgrowth inhibition, CNS development, synaptic plasticity, ER membrane morphology, and several other processes by interactions with several binding partners and multisubunit receptors [10,46,47]. Structural disorder could therefore allow different sets of interacting molecules to bind to the same sites within Nogo-A-Δ20 depending on the context, a model referred to as functional moonlighting [48]. Although no fully structured regions were found, we identified three segments within Nogo-A-Δ20 that appear to form transient and dynamical α-helical structures: 561 EAIQESL 567 , 639 EAMNVALKALGT 650 , and 693 SNYSEIAK 700 . All of these regions seem to be α-helical based on their positive SSP and negative Δ 3 J HNHα values, and they exhibit decreased flexibility as determined by HetNOE. Similar results were obtained for the very C-terminal four residues of Nogo-A-Δ20 (residues 722-725), which might represent the beginning of another α-helix in full-length Nogo-A. At the N-terminal boundary of Nogo-A-Δ20 (residues 548-560), flexibility is also reduced. However, the secondary structure of this region could not be determined unambiguously by SSP and Δ 3 J HNHα evaluation. Significant residual secondary structures are commonly found in IDPs, and they often resemble structural characteristics present in the bound state [49][50][51][52][53]. It has therefore been suggested that these residual structures are involved in initial molecular recognition [51,52]. One could speculate that the α-helical structures found in Nogo-A-Δ20 also serve as such recognition sparks, forming initial contact with binding partners. Adjacent unstructured regions could then confer higher specificity to the interaction. Strikingly, 561 EAIQESL 567 is located in direct juxtaposition with one of the most conserved domains of Nogo-A-Δ20 (residues 554-559) that harbours a β1-integrin binding motif (S1 Fig) [43]. Similarly, 639 EAMNVALK-ALGT 650 partially overlaps with the binding epitope for the Nogo-A-neutralizing antibody 11C7 (residues 630-640) that has been shown to enhance recovery from spinal cord injury in rats and macaques [54,55]. Forced dimerisation of Nogo-A-Δ20 has been reported to enhance the inhibitory properties of the protein [7,14]. The markedly elevated diffusion coefficients in the presence of TCEP support dimerisation through the formation of disulphide bridges of Nogo-A-Δ20 in a nonreducing environment. Dimerisation seems to be concentration dependent, as the diffusion coefficient of Nogo-A-Δ20 in the absence of TCEP decreased at higher concentrations of Nogo-A-Δ20. A larger fragment of human Nogo-A that contained Nogo-A-Δ20 has been shown to co-exist in two disulphide isomers by non-reducing SDS/PAGE [35]. Electrophoretic mobility was enhanced in each of these disulphide isomers as compared to the reduced fragment, indicating that both isomers harbour intra-molecular disulphide bridges. By mutational analysis, the authors were able to identify four conserved cysteines that are involved in this process, though the exact connectivity remains elusive [35]. Of these conserved cysteines, only one (corresponding to rat Cys574) is located within Nogo-A-Δ20, indicating a possible intramolecular disulphide bond with a cysteine outside of Nogo-A-Δ20. In the isolated Nogo-A-Δ20 fragment, this unpaired cysteine could contribute to non-physiological inter-molecular disulphide bridges resulting in dimerisation. Alternatively, it is possible that the second cysteine within Nogo-A-Δ20, Cys676, accounts for this observation, though it seems dispensable for overall folding [35]. Oxidation state-dependent dimerisation could be of physiological relevance for Nogo-A signalling. In order to impose its inhibitory effect on passing neurons, Nogo-A-Δ20 is presented on the surface of oligodendrocytes, where it is exposed to the oxidising environment of the extracellular space [5,7]. On the other hand, a different membrane topology is found at the ER, where Nogo-A-Δ20 faces the reducing milieu of the cytosol [7,11]. It can therefore be speculated that the oxidation state of Nogo-A-Δ20 at different cellular compartments contributes to its diverse functions, possibly through differential disulphide bonding and/or dimerisation. The connectivity of disulphide bridges in Nogo-A, as well as their individual contributions to folding, dimerisation, S1PR2 binding and inhibitory activity, will be the subject of future systematic mutational studies. No chemical shift perturbations could be observed upon titration of ECL peptides, and only very small exothermal signals were detected with ITC suggesting no specific high affinity binding. This is surprising, as ECL2 and ECL3 had been shown to bind Nogo-A-Δ20 with K D values of~280 nM and~350 nM, respectively, by microscale thermophoresis [12]. Microscale thermophoresis determines the diffusion coefficient of a labelled molecule as a function of the concentration of its binding partner. The diffusion coefficient is susceptible to various parameters such as buffer composition or size, charge, hydration shell or conformation of a molecule [56]. Therefore, not only molecular interactions are measured via microscale thermophoresis, but also conformational alterations or charge variations caused by slight changes in the buffer conditions within the titration experiment such as pH, which do not have to be induced by ligand binding. On the other hand, it should be noted that isolated ECLs are likely to assume different conformations than in the context of the whole GPCR. The physiologically relevant structure presumably depends on the relative positions of adjacent hydrophobic regions. Likewise, Nogo-A-Δ20 only represents a fragment of Nogo-A that might not include all structural features required for a physiological interaction. Future structural investigations should therefore concentrate on the full-length Nogo-A and S1PR2 proteins. In conclusion, we have shown that biologically active Nogo-A-Δ20, while unstructured in the majority of its sequence, contains three stretches with α-helical propensity. Whereas α-helices could be involved in initial recognition and presentation of disordered regions, structural flexibility of Nogo-A-Δ20 might be essential for specific interactions with the binding partners in cellular membranes, neuritic growth cones, at CNS synapses, and in the ER. We provide further evidence that dimerisation occurs in Nogo-A-Δ20, the physiological relevance of which needs to be further investigated. However, we could not detect structural changes of Nogo-A-Δ20 upon titration of isolated ECL2 or ECL3 by NMR spectroscopy, and only minor thermal heat exchanges were observed by ITC. It will be fascinating to gain more insight on the structural basis of this clinically highly relevant signalling node. Expression of Isotopically Labelled Nogo-A-Δ20 Rat Nogo-A-Δ20 (residues 544-725) was cloned into the pET28 vector containing a His 6 -tag at each terminus and a T7-tag between the N-terminal His 6 -tag and Nogo-A-Δ20 [7]. 15 N-or 13 C, 15 N-labelled Nogo-A-Δ20 was expressed in One Shot BL21 (DE3) strain of E. coli in M9 minimal medium with max. 4 g/L D-glucose-13 C 6 ( 13 C > 99%) or 8 g/L D-glucose-12 C 6 and 1 g/L 15 N-ammonium chloride ( 15 NH 4 Cl, 15 N > 98%) purchased from Sigma-Aldrich (Buchs, Switzerland). Bacteria were grown at 37°C at 100 rpm until the OD 590 reached 1.2, transferred to 30°C and induced with 1 mM IPTG. The fusion protein was expressed for 8 hours and cells were harvested by centrifugation. The wet pellet was stored at -80°C. Purification of Nogo-A-Δ20 All of the following purification steps were performed at 4°C. A frozen pellet of 1 L of bacterial culture was thawed on ice and resuspended in 50 mL lysis buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, 20 mM imidazole, pH 7.4). 0.5 mg/mL lysozyme, 0.5 mM PMSF, and 1 protease inhibitory tablet (Roche Diagnostics GmbH, Mannheim, Germany) were added. The lysate was stirred for 20 min. Cells were further disrupted by passing twice through a 110S microfluidizer (Microfluidics, Newton, Massachusetts, USA) at 40 PSI. The suspension was centrifuged at 40'000 rpm (125171 g) for 30 min (Optima L-90K Ultracentrifuge, rotor Ti-45, Beckman Coulter International, S.A., Nyon, Switzerland) to pellet cellular debris [7]. The supernatant of the centrifugation was bound to 3 mL Ni-NTA Agarose from Qiagen (Merck KGaA, Darmstadt, Germany) via batch mode during 2 h. The Ni-NTA was washed with 30 mL lysis buffer, eluted with ca. 5 mL elution buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, 500 mM imidazole, pH 7.4) via gravity flow and collected in 0.5 mL fractions. The elution buffer was exchanged to PBS buffer with a pre-packed and disposable PD-10 desalting column (GE Healthcare Life Sciences, Buckinghamshire, UK). To remove the N-terminal His 6 -tag, bovine thrombin (Sigma-Aldrich, Buchs, Switzerland) was added to the desalted sample with the ratio of 2 NIH units of thrombin per ca. 1 mg desalted Nogo-A-Δ20 for 1 hour. The cleaved fusion protein was purified on a Highload™ 26/60, Superdex™ 75 column using an Äkta FPLC system (prep grade, GE Healthcare, Uppsala, Sweden). To exclude batch-to-batch variations, 6 L of 15 N-labelled Nogo-A-Δ20 were expressed, purified, shock frozen in aliquots, and finally stored at -80°C until usage for ECL titration. CD spectroscopy CD measurements were carried out on a Jasco J815. The spectra were scanned from 260-198 nm at 20 nm/min with 1 nm band-pass, 4 seconds integration and averaged over 2 repetitions. The measurements of Nogo-A-Δ20 were executed in PBS at 25°C with a concentration of 10 μM. Dodecylphosphocholine (FC12; Affymetrix, Santa Clara, CA, USA) and ECL2 (see NMR section) were added to final concentrations of 6.67 mM and 10 μM, respectively. NMR spectroscopy The concentration of isotopically labelled Nogo-A-Δ20 for the NMR measurements was between 80-400 μM in PBS buffer containing 95% H 2 O and 5% D 2 O at pH 7.4. The experiments were recorded on 600 MHz, 700 MHz, or 900 MHz Bruker NMR spectrometers (Bruker BioSpin AG, Fällanden, Switzerland) equipped with either TCI or TXI cryoprobes. For the amino acid sequence assignment, a [ 15 N, 1 H]-HSQC and a set of four triple-resonance experiments were measured at 6°C. The chemical shifts of the amide proton, the amide nitrogen, the 13 [23], enabling assignment through proline residues. The difference of the chemical shifts of the measured δ 13 C α and δ 13 C β and random coil values [60] were calculated for the secondary chemical shift analysis. Composite values of Δδ 13 C α and ΔδC β were calculated using the SSP algorithm from Forman-Kay group [25]. The algorithm combines individual contributions of chemical shifts regarding their sensitivity to αand β-structure from different nuclei into a score. Hereby, the observed chemical shift differences of a residue are weighted against the expected chemical shift differences for a secondary structure. To minimize contributions from chemical shifts that are poor measures of secondary structures, e.g., glycines, the algorithm additionally averages the score over five residues. The final score of a residue ranges from 1 to -1, indicating fully formed α-helical or β-strand conformations, respectively. An intensity modulated [ 15 N, 1 H]-HSQC [61] was measured to obtain the 3 J HNHα scalar couplings (16 number of scans, 1 s relaxation delay, 2 τ = time for evolution of 3 J HNHα : 18 ms). The intensity ratios of the relation I m /I d = cos(π( 3 J HNHα )2 τ) were used for the calculation of the coupling constant 3 J HNHα , I m being the intensity of the modulated spectra and I d that of decoupled ones. The experimentally obtained 3 J HNHα was multiplied by a correction coefficient of the magnitude of 1.06 due to the different relaxation properties of the in-and antiphase magnetisation of the H N compared to the H α [61]. The secondary scalar couplings, Δ 3 J HNHα , were calculated by subtracting the corresponding random-coil values [29] from the experimentally measured 3 J HNHα data. Dynamics of Nogo-A-Δ20 were examined with a 15 N{ 1 H}-HetNOE experiment (8 number of scans, 6 s relaxation delay) [32]. The HetNOE was estimated by dividing I S , the intensity of the saturated spectrum, by I U , the intensity of the corresponding peak in the unsaturated spectrum. Error bars for the HetNOE plot were calculated using Gaussian error propagation [62]. To determine the diffusion coefficient of reduced and unreduced Nogo-A-Δ20, pulsed field gradient experiments [36] were measured (30[F2] × 16384[F1] complex data points, gradient between 5-75%, Δ of 200 ms, δ of 5.5 ms, 32 or 128 number of scans, 10 s relaxation delay, WATERGATE for water suppression). The sample constituted of either 560 μM in absence or presence of 2 mM tris(2-carboxylethyl)phosphine hydrochloride (TCEP, Sigma-Aldrich, Buchs, Switzerland) or 30 μM in absence or presence of 5 mM TCEP at pH 7.4 and 6°C. The experimental data points were fitted according to [36]. Gradient strength was calibrated with H 2 O, for which coefficient is known [63,64]. Linear regression and an statistical analysis of the slope differences were performed in Prism 5 (GraphPad Software, La Jolla, CA, USA), which follows a calculation method that is equivalent to ANCOVA [65]. For 15 N and 1 H frequencies, yielding a resolution of 0.04 ppm and 0.028 ppm, respectively. The combined 15 N and 1 H resolution was 0.07 ppm. ECL2 (peptide sequence NCLNQLEACSTVLPLYAKHYVL) and ECL3 (SILLLDSTCPVRACPVLYK) were purchased from JPT Peptide Technologies GmbH (Berlin, Germany). The concentration used for 15 N-labeled Nogo-A-Δ20 was 30 μM, 88 μM or 120 μM and the following molar ratios were measured: Nogo-A-Δ20: ECL2: 1: 1 and 1: 3, Nogo-A-Δ20: ECL3: 1: 3. The sample for ECL2 titration was measured at different pH values (pH 7.4 and 6.4) and different temperatures (6°C and 15°C). The Nogo-A-Δ20 sample for the ECL3 titration was measured at pH 7.4 at 6°C in the presence or absence of either 5 mM TCEP or 4 mM ZnCl 2 . The chemical shift differences (CSD) between the peaks of Nogo-A-Δ20 alone and those in presence of an ECL in the [ 15 N, 1 H]-HSQC were calculated using the following equation [66]: Isothermal Titration Calorimetry (ITC) Nogo-A-Δ20, as well as ECL2 and ECL3 of S1PR2 were dialysed against PBS (pH 7.4) at 4°C overnight. ITC experiments were carried out at 25°C on a MicroCal VP-ITC instrument (Malvern Instruments, Worcestershire, UK) with a cell volume of 1400 μL and a syringe volume of 300 μL. Each experiment consisted of an initial injection of 2 μL, followed by 29 injections of 10 μL. Stirring speed was 300 rpm. Nogo-A-Δ20 was 10 μM in the cell, whereas ECL peptides were 150 μM in the syringe. All data were analysed with the Origin software supplied by the manufacturer. CellProfiler software was employed to measure the sizes of only non-clumped cells [68]. Finally, non-linear regression was performed in Prism 5 (GraphPad Software, La Jolla, CA, USA).	v3-fos-license
2019-04-04T13:11:45.356Z	2015-03-02T00:00:00.000	94087844	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://figshare.com/articles/journal_contribution/Selective_Oxidation_of_Benzyl_Alcohols_to_Aldehydes_with_a_Salophen_Copper_II_Complex_and_i_tert_i_Butyl_Hydroperoxide_at_Room_Temperature/1320933/2/files/1928273.pdf", "pdf_hash": "53fe58f5ecec7dfc7585cdb299e478dd331159a8", "pdf_src": "TaylorAndFrancis", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:359", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "31bec1b3101a800b06cafaeeac9a8409c7ba68c0", "year": 2015 }	pes2o/s2orc	Selective Oxidation of Benzyl Alcohols to Aldehydes with a Salophen Copper(II) Complex and tert-Butyl Hydroperoxide at Room Temperature Abstract An efficient and selective oxidation of benzyl alcohols has been developed using a salophen copper(II) complex as the catalyst and tert-butyl hydroperoxide (TBHP) as the oxidant in the presence of base. Moderate to excellent yields of the corresponding benzaldehydes were obtained at room temperature without the carboxylic acids being formed. GRAPHICAL ABSTRACT INTRODUCTION As one of the most important raw materials, aldehydes are widely used in organic synthesis for the preparation of fine chemicals both in laboratory and industry. [1] Traditional methods for the synthesis of aldehydes mainly rely on the oxidation of alcohols, [2] organic halides, [3] amines, [4] alkenes, [5] and the methyl group on the aromatic ring. [6] Among these methods, the oxidation of alcohols is convenient and effective because of atom economy, available raw materials, and relative greater yields. [7] Many transition-metal compounds have been used for this purpose, such as vanadium (V), [8] cobalt (Co), [9] copper (Cu), [10] manganese (Mn), [11] ruthenium (Ru), [12] rhodium (Rh), [13] palladium (Pd), [14] iron (Fe), [15] and chromium (Cr). [16] In contrast to other metals, Cu is an abundant metal with less toxicity. [17] There are several reported methods in which Cu has been used as a cheap and "green" catalyst for the oxidation of alcohols. Copper catalytic systems employing 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) or dialkylazodicarboxylate as cocatalysts have emerged as some of the most effective methods in the oxidation of alcohols to aldehydes. [18] The firstgeneration copper-catalyzed aerobic oxidation protocol, which was developed by Riviere and Jallabert, [19] employed equivalents of CuCl and 1,10-phenanthroline (Phen). Subsequently, Marko and coworkers [20] developed a series of catalytic systems for aerobic alcohol oxidation using the catalytic amount of CuCl and Phen as the catalyst in combination with dialkylazodicarboxylates as redox-active cocatalysts, which exhibited the broadest scope of alcohol oxidation. On the other hand, the Cu=TEMPO system was also widely used for the oxidation of alcohols. [21] The related catalytic system, which consists of a copper salt with 2,2'-bipyridine as a ligand and TEMPO as a cocatalyst, could also enable the mild aerobic oxidation of primary alcohols to aldehydes. [22] Meanwhile, salen-type Schiff bases, including the salen and salophen Schiff bases, can be prepared by condensation between aldehydes and amines in different reaction conditions. [23] They are able to stabilize many different metals in various oxidation states with four coordinating sites and control the performance of metals in a large variety of useful catalytic transformations. [24] Schiff base metal complexes are of great importance for catalysis, [25] such as the asymmetric epoxidation of alkenes [26] and oxidation of alcohols. [27] Transition-metal Schiff base complexes that catalyzed oxidation of alcohols were attractive, especially for their more accessible synthesis conditions and versatile coordination structures. [16b] Salen-Cu(II) could be used in the oxidation of primary alcohols to the carboxylic acids in good yields. [27] However, when combined with TEMPO, the salen-Cu(II) complex could selectively oxidized the alcohols to the corresponding aldehydes at reflux temperature. [28] Because the system described requires the toxic TEMPO and high temperature, a mild, "green," and efficient catalytic system is still desirable. Herein, we developed an efficient method for the oxidation of benzyl alcohols to the corresponding aldehydes using a salophen copper(II) complex (Scheme 1) as the catalyst and tert-butyl hydroperoxide (TBHP) as the source of oxygen in the presence of base. To our delight, excellent yields and good selectivity were achieved for a variety of benzyl alcohols with no trace of corresponding carboxylic acid. RESULTS AND DISCUSSION The oxidation of benzyl alcohol was first investigated as the model reaction and the results are summarized in Table 1. Very poor yields were obtained when Scheme 1. Synthesis of the salophen copper(II) complex. SELECTIVE OXIDATION OF BENZYL ALCOHOLS no catalyst or base was used ( Table 1, entries 1 and 2), which indicated that both of them are necessary for the oxidation. To optimize the reaction conditions, the oxidant amount was varied from 0.6 to 2 equiv with other conditions remaining constant (Table 1, entries 6 and 10-18). A trend of increasing yield with oxidant amount was observed up to 1.1 equiv TBHP. There was still no carboxylic acid formed even when the oxidant amount was up to 2.0 equiv (Table 1, entry 6). It was worth noting that even when 0.6 equiv of TBHP was used for the oxidation (Table1, entry 10), the reaction provided the product in 91% yield. It is possible that oxygen may participate in the reaction as the reaction was conducted in the open air. The assumption was proved by the results from the controlled experiments (Table 1, entries 11 and 12). Under the same reaction conditions, O 2 was used as the oxidant but only 36% yield was obtained (Table 1, entry 13). The loading amount of catalyst was also examined ( Table 1, entries 6 and 19-23). The best yield was achieved when the amount of the catalyst was 2 mol%. The type and amount of the bases also played important influences on the reaction and 0.6 equiv NaOH resulted in the best yield ( To determine the application scope of this catalytic system, a wide range of benzyl alcohols were oxidized under the optimized conditions ( Table 2). All the benzyl alcohols employed were converted into the corresponding aldehydes with excellent yields and selectivity, and no carboxylic acids were detected with Reaction conditions: alcohols (0.5 mmol), salophen copper(II) complex (2 mol%), TBHP (1.1 equiv), and NaOH (0.6 equiv) in acetonitrile were stirred under room temperature in air overnight. b Isolated yield. c GC yield. SELECTIVE OXIDATION OF BENZYL ALCOHOLS high-performance liquid chromatography (HPLC) after the reaction completed. No obvious influence of the electronic effects was found in the reaction, and both benzyl alcohols containing the electron-withdrawing and electron-donating groups could produce the reaction with satisfactory yields (Table 2, entries 1-7). 2-Iodobenzyl alcohol provided a poor yield, which may be explained by the steric effects of the iodo substituent (Table 2, entry 10). In the case of 2-aminobenzyl alcohol ( Table 2, entry 11), an undesired by-product of imine was afforded, leading to a poor yield. The aliphatic primary alcohol exhibited low conversion and poor reactivity (Table 2, entries 20 and 21), but there was still no trace of carboxylic acid. Finally, a probable mechanism for this reaction has been proposed (Fig. 1). The role of the base is to deprotonate the alcohol and accelerate the formation of the benzyloxy-Cu(II) complex 1 by favoring the coordination of the resulting alcoholate to the salophen copper(II) complex. [22b,29c] The aldehydes were then obtained by the reaction of the benzyloxy-Cu(II) complex with the oxidant TBHP with release of the by-product H 2 O and the tert-butoxy-Cu(II) complex 2. Finally, the tert-butoxy-Cu(II) complex exchanged with the starting alcohol to release the tert-butyl alcohol and completed the catalytic cycle. [29] CONCLUSION In summary, a mild and selective oxidation method of benzyl alcohols to the corresponding aldehydes has been established when using the TBHP as the oxidant and a salophen copper(II) complex as the catalyst in the presence of NaOH at room temperature. In this protocol, a variety of benzyl alcohols are oxidized to the corresponding aldehydes in moderate to excellent yields and no overoxidation takes place. 1338 T. CHEN AND C. CAI EXPERIMENTAL All reagents were purchased from commercial sources and used without treatment; 70% TBHP in water was used. The products were purified by column chromatography over silica gel. 1 H NMR spectra were recorded on a Bruker AMX500 (500 MHz) spectrometer and tetramethylsilane (TMS) was used as a reference. A Nicolet IS-10 spectrometer was recorded for IR spectroscopy. Preparation of Salophen H 2 O-Phenylenediamine (108 mg, 1 mmol) in 5 mL MeOH was added to a stirred mixture of salicylaldehyde (244 mg, 2 mmol) in 10 mL MeOH. The resulting orange mixture was stirred overnight at room temperature. The solid product was collected by filtration, washed with cool alcohol, and dried in vacuo (256 mg, yield: 81%); 1 Preparation of Salophen Copper(II) Complex Solution of salophen H 2 ligand (189 mg, 0.5 mmol) in EtOH (10 mL) and Cu (OAc) 2 . H 2 O (99 mg, 0.5 mmol) in water (1 mL) were mixed and refluxed with vigorous stirring for 2 h. The resulting solution was then cooled to room temperature and filtered. After filtration, the solid product was washed with H 2 O, MeOH, and Et 2 O subsequently, then dried in vacuo to afford the desired copper complex (139 mg, yield 71%). IR: v (cm -1 ) ¼1602, 1519, 1334. Typical Procedure for the Oxidation of Alcohols Alcohol (0.5 mmol), salophen copper(II) complex (2 mol%), NaOH (0.6 equiv), and 70% TBHP in water (1.1 equiv) were dissolved in acetonitrile (5 mL), and the homogeneous solution was stirred at room temperature in air overnight. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was purified over silica gel by column chromatography (10-25% EtOAc in hexane). All the products were known compounds and were identified by comparison of their physical and spectra data with those of authentic samples. SUPPLEMENTAL MATERIAL Supplemental data for this article can be accessed on the publisher's website.	v3-fos-license
2020-01-15T14:08:20.640Z	2020-01-14T00:00:00.000	210192581	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://www.liebertpub.com/doi/pdf/10.1089/jop.2019.0079", "pdf_hash": "496ec45b8cae7e23b23ca6354b599c9997e08c6b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:370", "s2fieldsofstudy": [ "Biology", "Medicine" ], "sha1": "bdbbe3228e5c4971c48e1f8c46e03524769d3b54", "year": 2020 }	pes2o/s2orc	Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 μmol/L, latanoprost free acid (LAT-A) at 0.1 μmol/L, or prostaglandin F2α (PGF2α), at 0.1 μmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 μmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process. Introduction G laucoma is a neurodegenerative optical neuropathy that is characterized by the loss of retinal ganglion cells and their axons and is a leading cause of irreversible vision loss. 1,2 Intraocular pressure (IOP) reduction is currently the only evidence-based treatment strategy for glaucoma. Lowering of IOP using prostaglandin F (FP) receptor agonists (FP agonists), such as latanoprost, tafluprost, travoprost, and bimatoprost, is the current standard of care for patients with glaucoma and ocular hypertension. 3 However, prostaglandin-associated periorbitopathy (PAP) has been reported in FP agonist-treated patients with glaucoma. 4,5 PAP affects patient care in many ways, such as difficulty in IOP measurement, difficulty during surgery, and cosmetic concerns. 4,5 PAP is more frequent and more severe in bimatoprost users than in those using other FP agonists, 6 and it causes deepening of the upper eyelid sulcus (DUES) and pigmentation of the iris and skin surrounding the eye lid. 4 DUES is considered a cosmetic adversity of FP agonist treatments. [7][8][9] The long-term use of latanoprost has also been considered causative of DUES in case studies of patients with glaucoma. 10,11 Moreover, recent investigations suggest that DUES is induced by atrophy of orbital fat. 12 Prostaglandin E 2 (PGE 2 ) acts on a group of G-proteincoupled receptors, and the subtypes EP1, EP2, EP3, and EP4 have been shown to respond to PGE 2 . 13,14 Although PGE 2 has been shown to potently reduce IOP in a previous study, it was associated with adverse effects (AEs), such as flares of anterior chambers. 15 Thus, PGE 2 receptor agonists that reduce ocular hypertension with little or no AEs are being investigated globally. 16 We are currently developing omidenepag isopropyl (OMDI) as a new IOP-lowering ophthalmic solution. 17,18 This agent is a prodrug of the selective non-prostaglandin EP2 receptor agonist and was launched as a treatment for glaucoma and ocular hypertension first in Japan in 2018. OMDI is hydrolyzed by esterases to omidenepag (OMD) during corneal penetration, and the IOP-lowering effects of this drug are associated with increased outflow facility and uveoscleral outflow. 19 In a previous clinical study, topical applications of 0.002% OMDI significantly reduced IOP in patients with glaucoma, and all of the associated ocular AEs were mild in severity. 20 FP agonists, including latanoprost, inhibit adipogenesis by stimulating FP receptor in 3T3-L1 cells. 21 Prostaglandin F 2a (PGF 2a ) also inhibits adipocyte differentiation by binding the FP receptor. 22 Hence, DUES due to current antiglaucoma FP agonists likely follows inhibition of adipogenesis around the eyelid, followed by atrophy of orbital fat. Because the effects of the EP2 agonist OMD on adipocyte differentiation have not yet been demonstrated, we monitored adipocyte differentiation in 3T3-L1 cells treated with the pharmacologically active form of OMDI, and we made comparisons with the effects of other FP agonists. Methods Culture of CHO cells expressing mouse EP2 receptor and cAMP assays CHO cells stably expressing mouse EP2 (mEP2) receptor 23 and mock-CHO 24 were cultured in 24-well plates at 2 · 10 5 cells/well. Cells were then preincubated for 10 min at 37°C in N-(2-hydroxyethyl)piperazine-N¢-2-ethanesulfonic acid (HEPES)-buffered saline containing 10 mmol/L indomethacin, and reactions were started by the addition of OMD at 0.1, 1, 10, 100, 1,000, and 10,000 nmol/L, which was provided by Ube Industries, Ltd. (Yamaguchi, Japan). After incubation for 10 min at 37°C, reactions were terminated by the addition of 10% trichloroacetic acid. Subsequently, cAMP concentrations in cells were measured by using radioimmunoassays according to a cAMP assay system (Cyclic AMP kit Yamasa, Yamasa Corporation, Chiba, Japan). Cell viability assays Cell viability was determined by using MTS assays (CellTiter 96 Ò Aqueous One Solution Reagent; Promega, WI) with a Benchmark Plus Microplate Reader (Bio-Rad, CA) at an absorbance wavelength of 490 nm. On day 10 after initiation of differentiation, MTS reagent was added to each well and incubated for 30 min to measure the absorbance. The average absorbance of wells without cells was subtracted from absorbance values of cells. Data are presented as percentages of viable cells relative to controls. Oil Red O staining Oil Red O staining was performed by using a Lipid assay kit (Cosmo Bio, Sapporo, Japan) in accordance with the manufacturer's instructions. Briefly, differentiated 3T3-L1 cells were washed in Dulbecco's phosphate-buffered saline (D-PBS), fixed in 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 1 h, and stained with Oil Red O for 1 h. Oil Red Ostained cells were observed by using a microscope (IX70; Olympus, Tokyo, Japan). Six stained areas per well were then measured by using Win ROOF Ò ver. 5.8 (MITANI Corporation, Tokyo, Japan). Cell areas that were stained with LAT-A, PGF 2a , or OMD were expressed as percentages of those in untreated control cultures. Gene expression analysis Total RNA was isolated from 3T3-L1 cells on indicated differentiation days by using RNeasy mini Kits (QIAGEN, Hilden, Germany and Venlo, Netherlands). Quantities and qualities of isolated RNA were evaluated by using Nano-DropÔ (Thermo Fisher Scientific, Inc., MA). After dilution in RNase-free water, 20 ng/mL RNA samples were immediately reverse transcribed into cDNA by using Pri-meScriptÔ RT Master Mix reagent Kits (Takara, Shiga, Japan) in accordance with the manufacturer's instructions. Primers for mouse peroxisome proliferator-activated receptor g (PPARg; Pparg), CCAAT/enhancer-binding protein a (C/EBPa; Cebpa), and b (C/EBPb; Cebpb) (Takara) were used to quantify gene expression levels with Quan-tiFastÔ SYBR Ò Green PCR Kits (QIAGEN, Hilden, Germany) as described by the manufacturer (Table 2). Briefly, cDNA was amplified in the presence of SYBR Green PCR Master Mix in final reaction volumes of 20 mL per well by using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc., and Life Technologies, Inc., CA). Relative expression levels were calculated by using the standard DDCt method with 7500 Fast System SDS Software Version1.4 software (Applied Biosystems, CA). The housekeeping gene Gapdh was used as an internal control. Statistical analysis Fifty percent effective concentrations (EC 50 ) were calculated by using EXSUS (version 8.0; CAC Croit Co., Tokyo, Japan). Data were expressed as mean -SEM and were statistically analyzed by using EXSUS. Differences between drug treatment and control groups were identified by using Student's t-test, Wilcoxon test, Dunnett's test, or Steel test, and were considered significant when P < 0.05. mEP2 receptor agonist activity of OMD To investigate the agonistic activity of OMD toward the mEP2, we measured cAMP concentrations in CHO cells after treatments with OMD by using radioimmunoassays. OMD treatments (Fig. 1A) promoted cAMP production dose dependently (Fig. 1B), and they had an EC 50 value of 3.9 -0.49 nmol/L for the mEP2 receptor (n = 3) (Fig. 1C). The EC 50 value was calculated from 3 independent replicates. In addition, OMD did not promote the formation of cAMP in CHO cells in which the mEP2 receptor was not stably expressed (Fig. 1D). Effects of OMD and FP agonists on adipogenesis To evaluate the effects of OMD and FP agonists on adipogenesis, we performed Oil Red O staining of lipid droplets in 3T3-L1 cells after differentiation for 10 days. Marked increases in numbers of lipid droplets confirmed preadipocyte differentiation in control, as shown in previous report 21 (Fig. 3). Oil Red O staining patterns also differed significantly between cells treated with OMD and FP agonists (Fig. 3). Relative to control cells, stained areas comprised 12.6% -1.3% and 15.5% -1.5% of cell areas after treatments with LAT-A and PGF 2a , respectively. In addition, after treatments with OMD at 0.1, 1, 10, and 40 mmol/L, stained areas comprised 85.0% -6.6%, 93.9% -4.5%, 86.9% -2.7%, and 88.0% -5.9% of cell areas, respectively (Fig. 4). These data show that OMD treatments had no significant effects on adipogenesis. In contrast, the FP agonists LAT-A and PGF 2a significantly inhibited adipogenesis in 3T3-L1 cells. Adipogenic transcription factor expression during adipogenesis The adipogenic transcription factors Pparg and Cebp sequentially stimulate genetic changes that result in differentiation. 25,26 Herein, we confirmed these changes in transcription factor expression during adipogenesis in 3T3-L1 cells by using quantitative real-time polymerase chain reaction (PCR) analyses of Pparg, Cebpa, and Cebpb mRNAs. In time course experiments (Fig. 5) in differentiating 3T3-L1 cells, Pparg mRNA expression levels were increased by 1.4-fold at day 2, and by 1.8-fold at days 4 and 10. Cebpa expression was similarly increased by 2.9-fold at days 2 and 10, and by 3.2-fold at day 4, and that of Cebpb was increased by 2.1-fold by day 2. These results show early increases in Pparg and Cebpa mRNA expression levels during adipogenesis in 3T3-L1 cells. Cebpb was also transiently induced during the early stages of differentiation, as shown in previous studies. 25,26 Effects of OMD and FP agonists on mRNA expression of adipogenic transcription factors To investigate the effects of OMD and FP agonists on adipogenesis, we determined Pparg, Cebpa, and Cebpb expression levels during differentiation of 3T3-L1 cells. LAT-A and PGF 2a suppressed the expression of Pparg and Cebpa on days 2, 4, and 10 ( Figs. 6 and 7A). Cebpb expression levels were significantly lowered in LAT-A-and PGF 2a -treated cells on days 2 and 4, compared with those in control differentiating cells (Fig. 7B). In contrast, even at 40 mmol/L, OMD did not affect expression levels of these genes under the present conditions. Discussion In this study, we investigated the effects of the selective EP2 agonist OMD on adipogenesis and made comparisons with those of FP agonists in differentiating 3T3-L1 cells. OMD is a non-prostaglandin structure compound and OMDI, isopropyl ester of OMD, is an active pharmacological ingredient of ophthalmic solution for the treatment of glaucoma as an IOP-lowering agent. 17 After topical administration to ocular surfaces, OMDI is hydrolyzed by esterases to OMD during its corneal penetration. 18 During 2018, this drug was launched in Japan for the treatment of glaucoma and ocular hypertension. It is reported that DUES is one of the cosmetic AEs induced by FP agonists and induced by atrophy of orbital fat in the eyelid by inhibiting adipogenesis. 21 Although this AE was induced by stimulation of the FP receptor, no published studies demonstrate the effects of EP2 receptor agonists on adipogenesis. Overall, 3T3-L1 cells were originally developed by clonal expansion from murine Swiss 3T3 cells 27 and have been widely used as preadipocytes in studies of adipocyte differentiation. [27][28][29] In our hands, LAT-A and PGF 2a prevented the accumulation of lipid droplets in 3T3-L1 cells and inhibited adipogenesis (Figs. 3 and 4). In agreement, Taketani et al. showed that all FP agonists that are currently used as glaucoma 5. Gene expression analyses of adipogenic transcription factors during adipogenesis. Pparg, Cebpa, and Cebpb mRNA expression levels were determined by using real-time PCR, and comparisons were made with expression levels on day 0. P < 0.05, P < 0.01, compared with day 0 (Dunnett test). PCR, polymerase chain reaction. treatments, including latanoprost, inhibited adipogenesis by stimulating the FP receptor in 3T3-L1 cells. 21 In addition, FP agonists limited the induction of Pparg, Cebpa, and Cebpb during adipocyte differentiation (Figs. 6 and 7). Under normal conditions of adipogenesis, Cebpb is expressed early to transactivate Pparg and Cebpa, which are master transcriptional regulators of terminal adipocyte differentiation. 25,26 Further, it is reported that differentiation starts with the induction of Cebpb at an early stage, during which cells begin to express Pparg and Cebpa. 28,29 Our results are consistent with those of previous reports (Fig. 5). 28,29 These observations suggest that FP agonists prevent the accumulation of lipid droplets initially by inhibiting adipogenic Cebpb expression, and subsequently by limiting the associated increases in Pparg and Cebpa expression. A previous report suggested that C/EBPb was involved in cell proliferation. 25 We observed that Cebpb expression levels were significantly increased in LAT-A-treated cells on day 10, compared with those in control differentiating cells (Fig. 7B). The increase in cell viability in the LAT-Atreated group shown as in Fig. 2 may be due to the increase in Cebpb expression induced by LAT-A. Cebpb expression was also upregulated in the PGF 2a -treated group (Fig. 7B), although the cell viability did not increase in that group (Fig. 2). This discrepancy may arise from the receptor selectivity of LAT-A and PGF 2a . FIG. 6. Effects of OMD and FP agonists on Pparg mRNA expression on days 2, 4, and 10 of differentiation. Differences in Pparg expression between time points were quantified by using realtime PCR. P < 0.05, compared with control (Wilcoxon test). FIG. 7. Effects of OMD and FP agonists on Cebpa and Cebpb expression levels during the differentiation. Cebpa (A) and Cebpb (B) expression levels on days 2, 4, and 10 were quantified by using real-time PCR. *P < 0.05, compared with control (Wilcoxon test). In the present experiments, OMD did not affect lipid droplet accumulation or the expression levels of Pparg, Cebpa, and Cebpb, even at high concentrations of up to 40 mmol/L (Figs. 3,4,6, and 7). We also confirmed that EP2 and FP receptor mRNAs were expressed by 3T3-L1 cells under these conditions (data not shown), suggesting that OMD stimulates the EP2 receptor. Activated FP receptor initiates several intracellular events, including signaling through the phospholipase C/IP 3 R/Ca 2+ pathway. 30,31 PGF 2a also inhibits adipocyte differentiation via the Ga q -Ca 2+ -calcineurin-dependent signaling pathway. 32 Moreover, coupling of the EP2 receptor with G s leads to elevated cAMP concentrations. 14 Hence, multiple signaling pathways are involved in the downstream effects of FP and EP2 receptors, yet differences between these are considered central to the differing effects of the present receptor agonists on adipocyte differentiation. Previous report has shown that EP4 receptor stimulation increases cAMP and suppresses adipocyte differentiation, 33 although EP4 signaling also plays Ga i -mediated roles that are independent of cAMP. 34 Thus, the effects of EP4 receptor signaling on adipocyte differentiation may be mediated by additional cAMP independent mechanisms. Nonetheless, in contrast with EP4 agonists, the EP2 receptor agonist OMD did not suppress adipocyte differentiation, thus distinguishing the downstream effects of EP2 and EP4 receptors. The mechanism of DUES induced by FP agonists remains unclear, although the prevention of adipogenesis is considered a potentially major cause, as supported by several studies. 12,21 Choi et al. had reported that browning of adipocytes can be related to development of PAP. 35 Using human orbital adipose tissue samples, they reported that bimatoprost upregulates pathways involved in the browning of adipocytes via MAPK, PI3/Akt, and p38. 35 In addition, LAT-A activated the MAPKs extracellular signal-regulated kinase, p38, and c-Jun NH 2 -terminal kinase, 36 indicating that FP agonist-induced PAP is also caused by browning of adipocytes via kinase pathways. In this study, we focused on the effect of OMD on adipogenesis and the regulation of its transcriptional factors (Pparg, Cebpa, and Cebpb) compared with those of FP agonists. However, we may need to investigate the effect of OMD on browning of adipocytes and its related kinase pathways to comprehensively examine the influence of OMD on PAP in future experiments. In this study with 3T3-L1 cells, the highest dose of OMD (40 mmol/L) was equivalent to that of OMDI (0.002%) ophthalmic solution. 37 OMD has strong agonistic activity and selectivity for the human EP2 receptor (hEP2 EC 50 = 8.3 nmol/L). 18 We confirmed these agonistic activities of OMD toward the mEP2 receptor (mEP2 EC 50 = 3.9 nmol/L), and we showed that they are equivalent to those of hEP2 (Fig. 1C). Current results imply that OMDI ophthalmic solution has little to no effect on adipocyte differentiation in humans. Our findings using the 3T3-L1 cell line may have limited the extrapolation to human periocular or orbital adipose tissues. A previous report by Choi et al. using biopsied human orbital adipose tissues indicated that Pparg and Cebpa contributed to their adipogenic differentiation. 38 Further, they demonstrated that FP agonists inhibited the accumulation of intracytoplasmic lipid droplets by downregulation of Pparg and Cebpa, suggesting that FP agonists suppressed adipogenesis in human periocular and orbital adipose tissues in vivo. 38 Similarly, in our study using the 3T3-L1 cell line, we observed upregulation of Pparg and Cebpa with adipogenic differentiation (Fig. 5), and they were significantly inhibited by FP agonists (Figs. 6 and 7), implying that there is at least a partial overlap between the factors involved in adipogenesis in human periocular and orbital adipose tissues and the 3T3-L1 cell line at the transcriptional level. Although further studies are needed to clarify the effects of OMD in human samples, we think that we can estimate the effects of OMD on adipocyte differentiation in humans in the 3T3-L1 cell line compared with FP agonists. Long-term evidence in patients is needed, but current data suggest that OMDI does not induce DUES in glaucoma patients due to the different profile of OMD on gene expression related to adipogenesis in the eyelid fat tissue, unlike existing FP agonists.	v3-fos-license
2018-04-03T03:56:15.634Z	2017-10-26T00:00:00.000	22760887	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/292/51/21159.full.pdf", "pdf_hash": "b079389526e9752167f249bf51588db0bc4ef47d", "pdf_src": "Highwire", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:448", "s2fieldsofstudy": [ "Biology", "Chemistry", "Medicine" ], "sha1": "0ff28f056d422c523bf02e86480f9023007968a1", "year": 2017 }	pes2o/s2orc	Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure. Nicotinic acetylcholine receptors (nAChRs) 3 assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of ␣ (␣2-␣10) and ␤ (␤2-␤4) subunits (1)(2)(3)(4)(5). Many subtypes of these receptors can assemble with varying ratios of subunits, giving rise to multiple stoichiometries that exhibit different subcellular localization and functional properties (6 -9). In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds to and activates these receptors. It has been shown that nicotine influences the trafficking of nAChRs, resulting in increased expression on the cell surface (10 -14). It has also been proposed that nicotine and other ligands alter the assembly of the ␣4␤2 nicotinic receptor, resulting in a decrease in the low-sensitivity stoichiometry, (␣4) 3 (␤2) 2 , and an increase in the high-sensitivity stoichiometry, (␣4) 2 (␤2) 3 (8,(15)(16)(17)(18). Until now, no techniques were capable of specifically determining the stoichiometry of receptors in the ER versus those on the plasma membrane. We developed a single-molecule technique that allowed us to differentiate between receptors localized in the ER and plasma membrane to quantify the stoichiometry of individual receptors (Fig. 1). Single-molecule fluorescence measurements are widely utilized to investigate protein dynamics including the detection of conformational changes, stoichiometry, and protein mobility (17,19,20). Experiments with this level of detail are only feasible after purification of the protein from its physiological cellular environment (21,22). This type of purification is not achievable for many types of membrane proteins, which lose their structural and functional integrity when removed from their native cellular environment. This restricts single-molecule studies to a small number of membrane proteins. Other techniques, such as single-molecule pulldown, have also been used to capture proteins from cellular systems extending single-molecule studies to a wider range of proteins (19,23,24). Still, one of the primary limitations to applying these current approaches to membrane receptors is that organelle-specific information is lost during protein purification. Membrane proteins are synthesized in the ER and then trafficked to the cell surface through the secretory pathway. Assembly of individual subunits that comprise oligomeric membrane proteins also takes place in the ER prior to transport to the cell surface. Understanding drug-induced changes in the distribution of protein isoforms between the ER and plasma membrane is vital to determining how moderately different structural properties can vastly impact functional properties. Isolation of individual membrane receptors in cell-derived nanoscale vesicles composed of original membranes enables the separation of receptors based on organelle (25,26). Investigation of oligomeric proteins from specific organelles at a single-molecule level provides a way to distinguish between different structural and functional populations of these proteins, allowing the effect of changes in assembly on protein trafficking to be directly studied. Here we report a novel approach that enables us to perform organelle-specific single-molecule studies of membrane proteins. We can effectively select populations from the ER and the plasma membrane to quantify properties such as the distribution of stoichiometric assemblies of oligomeric proteins. We applied this technique to study nicotine-induced changes in the assembly of ␣4␤2 nAChRs in the ER and changes in trafficking to the cell surface. Ligand-induced up-regulation of ␣4␤2 receptors Nicotine and several other nicotinic receptor ligands have been shown to up-regulate the number of receptors on the cell surface. It has been hypothesized that this up-regulation is connected to changes in receptor stoichiometry (18,27). We first evaluated a series of ligands to determine whether they altered the expression and trafficking of ␣4␤2 by using a pH-sensitive fluorophore, super ecliptic pHluorin (SEP) (11,28,29). The SEP label was genetically incorporated into the protein sequence of the receptor so that it was on the luminal side of the ER and the extracellular side of the plasma membrane (Fig. 2, A and B). SEP is fluorescent at neutral pH and quenched at acidic pH. Thus, receptors in the ER and plasma membrane will exhibit fluorescence, whereas receptors in the Golgi and trafficking vesicles are not fluorescent. Using total internal refection fluorescence microscopy, we measured ligand-induced up-regulation of ␣4␤2 expression on the plasma membrane as an increase in plasma membrane integrated density (PMID) and a change in the distribution between the ER and plasma membrane (%PM) as compared with control cells (26,30). Exposure to nicotine or cytisine resulted in a 2.5-fold increase, varenicline yielded a 2-fold increase, and bupropion resulted in a 1.5-fold increase in ␣4␤2 expression on the cell surface (Fig. 2C). Additionally, the intracellular distribution of ␣4␤2 between the plasma membrane and peripheral ER (%PM) shifted toward the plasma membrane upon exposure to each of these ligands (Fig. 2D). Comparisons between the ligands showed that nicotine provides the highest level of up-regulation in terms of expression and distribution toward the plasma membrane. Ligand-induced changes in ␣4␤2 assembly To determine whether ligands that up-regulated nicotinic receptors also changed their assembly, we then generated whole-cell nanovesicles from cells expressing ␣4GFP ␤2wt nAChRs (Fig. 3A) and examined the distribution of the two possible ␣4␤2 isoforms, (␣4GFP) 2 (␤2) 3 and (␣4GFP) 3 (␤2) 2 . Nanovesicles were derived from cells both in the presence and absence of the nicotinic ligands. Single receptors were then isolated into membrane-derived vesicles via nitrogen cavitation, immobilized on a glass surface, and imaged using total internal reflection fluorescence (TIRF) microscopy. A representative image of isolated vesicles is shown in Fig. 3B. We then determined the number of photobleaching events (31-33) from the intensity time traces recorded for the fluorescence of each nanovesicles (Fig. 3, C and D). The assignment of the stoichiometric distribution is complicated by the fact that a small fraction of GFP exists in a non-fluorescent state (25,34). Additionally, the observed distribution of photobleaching events arises from a combination of (␣4GFP) 2 (␤2) 3 and (␣4GFP) 3 (␤2) 2 stoichiometries. To account for these factors, the observed distribution was fit to two binomial distributions, corresponding to the distributions of the photobleaching events of (␣4GFP) 2 (␤2) 3 and (␣4GFP) 3 (␤2) 2 . They were weighted iteratively to determine the contribution of each stoichiometry. Results from these unsorted vesicles provided a measure of the whole-cell distribution of ␣4␤2 stoichiometries. In the absence of any ligand, we observed a distribution of 41% (␣4) 2 (␤2) 3 and 59% (␣4) 3 (␤2) 2 (Fig. 4A). The presence of nicotine shifted the distribution of stoichiometry to 59% (␣4) 2 (␤2) 3 and 41% (␣4) 3 (␤2) 2 (Fig. 4B). A comparison of all ligands showed that each altered the assembly toward (␣4) 2 (␤2) 3 with cytisine increasing to 50%, varenicline increasing to 54%, and bupropion increasing to 55% Nitrogen cavitation is used to fragment the cells forming small membrane domains from cellular organelles. These membrane domains spontaneously form nanoscale vesicles. The domains and subsequent vesicles are small enough that there is a low probability of more than one receptor being encapsulated. The resulting vesicles have the same membrane properties as the organelle of origin, thus maintaining a physiological environment. Differences in the densities between the organelle membranes are used to separate them via gradient centrifugation. Vesicles are isolated on glass substrates for TIRF imaging. Altered stoichiometry of nAChRs of the high-sensitivity stoichiometry (Fig. 4, C-E). The observed distribution of bleaching steps is shown in Table 1. Organelle-specific stoichiometry of ␣4␤2 We isolated nanovesicles and then sorted them via gradient centrifugation into those derived from the ER and plasma membrane (Fig. 1). Differences in the density of endogenous ER and plasma membrane allow nanovesicles originating from these organelles to be separated using a density gradient (35). We verified the separation of vesicles using organelle-specific antibodies via Western blot analysis. The ER marker, anti-calnexin, was only found in the three fractions with the highest density, whereas the PM marker, anti-plasma membrane calcium ATPase (PMCA) or anti-Na ϩ /K ϩ ATPase was found in fractions with the lowest density (Fig. 5). We utilized this organelle-specific approach to distinguish between changes in receptor stoichiometry during assembly versus altered trafficking. Because nicotinic receptors are synthesized in the ER, differences in assembly are reflected in the stoichiometry of this population. Receptors are trafficked to the cell surface after assembly; thus, a change in the stoichiometry on the plasma membrane reflects preferential trafficking or increased stability on the cell surface. We performed separate single-molecule studies on both the ER and plasma membrane-specific nanovesicles. Single-molecule photobleaching analysis relies on the observation of single step bleaching events of GFP where each bleaching event corresponds to a single subunit. Our results using GFP-labeled ␣ subunits showed that the predominately expressed stoichiometry of ␣4␤2 nAChRs depends on the subcellular region. Receptors encapsulated in nanovesicles derived from the ER show that in the absence of nicotine, ␣4␤2 nAChRs predominately assemble with the low-sensitivity stoichiometry of (␣4) 3 (␤2) 2 . The distribution of photobleaching events from the ER resident ␣4␤2 nAChRs fit to a theoretical distribution showing that TIRF imaging of SEP, a pH-sensitive analog of GFP, is used to determine the expression and distribution of receptors between the ER and plasma membrane. SEP was genetically encoded into the ␣4 subunit to generate an ␣4-SEP construct. The ␣4-SEP ␤2-wt nicotinic receptors were expressed in N2a cells and imaged under TIRF. A, when the pH of the extracellular solution (ECS) was maintained at 7.4, receptors both in the ER and in the plasma membrane were observable. The observed fluorescence intensity is due to both the ER and plasma membrane receptor populations. B, when the extracellular solution was replaced with a pH 5.4 solution, all SEP on the plasma membrane transition to a non-fluorescent state, and only the receptors within the ER are visible. C, the integrated density of ␣4␤2 on the plasma membrane increased from ϳ2.5 ϫ 10 6 in the absence of any compound to 7 ϫ 10 6 in the presence of nicotine (Nic) or cytisine (Cyt). Varenicline (Var) and bupropion (Bup) both resulted in a 2-fold increase in the integrated density on the plasma membrane demonstrating ligand-induced up-regulation. D, the percentage of the receptors present on the plasma membrane increased from 21.5% for control cells to 30 -40% for all nicotinic receptor ligands, showing a shift in distribution of receptors toward the plasma membrane (n ϭ 61, 47, 42, 38, 51). The data are mean values Ϯ S.D.). ***, p Ͻ 0.001. Integrated density (average fluorescence intensity ϫ area) is the total gray values background within a region of interest that encompasses a cell. PMID is obtained by subtracting the integrated density of pH 5 image of a cell from pH 7 image of the same cell. Figure 4. Whole-cell evaluation of (␣4) 2 (␤2) 3 versus (␣4) 3 (␤2) 2 assembly upon exposure smoking cessation agents. Expected distributions of one, two, and three photobleaching steps were obtained by weighting two binomial distributions. A 2 goodness-of-fit test was used to verify expected and observed distributions of two and three GFP-labeled ␣4 subunits. A, in the absence of a pharmacological agent, the ␣4␤2 population exists as 41% (␣4) 2 (␤2) 3 and 59% (␣4) 3 (␤2) 2 . B, 500 nM nicotine alters the ratio of isoforms to 59% (␣4) 2 (␤2) 3 and 41% (␣4) 3 (␤2) 2 . C, 500 nM cytisine shifts the stoichiometry to 50% high-sensitivity receptors. D, 500 nM varenicline shifts the distribution to 54% high-sensitivity receptors. E, 500 nM bupropion shifts the stoichiometry to 55% high-sensitivity receptors, (␣4) 2 (␤2) 3 . The error bars for the subunit distribution are based on counting events and are calculated as the square root of the counts. Nicotine changes the stoichiometry of ␣4␤2 in the ER We next prepared ER and plasma membrane-specific vesicles from cells expressing ␣4␤2 in the presence of 500 nM nicotine. We observed a clear nicotine-induced shift in the stoichiometry of ER resident ␣4␤2 receptors (Fig. 6C). When nicotine was present, single-molecule bleaching step analysis showed the majority of endoplasmic ␣4␤2 assembled as the high-sensitivity isoform, fitting a 55% (␣4) 2 (␤2) 3 and 45% (␣4) 3 (␤2) 2 distribution (Fig. 6C). This shift from the stoichiometry seen in the absence of nicotine indicates that nicotine drives the assembly of the high-sensitivity isoform, (␣4) 2 (␤2) 3 . Although some groups have previously hypothesized that nicotine alters the assembly of ␣4␤2 receptors (8,9,36,37), these organelle-specific single-molecule studies allowed us to directly observe the process of nicotine altering the assembly of receptors in the ER for the first time. In addition to nicotine altering the assembly of ␣4␤2 within the ER, the percentage of the high-sensitivity (␣4) 2 (␤2) 3 stoichiometry on the plasma membrane was also increased (Fig. 6D). After exposure to nicotine, the distribution of photobleaching events obtained from plasma membraneresident ␣4␤2 nicotinic receptors was fit to a distribution of 70% (␣4) 2 (␤2) 3 and 30% (␣4) 3 (␤2) 2 (Fig. 6D). Discussion We have developed a new technique that allows us to perform organelle-specific single-molecule studies on membrane proteins. We utilized this novel method to determine that changes in nicotinic receptor stoichiometry related to nicotineinduced up-regulation (40) are likely driven by both changes in the assembly in the ER and the preferential trafficking of the high-sensitivity stoichiometry. The consequences of these changes are an increase in the number of receptors both in the ER and on the plasma membrane, as well as a shift in stoichiometric distribution toward the high-sensitivity assembly. Previous studies to measure ER specific changes in stoichiometry have primarily been limited by a lack of existing techniques that are capable of directly quantifying subcellular specific structural assemblies of complex proteins in a physiological cellular environment. The isolation of membrane proteins in organellespecific nanovesicles provides a snapshot of membrane protein assembly in each subcellular location at the time the vesicles are generated. We observed that nicotine, cytisine, varenicline, and bupropion all up-regulated the number of receptors on the cell surface. We also observed that these same compounds all altered the stoichiometry of ␣4␤2 nAChRs toward the highsensitivity stoichiometry. Nicotine induced the largest increase in membrane expression and the largest shift toward the high-sensitivity stoichiometry, (␣4) 2 (␤2) 3 . The only previous single-molecule study of ␣4␤2 stoichiometry on the plasma membrane showed that cytisine elicited a shift toward the lowsensitivity stoichiometry in contrast to our findings (17). This previous study only examined the stoichiometry in the very tip of filopodia projected into 150 -200-nm apertures. This restricted studies to a specialized surface domain likely accounting for the differences seen here. Our studies sample Control 767 53 386 328 54 Nicotine 192 22 112 58 9 Cytisine 357 41 187 130 17 Varenicline 833 94 459 280 30 Bupropion 1089 102 598 Altered stoichiometry of nAChRs the entire plasma membrane, providing a snap shot of the whole population trafficked to the cell surface. Our results indicate that a wide variety of molecules with different pharmacological properties including agonists, partial agonists, and antagonists all alter receptor assembly. This suggests a possible connection between ligand-induced up-regulation and changes in receptor assembly. We then performed organelle-specific single-molecule studies of ␣4␤2 nAChRs in the presence and absence of nicotine to resolve the connection between increased expression and changes in stoichiometry. Comparing the distribution between the low-sensitivity stoichiometry and high-sensitivity stoichiometry in the ER and plasma membrane showed a much larger fraction of receptors exhibiting the high-sensitivity stoichiometry on the plasma membrane. This strongly suggests that the high-sensitivity stoichiometry traffics to the cell surface more efficiently than the low-sensitivity stoichiometry. We also observed a shift in the ER stoichiometry toward the high-sen-sitivity isoform in the presence of nicotine. This suggests that nicotine induces an intracellular change in the assembly of the ␣4␤2 nAChR. Biased transfection can shift the production of the ␣4␤2 toward the high-sensitivity isoform. Single-molecule photobleaching event analysis of vesicles from biased transfections experiments confirmed a shift toward the high-sensitivity stoichiometry in vesicles originating from the ER. We observed an even larger shift toward the high-sensitivity stoichiometry on the plasma membrane. The presence of a higher proportion of the high-sensitivity subtype in the plasma membrane compared with the ER verifies the preferential trafficking of the (␣4) 2 (␤2) 3 stoichiometry from the ER to plasma membrane. Fig. 8 summarizes the fitted values from Fig. 6 to illustrate the shift in stoichiometry. The observed differences in organelle stoichiometry show that endogenous assembly in the ER favors the low-sensitivity stoichiometry but that the high-sensitivity isoform is preferentially trafficked from the ER to the plasma membrane. Despite having a lower fraction in the ER, this preferential trafficking results in a larger fraction of high-sensitivity receptors on the plasma membrane. Recent work by several groups has proposed that nicotine acts as a pharmacological chaperone either altering the assembly of nAChRs or influencing the trafficking. Previous studies have also shown that this increases the numbers of ␣4␤2 nAChRs in the ER and the plasma membrane. In these studies, we confirm that increased numbers of receptors in the ER and altered assembly likely play Figure 6. Single-molecule bleaching step analysis shows organelle-specific differences in ␣4␤2 nAChR isoforms. A, the observed ratio of vesicles showing one, two, or three steps was 0.057, 0.43, and 0.51, respectively (blue columns). These observed values were then fit to a 30:70 (high-sensitivity:lowsensitivity) stoichiometry. The fit was verified using a 2 goodness-of-fit analysis. B, the expression of ␣4␤2 nAChRs on the plasma membrane fit binomial distributions weighted for 56% (␣4) 2 (␤2) 3 and 44% (␣4) 3 (␤2) 2 . The observed fraction of vesicles showing one, two, or three bleaching steps was 0.12, 0.56, and 0.32, respectively. C, for ER resident receptors in the presence of nicotine, the observed fraction of one, two, and three bleaching steps were 0.086, 0.59, and 0.33, respectively. These observed values were then fit to a 55:45 distribution. D, the observed fraction of vesicles with one, two, or three bleaching steps were 0.11, 0.67, and 0.22, respectively. This was fit to a 70:30 distribution. The error bars for the subunit distribution are based on counting events and are calculated as the square root of the counts. Altered stoichiometry of nAChRs roles in plasma membrane up-regulation but are only part of the mechanism. Upon addition of nicotine, the assembly of subunits into a pentamer within the ER is altered to a higher ratio of high-sensitivity receptors that can then be efficiently trafficked to the cell surface. This suggests a mechanism of nicotine-induced plasma membrane up-regulation that is tied to increased numbers of receptors in the ER, preferential trafficking, and a change in assembly. The shift in assembly of ␣4␤2 nAChRs within the ER upon exposure to nicotine toward the preferentially trafficked high-sensitivity isoform is likely responsible in part for the nicotine-induced up-regulation that has been previously observed. It is possible that residues in the M1-M2 and M3-M4 loops on the intracellular side of each of the subunits of ␣4␤2 regulate preferential trafficking to the cell surface. These intracellular loops contain a number of ER retention and ER exit motifs, as well as sites that undergo post-translational modification in the secretory pathway (9,41). These same processes are also responsible for targeted trafficking to neuronal subcellular regions. Additionally, recycling from the Golgi back to the ER has been shown to be necessary for nicotine-induced upregulation of some nicotinic receptor subtypes (16). It is likely that differences in post-translational modification sites of the intracellular regions of ␣4 and ␤2 lead to the observed differences in trafficking between the two stoichiometries. Employment of our organelle-specific single-molecule method enabled the distinction between changes in trafficking compared with changes in assembly of ␣4␤2 nicotinic receptors to partially delineate the underlying mechanism of nicotine-induced up-regulation. Experimental procedures Plasmid constructs for fluorescently labeled nicotinic receptors (SEP and GFP) were generated as previously reported (26). The ␣4-SEP construct was made by fusing the DNA sequence of SEP to the 3Ј end of the DNA sequence of the ␣4 subunit. GFP constructs were made by inserting the label between the M 3 and M 4 transmembrane segments of the ␣4 subunit. Both constructs have been shown to produce functional receptors in previous studies (25,42,43). Cell culture Undifferentiated mouse neuroblastoma 2a (N2a) cells were employed to study the trafficking of ␣4␤2 nAChRs. N2a cells were cultured and maintained with an N2a growth medium (equal volume mixture of DMEM and OptiMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C with 5% CO 2 in a humidified incubator. Approximately 90,000 N2a cells were plated on a poly-D-lysine-coated glass-bottomed dish (35 mm in diameter; Cell E&G, San Diego, CA). The coated dish was prepared by incubating it with 0.1% poly-D-lysine in sterile deionized water at 37°C for 1 h. The unbound poly-D-lysine was removed by rinsing with sterile deionized water, and the dish was dried for 2 h in a biosafety hood. After 16 -24 h, the N2a cells were transfected with 500 ng The expected distribution of one, two, and three photobleaching steps was determined by weighting two binomial distributions, and the fit of expected and observed distribution was validated using a 2 goodness-of-fit test. The assigned weights represent the proportion of the high-and low-sensitivity stoichiometries. A, the observed photobleaching distribution of the receptors obtained from the whole-cell homogenate fit with the expected distribution obtained with 73% (␣4) 2 (␤2) 3 and 27% (␣4) 3 (␤2) 2 . B, the ER originated receptors exhibited a photobleaching step distribution which agreed with 67% (␣4) 2 (␤2) 3 and 33% (␣4) 3 (␤2) 2 stoichiometries. C, the stoichiometry for receptors from the plasma membrane was 82% (␣4) 2 (␤2) 3 and 18% (␣4) 3 (␤2) 2 . The error bars for the subunit distribution are based on counting events and are calculated as the square root of the counts. Figure 8. Organelle-specific single-molecule studies reveal a combination of endogenous preferential trafficking, and an intracellular increase in assembly may be responsible for nicotine-induced up-regulation. A, organelle-specific single-molecule photobleaching step studies of stoichiometry show that in the absence of nicotine, ␣4␤2 predominately assembles into the 3␣ stoichiometry (blue) (70%). B, in the absence of nicotine, the 2␣ stoichiometry (green) is preferentially trafficked to the cell surface, resulting in a higher proportion of receptors on the cell surface having the 2␣ stoichiometry. C, in the presence of nicotine, the intracellular assembly of ␣4␤2 is altered to favor the high-sensitivity, 2␣ isoform (green). D, the increase in availability of the preferentially trafficked stoichiometry, (␣4) 2 (␤2) 3 , leads to an even higher proportion of the 2␣ stoichiometry (green) on the plasma membrane (70%). Altered stoichiometry of nAChRs of each ␣4-SEP and ␤2-wt plasmids with 2 l of Lipofectamine 2000 as described previously (26). Briefly, cells were transfected for 24 h, followed by a 24-h incubation in growth media prior to imaging. Transfection mix was prepared by incubating a mixture of 250 l of OptiMEM and 2 l of Lipofectamine 2000 transfection agent for 5 min at room temperature, followed by a 25-min room temperature incubation upon combination with a mixture of 250 l of OptiMEM and 500 ng of each plasmid DNA. The 500 l of transfection mix was added to preplated cells in 1.5 ml OptiMEM. After 24 h, transfection medium was replaced with N2a growth medium for an additional 24-h incubation. Transfected cells were imaged 48 h after initial transfection. When applicable, 500 nM of each nicotinic ligand, (Ϫ)-nicotine hydrogen tartrate salt (Ͼ98%), bupropion hydrochloride (Ͼ98%), varenicline tartrate, or (Ϫ)-cytisine (Ͼ99%), was added to the transfection medium and replenished later in the growth medium. Transfection efficiency was generally 80% and was not significantly altered by the presence of any of the ligands. Total internal reflection fluorescence The TIRF microscope system employed to visualize SEP or GFP molecules was previously described (26,44). Briefly, a 488-nm DPSS laser excitation source was directed toward the back aperture of an objective (60ϫ, 1.49 NA) mounted on an inverted microscope (Olympus IX81). The angle of excitation light was adjusted to obtain total internal reflection through the objective using a stepper motor that translated the beam across the back aperture of the objective. The emission was collected though the objective, and a dichroic mirror was used to direct the light to an EMCCD camera (Andor). Receptor expression and distribution For in vivo fluorescence imaging studies, the growth medium of the transfected N2a cells was replaced with an extracellular solution (10 mM HEPES, 10 mM D-glucose, 150 mM NaCl, 4 mM KCl, 2 mM MgCl 2 , and 2 mM CaCl 2 ) of pH 7.4. The dish was then mounted on a translational stage, and the cells were located by exciting the SEP molecules with a 488-nm laser (ϳ1 milliwatt) source. Images of cells were captured using an EMCCD camera with a 200-ms exposure time. The extracellular solution was then replaced with an identical solution of pH 5.4, followed by a 10-min stage-top incubation before capturing images of the same cells. An open source software, ImageJ (National Institutes of Health) was employed to analyze the images. Background was subtracted using the rolling ball background subtraction with a diameter of 25 pixels. A freehand region of interest (ROI) was drawn around a cell, and an intensity based threshold was used to obtain an integrated density for each cell. The integrated density of the cell at pH 5.4 (ER ID) is subtracted from the integrated density of the same cell at pH 7.4 (total ID) to calculate the relative number of receptors on the plasma membrane or PMID. The percentage of receptors located on the plasma membrane within the TIRF region of excitation (%PM) is calculated by dividing the PMID by the total ID at pH 7.4, multiplied by 100. The data are reported as the means Ϯ S.D. Nanovesicle preparation HEK-293T cells were cultured and maintained with a growth medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C temperature with 5% CO 2 in a humidified incubator. Three million HEK-293T cells were plated in a Matrigel-coated T75 flask 16 -24 h prior to transfection. The cells were transfected with 14 l of Lipofectamine 2000 and 3.5 g of each plasmid as previously described (25,26). For biased expression experiments, a 1:10 transfection ratio of ␣4-GFP:␤2-wt using 1 g of ␣4-GFP and 10 g of ␤2-wt was employed. Briefly, a mixture of 250 l of OptiMEM and the above mentioned amount of ␣4-GFP and ␤2-wt plasmids was prepared. Separately, 14 l of Lipofectamine 2000 was added to 250 l of OptiMEM and incubated for 5 min at room temperature before being added to the DNA mixture. This new mixture was incubated at room temperature for 25 min. Afterward, the transfection mixture was added to the flask of HEK-293T cells. The following day, vesicles were prepared from transfected cells as previously described (25). Briefly, the cells underwent nitrogen cavitation at 250 p.s.i. for 5 min while suspended in 5 ml of sucrose-HEPES buffer supplemented with a protease inhibitor (250 mM sucrose, 10 mM HEPES, 1 Pierce protease inhibitor mini tablet per 10 ml of buffer (ThermoScientific), pH 7.5). Cell lysate was then centrifuged at 4000 ϫ g for 10 min. Supernatant was collected and centrifuged at 10,000 ϫ g for 20 min. Supernatant was again collected and centrifuged at 100,000 ϫ g for 1 h. The pellet was resuspended in 800 l of sucrose-HEPES buffer (250 mM sucrose, 10 mM HEPES, pH 7.5). Nanovesicles were stored at Ϫ80°C until use. Generation of ER and plasma membrane vesicles HEK-293T cells were transfected as described above. After transfection for 24 h, transfection mix was removed, and the cells were rinsed once with PBS. To generate nanoscale plasma membrane vesicles containing a single nAChR, transfected cells were first swollen for 20 min in a hypotonic solution (10 mM NaCl, 10 mM Tris-HCl, 1.5 mM MgCl 2 , 0.2 mM CaCl 2 , pH 7.4) at 0°C. To prepare both plasma membrane and ER-derived vesicles, the cells were treated with 5 ml of 1ϫ Versene (Invitrogen), incubated at 37°C for 5 min, and pelleted by centrifugation at 200 ϫ g for 5 min, as previously described. The cell pellet was resuspended in 3 ml of sucrose buffer plus protease inhibitors (250 mM sucrose, 10 mM HEPES, 1 Pierce protease inhibitor mini tablet per 10 ml of buffer (ThermoScientific), pH 7.5) before undergoing nitrogen cavitation in a nitrogen decompressor (Parr Instrument Company, Moline, IL). To generate ER nanovesicles, the cells were pressurized to ϳ250 p.s.i. for 20 min. At this pressure, plasma membrane rupturing is minimal, and therefore nanoscale vesicle formation from this organelle is negligible. To generate plasma membrane nanovesicles, the cells were pressurized to ϳ600 p.s.i. for 20 min. The cell lysate was collected and dispensed onto an OptiPrep gradient. Separation of organelle-specific vesicles Chemical & Scientific Corp., Westbury, NY) gradient was used Altered stoichiometry of nAChRs to purify organelle-specific nanovesicles. Gradient solutions of OptiPrep were prepared by diluting the 60% stock solution to 30,20, and 10% in sucrose-HEPES buffer (250 mM sucrose, 10 mM HEPES, pH 7.5) and stored at 4°C. The gradient was prepared in an Ultra-Clear centrifuge tube (Beckman Coulter), with 3 ml of the densest fraction added first. ER or plasma membrane nanovesicles containing cell lysate, based on nitrogen pressure during cavitation, was dispensed on top of the 10% fraction, before centrifugation at 112,000 ϫ g for 1.5 h. After centrifugation, nine 1-1.5-ml fractions, with density interfaces in the same fraction, were collected using a peristaltic pump. Tubing connected to the pump was vertically inserted into the centrifuge tube so that the highest density fraction is collected first. After fractionation, OptiPrep was removed from nanovesicles by centrifugation at 10,000 ϫ g for 1 h. Western blot analysis Resuspended OptiPrep fractions containing membrane proteins were ran on a prepackaged NuPAGE 4 -12% Bis-Tris gel (Life Technologies), followed by transfer to a nitrocellulose membrane. The membrane was first blocked for 1 h with a PBST solution (5% nonfat milk, 0.1% Tween in PBS). Primary antibodies specific for calnexin (Santa Cruz, calnexin antibody (H-70), catalog no. sc-11397) or PMCA (Santa Cruz, PMCA antibody (D-1), catalog no. sc-271193) were added to the membrane in a 1:1000 dilution and incubated overnight at 4°C. Endogenous calnexin is solely found in the membrane of the ER, whereas PMCA is expressed on the plasma membrane, thus providing a means to identify fractions that consist of exclusively ER or plasma membranes. After overnight incubation, primary antibodies were removed by four repeated 5-min washes with PBST. Secondary rabbit antibody (calnexin) or mouse antibody (PMCA) (Jackson ImmunoResearch) was added in a 1:5000 dilution and incubated for 1 h at room temperature, followed by another series of four repeated 5-min washes with PBST. Bands were visualized by addition of Western blotting substrate for chemiluminescence (Clarity; Bio-Rad) on a Chemi-Doc system (Bio-Rad). To validate these results, blots were repeated with a completely different set of antibodies. For primary antibodies, we used 1:2000 diluted rabbit monoclonal anti-calnexin (catalog no. ab92573, Abcam) for ER identification and 1:2000 diluted rabbit monoclonal anti-Na,K-ATPase (catalog no. ab76020, Abcam) for plasma membrane identification. In both sets of orthogonal studies, the ER and plasma membrane bands matched the expected molecular weights. Imaging nanovesicles A 35-mm glass-bottomed dish was cleaned by sonicating the dish in 5 M NaOH solution for 30 min at 45°C and then in 0.1 M HCl solution for 30 min at 45°C. The dish was rinsed with water, sprayed with 100% ethanol three times after each step, and then dried using compressed air. Finally, the dishes were treated in an oxygen plasma (21% oxygen for ϳ5 min). A biotinylated anti-GFP antibody functionalized glass-bottomed dish was prepared by incubating a cleaned dish at room temperature with 1 mg/ml Silane-PEG-Biotin in 95% ethanol for 30 min, 0.1 mg/ml NeutrAvidin in PBS (1ϫ PBS, pH 7.4) solution for 5 min, and finally 1 g/ml biotinylated anti-GFP antibody in PBS for 15 min. Between each of the steps, the dish was rinsed three times with 1ϫ PBS solution. Vesicles were immobilized on the biotinylated anti-GFP antibody functionalized dish by adding 50 -200-fold diluted vesicles in PBS for 30 min at room temperature. The unbound vesicles were removed by rinsing with PBS, and ϳ1 ml of PBS solution was added to the dish. The microscope setup employed to capture images for SEP based studies was also utilized to obtain movies of about 1000 frames (100 ms of exposure time) during 488-nm laser excitation (ϳ3 milliwatts). Data analysis A customized software package was written in Matlab to populate time traces from the movies collected with immobilized vesicles. Briefly, the first 10 frames of a movie were combined together to make a composite frame, which was utilized to find peaks with a user defined threshold level. A 3-pixel by 3-pixel ROI was selected for each peak position to obtain the mean intensity of the ROI. A 5-pixel by 5-pixel ring around the peak was selected, and the mean value of the pixels located on the ring was considered as background that was subtracted from the mean value of the ROI of the corresponding frame to obtain a background subtracted mean intensity of the ROI. Time traces for all peaks were stored in a temporary file. During the initial evaluation, a time trace of the temporary file was accepted if the difference of the mean of the intensities of first 20 frames and last 20 frames was more than twice the standard deviation of last 20 frames. All time traces for the qualified molecules were collected and stored for further analysis. A photobleaching step was counted only if it lasted at least 1 s and the intensity levels of a step and the next lower level had a difference of at least twice the standard deviation of the lower level. A time trace was considered to arise from a single-molecule if it showed at least one clear bleaching step. Each set of data was independently analyzed at least twice, and the results were compared. Data fitting The probability of observing a photobleaching event from a GFP molecule is less than 1. Therefore, a binomial distribution was employed to determine the distribution of the number of photobleaching events observed from a population of GFP-labeled receptors. A general equation for observing k number of photobleaching events from n number of GFP-labeled receptors can be written as, F͑k; n, p͒ ϭ n! k!͑n Ϫ k͒! p k ͑1 Ϫ p͒ nϪk (Eq. 1) where p is the probability of observing a photobleaching event from a GFP-labeled subunit, which has been previously determined as 0.90 (25). A matrix (M 2 ) with the probabilities of obtaining one, two, and three photobleaching events from two GFP containing ␣4␤2 nAChRs (i.e. (␣4-GFP) 2 (␤2-wt) 3 ) can be written as follows. Altered stoichiometry of nAChRs Similarly, matrix M 3 , containing the probabilities of observing one, two, and three photobleaching events from three GFP containing ␣4␤2 nAChRs (i.e. (␣4-GFP) 3 (␤2-wt) 2 ), can be expressed as shown in Equation 3. Because the probability of obtaining zero photobleaching events from two or three GFP-labeled ␣4␤2 nAChRs can be greater than zero, the probability distributions M 2 and M 3 were normalized as follows, Because the experimentally observed distribution emerged from a mixture of (␣4-GFP) 2 (␤2-wt) 3 and (␣4-GFP) 3 (␤2-wt) 2 stoichiometries, a theoretical probability distribution (T pd ) was computed by providing a weight to each normalized probability matrix as follows, where a 2 and a 3 are the weights assigned to MЈand MЈ 3 distributions, respectively, and a 2 ϩ a 3 ϭ 1. Therefore, a 2 and a 3 are the proportions of (␣4-GFP) 2 (␤2-wt) 3 and (␣4-GFP) 3 (␤2-wt) 2 stoichiometries, respectively. This probability distribution (T pd ) was multiplied by total number of observed one, two, and three photobleaching events to generate a theoretical distribution. A 2 goodness-of-fit test was employed to compare theoretical and observed distributions. The error bars for subunit distribution are based on counting events and are calculated as the square root of the counts (31). The values of a 2 and a 3 were iteratively assigned and a 2 goodness-of-fit test statistics was calculated for each set of a 2 and a 3 . A customized Matlab script was written to calculate a 2 and a 3 from the best 2 goodnessof-fit test statistics.	v3-fos-license
2020-11-18T14:07:01.001Z	2020-11-16T00:00:00.000	226987510	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.nature.com/articles/s41598-020-76986-3.pdf", "pdf_hash": "0c777b680342774c698ed321fad95726f4dff999", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:516", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "c0b7fe2bed1f28924a598af937d67d6fc1e88170", "year": 2020 }	pes2o/s2orc	Investigation of COSMO-SAC model for solubility and cocrystal formation of pharmaceutical compounds In this study, a predictive model named COSMO-SAC was investigated in solid/liquid equilibria for pharmaceutical compounds. The examined properties were the solubility of drug in the pure and mixed solvents, octanol/water partition coefficient, and cocrystal formation. The results of the original COSMO-SAC model (COSMO-SAC (2002)) was compared with a semi-predictive model named Flory–Huggins model and a revised version of the COSMO-SAC (COSMO-SAC (2010)). The results indicated the acceptable accuracy of the COSMO-SAC (2002) in the considered scope. The results emphasized on the suitability of the COSMO-SAC model for simple molecules containing C, H, and O by covalent and hydrogen bonding interactions. Applicability of the COSMO-SAC for more complicated molecules made of various functional groups such as COO and COOH doubly requires more modification in the COSMO-SAC. Methods COSMO file and sigma profile. As described before, the basis of the COSMO-SAC model is quantum mechanics through density function theory calculations. Several commercial or free software provide preliminary information for COSMO-SAC in the form of a text file called COSMO-file. Dmol 3 module in Materials Studio and academic free software GAMESS are few examples. In COSMO calculations, a molecule separates into several parts called segment and charge distributions over entire segments are calculated in order to neutralize whole molecule. Location of segments, segment areas and charge densities are the computed properties in COSMO file. In order to perform COSMO-SAC calculations, the following data must obtain from COSMO-file: (1) surface area ( A ) and cavity volume of the molecule ( V ), (2) location of segment (a vector with x, y and z coordination), its charge density ( σ * n ) and area ( A n (σ ) ). The mentioned information were modified in order to make the sigma profile ( p(σ ) ) required for COSMO-SAC calculations. Klamt et al. 4 introduced the following equation to average the charge densities from COSMO-file In the above equation, d mn is the distance between two segments n and m. The r n (segment radius) is obtained from segment area as follows: Mullins et al. 16 reported the value of r ave . The sigma profile defined as the probability of finding segments with charge density σ m : where n is determined from accounting the number of segments with specific charge density σ m and A(σ m ) is surface area with charge density σ m . Generally, for most molecules, charge density values range between − 0.025 to 0.025 ė A 2 . Four steps for generating the sigma profile are as below: 1. Consider 50 intervals by 0.001 increments in charge density range − 0.025 to 0. 025. 2. Each interval is defined by lower and upper bounds, σ left and σ right . Firstly, find the charge densities distributed at interval i and calculated their contributions according to: 3. Afterward, calculate probabilities at lower and upper bounds of interval i as below: (1) σ m = n σ * www.nature.com/scientificreports/ 4. The sigma profile is generated by plotting sigma values versus the calculated probabilities. As described in literature review, some authors divided the sigma profile into parts to have a better description of hydrogen-bonding (hb) interactions. Hsieh et al. 22 proposed to separate the sigma profile into non hydrogen bounding, hydroxyl group (OH) and non-hydroxyl group as follows equation (COSMO-SAC (2010)): where p NHB (σ m ) donates probabilities of all non-hydrogen bounding atoms, p OH (σ m ) shows probabilities of OH bounding and p OT (σ m ) determines F, N, and hydrogen atoms connected to F and N atoms. The above-mentioned contributions were determined as follows: where σ o is threshold for hydrogen bounding determination and its values is 0.007 ė A 2 . COSMO-SAC model. COSMO-SAC (2002). In the COSMO-SAC model, activity coefficients computed by solvation energy were obtained from ab initio solvation calculation at two steps: (1) the dissolution of a solute in the conductor, (2) conversion of the conductor into a real solvent. The activity coefficient of component i in solvent S in the COSMO-SAC ( γ i,S ) obtained by considering two contributions; combinatorial part. (γ C i,s ) and residual part(γ R i,s ) as follows 6 : The size and shape differences of the molecules are accounted in the combinatorial part and calculated by the Staverman-Guggenheim term as follows 26 : where θ i , φ i and l i are defined as follows: In the above expressions, q i and r i are related to cavity volume of component i ( V i ) and total surface area of molecule i ( A i ) obtained from the COSMO-file and defined as follows: where r o and q o are the normalized volume and normalized surface area. The residual part of the COSMO-SAC (2002) was defined as follows 6,17 : where n i , effective segment number of molecule i, is correlated with effective segment surface area ( a eff ) and surface area of molecule i ( A i ) according to below expression: where Ŵ(σ m ) is the segment activity coefficient and calculated from: www.nature.com/scientificreports/ The exchange energy �W(σ m , σ n ) is defined: The c hb and σ hb are the energy-type constant and cutoff value for hydrogen bonding interaction 16 . The σ acc and σ don are maximum and minimum values of σ m and σ n . α ′ accounts the misfit energy and the T and R are system temperature and the universal gas constant. The values of above mentioned parameters are reported in Mullins et al. 16 . In Eq. (16), the sigma profile for the mixture ( P S (σ ) ) are obtained from: COSMO-SAC (2010). After establishing NHB, OH, and OT sigma profiles, the segment activity coefficient calculates as follows: where subscript j shows pure liquid or mixture and subscript t denotes NHB, OH, and OT sites. The exchange energy has defined based on interaction between segments of different types, and is given by: In the above equation, φ is the volume fraction ( φ i = x i V i i x i V i ) and V is the molar volume.χ is the Flory-Huggins interaction parameter obtained from the Hansen solubility ( δ ) contributions in the forms non-polar (dispersion) forces (d), polar forces (p) and hydrogen-bonding (h) effects as follows 27 : The Hansen solubility parameters and their contributions were obtained by group contribution methods according to the following equations 28 : . Solid-liquid equilibria. In solid-liquid equilibria, the solid solubility in liquid phase is calculated according to the following expression: where x i and γ i stand the solubility and activity coefficient of compound i. The activity coefficient in the above expression was computed from the considered models as described before. H m , C P and T m represent the fusion enthalpy, the heat capacity of phase change between solid and liquid phases and the melting point temperature, respectively. In the current study, the second term of Eq. (27) was neglected ( C P = 0). Partition coefficient. When the equilibrium condition between two immiscible liquid phases establishes, the components distribute between two phases. The distribution of component i between two phases α and β measured by partition coefficient as follows 15 : where x α i and x where a and b are stoichiometric coefficient of substances A and B in the cocrystal. In the above equations, the K cc is solubility product and are computed by the following equation: The activity coefficients in Eq. (31) computed from the examined model. The solubility product (K CC ) is depend only on temperature and independent to solvent type. By knowing solubility product at single point, it can be applied to other conditions. After obtaining solubility product for desired system, the invariant points as intersections of cocrystal line and solubility line were computed by simultaneous solvation of Eqs. (27) and (31). Afterward, the cocrystal region is determined by varying drug mole fraction between two invariant points and obtaining API mole fraction from Eq. (31). Statistical analysis. In order to explore model precision in comparison to experimental data, several statistics were applied such as absolute average percentage deviation (% AAD), root mean square error (RMSE), mean square error (MSE), normalized root mean square error (NRMSE) and normalized mean square error (NMSE). MSE, NRMSE and NMSE were obtained from goodness of Fit function in MATLAB programming software. Absolute average percentage deviation was calculated as following equations: where cal are exp calculated and experimental data of desired properties and n is number of experimental data. The root mean square error (RMSE) was obtained as follows: Figures 1 and 2 compare sigma profiles generated in current studies for ibuprofen and acetyl salicylic acid in comparison to sigma profiles in the database provided by Mullins et al. 16 . Based www.nature.com/scientificreports/ on Figs. 1 and 2, the same trends between results in this study and Mullins et al. 16 were observed. The small departures between two curves originated from the software version and the sigma profile generation program. After generating the sigma profiles and providing the COSMO-SAC computation program for the activity coefficient, the solubilities in the binary and ternary systems were calculated and compared by experimental data obtained from the literature. 15 . The accuracy of these two COSMO-SAC models has been comprehensively examined through a very large dataset, containing 29,173 data points of infinite dilution activity coefficient and 139,921 VLE data points of 6940 binary mixtures 31 . The mentioned inconsistency arises from different universal constants implemented in sigma profile generation. The differences in investigated systems attribute the second reason for the observed inconsistency. It is interesting that the COSMO-SAC (2002) was obtained by only eight universal constant parameters without any further modifications. A list of considered pharmaceutical compounds and their physical properties and references for experimental data were presented in supplementary materials (Table S1). The Hansen solubility parameters, molar volumes for the Flory-Huggins model and the COSMO molar volume of the examined pharmaceutical compounds and solvents were presented on Table 1. Based on Table1, the molar volume obtained from group contribution method in Barton 28 and the COSMO calculations have some difference. Table 2 reports the COSMO-SAC (2002), the COSMO-SAC (2010), and the Flory-Huggins results for some pharmaceutical compounds categorized by the solvent type and sorted according to absolute average deviations (AAD%). The RMSE results for the COSMO-SAC (2002), the COSMO-SAC (2010), and the Flory-Huggins models were also reported in Table 2. Based on Table 2, the predictive model of the COSMO-SAC (2002) has a wide range of errors that are in agreement with errors reported by Hsieh et al. 15 . The COSMO-SAC (2010) and the Flory-Huggins have larger errors compared to the COSMO-SAC (2002). According to Table 2, pharmaceutical compounds containing H, C and O with the lowest hydrogen bonding numbers have the lower error. Besides, the structure of molecule has a remarkable influence on accuracy. In the case of acetaminophen and acetyl salicylic acid, by solvent replacement from ethanol to acetone, deterioration in model prediction was observed. The impact of eliminating F atom from flurbiprofen observes in the lower error reported for ibuprofen. Although borneol and isoborneol have the same chemical formula, the accuracy of the COSMO-SAC (2002) for them is entirely different. The above studies implied that molecular structure, atoms, and intermolecular interaction must be widely incorporated into the COSMO-SAC model. Since, the COSMO-SAC (2002) provides better approximations of solubility in examined systems, we prefer utilizing the original COSMO-SAC (2002) in our further investigation on the binary and ternary systems. Afterward, two www.nature.com/scientificreports/ models, the COSMO-SAC (2002) and the Flory-Huggins models were considered for the octanol/water partition coefficient and cocrystal formation. Afterward, the ternary systems of pharmaceutical compounds in binary solvents were also examined. On the basis of Table 2, two pharmaceutical compounds, acetaminophen and salicylic acid, were suggested. Acetaminophen consists of 20 atoms H, C, N, and O and two functional groups, OH and NH. Salicylic acid consists of 16 atoms H, C, and O, and two functional groups, OH and COOH. Figure 4 presents the comparison between the experimental and calculated solubilities of acetaminophen in ethanol/water mixtures as a function of ethanol mole fraction at two temperatures, 293.15 and 303.15 K. According to Fig. 4, a good agreement between experimental data and the COSMO-SAC calculations observe. The observed trends of the COSMO-SAC as a function temperature match with the reported experiments. www.nature.com/scientificreports/ Figure 5 shows the calculated solubility of salicylic acid in ethanol/ethyl acetate mixture compared to experimental data. On the basis of Fig. 5, a departure from experimental data was observed at higher ethyl acetate mole fraction. The ethyl acetate has a functional group COO which its interaction with COOH in salicylic acid has been ignored in the COSMO-SAC (2002). The octanol/water partition coefficients for some pharmaceutical compounds obtained from the COSMO-SAC model. In Table 3, the results of the octanol/water partition coefficient from the COSMO-SAC model compared to experimental data from the national library of medicine 34 Table 3, the various accuracies obtained regarding activity ratio in the octanol/water partition coefficient. In the octanol/water partition coefficient, if the errors in the numerator and denominator cancel each other out, a good accuracy between the COSMO-SAC computation and experiment is harvested. Otherwise, the discrepancies in obtained errors were seen. It is possible that the COSMO-SAC model fails for solubility prediction (such as dapsone) but presents a reasonable estimation of the octanol/water partition coefficient due to the above discussions. As observed from Table 3, the simple molecules made of H, C, and O by only hydrogen bonding Table 3, the octanol/water partition coefficients obtained from the Flory-Huggins model are farm from experimental data. In order to investigate a more complex system, a three-phases diagram of ternary system is explored by considering the sulfamethazine/salicylic acid cocrystal formation in methanol at 283.15 K, which studied by Ahuja et al. 35 . Details of calculation and methods were described in "Cocrystal formation" section. After performing the computation by the COSMO-SAC (2002), a triangular diagram of the considered system was plotted by a free software named ProSim Ternary Diagram. On the basis of Fig. 6 and experimental plots in Ahuja et al. 35 , some differences between experiments and the COSMO-SAC calculations were observed. The cocrystal region for SM/SA predicted by the COSMO-SAC is wider, while experimental data imply on the narrow region. The solubility line of SM in SA + ME mixture expanded in the COSMO-SAC model in comparison to experiments which interpreted by the COSMO-SAC ability in the considered system. The predicted solubility line of SA in the SM + SA is appropriately closer to the reported experimental data which indicates the good performance of the COSMO-SAC for SA. The reported inconsistencies in observed results originated from molecular structure, constituent atoms, and their interactions. The electronegative atoms S and N in sulfamethazine create the observed discrepancies, while their contributions were not considered in the COSMO-SAC (2002) model. The ternary phase diagram carbamazepine (CBZ)/acetylsalicylic acid (ASA) in ethanol (ET) at 298.15 K were computed by the COSMO-SAC (2002) and plotted in Fig. 7. Veith et al. 29 studied the CBZ/ASA/ET by PC-SAFT EOS. According to Veith et al. 29 , the PC-SAFT EOS without binary interaction parameters estimated the narrow www.nature.com/scientificreports/ Conclusions The COSMO-SAC as a predictive model has been gained a great attention in thermodynamic modeling and phase equilibria considerations. The eight universal parameters and predefined atomic radiuses for C, H, O, S, N, F, and Cl are the general basis of the COSMO-SAC model. In the current study, the COSMO-SAC model implemented in solid-liquid phase equilibria in form of solubility data in binary and ternary systems, octanol/ water partition coefficient, and cocrystal studies. For more comparison, the COSMO-SAC model was also compared with the Flory-Huggins model. The obtained results implied that molecular structure, constituent atoms, functional group, and their interactions have remarkable impacts on the obtained results. In general, the simple molecules made of atoms H, C, and O under special condition, atom N by simple covalent and hydrogen bonding interactions can be deliberated by the COSMO-SAC model. The presence of other atoms such as F and S and other functional groups such as COO and COOH made complex systems. This complexity provides some opportunities to modify the original the COSMO-SAC model. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.	v3-fos-license
2020-05-02T13:03:52.488Z	2020-04-30T00:00:00.000	218470637	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://insight.jci.org/articles/view/137017/files/pdf", "pdf_hash": "4cac2a14339dc2acb6f9d1164e168f910881561a", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:518", "s2fieldsofstudy": [ "Biology", "Medicine" ], "sha1": "f1c3830e312157b2171f3f118185051f12ba348e", "year": 2020 }	pes2o/s2orc	Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells secretion, while activation of G q signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca 2+ . The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells. The 3D multicellular human islet architecture, while essential for islet cell function presents experimental challenges for mechanistic studies of intracellular signaling pathways. Using primary human islets, we developed a pseudoislet system that resembles native human islets in morphology, cellular composition, cell identity, and dynamic insulin and glucagon secretion. This system allows for efficient virally mediated genetic manipulation in almost all cells in the pseudoislet. To evaluate the coordination between intracellular signals and islet hormone secretion, we developed an integrated system consisting of pseudoislets and a microfluidic device that enables studies of islet intracellular signaling using genetically encoded biosensors in conjunction with hormone secretion. Furthermore, we used this integrated approach to define aspects of human islet biology by investigating GPCR signaling pathways using DREADDs and a calcium biosensor. Despite α and β cells both being excitable secretory cells and sharing many common developmen-tal and signaling components, this experimental approach allowed us to demonstrate similar and distinct responses to activation of GPCR signaling pathways, highlighting the uniqueness in each cell’s molecular machinery. The activation of G i signaling was inhibitory in β and α cells, resulting in reduced insulin and glucagon secretion, respectively, and showed a more substantial affect in β cells, where this signaling blunt-ed insulin response to both a glucose ramp and to KCl-mediated depolarization. Interestingly, direct KCl depolarization was not sufficient to overcome these inhibitory effects in either β or α cells, suggesting that reduced cAMP via the inhibition of adenylyl cyclase, in addition to cAMP-independent pathways (48), plays a role in both insulin and glucagon secretion. These results align well with those of recent studies in β cells, suggesting that cAMP tone is crucial for insulin secretion, and observations in α cells highlighting cAMP as a key mediator of glucagon secretion (13, 17, 19, 49). There were major differences in response to activation of G q signaling in β cells compared with α cells. 1% 2 mM in 5% at 37 o for <24 hours before beginning studies. This study used data from the Organ Procurement and Transplantation Network (OPTN) that was in part compiled from the data hub accessible to IIDP-affiliated investigators through the IIDP portal (https://iidp.coh.org/secure/isletavail). The OPTN data system includes data on all donors, wait-listed candidates, and transplant recipients in the US, submitted by the members of OPTN. The Health Resources and Services Administration of the US Department of Health and Human Services provides oversight of the activities of the OPTN contractor. The data reported here have been supplied by UNOS as the contractor for OPTN. The interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an official policy of or interpretation by the OPTN or the US government. Introduction Pancreatic islets of Langerhans, small collections of specialized endocrine cells interspersed throughout the pancreas, control glucose homeostasis. Islets are composed primarily of β, α, and δ cells but also include supporting cells, such as endothelial cells, nerve fibers, and immune cells. Insulin, secreted from the β cells, lowers blood glucose by stimulating glucose uptake in peripheral tissues, while glucagon, secreted from α cells, raises blood glucose through its actions in the liver. Importantly, β and/or α cell dysfunction is a key component of all forms of diabetes mellitus (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Thus, an improved understanding of the pathways governing the coordinated hormone secretion in human islets may provide insight into how these may become dysregulated in diabetes. In β cells, the central pathway of insulin secretion involves glucose entry via glucose transporters where it is metabolized inside the cell, resulting in an increased ATP/ADP ratio. This shift closes ATP-sensitive potassium channels, depolarizing the cell membrane and opening voltage-dependent calcium channels where calcium influx is a trigger of insulin granule exocytosis (12). In α cells, the pathway of glucose inhibition of glucagon secretion is not clearly defined, with both intrinsic and paracrine mechanisms proposed (13)(14)(15). Furthermore, gap junctional coupling and paracrine signaling between Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of G i and G q GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of G i signaling reduced insulin and glucagon secretion, while activation of G q signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca 2+ . The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells. islet endocrine cells and within the 3D islet architecture are critical for islet function, as individual α or β cells do not show the same coordinated secretion pattern seen in intact islets (16)(17)(18)(19)(20). The 3D islet architecture, while essential for function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Furthermore, human islets show a number of key differences from rodent islets, including their endocrine cell composition and arrangement, glucose set point, and both basal and stimulated insulin and glucagon secretion, highlighting the importance of studying signaling pathways in primary human cells (21)(22)(23)(24). To study signaling pathways in primary human islet cells within the context of their 3D arrangement, we developed an integrated approach that consists of (a) human pseudoislets closely mimicking native human islet biology and allowing for efficient genetic manipulation and (b) a microfluidic system with the synchronous assessment of intracellular signaling dynamics and both insulin and glucagon secretion. Using this experimental approach, we demonstrate differences in G q and G i signaling pathways between human β and α cells. Human pseudoislets resemble native human islets and facilitate virally mediated manipulation of human islet cells. To establish an approach that would allow manipulation of human islets, we adapted a system where in human islets are dispersed into single cells and then reaggregated into pseudoislets (25-29) ( Figure 1A and see Vanderbilt Pseudoislet Protocol in Supplemental Information for detailed protocol; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.137017DS1). To optimize the formation and function of human pseudoislets, we investigated two different systems to create pseudoislets, a modified hanging drop system (30,31) and an ultralow attachment microwell system. We found that both systems generated pseudoislets of comparable quality and function (Supplemental Figure 1, A and B) and thus combined groups for comparisons between native islets and pseudoislets. A key determinant of pseudoislet quality was the use of a nutrient-and growth factor-enriched media (termed Vanderbilt pseudoislet media; see Vanderbilt Pseudoislet Protocol in Supplemental Information for detailed protocol). Pseudoislet morphology, size, and dithizone (DTZ) uptake resembled that of normal human islets (Figure 1, B-D). Pseudoislet size was controlled to between 150 and 200 μm in diameter by adjusting the seeding cell density and thus resembled the size of an average native human islet. Compared with native islets from the same donor cultured in parallel using the same pseudoislet media, pseudoislets had similar insulin and glucagon content, though insulin content was reduced in pseudoislets from some donors ( Figure 1E). Endocrine cell composition was also similar, with the ratio of β, α, and δ cells in pseudoislets unchanged compared with that in cultured native islets from the same donor (Figure 1, F and G). As the primary function of the pancreatic islet is to sense glucose and other nutrients and dynamically respond with coordinated hormone secretion, we assessed the function of pseudoislets compared with native islets by perifusion. We used the standard perifusion (herein referred to as macroperifusion) approach of the Human Islet Phenotyping Program of the Integrated Islet Distribution Program (IIDP), which has assessed nearly 300 human islet preparations. In this system, approximately 250 islet equivalents (IEQs) are loaded into a chamber and exposed to basal glucose (5.6 mM glucose; white) or various secretagogues (16.7 mM glucose, 16.7 mM glucose and 100 μM isobutylmethylxanthine [IBMX], 1.7 mM glucose and 1 μM epinephrine, 20 mM potassium chloride [KCl]; yellow) ( Figure 1H). Pseudoislet insulin secretion was very similar to that of native islets in biphasic response to glucose, cAMP-evoked potentiation, epinephrine-mediated inhibition, and KCl-mediated depolarization ( Figure 1H). Pseudoislets and native islets also had comparable glucagon secretion, which was inhibited by high glucose and stimulated by cAMP-mediated processes (IBMX and epinephrine) and depolarization (KCl) ( Figure 1I). Compared with native islets on the day of arrival, pseudoislets largely maintained both insulin and glucagon secretion after 6 days of culture, with the exception of a slightly diminished second-phase glucose-stimulated insulin secretion and an enhanced glucagon response to epinephrine in cultured native islets and pseudoislets (Supplemental Figure 1, C-N). These results demonstrate that, after dispersion into the single-cell state, human islet cells can reassemble and reestablish intraislet connections crucial for coordinated hormone release across multiple signaling pathways. Interestingly, the islet architecture of both native whole islets and pseudoislets cultured for 6 days showed β cells primarily on the islet periphery, with α cells and δ cells situated within an interior layer. Furthermore, the core of both the cultured native islets and pseudoislets consisted largely of extracellular Additionally, the islet cell arrangement suggests that extracellular matrix and endothelial cells may facilitate pseudoislet assembly. Proliferation, as assessed by Ki67, was low in both native and pseudoislets, with β cells below 0.5% and α cells around 1% (Figure 2, A and D). Similarly, apoptosis, as assessed by TUNEL, was very low (<0.5%) in pseudoislets and cultured human islets ( Figure 2, A and E). Interestingly, endothelial cells appear to have greater turnover, as evidenced by the presence of both Ki67 and TUNEL staining in the core of both native islets and pseudoislets ( Figure 2A). To assess markers of α and β cell identity in pseudoislets, we investigated expression of several key islet-enriched transcription factors. The expression of β (PDX1, NKX6.1) and α cell markers (MAFB, ARX) as well as those expressed in both cell types (PAX6, NKX2.2) was maintained in pseudoislets when compared with native islets (Figure 2, F-J), indicating that the process of dispersion and reaggregation does not affect islet cell identity. The 3D structure of intact islets makes virally mediated manipulation of human islet cells challenging due to poor viral penetration into the center of the islet. We adopted the pseudoislet system to overcome this challenge by transducing the dispersed single islet cells before reaggregation ( Figure 3A). To optimize transduction efficiency and subsequent pseudoislet formation, we incubated with adenovirus for 2 hours in Vanderbilt pseudoislet media at a multiplicity of infection of 500. Transducing pseudoislets with control adenovirus did not affect pseudoislet morphology or function and achieved high transduction efficiency of β and α cells throughout the entire pseudoislet (Supplemental Figure 2, A-E). Interestingly, β cells showed a higher transduction efficiency (90%) than α cells (70%), suggesting that α cells may be inherently more difficult to transduce with adenovirus (Supplemental Figure 2B). Activation of G i signaling reduces insulin and glucagon secretion. To investigate the value of this experimental approach, we sought to perturb islet gene expression and then assess islet cell function. We chose to alter GPCR signaling in islet cells because GPCRs are known to modulate islet hormone secretion (32,33). GPCRs, a broad class of integral membrane proteins, mediate extracellular messages to intracellular signaling through activation of heterotrimeric G proteins, which can be broadly classified into distinct families based on the G α subunit, including G i -coupled and G q -coupled GPCRs (34). An estimated 30%-50% of clinically approved drugs target or signal through GPCRs, including multiple used for diabetes treatment (35,36). Studying GPCR signaling with endogenous receptors and ligands can be complicated by a lack of specificity -ligands that can activate multiple receptors or receptors that can be activated by multiple ligands. To overcome these limitations, we used the DREADD technology (37). DREADDs are GPCRs with specific point mutations that render them unresponsive to their endogenous ligand. Instead, they can be selectively activated by the otherwise inert ligand, clozapine-N-oxide (CNO), thus providing a selective and inducible model of GPCR signaling (37,38). DREADDs are commonly used in neuroscience as molecular switches to activate or repress neurons with G q or G i signaling, respectively (39). In contrast, there have been comparatively very few studies using DREADDs in the field of metabolism, but there have been investigations of the G q and G s DREADD in mouse β cells and the G i DREADD in mouse α cells (16,40). The G s -coupled DREADD has been reported to be leaky and have basal activation, and thus, we chose here to focus on the 2 most commonly used DREADDs, G i and G q , to demonstrate how this experimental approach can be used. To our knowledge this is the first study to use this powerful technology in human islets. To investigate G i -coupled GPCR signaling, we introduced adenovirus encoding hM4Di (Ad-CMV-hM-4Di-mCherry), a G i DREADD, into dispersed human islet cells, allowed reaggregation into pseudoislets and then tested the effect of activated G i signaling ( Figure 3A). G i -coupled GPCRs signal by inhibiting adenylyl cyclase, thus reducing cAMP, and by activating inwardly rectifying potassium channels ( Figure 3B). and somatostatin (SOM; δ cells). Scale bar: 50 μm. (G) Quantification of relative endocrine cell composition of native islets and pseudoislets; n = 4 donors; P > 0.05. Insulin (H) and glucagon (I) secretory response to various secretagogues measured by perifusion of native islets and pseudoislets from the same donor (n = 5). G 5.6, 5.6 mM glucose; G 16.7, 16.7 mM glucose; G 16.7 + IBMX 100, 16.7 mM glucose with 100 μM isobutylmethylxanthine (IBMX); G1.7 + Epi 1, 1.7 mM glucose and 1 μM epinephrine; KCl 20, 20 mM potassium chloride (KCl). Wilcoxon matched-pairs signed-rank test was used to analyze statistical significance in E and G. H and I were analyzed by 2-way ANOVA; P > 0.05. The area under the curve for each secretagogue was compared by 1-way ANOVA with Dunn's multiple comparison test (Supplemental Figure 1, E-H and J-M). Data are represented as mean ± SEM. Endogenous GPCRs, which couple to G i proteins, include the somatostatin receptor in all islet cells as well as the α 2 adrenergic receptor in β cells (32,33). CNO (10 μM) had no effect on insulin or glucagon secretion in mCherry-expressing pseudoislets (Supplemental Figure 2, F and G), thus we compared the dynamic hormone secretion of hM4Di-expressing pseudoislets with and without CNO in response to a glucose ramp (2 mM glucose, 7 mM glucose, 11 mM glucose, 20 mM glucose; gray) and depolarization by KCl (20 mM; yellow) by perifusion (Figure 3, C and G). Activation of G i signaling had clear inhibitory effects on insulin secretion by β cells at low glucose, which became more prominent with progressively higher glucose concentrations (gray shading; Figure 3, C-E). Furthermore, bypassing glucose metabolism Schematic of the G i -coupled GPCR signaling pathway. CNO, clozapine-N-oxide; AC, adenylyl cyclase; ATP, adenosine triphosphate; GIRK, G protein-coupled inwardly rectifying potassium channel; K + , potassium ion. (C) Dynamic insulin secretion assessed by macroperifusion in response to low glucose (G 2, 2 mM glucose; white), glucose ramp (G 7, 7 mM; G 11, 11 mM; G 20, 20 mM glucose; gray), and KCl-mediated depolarization (KCl 20, 20 mM potassium chloride in the presence of G 2 or G 11; yellow) in the absence (blue trace) or presence of CNO (red trace); n = 4 donors/each. 10 μM CNO was added after the first period of 2 mM glucose, as indicated by a vertical red arrow and then continuously administered for the duration of the experiment (red trace). Note the split of y axis to visualize differences between traces at G 2 ± CNO. (D-F) Insulin secretion was integrated by calculating the area under the curve (AUC) for response to the low glucose (white), glucose ramp (gray), and KCl-mediated depolarization (yellow). Baseline was set to the average value of each trace from 0 to 21 minutes (before CNO addition). (G-J) Glucagon secretion was analyzed in parallel with insulin as described above. Insulin and glucagon secretory traces in C and G, respectively, were compared in the absence versus presence of CNO by 2-way ANOVA; ***P < 0.0001 for both insulin and glucagon secretion. Area under the curve of insulin (D-F) and glucagon responses (H-J) to low glucose, glucose ramp, and KCl-mediated depolarization were compared in the absence versus presence of CNO by Mann-Whitney test; P < 0.05. Data are represented as mean ± SEM. by directly activating β cells via depolarization with potassium chloride did not overcome this inhibition by G i signaling (yellow shading; Figure 3, C and F). Together, these data demonstrate that in human β cells G i signaling significantly attenuates, but does not completely prevent, insulin secretion and that this effect, at least in part, occurs downstream of glucose metabolism. The activation of G i signaling also had inhibitory effects on glucagon secretion (Figure 3, G-J). We did not observe a substantial inhibition of glucagon secretion in the hM4Di and hM4Di+CNO group in response to glucose, but activation of G i signaling with CNO caused a clear reduction in glucagon secretion, and secretion remained lower in the hM4Di+CNO group than in control hM4Di pseudoislets. When stimulated with potassium chloride, pseudoislets with activated G i signaling increased glucagon secretion but not to the level of controls. This demonstrates that the inhibitory effects of G i signaling persist even if the α cell is directly activated by depolarization. Thus, in α cells, activation of G i signaling reduces glucagon secretion across a range of glucose levels and when the cell is depolarized by potassium chloride. Activation of G q signaling greatly stimulates glucagon and somatostatin secretion but has both stimulatory and inhibitory effects on insulin secretion. G q -coupled GPCRs signal through phospholipase C, leading to IP 3 -mediated Ca 2+ release from the endoplasmic reticulum ( Figure 4A). Endogenous GPCRs, which couple to G q proteins in islets, include the M 3 muscarinic receptor and the free fatty acid receptor FFAR (also known as GPR40) (32,33). To investigate G q -coupled GPCR signaling, we introduced hM3Dq (Ad-CMV-hM3Dq-mCherry), a G q DREADD, into dispersed human islet cells, allowed reaggregation, and assessed hM3Dq-expressing pseudoislets by perifusion. When CNO was added to activate G q signaling, there was an acute increase in insulin secretion. However, this was not sustained, as insulin secretion fell quickly back to baseline, highlighting a dynamic response to G q signaling in β cells (Figure 4, B-E). Furthermore, continued G q activation inhibited glucose-stimulated insulin secretion, suggesting that in certain scenarios G q signaling may override the ability of glucose to stimulate insulin secretion. These results highlight the value of assessing hormone secretion in the dynamic perifusion system. Finally, G q activation reduced, but did not completely prevent, insulin secretion in response to direct depolarization with potassium chloride, indicating that the inhibitory effects cannot be overcome by bypassing glucose metabolism and suggesting that they occur downstream of the K ATP channel. Together, these data indicate that activated G q signaling can have both stimulatory and inhibitory effects on human β cells. In contrast, activation of G q signaling in α cells robustly increased glucagon secretion in low glucose, and it remained elevated with continued CNO exposure during glucose ramp as well as in the presence of potassium chloride (Figure 4, F-I). This indicates that in contrast to the β cells, activation of G q signaling in α cells robustly stimulates glucagon secretion, and this response is sustained across a glucose ramp and during KCl-mediated depolarization. Given the differing responses in β and α cells and the potential for paracrine signaling, we sought to measure somatostatin secretion and elucidate the effect of G q activation in δ cells. The relatively low abundance of δ cells in the native islets and pseudoislets (approximately 5%) prevented detection of somatostatin in the perifusion and microperifusion experiments (below assay sensitivity), so we tested the effect of CNO in low (2 mM) and high (11 mM) glucose in the context of static incubation. In glucose alone, somatostatin secretion was below the assay detection limit in 3 of the 4 donors tested; in contrast, activation of G q signaling increased somatostatin secretion in both low and high glucose (Supplemental Figure 3, A-D), showing that G q signaling robustly stimulates δ cells. Integration of the pseudoislet system with genetically encoded biosensor and microfluidic device allows synchronous measurement of intracellular signals and hormone secretion. While conventional macroperifusion systems, including the perifusion system used in this study, reliably assess islet hormone secretory profiles (3,6,7,41,42), their configuration does not allow coupling with imaging systems to measure intracellular signaling. To overcome this challenge, we developed an integrated microperifusion system consisting of pseudoislets and a microfluidic device that enables studies of islet intracellular signaling using genetically encoded biosensors in conjunction with hormone secretion ( Figure 5A and Supplemental Figure 4, A-C). The microfluidic device (Supplemental Figure 4A) (43) is made of bioinert and nonabsorbent materials, with optimized design for nutrient delivery, synchronous islet imaging by confocal microscopy, and collection of effluent fractions for analysis of insulin and glucagon secretion. The microperifusion system uses smaller volumes, slower flow rates, and fewer islets than our conventional macroperifusion system (Supplemental Figure 4, D-F). To investigate the dual effects of activated G q signaling on insulin secretion, we cotransduced pseudoislets with hM3Dq and GCaMP6f (Supplemental Figure 4C), a calcium biosensor (Ad-CMV-GCaMP6f), as the G q pathway conventionally signals through intracellular Ca 2+ ( Figure 4A). In the absence of CNO, hM3Dq-expressing pseudoislets had stepwise increases in GCaMP6f relative intensity as glucose increased, corresponding to increasing intracellular Ca 2+ and highlighting the added value of the system ( Figure 5B). This intracellular Ca 2+ response to stepwise glucose increase was accompanied by increasing insulin secretion ( Figure 5C), but the first phase of insulin secretion was not as clearly resolved as in the macroperifusion ( Figure 4B). Since there are differences in the design of the macroperifusion and microperifusion systems, we used multiphysics computational modeling with finite element analysis (44,45) to model the insulin secretion dynamics of the two systems (Supplemental Figure 4, H and I). This modeling accurately predicted the Figure 3; n = 4 donors/each. (F-I) Glucagon secretion was analyzed in parallel with insulin, as described in Figure 3. Insulin and glucagon secretory traces in B and F, respectively, were compared in the absence versus presence of CNO by 2-way ANOVA; ***P < 0.0001 for both insulin and glucagon secretion. Area under the curve of insulin (C-E) and glucagon responses (G-I) to each stimulus were compared in the absence versus presence of CNO by Mann-Whitney test; P < 0.05. Data are represented as mean ± SEM. overall shape of each insulin secretory trace, with the macroperifusion showing a "saw-tooth" pattern (Supplemental Figure 4H) while the microperifusion had a more progressive increase (Supplemental Figure 4I). Using this approach, we found that differences in the insulin secretory profiles were primarily due to the different fluid dynamics and experimental parameters between the two perifusion systems, especially the experimental time for each stimulus and the flow rate. Overall, this analysis demonstrates how perifusion parameters can affect insulin secretory pattern and indicates the strength of using complementary approaches. It also emphasizes the importance of validating new microperifusion devices (46,47) by comparing these with macroperifusion systems that have been used for many years by many laboratories. When G q signaling was activated with CNO, we again saw a transient stimulation of insulin secretion at low glucose followed by relative inhibition through the glucose ramp, while glucagon secretion from α cells was stimulated throughout the entire perifusion, independently of glucose concentration ( Figure 5, C, D, and G-J). Furthermore, the Ca 2+ dynamics in response to G q activation were consistent with the insulin secretory trace showing a rapid but short-lived increase in intracellular Ca 2+ . Interestingly, the Ca 2+ signal remained elevated above baseline but did not significantly increase with rising glucose (Figure 5, B, E, and F). This indicates that the dual effects of G q signaling on insulin secretion in β cells are largely mediated by changes in intracellular Ca 2+ levels. Figure 5. Pseudoislet system integrated with microfluidic device allows for coregistration of hormone secretion and intracellular signaling dynamics. (A) Schematic of pseudoislet system integration with a microfluidic device to allow for synchronous detection of intracellular signaling dynamics by the genetically encoded GCaMP6f biosensor and confocal microscopy and collection of microperifusion efflux for hormone analysis. Dynamic changes in GCaMP6f relative intensity (B), insulin secretion (C), and glucagon secretion (D) assessed during microperifusion in response to a low glucose (G 2 -2 mM glucose; white), glucose ramp (G 7, 7 mM; G 11, 11 mM; and G 20, 20 mM glucose; gray) and in the absence (blue trace) or presence of CNO (red trace); n = 3 donors/ each. 10 μM CNO was added after the first period of 2 mM glucose, as indicated by a vertical red arrow and then continuously administered for the duration of the experiment (red trace). See Supplemental Videos 1 and 2 for representative visualization of each experiment. Calcium signal (E and F) and insulin (G and H) and glucagon (I and J) secretion was integrated by calculating the area under the curve (AUC) for response to the low glucose (white) and glucose ramp (gray). Baseline was set to the average value of each trace from 0 to 8 minutes (before CNO addition). Calcium and hormone traces in B-D were compared in the absence versus presence of CNO by 2-way ANOVA; P < 0.05 for calcium trace, **P < 0.0001 for both insulin and glucagon secretion. Area under the curve of calcium (E and F), insulin (G and H), and glucagon responses (I and J) to low glucose and glucose ramp were compared in the absence versus presence of CNO by Mann-Whitney test; P < 0.05, **P < 0.01. Data are represented as mean ± SEM. Discussion The 3D multicellular human islet architecture, while essential for islet cell function presents experimental challenges for mechanistic studies of intracellular signaling pathways. Using primary human islets, we developed a pseudoislet system that resembles native human islets in morphology, cellular composition, cell identity, and dynamic insulin and glucagon secretion. This system allows for efficient virally mediated genetic manipulation in almost all cells in the pseudoislet. To evaluate the coordination between intracellular signals and islet hormone secretion, we developed an integrated system consisting of pseudoislets and a microfluidic device that enables studies of islet intracellular signaling using genetically encoded biosensors in conjunction with hormone secretion. Furthermore, we used this integrated approach to define aspects of human islet biology by investigating GPCR signaling pathways using DREADDs and a calcium biosensor. Despite α and β cells both being excitable secretory cells and sharing many common developmental and signaling components, this experimental approach allowed us to demonstrate similar and distinct responses to activation of GPCR signaling pathways, highlighting the uniqueness in each cell's molecular machinery. The activation of G i signaling was inhibitory in β and α cells, resulting in reduced insulin and glucagon secretion, respectively, and showed a more substantial affect in β cells, where this signaling blunted insulin response to both a glucose ramp and to KCl-mediated depolarization. Interestingly, direct KCl depolarization was not sufficient to overcome these inhibitory effects in either β or α cells, suggesting that reduced cAMP via the inhibition of adenylyl cyclase, in addition to cAMP-independent pathways (48), plays a role in both insulin and glucagon secretion. These results align well with those of recent studies in β cells, suggesting that cAMP tone is crucial for insulin secretion, and observations in α cells highlighting cAMP as a key mediator of glucagon secretion (13,17,19,49). There were major differences in response to activation of G q signaling in β cells compared with α cells. In α cells, the activated G q signaling elicited a robust and sustained increase in glucagon secretion in the presence of a glucose ramp and potassium chloride. In contrast, G q signaling in β cells had a transient stimulatory effect in low glucose and then inhibitory effects on both insulin and intracellular Ca 2+ levels, with sustained activation during glucose ramp. Interestingly, previous studies of acetylcholine signaling have also reported dual effects on Ca 2+ dynamics in β cells depending on the length of stimulation (50). This signaling was thought to be mediated through the muscarinic acetylcholine receptor M 3 (from which the hM3Dq DREADD is based). Overall, these results suggest a negative feedback or protective mechanism that prevents sustained insulin release from β cells in response to G q signaling that is not active in α cells under similar circumstances. There are limitations and caveats to the current study. First, our approach resulted in DREADD expression in all cell types. Although we can distinguish the effects on islet α and β cells through their distinct hormone secretion, it is possible that complex paracrine signaling contributes to the results described here. Future modifications of this system could incorporate cell-specific promoters to target a particular islet cell type. Second, the DREADDs are likely expressed at higher levels than endogenous GPCRs. To mitigate this, we used the appropriate DREADD-expressing pseudoislets as our controls and were encouraged to see normal secretory responses in these control pseudoislets. Third, while there is some concern that CNO can be reverse metabolized in vivo into clozapine, which could potentially have off-target effects (51), this is unlikely in our in vitro system. We also verified that CNO had no effect on mCherry-expressing pseudoislets, making it unlikely that CNO itself is competitively inhibiting endogenous receptors in human islets. Fourth, we used a CNO concentration of 10 μM for all of our experiments, a standard concentration used for in vitro assays (16,52), but it is possible that islet cells may show dose-dependent effects. Finally, this is an in vitro study focused on acute functional effects of these pathways on human islets, and chronic in vivo studies of these pathways may show different results. For example, in mouse β cells, chronic in vivo activation of G q pathways using the DREADD system lead to an increase in β cell function and mass (53) while inhibition of G i signaling with β cell-specific pertussis toxin expression affected only function (54). Future work could involve transplantation of DREADD-expressing pseudoislets into immunodeficient mice to study the effect of activating these pathways on human islets in vivo (55). Overall, these findings demonstrate the utility of the pseudoislet system for its ability to manipulate human islets. Other approaches include inducible pluripotent stem cells that allow similar genetic manipulation. However, it is unclear if these approaches create entirely normal human islet cells. We show in this system that α and β cells in pseudoislets maintain their fully differentiated state as well as their dynamic responsiveness to glucose and other stimuli. Additionally, this approach allows for the study of all islet cells within the context of other cell types and 3D assembly. While our data suggest that breaking down and rebuilding the islet does not impair paracrine interactions, this could be further evaluated by looking at secretion in response to factors that exclusively rely on paracrine interactions such as ghrelin or certain amino acids (17,56,57). Ultimately, the integration of the pseudoislet approach with a microfluidic perifusion system and livecell imaging provides a powerful experimental platform to gain insight into human islet biology and the mechanisms controlling regulated islet hormone secretion, which currently limit the development of novel therapeutic approaches. Here, we focus on virally mediated gene expression to alter signaling pathways, but this system could be adapted to accommodate technologies such as CRISPR. Furthermore, after islet dispersion into single cells, techniques to purify live-cell populations such as FACS with cell surface antibodies (41,58) could be incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation. Combined with accurate cell-specific targeting, this approach would allow the measurement of intracellular dynamics at the individual cell level and distinguish intracellular responses of islet endocrine cells to stimuli. Methods Human islet isolation. Human islets (n = 24 preparations, Supplemental Table 1) were obtained through partnerships with the IIDP (https://iidp.coh.org/), Alberta Diabetes Institute IsletCore (https://www.epicore. ualberta.ca/IsletCore/), and Human Pancreas Analysis Program (https://hpap.pmacs.upenn.edu/) or isolated at the Institute of Cellular Therapeutics of the Allegheny Health Network. Assessment of human islet function was performed by islet macroperifusion assay on the day of arrival, as previously described (42) This study used data from the Organ Procurement and Transplantation Network (OPTN) that was in part compiled from the data hub accessible to IIDP-affiliated investigators through the IIDP portal (https://iidp.coh.org/secure/isletavail). The OPTN data system includes data on all donors, wait-listed candidates, and transplant recipients in the US, submitted by the members of OPTN. The Health Resources and Services Administration of the US Department of Health and Human Services provides oversight of the activities of the OPTN contractor. The data reported here have been supplied by UNOS as the contractor for OPTN. The interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an official policy of or interpretation by the OPTN or the US government. Pseudoislet formation. See Vanderbilt Pseudoislet Protocol in Supplemental Information for the detailed pseudoislet formation protocol. Briefly, human islets were handpicked to purity and then dispersed with HyClone trypsin (Thermo Scientific). Islet cells were counted and then seeded at 2000 cells per well in Cell-Carrier Spheroid Ultra-low attachment microplates (PerkinElmer) or 2500 cells per drop in GravityPLUS Hanging Drop System (InSphero) in enriched Vanderbilt pseudoislet media (see Vanderbilt Pseudoislet Protocol in Supplemental Information for detailed protocol). Cells were allowed to reaggregate for 6 days before being harvested and studied. Immunohistochemical analysis. Immunohistochemical analysis of islets was performed by whole mount or on 8-μm cryosections of islets embedded in collagen gels as previously described (3,21). Primary antibodies against all antigens and their working dilutions are listed in Supplemental Table 2. Apoptosis was assessed by TUNEL (MilliporeSigma, S7165) following the manufacturer's instructions. Digital images were acquired with a Zeiss LSM 880 or LSM 510 laser-scanning confocal microscope (Zeiss Microscopy Ltd) or ScanScope FL (Aperio/Leica Biosystems). Images were analyzed using HALO Image Analysis Software (Indica Labs) or MetaMorph v7.1 (Molecular Devices LLC). Assessment of islet function in vitro by static incubation. Pseudoislets (10-20 IEQs/well) were placed in 2 mL DMEM (media, 2 mM glucose) of a 12-well plate (351143, Corning) and allowed to equilibrate for 30 minutes and then were transferred to media containing the stimuli of interest for 40 minutes. Assessment of islet function by macroperifusion. Function of native islets and pseudoislets was studied in a dynamic cell perifusion system at a perifusate flow rate of 1 mL/min as described previously (3,42) using approximately 250 IEQs/chamber. The effluent was collected at 3-minute intervals using an automatic fraction collector. Insulin and glucagon concentrations in each perifusion fraction and islet extracts were measured by radioimmunoassay (insulin, RI-13K; glucagon, GL-32K, MilliporeSigma). Microperifusion platform. The microperifusion platform ( Figure 5 and Supplemental Figure 4) is based on a previously published microfluidic device with modifications (43). Design modifications were incorporated using SolidWorks 2018 3D computer-aided design (CAD) software. Microfluidic devices were machined, according to the CAD models, using a computer numerical controlled milling machine (MDX-540, Roland) from poly(methyl methacrylate) workpieces. To reduce the optical working distance, through holes were milled into the culture wells and a no. 1.5 glass coverslip was bonded to the bottom component of the microfluidic device using a silicone adhesive (7615A21, McMaster-Carr). Custom gaskets were fabricated using a 2-part silicone epoxy (Duraseal 1533, Cotronics Corp.) and bonded into the top component of the device using a specialized polyester adhesive (PS-1340, Polymer Science). The 2 components of the microfluidic device (Supplemental Figure 4A) were assembled in a commercially available device holder (Fluidic Connect PRO with 4515 Inserts, Micronit Microfluidics), which creates a sealed system and introduces fluidic connections to a peristaltic pump (P720, Instech) though 0.01-inch FEP tubing (IDEX, 1527L) and a low-volume bubble trap (Omnifit, 006BT) placed in the fluid line just before the device inlet to prevent bubbles from entering the system (see Supplemental Figure 4B for microperifusion assembly). Assessment of pseudoislets by microperifusion. The microperifusion apparatus was contained in a temperature-controlled incubator (37°C) fitted to a Zeiss LSM 880 laser-scanning confocal microscope (Zeiss Microscopy Ltd) (Supplemental Figure 4B). Pseudoislets (~25 IEQs/chamber) were loaded in a prewetted well, imaged with a stereomicroscope to determine loaded IEQ, and perifused at 100 μL/ min flow rate with Krebs-Ringer buffer containing 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl 2 , 1 mM MgCl 2 , 25 mM HEPES, 0.1% BSA, pH 7.4, at 37°C. Perifusion fractions were collected at 2-minute intervals following a 20-minute equilibration period in 2 mM glucose using a fraction collector (model 2110, Bio-Rad) and analyzed for insulin and glucagon concentration by RIA (insulin, RI-13K; glucagon, GL-32K, MilliporeSigma). GCaMP6f biosensor was excited at 488 nm and fluorescence emission detected at 493-574 nm. Images were acquired at 15-μm depth every 5 seconds using a ×20/0.80 Plan-Apochromat objective. Image analysis was performed with MetaMorph v7.1 software (Molecular Devices). Pseudoislets in the field of view (3-7 pseudoislets/field) were annotated using the region-of-interest tool. The GCaMP6f fluorescence intensity recorded for each time point was measured across annotated pseudoislet regions and normalized to the baseline fluorescence intensity acquired over the 60 seconds in 2 mM glucose before stimulation. The calcium, insulin, and glucagon traces were averaged from 5 microperifusion experiments from 3 independent donors. Fluid dynamics and mass transport modeling. 2D finite element method (FEM) models, which incorporate fluid dynamics, mass transport, and islet physiology, were developed for the macroperifusion and microperifusion platforms and implemented in COMSOL Multiphysics Modeling Software (release version 5.0). Fluid dynamics were governed by the Navier-Stokes equation for incompressible Newtonian fluid flow. Convective and diffusive transport of oxygen, glucose, and insulin were governed by the generic equation for transport of a diluted species in the chemical species transport module. Islet physiology was based on Hill (generalized Michaelis-Menten) kinetics using local concentrations of glucose and oxygen, as previously described (44,45). The geometry of the macroperifusion platform was modeled as the 2D cross section of a cylindrical tube with fluid flowing from bottom to top (Supplemental Figure 4D). The geometry of the microperifusion platform was modeled as a 2D cross section of the microfluidic device with fluid flow from left to right (Supplemental Figure 4E). In both the macroperifusion and microperifusion models, 5 islets with a diameter of 150 μm (5 IEQs) were placed in the flow path. FEM models were solved as a time-dependent problem, allowing for intermediate time steps that corresponding with fraction collection time during macroperifusion and microperifusion. A list of the parameters used in the computational models is provided in Supplemental Table 3. Statistics. Data are expressed as mean ± SEM. A P value of less than 0.05 was considered significant. Analyses of area under the curve and statistical comparisons (Mann-Whitney test, Wilcoxon matchedpairs signed-rank test, and 1-and 2-way ANOVA) were performed using Prism v8 software (GraphPad). Statistical details of experiments are described in the figure legends and the Results. Study approval. The Vanderbilt University Institutional Review Board does not classify deidentified human pancreatic specimens as human subject research. Author contributions JTW, RH, HAN, MI, JRL, ΑA, MB, and ACP conceived and designed the experiments. JTW, RH, HAN, GP, RA, CR, DCS, MI, PW, AGO, RB, and MB performed experiments or analyzed the data and interpreted results. JTW, MB, and ACP wrote the manuscript. All authors reviewed, edited, and approved the final version.	v3-fos-license
2016-05-04T20:20:58.661Z	2011-02-19T00:00:00.000	208929944	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://journals.iucr.org/e/issues/2011/03/00/tk2720/tk2720.pdf", "pdf_hash": "1367a5780d42c5b2989c1e328f917d0b8660b099", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:529", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "1343a0593b2dd180eb71d7a9aa6420bfc2961132", "year": 2011 }	pes2o/s2orc	2-Bromo-2-methyl-N-(4-nitrophenyl)propanamide The title compound, C10H11BrN2O3, exhibits a small twist between the amide residue and benzene ring [the C—N—C—C torsion angle = 12.7 (4)°]. The crystal structure is stabilized by weak N—H⋯O, C—H⋯Br and C—H⋯O interactions. These lead to supramolecular layers in the bc plane. The title compound, C 10 H 11 BrN 2 O 3 , exhibits a small twist between the amide residue and benzene ring [the C-N-C-C torsion angle = 12.7 (4) ]. The crystal structure is stabilized by weak N-HÁ Á ÁO, C-HÁ Á ÁBr and C-HÁ Á ÁO interactions. These lead to supramolecular layers in the bc plane. RMF is grateful to the Spanish Research Council (CSIC) for the use of a free-of-charge licence to the Cambridge Structural Database. RMF and FZ also thank the Universidad del Valle, Colombia, and the Instituto de Química de Sã o Carlos, USP, Brasil for partial financial support. RLAH thanks CNPq for partial financial support. Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: TK2720). most initiators for ATRP processes are alkyl halides (Matyjaszewski & Xia, 2001). The C4-N1-C5-C6 torsion angle is 12.7 (4)°, indicating a small twist between the benzene ring and the amide. An intramolecular C-H···O interaction is observed (see Table 1). Experimental The initial reagents were purchased from Aldrich Chemical Co. and were used as received. In a 100 mL round bottom flask 4-nitroaniline (3.258 mmol, 0.450 g), triethylamine (0.653 mmol, 0.066 g) were mixed, then a solution of 2-bromo isobuturyl bromide (0.704 g) in anhydrous THF (5 mL) was added drop wise, under an argon stream. The reaction was carried out in a dry bag overnight under magnetic stirring. The solid was filtered off and dichloromethane (20 mL) added to the organic phase which was washed with brine (40 mL) followed by water (10 mL). The solution was concentrated at low pressure affording colourless crystals and recrystalized from a solution of hexane and ethyl acetate (80:20). M. pt. 356 (1) K. Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.	v3-fos-license
2021-06-22T17:56:18.436Z	2021-03-01T00:00:00.000	235502386	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.scielo.br/j/abmvz/a/HQyXRQTbvrR5CWF3dKg4fgC/?format=pdf&lang=en", "pdf_hash": "fc67df40dbb787639e9d83b587da8d4eb0d3f340", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:539", "s2fieldsofstudy": [ "Biology" ], "sha1": "132ad7d8b775750c3eb396c6295c9e0c6c44f5dd", "year": 2021 }	pes2o/s2orc	Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dosedependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10μg/mL). Flow cytometry analysis revealed that melittin at 2μg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2μg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5μg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells. INTRODUCTION Apitoxin or bee venom is secreted by a specialized gland present in the worker bees and confined in a vesicle until the moment of stinging (Benton et al., 1963). It is a complex mixture of nitrogenous compounds, containing several biologically active components, including enzymes, peptides, and biogenic amines, conferring a wide variety of allergic and pharmacological properties, such as anti-inflammatory, antimicrobial, and antitumor activities (Cardoso et al., 2003;Abreu et al., 2010). Melittin is a highly active water-soluble toxic peptide, with only 26 amino acids in its conformation, present in the venom of honeybees and contributes to about 50% of its dry weight (Cruz-Landim and Abdalla, 2002). In the venous vesicle, melittin is arranged in a tetrameric form, which gives it a low toxicity (Cardoso et al., 2003). However, after being released, it dissociates into a highly toxic monomeric form. In addition to it, phospholipase A2 is also present in the venom, which further amplifies the catalytic actions of melittin (Cardoso et al., 2003;Koumanov et al., 2003). Melittin exerts a rapid cytolytic action by destabilizing the membranes and releasing the cytoplasmic content of various cell types (Dempsey, 1990). The hemolytic activity, being a characteristic biological effect, is used to detect the peptide in poisonous extracts (Tosteson et al., 1985). Its lithic capacity is not only restricted to animal cells as it also exerts antibacterial and antifungal activities (Ashthana et al., 2004). The objective of this study was to examine the effects of melittin in MDBK (Madin-Darby Bovine Kidney) cells by analysis of mitochondrial functionality, apoptosis potential, necrosis, ROS production, lipid peroxidation, DNA fragmentation and cell membrane destabilization. (KASVI®, Brazil). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA) and added to E-MEM (10%) when the need for cell multiplication. Dimethyl sulfoxide (DMSO), MTT (3-(4,5-dimethylthiazol-2yl)-2-5-diphenyl-2H-tetrazolate reagent), as well as other reagents used in flow cytometry and confocal microscopy, were commercially purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Melittin Melittin was dissolved in sterile E-MEM at a concentration of 1mg/mL and stored at -20°C. The different concentrations used in the experiments were made by diluting the stock solution in E-MEM. MDBK cells were maintained in a humid incubator at 37°C and 5% CO2 in cell culture bottles with E-MEM supplemented with 10% FBS. After the establishment of the monolayer and attaining approximately 80% confluency, the cells were transferred to 96 well plates (100μL/well) at a concentration of 3 × 10 4 cells/mL. After 24h in the incubator, the medium was carefully aspirated and melittin was added to the wells (100μL/well) at different concentrations (1 to 10μg/mL) in six replicates. All plates were incubated for 72h under the same conditions until the time of reading. MDBK cells maintained in E-MEM without exposure to melittin were used as controls. The MTT reduction assay is used to determine cell viability through mitochondrial functionality (Mosmann, 1983). After an exposure to melittin, the supernatant was carefully aspirated and 50μL/well of 1mg/mL MTT solution was transferred. Then, the plates were incubated for another 4h under the same condition, the supernatant was removed and 100μL/well of DMSO was added to solubilize the generated formazan crystals. After 15min of constant stirring, the plates were read spectrophotometrically at 540nm. The 50% and 90% cytotoxic concentrations (CC) were calculated by the formula (AT/AC) × 100, where AT indicates the mean absorbance of the wells where cells received treatment and AC the mean of the control wells where cells did not receive melittin. For flow cytometry, MDBK cells, cultured and exposed to melittin at different concentrations (1.0 to 2.5μg/mL) for 72h, were subjected to the specific reagent for each analysis and then trypsinized, resuspended in 100μL of E-MEM, and stored under refrigeration. The analysis was performed on Attune Acoustic Focusing Cytometer® (Applied Biosystems) and the results were evaluated using the Attune Cytometric Software version 2.1. Hoechst 33342 fluorescent probe (2mM) was used to separate MDBK (Hoechst positive) cells from the cell debris (negative hoechst). Cell populations were detected by a VL1 photomultiplier (450/40 filter). Twenty-thousand events were analyzed per sample with a flow rate of 50μL/s. For the detection of MDBK cell population, an FSC x SSC scatter plot was constructed. Arq About apoptosis/necrosis: after exposure to melittin, 2μL of fluorescein isothiocyanateconjugated Annexin V antibody (FITC) was added and the cells were incubated for 1h. After, propidium iodide (PI, 50μg/mL) was added with further incubation for 10min (Masango et al., 2015). The cells were removed from the plate and placed under refrigeration for readings in the flow cytometer. The viable cells were not labeled with fluorophores (FITC-, PI-), while apoptotic cells outsource phosphatidylserine which is recognized by Annexin V (FITC+, PI-), and the necrotic cells due to the ruptured nuclear membrane, show binding with PI (FITC-, PI+ and FITC+, PI+). The fluorescence was read through the photomultiplier BL3 (640 LP filter). The results were expressed as the percentage of the cell populations as calculated by the formula: (number of positive events/total number of events) × 100. The Mitochondrial membrane potential (MMP) analysis was performed using rhodamine 123 fluorescent dye which concentrates on the active mitochondrial membranes when electrons are donated to the respiratory chain. After exposure of the MDBK cells to melittin, rhodamine 123 (100nM) was added into the plates and maintained for 1h, and the excess reagent was aspirated. The cells were analyzed for the fluorescence intensity emitted in more active (higher concentration of fluorescence, greater accumulation of rhodamine) and less active mitochondria (less fluorescence, less accumulation of rhodamine) (Gillan et al., 2005). Rhodamine 123 fluorescence was read through the BL1 photomultiplier (530/30 filter). The expressed data refer to the percentages of cells with low MMP, calculated by the formula: (number of cells with low MMP/total cell number) × 100. In order to analyze the intracellular production of reactive oxygen species (ROS) the oxidation of the fluorescent dye 2', 7' dichlorofluorescein diacetate (H2DCF-DA) by the intracellular ROS was monitored. h2DCF-DA (1 mM) was added and retained for 1h to the cells after exposure to melittin. The fluorescence emitted was read by the photomultiplier BL1 (530/30 filter) and the data were expressed by the mean green fluorescence intensity ±standard error (Domínguez-Rebolledo et al., 2011). The lipid peroxidation of cell membranes was evaluated by the lipophilic fluorophore probe C11-BODIPY581/591, which is analogous to unsaturated fatty acids. The fluorescence of this probe changes after the lipidic peroxidation, as it emits red fluorescence when present on intact membranes but emits orange to green shades when membranes are attacked by oxidative radicals (Aitken et al., 2007). C11-BODIPY581/591 was added to the cells for 2h, and it was aspirated and the concentrations of melittin were added. After incubation for 72h, the cells were trypsinized and subjected to read fluorescently. The results are expressed as the percentage of cell shaving peroxidized lipids at membranes. The results were obtained using the formula: (number of events with red fluorescence/total number of events) × 100. For DNA fragmentation, after exposure to melittin, the cells were treated for 5min with the fluorescent probe acridine orange (AO) (Ojeda et al., 1992). AO is inserted into the double-stranded DNA as a monomer but can also aggregate to single-stranded DNA. The monomeric AO bound to the intact DNA emits green fluorescence, while the aggregated AO emits orange to red fluorescence (Hoshi et al., 1996). The results were expressed as percentages of cells with DNA fragmentation, calculated by the formula: (number of cells with orange fluorescence/total number of cells) × 100. The use of confocal microscopy: MDBK cells were grown on 0.13mm thick glass cover plates in 24-well plates. After the establishment of the monolayer, the cells were exposed to different concentrations of melittin ranging from 1.0 to 2.5μg/mL for 72h. The specific reagents were then added to the wells for the given time. The supernatants were aspirated, and the coverslips removed from the wells of the plate with the aid of forceps and placed on a slide for analysis under confocal microscopy (Inverted Spectral Laser Scanning Confocal Microscope, Leica, TCS SP8). The destabilization of the plasma membranes was evaluated by the fluorescent and hydrophobic probe merocyanine-540 that presents tropism and intensely blends the membranes that have a high disorder of their components and that have lost their typical asymmetry (translocation of the phospholipids in lipid bilayer) (Langner and Hui, 1993). The analysis was combined with the addition of YO-PRO-1, a semipermeable probe that binds to cellular DNA, enabling a viability analysis associated with the membrane state (Thomas et al., 2006). For this analysis, after culturing the cells and exposing to melittin, merocyanine-540 (2.7 μM) and YO-PRO-1 (25 nM) were added to the wells for 10 min before visualizing under confocal microscope (the method was adapted from Peña et al., 2004). In addition, Hoechst 33342 was added in the same manner as already described. The intensity of red fluorescence emitted by merocyanine-540 indicates the levels of membrane destabilization and green fluorescence emitted by YO-PRO-1 indicates the cells with the labeled DNA due to the high degree of membrane disorganization. The data obtained by the MTT assay are represented by the mean ± standard deviation (SD). Analysis of variance (ANOVA, Tukey's test) was performed to evaluate the differences between the treatment groups and the control group. The flow cytometry data were subjected to analysis of variance (ANOVA, LSD test) for comparison of the groups. Pearson's correlations were performed to evaluate associations between the analyzed variables. In all cases, Statistix statistical software version 10.0® was used and significant results were considered when P<0.05. RESULTS AND DISCUSSION The MTT assay revealed a decrease in mitochondrial functionality of up to 78.24 ±3.59% (P<0.01, n = 6) for MDBK cells at a concentration of 10μg/mL of melittin in a dose-dependent manner ( Table 1). The lowest concentrations (1 and 2μg/mL) showed the highest mitochondrial activity rates (0.97% ±0.04% and 0.94% ±0.03%, respectively) and did not differ from the control, whereas all other concentrations of melittin decreased the mitochondrial activity of exposed cells (P<0.05, n = 6). In this context, an active state of mitochondria does not always reflect cellular health, since their activity is also increased in cases of cellular injury. This organelle may act as a pro-apoptotic signal after a damage to its membrane with consequent permeabilization and release of pro-apoptotic molecules present therein (Grivicich et al., 2007). Our results are in agreement with previous findings reported by Zhou et al. (2013), who described only 6.46% ±1.83% (P<0.05, n = 6) inhepG2 cells exposed to melittin at 10.0μg/mL. Results obtained by flow cytometry analysis indicated damage to cells, which were not detectable by the MTT test, as the latter estimates cell viability only by mitochondrial activity. The rates of apoptosis and necrosis, as analyzed by flow cytometry, are given in Figure 1. Other physiological changes in the cells were demonstrated by flow cytometry analysis (Table 2). When the cells were exposed to higher concentrations of melittin, the necrotic cell rates were increased. Regarding the cell necrosis, the control cells showed 20.9% ±0.8% of cellular necrosis. Zhou et al. (2013), by a flow cytometric analysis after an exposure of hepG2 cells (derived from hepatocarcinoma) to melittin (0, 1, 2, and 5μg/mL), found necrosis rates of 0%, 6.8%, 1.6%, and 0.8%, respectively. The treatments with 1 and 1.5μg/mL of melittin did not show difference from the control. However, there was statistically significant difference (P<0.05) at the highest concentrations of melittin, as 39.4% ±1.5% necrotic cells were seen at 2μg/mL of melittin (non-toxic to MTT reduction) and 69.7% ±1.8% at 2.5μg/mL of melittin. Zhou et al. (2013) results demonstrated lower rates of cellular necrosis than those found in this study, but it should be considered that in their study, the necrosis rate of the control group was subtracted from all other groups. Furthermore, the cells studied by these authors are derived from hepatocarcinoma and were exposed to melittin for 24h while in our study MDBK cells were exposed to melittin for 72h. DNA fragmentation was also evaluated by flow cytometry which revealed that MDBK cells exposed to 2.5μg/mL of melittin (4.8% ±0.4%) showed the maximum impact of melittin toxicity. The difference was statistically significant when compared to the cell control (2.57% ±0.77%) (P<0.05) (Table B.2); however, interestingly, the DNA fragmentation rate (0.6% ±0.06%) at 1μg/mL of melittin was lower than cell control. An exposure to melittin caused oxidative stress in MDBK cells by increasing the intracellular production of ROS. The cells exposed to 2.5μg/mL of melittin presented a mean of 43360 ±12289 LI and differed statistically (P<0.05) from the control (17237 ±5017.3) and from other concentrations. The elevated level of intracellular ROS indicates a biochemical imbalance to the point of altering the natural balance between prooxidants and antioxidants. The changes in the redox balance of biological systems such as cells, organelles, and tissues can cause oxidative stress (Schafer and Buettner, 2001). There were no other changes in mitochondrial membrane potential (MMP) patterns. All treatments resulted in an increase in MMP of MDBK cells, but there was a statistical difference (P<0.05) between control (16.07%±0.69%) and treatment with 2μg/mL (17.8%±0.67%). Also, LPO showed a significant correlation with MMP (r = 0.4149, P<0.05), suggesting an increase in mitochondrial functions in an attempt to revert the stress caused to the cells by melittin. The rate of apoptosis could still be influenced by the release of pro-apoptotic mitochondrial messengers but there was a low and non-significant correlation between MMP and apoptosis (r = 0.1238, P>0.05). The action of melittin occurs mainly on phospholipid membranes of the cells as described by Terwilliger et al. (1982), causing permeabilization, artificial pore formation, disruption, and lysis (Lee et al., 2008;Zhang et al., 2011). The mechanisms of action proposed on the membranes were explained by a "barrel model" and "carpet model". In the first model, the aggregates of melittin are perpendicularly formed on the surface of the membrane, resulting in membrane rupture in the form of toroidal pores (or in the form of a barrel). In the carpet model, melittin is distributed on the surface of the membrane in a parallel fashion, disorganizing the lipid bilayer and causes permeabilization (Bechinger, 1999;Gordon-Grossman et al., 2012). Both proposed models suggest the formation of pores or permeabilization of the membranes, explaining the appearance of necrotic cells after exposure to melittin and amplified in a proportion to the concentration of it. While analyzing the destabilization of the phospholipid membranes of MDBK cells exposed to melittin with the aid of confocal microscopy, the microphotographs revealed greater destabilization concomitant with the increase in the concentration of melittin (Figure 2), according to the results obtained by flow cytometric analysis for lipid peroxidation. As shown in Table 2, the increase in the LPO of membranes was directly proportional to the concentration of melittin, which showed statistically significant difference from the control (P<0.05). In addition to the already known mechanisms of action of melittin directly on the lipid membranes (barrel and carpet models), the LPO may also be caused by ahigh production intracellular ROS. Lipid peroxidation is a chain reaction that starts by attacking lipids by an ROS that has sufficient reactivity to sequester a hydrogen atom from a methylene (CH-2) group. The termination of this process is marked by the propagation of lipid and peroxyl radicals produced until they destroy themselves (Ferreira and Matsubara, 1997). Basically, LPO involves incorporating molecular oxygen into a fatty acid to produce a lipid hydroperoxide as the starting primary product. The LPO process occurs in several stages and with numerous possibilities of chemical reactions, which makes it difficult to understand and evaluate the process as a whole (Lima and Abdalla, 2001). Figure 2. Membrane destabilization. Microphotographs were obtained by confocal microscopy of MDBK cells exposed to melittin (0, 1.0, 1.5, 2.0 and 2.5μg/mL) for 72h. An exposure to melittin elevates cellular metabolism. The increase in the concentration of melittin resulted in a concomitant increase in the levels of ROS, LPO, MMP, and cellular necrosis but the rate of apoptosis was decreased, which was inversely proportional to the rate of necrosis (r = -0.7049, P<0.001). The free radicals attack the cell itself and the lipids in the cell membrane are peroxidized, which explains the correlation between ROS and LPO (r = 0.4158, P<0.05). The peroxidation of lipids resulted in the destabilization of membranes, as shown in Figure 2, making possible the cyclization of phosphatidylserine, characterizing an apoptotic process. This mechanism explains the observed correlation between LPO and the rate of apoptosis (r = 0.4978, p<0.05). The correlations between the analyzed variables are shown in Figure3, which may contribute for the elucidation on the mechanism of action of melittin on MDBK cells. Figure 3. Pearson's correlations between the cellular function parameters as evaluated in MDBK cells exposed to melittin for 72h. CONCLUSION In this study, different analyses were performed in order to elucidate the action mechanisms of melittin toxicity on MDBK cells. The toxicity presented dose-dependent characteristics. Melittin at 2μg/mL had the highest necrosis rate for the cells and lipoperoxidation of the membranes. The highest ROS production occurred when the cells were exposed to 2.5μg/mL melittin, and this concentration also increased DNA fragmentation. There was a significative and positive correlation between the lipoperoxidation of membranes with ROS, mitochondrial functionality and apoptosis.	v3-fos-license
2018-04-03T00:31:46.739Z	2004-10-01T00:00:00.000	12614135	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/279/40/41275.full.pdf", "pdf_hash": "df8a9618ce9e12242323cac2ef82e527ea382944", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:657", "s2fieldsofstudy": [ "Biology" ], "sha1": "c897de58b1ae1fb916f80f75c89ecf4ce00cafda", "year": 2004 }	pes2o/s2orc	Hypoxia-induced nucleophosmin protects cell death through inhibition of p53. Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival. Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1␣. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxiainduced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival. Hypoxia is a physiological stress that can activate the cell death program and thus select for cells resistant to hypoxiainduced apoptosis. Indeed, many tumors can grow in hypoxic microenvironments, which is often associated with poor prognosis and less response to cancer therapy (1). While most tumor cells retain the ability to undergo apoptosis in response to hypoxic stress, they can become adaptive to hypoxia by increasing synthesis of factors that promote cell survival and proliferation (2). The transcriptional factor hypoxia-inducible factor-1␣ (HIF-1␣) 1 is a central mediator of hypoxic response (3). Under normoxic conditions, Hif-1␣ is rapidly degraded by the proteosome after being targeted for ubiquitination, a process that is dependent on the tumor suppressor von Hippel-Lindau protein (4). However, Hif-1␣ is stabilized under hypoxic conditions, which in turn transactivates a variety of genes in the adaptive response (3). Hif-1␣ has been shown to play essential roles in hypoxia-mediated apoptosis, cell proliferation, and tumor angiogenesis (5). Nucleophosmin (NPM) is a multifunctional protein initially characterized as a nucleolar protein functioning in the processing and transport of ribosomal RNA (6). NPM is found to be more abundant in tumor and growing cells than in normal resting cells (6 -15). In fact, NPM has been proposed as a tumor marker for human colon (9), ovarian (10), prostate (11), and gastric (12) cancers because NPM expression is markedly higher in these tumor cells than in the corresponding normal cells. NPM is also identified as a major gene product required for stem cell development (stemcell.princeton.edu). Conversely, NPM expression is down-regulated in cells undergoing differentiation or apoptosis. For example, NPM mRNA is decreased in HT29-D4 colon carcinoma cells treated in vitro to undergo differentiation (13). The levels of NPM were significantly lower in the WEHI-231 B lymphoma cells and the human T cell leukemia Jurkat cells treated to undergo growth rest or apoptosis, as compared with untreated cells (14,15). Similarly, repression of NPM expression by antisense strategy potentiated drug-induced apoptosis in the human HL60 leukemia cells (16). NPM appears to be the target for certain transforming oncogenes. Indeed, Zeller et al. (17) used DNA microarray technology for gene expression analysis to examine target sequences in human genome by the oncogenic transcription factor c-Myc and identified NPM as a Myc-responsive gene. NPM expression was 3.5-fold higher in myc-overexpressing avian bursal neoplasia than in normal bursa (18). NPM is also frequently found in the chromosomal translocation associated with several hematopoietic malignancies, such as acute promyelocytic leukemia (19), anaplastic large cell lymphomas (20), and myelodysplasia/acute myeloid leukemia (21). We have characterized the response of NPM to hypoxia in human normal and cancer cells and demonstrated that NPM is strongly induced in response to hypoxia and protects cell death likely through inhibition of p53 activation. EXPERIMENTAL PROCEDURES Cell Culture and Treatments-Normal lymphoblasts derived from a healthy donor were maintained in RPMI medium 1640 (Invitrogen) supplemented with 15% fetal bovine serum (FCS). Human embryonic kidney 293 cells (HEK293) and human normal fibroblasts derived from a healthy donor were grown in Dulbecco's modified Eagle's medium with 10% FCS and ␣-minimal essential medium with 20% FCS, respectively. The human cancer cell lines used in this investigation were: HCT116 (colon cancer), MCF-7 (breast cancer), PC-3 (prostate cancer), K562 (chronic myelogenous leukemia), and HL60 (promyelocytic leukemia). These cells were maintained in various media in accordance to the requirements. Two sets of cells approaching confluence were incubated in parallel at 37°C in normoxia (humidified air with 5% CO 2 ) or hypoxia within a modular incubator chamber (BioSpherix, Redfield, NY) filled with 0.1% O 2 , 5% CO 2 , and balance N 2 . Immunoprecipitation and Immunoblotting-Whole cell extracts (5 * This work was supported by an American Cancer Society (Ohio Division) support grant, a Fanconi Anemia Research Fund grant, a Trustee grant from the Cincinnati Children's Hospital Medical Center, and National Institutes of Health Grant R01 CA109641 (to Q. P.). 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. RNA Isolation and RT-PCR-Total RNA was prepared with RNeasy kit (Qiagen) following the manufacturer's procedure. Reverse transcription was performed with random hexamers and Superscript II RT (Invitrogen) and was carried out at 42°C for 60 min and stopped at 95°C for 5 min. These reactions were followed by PCR using the following procedure: 95°C for 5 min; 15, 20, or 30 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min. For each primer pair (oligonucleotide sequences available on request), PCR was performed to obtain linearity between the amount of input RT product (1, 2, and 4 l) and output PCR product. NPM Transcription Studies-A nucleotide fragment (Ϫ587 to ϩ4 nucleotide of human NPM sequence (GenBank TM accession number BC009623)) encompassing the basal elements of human NPM promoter was cloned into the NcoI and SmaI sites of a PGL-3 vector (Promega) containing the luciferase reporter. The three mutant NPM promoter constructs were made by changing the consensus HRE 5Ј-RCGTG-3Ј to 5Ј-RAAAG-3Ј in each of the three putative HREs in the NPM promoter ( RNA Interference-Two small interfering RNA (siRNA) oligonucleotide duplexes (150 nM each), one targeting nucleotides 103-125 relative to the translation initiation codon of human NPM (22) and the other serving as the control, were transfected into cells with Oligofectamine (Invitrogen) according to the manufacturer's protocol. The transfected cells were incubated for 6 h under normoxic conditions followed by 16 h under hypoxic conditions. Analysis of Apoptosis-Cell viability was measured by trypan blue exclusion analysis. To quantify apoptotic cells, we used a polyclonal antibody to the active form of caspase 3 in a flow cytometric assay to detect cells in the early stages of apoptosis. RESULTS AND DISCUSSION NPM Protein Is Induced by Hypoxia-Expression of NPM is induced by stresses like DNA-damaging UV irradiation (23) and oncogenic insults (17,18). We thus examined NPM expression in hypoxic normal human lymphoblast and fibroblast cells. We found that the level of NPM protein was increased significantly in response to hypoxia after 6 h and last over a 24-h period (Fig. 1A, top panel). This coincides with hypoxia induction of HIF-1␣ (middle panel). NPM, as well as HIF-1␣, was also induced in cells treated with CoCl 2 , a chemical mimic of hypoxia (Fig. 1A, lanes 5 and 10; Ref. 24). Because high levels of NPM expression have been found in a variety of human cancers (7, 9 -15) and because many tumors contain hypoxic microenvironments (1), we wished to determine whether the NPM protein was elevated in hypoxic cancer cells. Five human cancer cell lines, HCT116 (colon cancer), MCF-7 (breast cancer), PC-3 (prostate cancer), K562 (chronic myelogenous leukemia), and HL60 (promyelocytic leukemia), were subjected to normoxia or hypoxia for 12 h before lysis. As shown in Fig. 1B, NPM expression was significantly increased in all tested cancer cell lines exposed to hypoxic conditions compared with cells incubated in normaxic conditions. Given that most cancer cells retain the ability to undergo hypoxiainduced cell death (25), elevated NPM expression in response to hypoxia by leukemia and other cancers raises the expectation that tumor cells may require increased expression of NPM to maintain cell survival under hypoxic conditions, and targeting this molecule may prove useful for cancer prevention and treatments. Hypoxia-induced Expression of NPM mRNA-To gain insight into the molecular basis for NPM induction in response to hypoxia, we performed RT-PCR analysis with mRNA extracted from HEK293 cells incubated in normoxia or hypoxia conditions. A significant elevation of NPM transcripts (2-4-fold) was observed at 6 h of hypoxia with peak induction at 12 h ( Fig. 2A). Hypoxia did not induce transcription of HIF-1a, as observed previously (26). However, the induction NPM transcription was concordant with that of VEGF, a well characterized hypoxiainducible gene (26). Thus, these results suggest a role for transcriptional control in the induction of NPM by hypoxia. NPM Promoter Is Responsive to Hypoxia and Activated by HIF-1␣-To identify cis-acting DNA sequences that respond to hypoxia, we isolated a 587-bp fragment of the 5Ј-flanking region of the human NPM gene (Fig. 2B). Promoter assays were performed in HEK293 cells with various luciferase reporter plasmids under normoxic and hypoxic conditions. As shown in Fig. 2C, luciferase expression driven by the NPM promoter (NPM-Luc) was increased 4.8-and 3.2-fold in response to hypoxia and by co-transfection with pcDNA3-HIF-1␣ under normoxia conditions, respectively. In contrast, significant luciferase activity was not observed in cells transfected with a luciferase plasmid containing no promoter (pGL3-Basic) under hypoxic conditions or co-transfected with pcDNA3-HIF-1␣ under normoxia conditions (Fig. 2C). Mutation of the HRE located 395 bp upstream of the NPM translation start codon (HRE1-AAA), but not the other two HRE mutants (HRE2-AAA and HRE3-AAA), abolished the increase in luciferase activity in cells exposed to hypoxia or cotransfected with pcDNA-HIF-1␣ (Fig. 2C). Collectively, these results demonstrate that NPM promoter is responsive to hypoxia and can be activated by HIF-1␣. NPM Protects against Hypoxic Cell Death of WT but Not p53-null Cells-To assess the physiological role of the inductive response of NPM to hypoxia, we analyzed hypoxia-induced apoptosis in the presence of NPM overexpression and a reduced level of intracellular NPM. We first applied RNA interference to down-regulate the expression in human breast cancer MCF-7 cells and determined the effect on hypoxia-mediated apoptosis. We transfected MCF-7 cells with either a 21-bp NPM-siRNA or a control RNA duplex with the same NPM sequence in the opposite orientation. Transfection with NPM-siRNA resulted in significant reduction of the intracellular NPM proteins (Fig. 3A, lanes 3, 6, and 9), while the control RNA duplex had no effect on NPM protein level (lanes 2, 5, and 8). The effect of NPM knockdown was analyzed by a flow cytometric method for hypoxia-induced caspase-3 activity (early apoptosis). Control or mock-transfected cells exposed to hypoxia showed slight increase in apoptotic rates compared with the corresponding groups cultured under normoxic condi-FIG. 2. Hypoxia-induced transcriptional up-regulation of NPM. A, first strand cDNA was synthesized from the total RNAs extracted from normal lymphoblasts incubated under hypoxic conditions and subjected to RT-PCR analysis. Shown is a RT-PCR result obtained with 30 cycles of amplification of cDNA encoding NPM, HIF-1␣, VEGF, and 28 S rRNA, respectively. B, the promoter sequence of NPM from HEK293 cells with three consensus HREs (highlighted in gray). The translation start codon at position ϩ1 is in boldface. The 5Ј end of the mRNA is indicated by an arrow, and a putative TATA box is underlined. C, luciferase reporter constructs containing no promoter (pGL3-Basic), the WT NPM promoter, or each of the three mutant NPM promoters was transfected into HEK293 cells along with pcDNA3 (vector) or pcDNA3-HIF-1␣, and the transfectants were incubated under normoxic or hypoxic conditions. After 24 h of co-transfection, luciferase activities were determined. The values represent the average luciferase activity of three independent experiments; bars indicate standard error. tions (Fig. 3B). However, a significant increase of caspase-3 activity (2-3-fold) was observed in hypoxic cells expressing the NPM-siRNA, as compared with the control or mock-transfected hypoxic cells (Fig. 3B). We next tested whether NPM overexpression could protect cell death induced by hypoxia. Because NPM has been shown to interact with the tumor suppressor p53 (22), we wished to determine whether the NPM protection against hypoxia-induced cell death involves p53. The genetically matched p53 WT and null mouse embryonic fibroblasts were transfected with vectors expressing WT NPM or a mutant variant with a deletion of the C-terminal 120 amino acids of NPM (NPM⌬C) that is required for binding to p53 (22). High levels of FLAG-tagged NPM or NPM mutant proteins were achieved in the transfectants (Fig. 3C), whereas no FLAG-tagged proteins were detected in control vector samples (Fig. 3C, lanes 1, 4, 7, and 10). We then tested the transfectants for sensitivity to hypoxia. Overexpression of WT NPM in p53 WT cells increased cell survival in hypoxic conditions by nearly 2-fold, as compared with the control vector transfected p53 ϩ/ϩ cells, whereas WT cells transfected with the vectors expressing the mutant NPM⌬C deficient in p53 binding were as sensitive to hypoxia as those transfected with control vectors. In p53 Ϫ/Ϫ cells, overexpression of neither WT NPM nor the mutant NPM⌬C had significant effect on hypoxia-induced cell death compared with the control vector transfected p53 Ϫ/Ϫ cells. Taken together, these results indicate that NPM protects cells from hypoxiainduced cell death through a mechanism involving p53. Hypoxia-induced NPM Inhibits Phosphorylation of p53 Ser-15 -Because we observed no increased protection against hypoxiainduced cell death in NPM-overexpressing p53 Ϫ/Ϫ cells, we reasoned that NPM protects against hypoxic cell death by inhibiting p53 activity. It has been shown that hypoxia induces phosphorylation at the Ser-15 residue of p53 (P-p53 Ser-15 ), which is critical for p53 transactivation and subsequent apoptotic signal transduction (27,28). We thus determined whether hypoxia-induced NPM affected p53 activity. Normal human fibroblasts transfected with control (empty vector; Fig. 4A, lanes 1 and 2), WT NPM (lanes 3 and 4), mutant NPM⌬C (lanes 5 and 6) vectors, or siNPM (lanes 7 and 8) duplexes were incubated in normxia or hypoxia for 16 h. Hypoxia induced p53 Ser-15 phosphorylation, resulting in the up-regulation of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 (Fig. 4A, lane 2). Strikingly, overexpression of NPM significantly reduced P-p53 Ser-15 and p21 (lane 4), whereas overexpression of the mutant NPM⌬C, which lacks the p53-interacting domain (22), failed to inhibit p53 activation (lane 6). Suppression of NPM expression by siNPM increased the level of P-p53 Ser-15 and p21 (lane 8). We next asked whether inhibition of p53 activation was due to interaction between the two proteins. Indeed, we found that hypoxia increased association of endogenous NPM with p53 (Fig. 4B, compare lane 3 and lane 4). It appeared that NPM interacted with both total p53 and P-p53 Ser-15 . While forced expression of exogenous NPM under hypoxic conditions did not further enhanced its association with p53 (Fig. 4B, lower panel, lane 5), it did significantly reduced the bound P-p53 Ser-15 (Fig. 4B, upper panel, lane 5). Overexpression of the mutant NPM⌬C had no effect, consistent with its defect in interaction with p53 (22). Transfection of the cells with either siNPM (lane 7) or the control siRNA (lane 6) did not appear to have significant effect on the NPM-p53 interaction, although we observed increased P-p53 Ser-15 bound to the endogenous NPM (Fig. 4B, upper panel, lane 8). These results collectively demonstrate that NPM plays a role in regulation of hypoxiainduced p53 activity, possibly by directly binding to the tumor suppressor. Hypoxia plays an important role in many pathological processes such as ischemic stroke and tumor progression. Cells respond to hypoxia by expressing a variety of gene products to adapt to altered environments or to exploit for a survival/ proliferative advantage (1). Here we have demonstrated that hypoxia induces the expression of NPM, a protein frequently overexpressed in a variety of human malignancies. We have also shown that the NPM promoter encodes a functional HRE that can be activated by hypoxia or by forced expression of HIF-1␣. Suppression of NPM expression by siRNA targeting NPM increases hypoxia-induced apoptosis. Induction of NPM in response to hypoxia has important physiological relevance because hypoxic stress requires inductive expression of antiapoptotic genes like NPM to direct the cell toward the survival/ proliferative state instead of the apoptotic state. In addition, our study demonstrates that hypoxic cells overexpressing NPM are more resistant to apoptosis possibly through inhibition of p53, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Whole cell lysates (5 mg of total proteins) were immunoprecipitated with anti-NPM antibodies conjugated to the paramagnetic Dynabeads, and the anti-NPM immunocomplexes were analyzed by immunoblotting with antibodies specific for phospho-p53 Ser-15 (P-p53 Ser-15 ; upper) or total p53 (lower). Lanes 1 and 2 contain 100 g of extracts from empty vector-transfected cells incubated in normoxia and hypoxia, respectively.	v3-fos-license
2017-05-09T23:38:30.997Z	2003-10-31T00:00:00.000	50989490	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1424-8220/3/10/451/pdf?version=1403299858", "pdf_hash": "743cfe0b71c54f5c07f563bae04f8e17de04999e", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:671", "s2fieldsofstudy": [ "Environmental Science", "Biology", "Engineering", "Chemistry" ], "sha1": "743cfe0b71c54f5c07f563bae04f8e17de04999e", "year": 2003 }	pes2o/s2orc	Semiconductor Metal Oxide Sensors in Water and Water Based Biological Systems The results of implementation of In2O3-based semiconductor sensors for oxygen concentration evaluation in water and the LB-nutrient media (15.5 g/l Luria Broth Base, Miller (Sigma, Lot-1900) and NaCl) without bacteria and with .coli bacteria before and after UV-irradiation are presented. Introduction Effective implementation of semiconductor metal oxide sensors (SMOS) for air and gas media analysis may find logical extension.There are good options to use these sensors for analysis of various gas components dissolved in polar liquids (for instance, in water or liquid phase biological systems).Such possibility results from an increase of electron transfer rate between adsorbed gas molecules and semiconductor surface, caused by polar media.That is why theoretic options exist for development of a method for gas analysis at a temperature of a liquid system.Such method could not only simplify a measurement process, but also improve sensor sensitivity as a result of increase of gas molecules adsorption rate under the conditions of temperature decrease. Though advantages of semiconductor metal oxide sensors are explicit (i.e.high sensitivity, operating speed, small sizes, options for real time measurements, comparatively low cost), only few papers were thitherto published on the implementation of this method for gas analysis in liquid media. Experimental approach was presented in the series of papers of Miasnikov and co-authors [1][2][3][4], in which the semiconductor sensors technique for gas analysis in polar liquids was developed.According to these papers, the trend has been toward intensification of the processes of adsorption and desorption of gases and radical particles on the semiconductor surfaces (ZnO, TiO 2 ) in polar liquids without additional heating, which is necessary in the case of gas media.Equations are proposed [3], which interlink sensor electrophysical parameters with oxygen concentration in a liquid. It should be emphasised that the results in [1][2][3][4] were obtained in the sells, separated from environment, and in the low concentration range of dissolved oxygen (2,24E -03 -4,80E -7 µg/l).But the most important, as a practical matter, liquid phase systems, i. e biochemical liquids, such as blood, cytoplasm and others, constitute open systems with wide range of oxygen concentrations, as compared to concentrations investigated in the above papers.That is why our understanding is that SMOS-based biosensors have good prospects of implementation for open systems at high concentrations of dissolved oxygen.We should also like to emphasise that direct extrapolation would be unfounded of the results, obtained in [1][2][3][4], to the open liquid phase systems.Special investigations should be carried out for reconciliation of the conclusions, made in [1][2][3][4], with the results, which would be obtained for the open systems at high concentration of dissolved oxygen.Bio-sensors seem to be the efficient in this direction, and special research should be carried out with the use of bio-system models for biosensors development on the basis of SMOS. When choosing test model for our investigations we were guided by one of the most important problem of medical diagnostics of virus infections, which is connected to early virus recognition in human organism.This problem could be solved by means of assessment of the quantity of gas phase vital function products of the microorganisms (including oxygen) in a nutrient medium at a stage, that forerun active growth of the above microorganisms.With this purpose we have developed sensitive semiconductor metal oxide sensors for evaluation of micro alterations of oxygen concentrations in a nutrient medium, when it contains pathogen bacteria. In our paper the results are presented of implementation of In 2 O 3 -based semiconductor sensors for oxygen concentration evaluation in the LB-nutrient media (15.5 g/l Luria Broth Base, Miller (Sigma, Lot-1900) and NaCl) without bacteria and with .colibacteria before and after UV-irradiation. Experimental procedure Semiconductor sensors for measurement of dissolved oxygen concentration in the water represent insulating substrate with measurement Pt-electrodes, applied by cathodic sputtering method, and In 2 O 3 sensitive semiconductor layer (width 1 m) applied according to special technology, providing good adhesion and water resistance. .coli 600-lux culture was used, which contains multy-copy plasmid with luciferase gene (luminosity range from 400 to 480 nm).The cells were cultivated at 33 0 in LB-nutrient media, produced with the following contents: 15.5 g/l Luria Broth Base, Miller (Sigma, Lot-1900) with addition of N Cl..coliC600-lux culture, containing multy-copy plasmid with luciferase gene with LEX-A promotor, was chosen in such a way that produced system provide oxygen self-sufficiency. Sensor calibration was carried out in the isolated calibration cell for all water systems.Nitrogen, helium or argon was used as carrier gas for extrinsic oxygen.Gas mixtures with predetermined concentrations by means of dynamic generator of standard mixtures ( -3 brand).Gas mixture was delivered to calibration cell by bubbling through glass tube with Shott filter.Output sensor response was transferred from the secondary actuator to PC through L-card interface. Results and Discussion Results of sensor calibration in pure water, in NaCl (0,5g/l) water solution and in LB-nutrient media are presented in Fig 1 .As it was revealed in [3], under stationary operation condition of a sensor in separated sell at low oxygen concentration, oxygen concentration dependence of electrocunductivity is as follows: where σ 0 and σ m -initial (before oxygen adsorption) and stationary (after achievement of oxygen adsorption-desorption process) sensor electroconductivity, -constant, which is proportional to the ratio of constants of oxygen adsorption-desorption ( 1 and 2 ); -concentration of dissolved oxygen; -partial oxygen pressure over liquid surface; α -Bunzen factor.Calibration curves for all three systems (Fig. 1) are described by formula1 (1) within the range of experimental error.Observed sensor sensitivity in NaCl solution is higher as compared to pure water (Fig. 1).The same effect, though at less extent (Fig. 1), is observed in LB-nutrient media containing 0,5 g/l NaCl and organic admixtures.We have carried out a series of experiments on low NaCl concentration influence on oxygen sensitivity of the sensor in an open water system.Obtained results are presented in Fig. 2. As it is shown in Fig. 2, the sensitivity of the sensor increases with NaCl concentration increase, which is in good agreement with the data of [4], which was obtained for closed cell.As it was already mentioned, the major objective of our investigation is to show that semiconductor metal oxide sensors can be successfully implemented in biological liquids for detection of micro changes of dissolved oxygen concentration, resulted from bacteria vital function at various stages of its growth.With the help of sensor we intended to reveal the dependence between dissolved oxygen concentration changes and state of bacteria, i.e. their viability level.With the purpose of changes in bacteria state they were subjected to UV-irradiation, which influenced upon their viability.Beforehand the influence of UV-irradiation upon LB-nutrient media was investigated (Fig. 3).It is shown that during first minutes of UV-irradiation, the kinetic of sensor response changes strongly.In the inset in Fig. 3 the result of subtraction of sensor responses before and after UVirradiation is presented.The result of subtraction may be explained as the rise of new donor signal (after UV-irradiation of LB-media) on the background of unchanged oxygen sensor response.The above donor signal completely disappears 10-15 minutes after UV-irradiation.The only possible explanation of this fact is the appearance of fragments of organic molecules with short life period in LB-media, as a result of UV-irradiation. It is well known that .colicells are characterised by complicated mode of damage repair, which was elaborated during evolution process.In the case of damage in DNA molecules, resulted from various chemical or physical factors, including UV-irradiation, the SOS-lux system is being launched.This system is based upon luminescent bacteria ability to radiate visible light as a result of luciferase catalysis.This process is accompanied by oxygen absoption and is described by the below scheme: where FNMH 2 -unsaturated flavionic mononucleotid; RCHO -long oil polymer aldehyde; RCOOHlong oil polymer acid.So, after irradiation in the culture methabolic processes are launched, which are accompanied by decrease of oxygen absorption.But simultaneously the system is launched of DNA repairing in .colibacteria cells, which increases oxygen absorption. With the purpose of investigation UV-irradiation influence upon change of dissolved oxygen concentration in LB-media, containing .colibacteria, the test specimens of bacteria culture were prepared and stored at room temperature (20 ).Metabolism rate substantially depends upon temperature.Optimal temperature value is higher than 30 .That's why at 20 the rate of cells division process is low or equals to zero.If the division process takes place, it should influence upon dissolved oxygen concentration.Slow oxygen concentration decrease was indicated by means of sensor in the test specimens of the bacteria culture (Fig. 4). Then test specimen was placed into UV-irradiation chamber (wave length 10-400 nm, irradiation time 3 minutes, integral flow density 250 watt-second/m 2 ).During the period of specimen irradiation, the sensor was placed into pure nutrient medium.5 seconds after the completion of test specimen irradiation, the measurements of concentration of dissolved oxygen were started. Fig. 4 presents kinetic curves concentration changes of dissolved oxygen in the test specimen after irradiation, which was obtained by means of semiconductor sensor.The comparison of the curves reveals that sensor response in the specimen after UV-irradiation decreases 3 times quicker than sensor response in the test specimen of the same culture before irradiation.Just this result was should be realised in case of validity of scheme 2. The curve of the irradiated specimen (bottom curve, Fig. 4) also presents a some peculiarity, which reveals at the same time interval as additional donor signal in the LB-pure nutrient medium (Fig. 3).So, we can assume that to parallel processes take place, and their influence upon the sensor response kinetics differs one from another.First of all, it is quick decrease of dissolved oxygen concentration in the specimen after irradiation, which results from increase of oxygen absorption by .colibacteria through the launch of SOS-reparation mechanism.Secondly, it is creation of high molecular radicals LB-media and their further recombination and disappearance. Conclusion In our research we demonstrated and proved availability of options for implementation of semiconductor metal oxide sensors (SMOS) in various liquid phase systems for evaluation of dissolved oxygen concentration changes under real time conditions.The proposed techniques present high sensitivity, which cannot be achieved by other techniques.This allows investigating kinetics of the complicated biological processes, such as bacteria sells division (transfer to the logarithmic growth phase, and cells reparation processes, which result from UV irradiation. We can state with assurance that semiconductor sensor techniques has good prospects in the area of biological and biochemical systems, and its further development will become substantial contribution to bio-sensor science. Authors express their gratitude to Dr. Alipov E.D. (MIFI) for his kind co-operation during arrangement of the investigations and for supply of LB-nutrient media and bacteria culture. Figure 1 . Figure 1.Calibration curves for semiconductor sensor : in water, in NaCl water solution, in LBnutrient media. Figure 2 . Figure 2. NaCl concentration dependence of semiconductor sensor.The arrow mark NaCl concentration in LB-nutrient media. Figure 3 . Figure 3. Kinetics of sensor response in LB-nutrient media before and after UV-irradiation.Inset shows the result of subtraction of these curves. Figure 4 . Figure 4. Kinetics of sensor response in LB-nutrient media with .colibacteria before and after UV-irradiation.	v3-fos-license
2019-03-31T10:48:41.762Z	2018-01-04T00:00:00.000	92981822	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2306-5710/4/1/3/pdf", "pdf_hash": "9536471e092ca440108c7e1e60b32da5d46a366e", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:677", "s2fieldsofstudy": [ "Environmental Science" ], "sha1": "0bf82fabbce9ddf505927616524f55a0ebb16b0a", "year": 2018 }	pes2o/s2orc	Effect of Different Glass Shapes and Size on the Time Course of Dissolved Oxygen in Wines during Simulated Tasting : The different shapes and sizes of wine glass are claimed to balance the different wine aromas in the headspace, enhancing the olfactory perception and providing an adequate level of oxygenation. Although the measurement of dissolved oxygen in winemaking has recently received much focus, the role of oxygen in wine tasting needs to be further disclosed. This preliminary study aims to explore, for the ﬁrst time, the effect of swirling glasses of different shapes and sizes on the oxygen content of wine. Experimental trials were designed to simulate real wine tasting conditions. The O 2 content after glass swirling was affected to a considerable extent by both the type of wine and the glass shape. A lack of correlation between the shape parameters of ﬁve glasses and the O 2 content in wine was found which suggests that the nonequilibrium condition can occur during wine tasting. The International Standard Organisation (ISO) glass—considered to be optimal for the wine tasting—allowed less wine oxygenation than any other glass shapes; and the apparent superiority of the ISO glass is tentatively attributed to the more stable oxygen content with time; i.e., less variability in oxygen content than any other glass shape. Introduction Although the critical role of oxygen in enology has recently been disclosed from a chemistry [1] and winemaking point of view [2][3][4], little information is available from the sensory perspective. Before tasting, the glass of wine is usually "swirled" by holding the glass by the stem and gently rotating it. This action, technically called 'orbital shaking', increases the surface area of the wine by spreading it over the inner part of the glass and consequently enabling some evaporation to take place [5]. Moreover, it is also expected to draw in some oxygen from the air. The ingress estimated on the undisturbed surface of a wine is about 200 mg/h/m 2 [6]. The physics of wine swirling was recently investigated with an elegant fluid dynamic approach, which modelled the pumping mechanism induced by the wave propagation along the glass wall [7]. Three factors seemed to determine whether the team spotted one big wave in the wine or several smaller ripples: (i) the ratio of the level of wine poured in to the diameter of the glass; (ii) the ratio of the diameter of the glass to the width of the circular shaking; (iii) and the ratio of the forces acting on the wine. From a practical point of view, these findings suggest that the mixing and oxygenation may be optimized with an appropriate choice of shaking diameter (d) and rotation speed (rpm). In this view, the glass shape parameters ( Figure S1) can play a key role as they may influence the perceived volume of wine [8], and the perception of wine odors [9][10][11], and color [12][13][14], and therefore the consumer's preference [15,16] as well. Moreover, with time, the glass shape affects the change of headspace chemical composition of wine poured inside, and the D ratio (i.e., maximum diameter divided by opening diameter) seems to be the most important parameter relating glass shape to headspace composition [17]. Considering that both consumers and professional wine tasters usually swirl the glass of wine for approx. 10 to 20 s to unlock odors, there is a need for information to disclose the effect of glass shape on the oxygen content of wines during simulated tasting condition. This preliminary trial aimed to study whether the glass shape can affect the oxygen content in wine under both static and dynamic conditions (i.e., swirling), the latter to simulate the standard procedure of sensory evaluation of wine. The use of optical oxygen sensors (also called minisensors) allowed for the first time the on-line non-invasive and non-destructive oxygen measurements in a glass under dynamic conditions. Samples Both red and white wines were selected for this study, including (i) a Rebola 'Nita' white wine Colli di Rimini DOP 2013, (ii) a Sangiovese red wine Carlo Leo Romagna DOP 2013, and (iii) a Cabernet Sauvignon 'Tano' Colli di Rimini DOP 2013 (Az. Agric. Le Calastre, Rimini, Italy). Sangiovese is the main red grape in Italy, Cabernet Sauvignon is a well know international grape variety, and Rebola is an emerging local white grape variety of great interest as well. The bottled wines were provided by the producer and were stored at room temperature (20 ± 1 • C) until the swirling trials. Preliminary characterization of wine composition (Table S1) was carried out according to endorsed methods (RESOLUTION OIV/OENO 390/2010). Oxygen Measurement Non-invasive dissolved oxygen (DO) in wine was measured using an OXY-4 oxygen meter (PreSens GmbH, Regensburg, Germany) equipped with a polymer optical fiber and PSt3 spot, also called minisensor (Presens GmbH). The PSt3 spot had a thickness of 1 mm, a diameter of 4 mm, and a response time (t 90 , the time for 90% of the change in signal to occur) of 10 s. In each glass, one minisensor was glued 5 mm below the wine level and calibrated with water at a controlled temperature according to the manufacturer's instructions ( Figure S2). In each bottle, once uncorked, the dissolved O 2 content of wine was directly measured with an oxygen-dipping probe (Presens GmbH) placed in the middle of the bottle in a dynamic regime (i.e., with stirring). Before bottling the wines, three glass bottles (750 mL) were equipped with two minisensors each to ascertain both the headspace and the dissolved O 2 content in static regime (i.e., bottled wines). Glasses Six different wine glasses were selected for this study, including the ISO official-tasting glass (Bormioli, Parma, Italy) (Table 1; Figure 1). Glass parameters were measured according to the literature (Hirson, Heymann and Ebeler 2012) and each glass was filled with a fixed volume of wine (50 mL) to fit the conditions commonly used during professional wine tasting [18]. Swirling Trials To simulate the gesture of hand swirling during wine tasting, each glass of wine was placed onto an orbital shaker (model 709/R ASAL, Cernusco s/N, Italy) with a shaking diameter of 3 cm and a shaking speed of 150 rpm (this value was optimized with preliminary screening trails from 100 to 250 rpm). Each glass of wine was swirled for 10, 20 and 40 s, using independent wine samples. Table 1 for details). Experimental Design and Statistical Analysis The four variables (6 glass types, 3 wines, 3-time readings, 3 replicates for each glass) required 162 independent measurements. Statistical analysis was based on a paired t-test (p-level < 0.05) using the Unscrambler X (v. 10.3, Camo ASA, Oslo, Norway). Results and Discussion The oxygen content was measured (i) before and after opening the bottle of wine and (ii) before and during the glass swirling trails, for which protocol was designed to simulate the usual wine tasting by consumers and experts. As expected, the concentration of oxygen in bottled wine was very low with an average range from 4 to 14 µg/L for headspace O2, and in the range of 3 to 29 µg/L for dissolved O2 in wine. In bottled wine, the rate of O2 dissolution was less than the consumption; however, once the bottles are opened the wine comes into contact with air and the oxygen content is expected to rise. In fact, soon after opening the O2 content in bottled wine increased regularly up to 0.99 mg/L in 15 min (Figure 2). These findings are consistent with the initial oxygen absorption capacity of Madiran red wines with pH 3.78 [19]. The O2 accumulation in wine implied that the rate of oxygen dissolution was higher than the rate of its uptake. The latter can be (indirectly) measured by the drop in SO2. According to Boulton [20], the oxygen consumption reactions in wine involve the phenolic compounds as the main substrates and the oxygen consumption is the rate-limiting reaction. The rates of this reaction are first order in oxygen concentration and catalyzed by ferrous ion; therefore, the rate constant would be related to the ferrous ion concentration, but the rate law would depend only on the oxygen concentration. The amount of oxygen found after 15 min most likely increases the wine redox value of ca. 25 mV and will theoretically consume ca. 4 mg/L of sulfur dioxide [21]; both parameters could affect the sensory properties of wines to some extent [4,13]. Table 1 for details). Experimental Design and Statistical Analysis The four variables (6 glass types, 3 wines, 3-time readings, 3 replicates for each glass) required 162 independent measurements. Statistical analysis was based on a paired t-test (p-level < 0.05) using the Unscrambler X (v. 10.3, Camo ASA, Oslo, Norway). Results and Discussion The oxygen content was measured (i) before and after opening the bottle of wine and (ii) before and during the glass swirling trails, for which protocol was designed to simulate the usual wine tasting by consumers and experts. As expected, the concentration of oxygen in bottled wine was very low with an average range from 4 to 14 µg/L for headspace O 2 , and in the range of 3 to 29 µg/L for dissolved O 2 in wine. In bottled wine, the rate of O 2 dissolution was less than the consumption; however, once the bottles are opened the wine comes into contact with air and the oxygen content is expected to rise. In fact, soon after opening the O 2 content in bottled wine increased regularly up to 0.99 mg/L in 15 min (Figure 2). These findings are consistent with the initial oxygen absorption capacity of Madiran red wines with pH 3.78 [19]. The O 2 accumulation in wine implied that the rate of oxygen dissolution was higher than the rate of its uptake. The latter can be (indirectly) measured by the drop in SO 2 . According to Boulton [20], the oxygen consumption reactions in wine involve the phenolic compounds as the main substrates and the oxygen consumption is the rate-limiting reaction. The rates of this reaction are first order in oxygen concentration and catalyzed by ferrous ion; therefore, the rate constant would be related to the ferrous ion concentration, but the rate law would depend only on the oxygen concentration. The amount of oxygen found after 15 min most likely increases the wine redox value of ca. 25 mV and will theoretically consume ca. 4 mg/L of sulfur dioxide [21]; both parameters could affect the sensory properties of wines to some extent [4,13]. As time progresses, the dissolved oxygen content in wine is expected to reach a plateau approaching the saturation value of ca. 8.6 mg/L at 20 °C [22]. This postulate was confirmed by monitoring with time the dissolved O2 in a wine glass under firm conditions, i.e., unstirred, unshaken, directly after pouring. (Figure 3). The glass n. 5 showed a very fast O2 intake followed by the glass n. 4, whereas the wine poured on the ISO glass (No. 1), commonly used in professional wine tasting, showed the lowest and most stable O2 content. Although dissolved oxygen mostly depends on the surface area of wine exposed and the exposure time, a lack of significant correlation was found between the glass shape parameters and the O2 content in wine. To explain this result, the hypotheses of nonequilibrium conditions and complex interaction among parameters were formulated. Table 1 for details). As time progresses, the dissolved oxygen content in wine is expected to reach a plateau approaching the saturation value of ca. 8.6 mg/L at 20 • C [22]. This postulate was confirmed by monitoring with time the dissolved O 2 in a wine glass under firm conditions, i.e., unstirred, unshaken, directly after pouring (Figure 3). The glass n. 5 showed a very fast O 2 intake followed by the glass n. 4, whereas the wine poured on the ISO glass (No. 1), commonly used in professional wine tasting, showed the lowest and most stable O 2 content. Although dissolved oxygen mostly depends on the surface area of wine exposed and the exposure time, a lack of significant correlation was found between the glass shape parameters and the O 2 content in wine. To explain this result, the hypotheses of nonequilibrium conditions and complex interaction among parameters were formulated. As time progresses, the dissolved oxygen content in wine is expected to reach a plateau approaching the saturation value of ca. 8.6 mg/L at 20 °C [22]. This postulate was confirmed by monitoring with time the dissolved O2 in a wine glass under firm conditions, i.e., unstirred, unshaken, directly after pouring. (Figure 3). The glass n. 5 showed a very fast O2 intake followed by the glass n. 4, whereas the wine poured on the ISO glass (No. 1), commonly used in professional wine tasting, showed the lowest and most stable O2 content. Although dissolved oxygen mostly depends on the surface area of wine exposed and the exposure time, a lack of significant correlation was found between the glass shape parameters and the O2 content in wine. To explain this result, the hypotheses of nonequilibrium conditions and complex interaction among parameters were formulated. Table 1 for details). Figure 3. Time course of oxygen dissolution in wine glass under firm conditions. Legend: lines 1-6 refer to different glass shapes (see Table 1 for details). For any specific wine, the variation of the dissolved oxygen concentration with time can be arranged as: where: k L a: volumetric mass transfer coefficient (T −1 ); O 2 Dissolved oxygen concentration in wine (mg O 2 L −1 ); O * 2 Oxygen equilibrium concentration in wine (mg O 2 L −1 ). Therefore, the volumetric mass transfer coefficient is the aggregate result of both contributions: the resistance to mass transport in the liquid side (k L ) and the interfacial area (a). The oxygen transfer rate will decrease during the period of aeration as O 2 approaches O * 2 due to the decline in the driving force (O * 2 − O 2 ). The experimental k La values of wines in opened bottle and glasses were tentatively estimated upon monitoring the increase in the dissolved oxygen concentration of a wine during aeration and agitation and by plotting of oxygen deficit (O * 2 − O 2 ) versus time (t) on a semilogarithmic graph. The slope of the regression line determined the overall mass transfer coefficient (k L a), which showed 5 times variation in the range 0.0001-0.0005 s −1 (Table 1), with k La of wine in open bottle at the low value of 0.0001 s −1 . As the estimated k La values refer to static conditions (i.e., non-agitated vessels-bottle or glass-and lack of gas supply, e.g., micro-oxygenation) the current findings are consistent with data from the literature. The mass transfer coefficient (k La ) in the aerated model wine solution range between 0.00145 s −1 [23] and 0.013 s −1 [24], the values of which are affected by several parameters, including the oxygen gas flow rate and the intensity of agitation. The best estimate of the rotational speeds effect is given in terms of liquid side mass transfer coefficient (k L ) which increases more than 9 times as rotational speed increases from 50 to 120 rpm [25]. Remarkably, k La is found to increase following a power law with exponent rising from 0.5 to 3 due to significant increases in the interfacial area due to vortex [26]. During professional wine tasting for appellation certification, the wine is held in a glass for about 5-10 min and swirled for approx. 5-30 s before tasting. In this view, the swirling trails were designed to verify the likely occurrence of nonequilibrium conditions during the sensory evaluation of wine. The initial O 2 content at time zero (T 0 ) was always measured before each swirling trials for every glass shape. The O 2 content after glass swirling was affected to a considerable extent by both the type of wine and the glass shape. Short swirling time (up to 20 s) most often decreased the dissolved oxygen in glass wine (Table 2), while O 2 significantly increased at T 40 in Rebola white and Sangiovese red wines for all glass shape, except for glass n. 1 in Sangiovese trial. In contrast, the O 2 content in Cabernet Sauvignon wine increased only in glass n. 5 and n. 6 at T 40 . It seems that the Cabernet Sauvignon would require more time to enhance the O 2 content compared to the other two wines, which are lower in polyphenolic compounds and SO 2 . According to the Henry's law, the oxygen uptake resulting from the presence of antioxidants in wine is rapid and follows a largely exponential form [20]. The alternative hypothesis that O 2 is initially stripped out from solution by the release of CO 2 is postulated. Clearly, the ISO glass (No. 1)-usually considered to be optimal for wine tasting-allowed less wine oxygenation than any other glass shape. Considering that the O 2 content of wine most likely affects the performance of sensory evaluation, based on our findings, the apparent superiority of the ISO glass is tentatively attributed to the more stable oxygen content with time, i.e., less variable than any other glass shape. The current results under the dynamic regime substantiate the findings of Venturi et al. [27] who also investigated the influence of glass shape on the dissolved oxygen content of a rosé wine under static conditions-using the polarographic ADI dO 2 sensor-for which equilibration time was found to be approx. 1 h. In conclusion, the current preliminary study showed that pouring wine into a glass considerably affects the O 2 content and the likely occurrence of nonequilibrium condition requires a careful standardization procedure during a real wine tasting. Table 2. Heatmap plot of dissolved oxygen (mg/L) in wine glasses at time zero (T 0 ) and after 10, 20 and 40 s (T 10 , T 20 , T 40 ). Legend: glass 1-6 refers to different shape (see Table 1 for details). The symbol in letters (a, b) refers to significant paired difference between samples (p-level = 0.05) if occurred. Figure S1: Glass parameters, Table S1: Chemical composition of wines, Figure S2: Experimental online measurement. Figure S1. Glass parameters. Figure S2. Experimental online measurement.	v3-fos-license
2018-12-18T05:21:16.605Z	2004-01-01T00:00:00.000	56377194	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://downloads.hindawi.com/journals/jspec/2004/906243.pdf", "pdf_hash": "962ebebec1c0c56e9df5094fac062db29667f465", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:688", "s2fieldsofstudy": [ "Medicine", "Materials Science", "Physics" ], "sha1": "962ebebec1c0c56e9df5094fac062db29667f465", "year": 2004 }	pes2o/s2orc	Human breast tissue characterisation with small-angle X-ray scattering Small-angle X-ray scattering (SAXS) patterns from breast tissue samples are compared with their histology. Formalin fixed human breast tissue specimens containing ductal and lobular carcinoma were studied. Histo-pathological information is compared with the scattering data, and there is a clear spatial correlation. Supra-molecular organisation of collagen fibrils is modelled and the model is used to create scattering maps. The model parameters include the axial periodicity (d-spacing), radius and packing of the fibrils, and these are derived from comparison with the experimental scattering patterns. The d-spacing is to 0.5% larger in malignant zones of the tumours than in the healthy zones. There are also characteristic differences in the fibril diameter and packing. Introduction Breast cancer is one of the principal causes of death among women in developed countries [5].The mortality of the disease is considerably reduced, if tumours are found at an early stage of growth [9].Frequently the disease may be fully symptomless, and therefore mammographic screening is carried out among women over 50 years old in many countries.Nevertheless, in young dense breast, the cases of false positives and/or missed tumours, false negatives, are frequent [12], especially in the case of the lobular carcinoma, and/or in the absence of micro-calcifications or masses of different densities. Mammography is based on the absorption of X-rays in the tissues, and it reveals changes in density and morphology.The absorption contrast is weak, and the mammographic signs of cancer are subtle, and new methods for imaging of breast cancer are being developed [13].In particular, the contrast in soft tissues such as mammary gland is much more enhanced with phase contrast methods. The nature of the breast tumours is indicated either by morphology (in the core needle biopsies) or radiographic signs (in the mammograms).In the case of malignant diagnosis an increasingly employed treatment is conserving surgery in combination with chemotherapy, hormone treatment or postoperative radiotherapy.However, before proceeding to definitive treatment, the confirmation of diagnosis is always done by the histological biopsy.In a core needle biopsy, the needle is introduced into the tissue and a little piece of the tumour is extracted.Then the biopsy is examined by histo-pathological methods in order to determine the nature of the tumour. Breast tumour growth (hyperplasia) is closely related to collagenosis, and connective tissue may wrap the tumour and encapsulate it in the healthy tissue.Fibrillar collagen is newly formed in the tumours, and it has a supra-molecular structure different from the one found in healthy tissues [10,11].Smallangle X-ray scattering (SAXS) is an efficient technique for retrieving the supra-molecular structure of the breast tissues, especially those rich in collagen [4].Different "signatures" of the SAXS patterns of the tissues can be systematically studied and the scattering signals can be mapped and compared with histo-pathology of the samples. Small-angle X-ray scattering SAXS is a powerful method to determine structural features of molecular systems in soft tissue and their different degrees of organisation.The scattering vector k is defined by two unit vectors that indicate the incident (s o ) and the scattered beam (s), respectively (Fig. 1), The modulus of the scattering vector is or alternatively, Here θ is half of the scattering angle.Both k and s will be used in the following.The Bragg Law gives the condition for constructive interference of scattering from a structure that has a periodicity d in the direction of the scattering vector, where n = 1, 2, 3, . . .are the different orders.Combining Eqs (3) and ( 4), This equation is particularly useful, because it gives the real space periodicity d in terms of the positions of the diffraction maxima, s. The scattering amplitude in the units of the electron scattering length (electron classical radius) is the so-called structure factor, and ρ is the electron density.Basically, the scattering amplitude is the Fourier transform of the electron density of the scatterer.The observable quantity is the intensity, which is the Fourier transform of the autocorrelation function P (r) of the electron density, The intensity distribution of X-ray scattering contains information about the structure of the object on many different levels.Periodicity in the atomic scale produces maxima at large values of s, while the intensity modulations due to macromolecules and their assemblies are seen at small s, i.e., in the SAXS regime.Typical length scales for these objects are from a few nanometers to several hundred nanometers, so that with 0.1 nm radiation the SAXS pattern of a tissue sample is observed at scattering angles of one degree and less.Specific formulae for SAXS can be given only for isolated, randomly oriented independent objects, but some of the results are valid under quite general conditions [6].At the limit of forward scattering the intensity is proportional to the square of the Fourier transform of the shape function of the object, i.e., the object size can be deduced from the intensity at very small k.Another important general result is the Porod law, which gives the intensity of scattering at the large values of k in the SAXS pattern.For a 3-dimensional object with a smooth surface the asymptotic form of intensity is Here ∆ρ is the density difference between the object and its surroundings, and S is the surface area of the scatterers per unit mass.Similar results are obtained for thin disks and long rods, for which the power law exponents are −2 and −1, respectively.For the present case, particularly relevant are polymer chains, which are locally rod-like but become coils over large distances.For these the power law exponent is −2 or −5/3.Tissues are made of hierarchical molecular and supra-molecular structures, so that the requirement of independent scatterers is not met.However, realistic models can be constructed and the corresponding diffraction patterns can be calculated.Model parameters are obtained from comparison with the experimental data, so that the tissues may be characterised by these parameters.In the following, a model is presented for one of the most ordered tissue components, namely fibrillar collagen. Collagen Collagen is a protein, a polypeptide chain of amino acids, where every 3rd residue is Glycine (Gly-Xxx-Yyy).Collagen type I and III are found in the connective tissue of breast, and both types are fibrillar [8].The molecule of fibrillar collagen consists of a triple α-helix, coiled up as a rope [7].Collagen type I has two identical chains, and the third one is different.Collagen III has all three identical α-chains.In fibrils, collagen molecules pack laterally to each other with hydrogen bonds, in an approximately hexagonal close-packed (hcp) structure [14].Longitudinally, collagen molecules bind to each other in a staggered arrangement.This is a periodic structure, repeated along the fibril axis.The fibrils eventually pack together also in a near-hcp arrangement.This is the supra-molecular structure that gives rise to a characteristic SAXS pattern of collagen. Collagen fibrils in healthy breast tissue have a radius of about 90 nm, have an inter-fibrillar distance of approximately 100 nm, and the axial periodicity (d-spacing) is approximately 65 nm. SAXS from collagen Scattering from a single collagen fibril is well known [14], and also scattering from closely packed fibrils [2,3].For scattering from a single fibril one must consider two cases: well-oriented (referred to the beam direction) and randomly oriented fibrils.The SAXS pattern is obtained as a linear combination of the contributions from the orientation and size distributions of the fibrils.The contribution from welloriented fibrils can be divided into equatorial and meridional directions.In the equatorial direction there is intensity modulation, which can be described by Bessel functions, and these provide estimates for the distribution of the fibril radii.In the meridional direction many orders of distinct Bragg reflections are observed, and these give the axial d-spacing.The contribution of the randomly oriented fibrils is that of scattering from long rods, which falls off as k −1 .The fibrils are closely packed, and the interference gives rise to a broad maximum at small k-values in the equatorial direction.In spite of its simplicity, this model gives a good description of the observed SAXS pattern [4]. Collagen degradation SAXS patterns give information about the supra-molecular arrangement of collagen, so can be used to identify modifications of such structures.Tissues containing collagen can be classified by the fibril diameter, their packing, and by their axial periodicity.It has been demonstrated that collagen fibrils in healthy tissues have molecular characteristics different from those in malignant tumours [10,11].The SAXS patterns from collagen reflect these differences.For instance, the radii of the fibrils and distances between them are larger in the benign case than in the malignant case, while the axial periodicity in the malignant lesions is about 1% bigger than in healthy tissue [4]. Another important indication that changes take place in the collagen when cancer develops is the change in the average scattered intensity in certain regions of the SAXS pattern.For instance, the background intensity between the 5th and 6th collagen peaks is significantly higher in the case of invaded collagen1 than in the case of totally healthy collagen from the same tumour.Other degradation indicators are also possible to measure, but those are not so evident.For instance, there is a loss of sharpness of the collagen peaks, which reduce their maximum intensity with respect to the background. Experiment The experiment was carried out at ID02 High Brilliance beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.The ESRF is a 6 GeV third generation synchrotron radiation source, which offers a wide range of possibilities to X-ray research.In our case, an undulator was used as the source of radiation, which was monochromatized using Bragg reflections from perfect silicon crystals and focused with a toroidal mirror.A two-dimensional position sensitive detector2 was used to acquire the scattering patterns.The integration time was typically of 20-50 milliseconds.The set-up is shown schematically in Fig. 2. The samples were formalin fixed, and they were sealed hermetically in a holder between Kapton3 foils.The diameter of the samples was 20 mm, and the thickness 1 mm.The samples were scanned through a 200 µm beam in steps of 1 mm to 0.25 mm. Sample preparation The samples used in this work are human breast tumours and tissues from surgically excised specimens.These excised specimens were flash frozen in liquid nitrogen and stored in freezer at −80 • C, until their use.Cylindrical pieces, containing tumour and non-tumour tissue were cut off from the specimens while frozen.Several transversal slices were cut from each cylinder.Three holes parallel to the cylinder axis were made using a hollow needle.Surgical marking dyes were introduced in the holes, which were used as reference marks.Immediately after, a thin slice was cut off, let defrost and submerged in formalin 4 .As soon as the sample was formalin fixed, it was processed for histological examination5 by a pathologist.The histology of this slice was used as guide to select SAXS measurement points during the experiment.The actual SAXS samples were cut off from the cylindrical pieces right next to the one used for histology, and submerged in formalin.The two slices, guide and sample, are adjacent, so that the histology provides a map of the sample.The colour marks were present in both guide and sample slices, which helped orienting them correctly. After the SAXS measurement, the samples (already formalin fixed) were introduced in pathological processing cassettes and prepared for the final histo-pathological examination.In this way, a one-to-one correspondence between the classifications by pathology and SAXS patterns was obtained. This research work was performed in accordance with the ethical regulations of the hospital. Data acquisition and mapping The scattering patterns were recorded with the CCD and corrected to spatial distortions and normalised to correspond to the same sample thickness.One-dimensional scattering curves were extracted after azimuthal integration of the two-dimensional patterns. Some of these scattering patterns are shown in Fig. 3.The differences in the SAXS patterns are clear enough to easily identify tissues. In order to compare the histology with the scattering patterns, some of the indicators of collagen degradation obtained from the SAXS patterns were used to construct maps of these indicators.The coordinates of measurement spots were retrieved from the positions of the motors, calibrated to the sample holder frame before the experiment started.The colour marks provided reference for sample position in the holder frame, so that the exact location of the measurement point was known. The d-spacing was one of the indicators used for mapping.In this experiment the samples were formalin fixed, and the fixation process removes water from the samples, which may change the axial period.The position of the fifth order collagen peak, in terms of the scattering vector s, was retrieved from the Fig. 3. Tissue characterisation using SAXS patterns.The featureless curve of lowest intensity corresponds to the healthy adipose tissue (fat).Adipose tissue invaded by cancer has diffraction peaks due to the presence of newly formed collagen among the cancer cells (invaded fat).Healthy collagen scattering curve shows the typical collagen peaks, as well as features related to the size and packing of the collagen fibrils.The intensity of scattering from collagen invaded by cancer cells is clearly higher.It preserves, though, the collagen structures in general, but somewhat different.These differences arise from changes in the supra-molecular structure of the collagen.The highest intensity is observed from a necrotic region of a tumour, and the pattern is structureless with a k −2 fall-off of intensity, suggesting disintegrated polypeptide coils with a large specific surface.scattering patterns of all the measurement spots in every sample (see Fig. 4).From these positions the axial d-spacing of the collagen fibrils was determined (cf.Eq. ( 5)), In general, the values of d were 0.3 to 0.4 nm smaller than those measured from fresh samples [4]. The second indicator of collagen degradation is the background intensity between collagen peaks (see Fig. 4).It is seen from Eq. ( 8) that the intensity increases with the surface area of scatterers per unit of volume, and with an increase of the contrast of electron density.Collagen fibrils may suffer of "peelingoff" when they degrade, which increases their surface per unit volume.An observed effect of degradation is the reduction of the fibril diameter.The average intensity in a certain range of the SAXS pattern is plotted in a selected region of the sample.The range is chosen to be the background between two collagen peaks in the regime where the Porod law applies.The range chosen was between the 5th and the 6th collagen peaks (or s in the range 0.08-0.09nm −1 ).In this case, the lower intensities correspond to the adipose tissue, the medium intensities to the healthy collagen, slightly higher intensities to the invaded collagen and the highest intensities to the necrotic tissues.Using the motors positions as reference and the histology, it is possible to build maps of the scattered intensity along certain regions (cf.Figs 5a and 5b). The axial period of collagen fibrils was measured from all the SAXS patterns obtained from all the samples used in this work.All of them, but two, were histologically classified as carcinoma, either lobular or ductal.However, depending on the way the specimens were prepared, one can find more or less healthy tissue from the tumor surroundings.For this reason, samples marked as malignant in a general pathological diagnosis might be locally benign and, therefore, they present general characteristics of benign tissues.Two examples of this can be found in samples E/16C and G/61B, Table 2.These samples are both parts of the same tumor, a ductal carcinoma.Their histology shows a majority of benign tissue, with some islands of in situ6 carcinoma and sporadic invasive cells.Therefore, the axial period from these samples is about 0.3 nm shorter than in the rest of the sample. The samples used for this experiment were formalin fixed.It was already noted that formalin fixation removes water from the tissues, and reduces the axial period of the collagen fibrils.However, the differences in the axial period are systematically the same as in fresh samples: the period is longer in invaded collagen than in the healthy one.In the earlier work with fresh samples [4], the difference in the axial period was 0.3 nm, while in the present case the difference is 0.2 nm on the average (cf.Tables 1 and 2). Conclusions We can conclude that the SAXS signal is unique for a given tissue and that it is very useful in the characterisation of breast tissues and their pathologies.The supra-molecular structure of the collagen can be described by a few well-defined parameters, which can be retrieved from the SAXS patterns.There are differences in these parameters between malignant and benign tumours or healthy tissue, and though small, these differences allow tissue classification.The differences in the collagen structure correlate closely with the histopathology of the tissues at the spatial resolution of this work (200 µm beam at 0.25 mm scan step).The formalin fixation is found to change the absolute d-spacing of the collagen fibrils, but it does not interfere in the relative differences between healthy and invaded tissues.This is of great value, since the transport and conservation of samples is greatly facilitated by formalin fixation. Automatic determination of the axial collagen period is difficult, because the differences between different tissues are small.On the other hand, there are large differences in the background intensity and area of the collagen peaks between different tissues, as seen Fig. 3.The average intensity may rise by a factor 5 from healthy to invaded fat, and by more than a factor 10 from healthy collagen to necrotic tissues.These differences can be used for tissue mapping, as shown in Fig. 5.Much systematic work is needed to compare maps of the characteristic parameters of SAXS patterns with histo-pathology of the samples.It is expected that such comparisons provide new information on changes of tissue structures on the molecular level during cancer growth. Fig. 1 . Fig. 1.Geometry of elastic scattering of X-rays indicated by unit vectors so and s.The scattering vector k = (2π/λ)(s − so) and the scattering angle is 2θ. Fig. 2 . Fig. 2. Schematic presentation of the experiment set-up.An undulator (Un) is used as the X-ray source at ID02 beamline of the ESRF.After several optical components (Op), the beam is monochromatic and focused.The sample position and detector are indicated by S and D, respectively. Fig. 4 . Fig. 4. Schematic scattering pattern of collagen between the 3rd and 7th axial peaks.The area of the third collagen peak (A), the position of the third and fifth peaks (s 3 , s 5 ), and the intensity between the peaks (I) are used as indicators. Fig. 5 . Fig. 5. (a) Mapping of a sample containing islands of necrotic tissue encapsulated in collagen (ductal carcinoma).Top panel, the area under the 3rd collagen peak (cf.Fig. 4).Step size is 0.25 mm.The bottom panel shows the corresponding variation of the intensity of between the 5rd and 6th collagen peaks.Collagen peaks are small or disappear when the background scattered intensity is high, and vice versa.(b): Histology of the sample.The area where the SAXS patterns were recorded is shown by the rectangle, and the scan numbers are indicated (57 to 89).Pink stain indicates collagen, and yellowish stained tissues are necrotic. Table 2 Collagen d spacing measured from formalin fixed tissues, averaged for all the points	v3-fos-license
2021-01-18T14:24:06.440Z	2021-01-18T00:00:00.000	231629480	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fpls.2020.613936/pdf", "pdf_hash": "f243650dc97521fc7f125f8cf9e2f6648214143a", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:689", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "sha1": "f243650dc97521fc7f125f8cf9e2f6648214143a", "year": 2020 }	pes2o/s2orc	Sodium Influx and Potassium Efflux Currents in Sunflower Root Cells Under High Salinity Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+. INTRODUCTION Sunflower (Helianthus annuus L.) is an important crop that is widely used in the oil industry and animal feeding. Global sunflower production increased more than twice since 2000 (Pilorgé, 2020). It is the third highest oilseed produced in the world, the fourth vegetable oil and the third protein feed source among oilseed crops. Although sunflower plants exhibit medium salt Na + influx through NSCCs can be inhibited by increased external [Ca 2+ ] . This phenomenon is widely used in agriculture to ameliorate NaCl toxicity (Bressan et al., 1998). We have previously found that high external Ca 2+ levels inhibit both Na + entry and K + efflux channels, thereby blocking both Na + toxic influx and loss of K + (Shabala et al., 2006). In the recent past, blockade of Na + influx by external Ca 2+ has only been investigated in Arabidopsis thaliana Shabala et al., 2006). Therefore, it is still unclear whether other plants share this mechanism. In the present investigations, using patch-clamp electrophysiology, we have characterized the NSCC-like Na + conductance and determined its Ca 2+ sensitivity in root protoplasts of H. annuus L. seedlings (hybrid KBSH-53), which is widely cultivated in arid regions of India. To our knowledge, this is the first electrophysiological study of any ion currents in sunflower as well as properties of Na + influx and K + efflux conductances in this species. The hydroponic cultivation system was used for sunflower root growth measurements. Germinated seeds (germination: 2 days on wetted filter paper) were cultivated during 7 days in vertical polycarbonate sheets. Each root was directed to a separate channel of polycarbonate sheets in order to prevent root entanglement (Green House Polycarbonate Sheets; Greenhouse Megastore, United States). Polycarbonate sheets were mounted vertically in large square glass vessel and dipped into the medium (volume: 2 L), which was stirred with a stream of air (air compressor Barbus Aquael OXYPRO; China). The medium contained 5% original Murashige and Skoog nutrient composition (MS; Duchefa #M0221; Murashige and Skoog, 1962), рН 6.0 (adjusted by KOH). Treatments (CaCl 2 , NaCl, etc.) were added to this medium as required. All growth solutions were replaced every day (for freshness). Root length (main root) was measured after 7 days of treatment. Patch-Clamp Measurements Conventional patch-clamp and protoplast isolation techniques were used Demidchik et al., 2010). The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsm kg −1 , with D-sorbitol. Other salines are indicated in figure legends. A freshly prepared mixture of this solution was applied in whole-cell outside-out patches. The pipette solution (PS) contained the following composition (mM): 70 KGluconate, 10 KCl, 1 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA) and 0.475 mM CaCl 2 (10 nM free Ca 2+ ), pH 7.2 with 2 Tris, and 1 MES. To examine the sensitivity of wholecell outward current to cation channel blockers (TEA + and Gd 3+ ), 10 mM TEACl or 100 μM GdCl 3 were added to the bathing solution. The size of protoplasts was measured using Nikon NIS-Elements software and used to calculate the mA/ m 2 current densities. Typical transmembrane currents are from the same cell (including Gd 3+ blockade test). Liquid junction potentials were calculated by JPCalc, which is included in Axon Clampex 10.6 software (Molecular Devices, United States) and corrected. The voltage was held at −90 mV, then square 7.6 s-long or 1.5 s-long depolarizing or hyperpolarizing voltage pulses were applied. Currents were measured using PC-ONE Patch/ Whole Cell Clamp (CORNERSTONE Series) amplifier (Dagan Corporation, United States) controlled by Digidata 1,320/Clampex 10.6 (Molecular Devices, United States). Current-voltage (I-V) and other curves were plotted and analyzed using SigmaPlot 10.0 (Systat Software Inc., United States). Effect of Ca 2+ on Sunflower Seedling Growth in High Salinity Conditions Root growth tests were carried out using seedlings of H. annuus L. KBSH-53 in vertical hydroponic chambers in controlled environment (Figure 1). The effect of 80 and 120 mM NaCl on the length of main root was examined (preliminary tests showed that 40 mM NaCl did not modify plant growth). Measurements were carried out against two levels of Ca 2+ (0.2 and 2 mM) in the cultivation solution containing 5% MS (original composition). Growth in NaClfree solutions (control conditions) containing 2 mM CaCl 2 was approximately 25% slower than growth on the background of 0.2 mM CaCl 2 (p = 0.007; seven independent trials; each trial included 9-10 plants). Addition of 80 mM NaCl along with 0.2 mM CaCl 2 resulted in approximately 5-fold decrease of root length (p < 0.001; 12 independent trials; each trial included 9-10 plants). At the same time, 120 mM NaCl induced 6.5-fold delay in growth. Increase of external Ca 2+ level from 0.2 to 2 mM significantly improved plant growth in the presence of NaCl. In this case, application of 80 NaCl did not induce statistically significant decrease of root length (p = 0.235; 11 independent trials; each trial included 8-10 plants) while the effect of 120 mM NaCl was twice smaller (p = 0.008; eight independent trials; each trial contained eight plants; comparison with 0.2 mM CaCl 2 ). Overall, these data show that Ca 2+ (the physiological range) has a strong ameliorative effect on the growth of H. annuus L. KBSH-53 roots in salinized conditions. Protoplast Isolation and Obtaining Gigaohmic Resistance Patches No reports have been published about transmembrane currents of H. annuus L. or protocols for protoplast isolation for patchclamp tests in this species. To our knowledge, several attempts have been made to isolate sunflower protoplasts suitable for patch-clamp studies but none of them were successful for implementation in routine electrophysiological practice. In most cases, protoplast isolation from sunflower required overnight treatment by enzymes and did not yield viable protoplasts from any tissues apart from hypocotyl (Lenee and Chupeau, 1986;Kativat et al., 2012). We have developed protocols for H. annuus L. root patch-clamp analyses that were based on previous protocols elaborated for A. thaliana and Triticum aestivum Demidchik et al., 2010;Straltsova et al., 2015;Sosan et al., 2016;Makavitskaya et al., 2018). Ten osmolality levels were examined (300-750 mOsm kg −1 ; 50 mOsm kg −1 step) in 10 replicates. Round shaped viable protoplasts were observed only at 600 and 650 mOsm kg −1 but the density of viable protoplasts was approximately six times higher at 600 mOsm kg −1 comparing to 650 mOsm kg −1 (up to 55 ± 4 viable protoplasts per 1 ml of the enzyme solution; mean ± SE; n = 10). Experimental work on protoplasts reported here was carried out using the osmolality level of 600 mOsm kg −1 . We have previously developed techniques and voltage-clamp protocols for the patch-clamp analysis of inwardly-and outwardlydirected conductances in higher plants, including Na + -conducting NSCCs Demidchik et al., 2010). The probability rate of observing "gigaohmic" contact required for patch-clamp measurements in sunflower protoplasts was low (2,750 protoplasts were patch-clamped; "gigaohmic" contact formed in 409 protoplasts). Approximately one-third of these protoplasts survived after addition of high NaCl concentration and maintained gigaohmic pipette resistance (139 protoplasts). A number of methods for improving patch stability were applied (different levels of external Ca 2+ , H + , use of Na + instead K + in the patch-clamp pipette, additional pipette polishing, hydrophobic coating, etc.) but none of these significantly improved the "gigaseal. " Currents of Sunflower Root Protoplasts in Control Conditions and in Presence of NaCl Protoplasts were patch-clamped in the sealing solution containing 20 mM CaCl 2 and 0.3 mM KCl (pH 6.0) using pipettes filled with the solution comprising of 70 mM KGluconate and 10 mM KCl (pH 7.2, 100 nM Ca 2+ ). High external Ca 2+ allowed gigaseal formation , while high intracellular (pipette) K + "mimicked" cellular K + level (Demidchik, 2014). Potassium gluconate (70 mM) in the pipette solution was used instead of KCl to avoid Cl − efflux currents, which can overlap with Na + influx conductance. Gluconate is a poorly permeable organic anion that minimizes anion efflux currents in patch-clamped root protoplasts (Makavitskaya et al., 2018). In these conditions, a moderate negative inwardly directed current was measured (Figure 2A). This current was voltage-independent and sensitive to 100 μM Gd 3+ (77.3 ± 4.5% decrease of the amplitude; ±SE; n = 5; data not shown). It showed very rapid ("instantaneous") activation kinetics. When external CaCl 2 was decreased from 20 to 0.2 mM, this current decreased by five times, demonstrating that it was mediated by Ca 2+ influx (Figures 2-4, 5; p < 0.001; n = 5). These Ca 2+ currents were similar to NSCC-mediated Ca 2+ currents previously reported in A. thaliana root protoplasts . It should be noted that in Arabidopsis, NSCCs mediating these currents were Na + -permeable . Addition of NaCl to patchclamped protoplasts in the presence of 20 mM extracellular Ca 2+ did not induce inwardly directed current (as expected for Na + influx NSCCs). The reversal potential was −91 ± 4 mV (20 mM CaCl 2 ; ±SE; n = 11) and it was not modified by NaCl addition (Figure 2). The outward current measured in the presence of 20 mM external CaCl 2 was significantly blocked by the addition of NaCl to the bathing solution (Figure 2). The outwardly directed conductance dropped three times when 80 mM NaCl was added (Figure 5). In the conditions used in the work, the outward current could be mediated by K + efflux through KORs or by Cl − influx via anion channels (Demidcik et al., 2002(Demidcik et al., , 2014de Angeli et al., 2007;Demidchik, 2012;Hedrich, 2012). However, only K + currents can be blocked by Na + because the anion channels are insensitive to this and other alkali metals (Barbier-Brygoo et al., 2000). Moreover, the addition of K + channel blocker TEA + (30 mM TEACl) inside the patchclamp pipette instead of 80 mM K + (70 mM KGluc and 10 mM KCl) decreased the outward current by 8-9 times (p < 0.001; n = 5; data not shown) demonstrating that this current was mediated by KORs. The time-dependent component of the outward K + current was inhibited after the addition of NaCl to the bathing solution while instantaneous current remained very similar (Figure 2). It can be thus hypothesized that the residual outward current was mediated by anion channel-catalyzed Cl − influx or K + efflux via NSCCs (previously described in Shabala et al., 2006). The maximal reduction of the outward current was 4.3, as measured in the presence of 80 NaCl at 7.6 depolarizing pulses . Statistically significant (p < 0.01; ANOVA test) difference between "2 mM CaCl 2 " (circles) and "40 mM NaCl" (squares) was found at all voltage values apart from −105 mV. The difference between "2 mM CaCl 2 " (circles) and "80 mM NaCl" (triangles) was statistically significant at all voltage values (p < 0.01; ANOVA test). The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsM, with D-sorbitol. The pipette solution contained 70 mM K gluconate, 10 mM KCl; 100 nM Ca 2+ was adjusted with 1 mM BAPTA and 0.475 mM CaCl 2 , pH 7.2 with 2 mM Tris, 1 mM MES. 100 μM GdCl 3 was added to the bathing solution on the background of 2 mM CaCl 2 and 80 mM NaCl for 5 min before recording current-voltage curves. Frontiers in Plant Science \| www.frontiersin.org (Figure 2). This reduction was 3.2 times as calculated for 1.5-s-long segments of depolarising pulses (directly comparable with pulses used in Figures 3, 4). These results demonstrate a high sensitivity of KOR to Na + and suggest a relatively low sensitivity of KOR to external Ca 2+ in salt-tolerant sunflower. Sodium Influx Currents in Sunflower Root Protoplasts Under Low External Ca 2+ Calcium ions are blockers of plant Na + -permeable NSCCs Shabala et al., 2006). This may be the reason for no detection of Na + influx conductance in the presence of 20 mM CaCl 2 (Figure 2). However, the decrease of external Ca 2+ from 20 to 2 mM (typical soil solution level of Ca 2+ ; White and Broadley, 2003;Marschner, 2011) resulted in the increase in the inward Na + current, which correlated with a shift of reversal potential to more positive values (from −86.6 ± 3.2 mV in control to −49.8 ± 2.5 mV at 40 mM NaCl and −30.1 ± 1.8 mV at 80 mM NaCl; ±SE; n = 6-11), consistent with currents being dominated by the movement of Na + (Figure 3). This can be interpreted as weakening the Ca 2+ -induced blockade of the NSCCs. Sodium influx current showed an "instantaneous" kinetics and was voltage-independent. The shift of the reversal potential in response to NaCl to more positive values decreased the KOR-mediated outwardlydirected currents (as the activation curve moved positive). Moreover, the decrease of the external CaCl 2 destabilized patches and caused a breakdown at depolarization that did not allow depolarizing pulses longer than 1.5 s (note: 7.6 s-long pulses were applied at 20 mM CaCl 2 to record full activation of KORs). In this regard, the measurements were limited to shorter segments of the outwardly-directed K + currents (Figure 3; see also calculation of conductance change in Figure 5), and it was not possible to fully compare the data with those shown in Figure 2. The obtained data demonstrated that an addition of NaCl (both 40 and 80 mM), in the presence of 2 mM CaCl 2 , inhibited the outwardly-directed currents slightly weaker than in the presence of 20 mM CaCl 2 (Figures 3, 5). The time-dependent component of the current was almost fully inhibited. . The difference between "0.2 mM CaCl 2 " (circles) and "40 mM NaCl" (squares) as well as between "0.2 mM CaCl 2 " (circles) and "80 mM NaCl" (triangles) was statistically significant at all voltage values (p < 0.01; ANOVA test). Statistically significant (p < 0.01; ANOVA test) difference between "40 mM NaCl" (squares) and "80 mM NaCl" (triangles) was found at a voltage of more negative than −80 mV. The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsM, with D-sorbitol. The pipette solution contained 70 mM K gluconate, 10 mM KCl; 100 nM Ca 2+ was adjusted with 1 mM BAPTA and 0.475 mM CaCl 2 , pH 7.2 with 2 mM Tris, 1 mM MES. The addition of 100 μM Gd 3+ , which is a non-specific blocker of NSCCs and other plant cation channels (Demidchik and Maathuis, 2007) to the bathing solution containing 2 mM CaCl 2 and 80 mM NaCl, caused a very strong inhibition of both inward and outward currents (5-6-fold decrease of currents; Figure 3). This indicates that both currents were mediated by cation channels (not by anion channels). Lowering the external CaCl 2 from 2 to 0.2 mM in the presence of 40 or 80 mM NaCl resulted in further increase in inwardly-directed voltage-independent Na + current (Figure 4). The reversal potential values measured after the addition of 40 and 80 mM NaCl were − 37.4 ± 2.9 mV and −26.5 ± 3.2 mV, respectively (±SE; n = 5). These values were more positive compared to those measured at 2 mM Ca 2+ , suggesting that it was due to increased permeability to Na + (in conditions of external 40 or 80 mM Na + , Na + reversal potential is positive). The outwardly-directed K + efflux conductance was equally blocked by 40 and 80 mM NaCl in the presence of 0.2 mM CaCl 2 , suggesting the saturation of the blockade at 40 mM NaCl or lower level of salt (Figures 4, 5). Interestingly, the time-dependent current component was almost fully blocked, when 40 or 80 mM NaCl were added on the background of 0.2 mM CaCl 2 . DISCUSSION Overall, data reported here demonstrate for the first time that H. annuus root plasma membrane has a set of ionic conductances dominated by NSCCs and KORs. Similar conductances were previously recorded in the plasma membranes of root protoplasts isolated from A. thaliana (Maathuis and Sanders, 2001;Demidchik et al., , 2010Shabala et al., 2006), Thellungiella halophila (Volkov et al., 2004;Volkov and Amtmann, 2006), Pisum sativum (Zepeda-Jazo et al., 2011), T. aestivum (Straltsova et al., 2015) and other species (Demidchik, 2014). To our knowledge, this work is the first patch-clamp and voltageclamp study on sunflower. It should be noted that previous works have touched on the topic of sunflower electrophysiology only in terms of measurements of membrane potential (Stankovic et al., 1997). Figures 2-4). G max is the maximal value of conductance measured calculated in an individual experiment (set of IV curves). Experimental conditions and ionic species in external and pipette solutions are same as in Figures 2-4. Data are mean ± SE (n = 5-11; *** p < 0.0001; ANOVA test; comparison to control; no significant difference where unmarked). In this investigation, the Helianthus Na + influx currents were also measured and analyzed (Figures 2-4). These currents showed voltage-independent activation, lack of time-dependent component and high sensitivity to Gd 3+ . These properties are fully in line with the characteristics of Na + -permeable NSCCs previously measured in A. thaliana (Maathuis and Sanders, 2001;Shabala et al., 2006) and T. halophile (Volkov et al., 2004;Volkov and Amtmann, 2006). However, sunflower Na + -permeable NSCCs showed a much weaker response to the decrease of extracellular Ca 2+ as compared to Arabidopsis or Thellungiella in the range of physiological Ca 2+ levels (2-0.2 mM). Thus, sunflower NSCCs has smaller Na + current density (and potentially lower number of channels per same membrane area) than Arabidopsis or Thellungiella at physiological extracellular [Ca 2+ ], potentially preventing toxic Na + influx and cell reactions induced by NaCl. This makes it possible to assume that Ca 2+ could cause greater inhibition of NSCCs in sunflower roots. Interestingly, the response of Na + influx to Ca 2+ correlated well with Ca 2+ -induced protection of root growth in sunflower seedlings treated with NaCl at different external [Ca 2+ ] (Figure 1). Growth inhibition by 80 mM NaCl was prevented by 2 mM CaCl 2 while the treatment with 0.2 CaCl 2 was not effective (Figure 1). Results presented here also demonstrate a high sensitivity of KOR to Na + and suggest a relatively low sensitivity of KOR to external Ca 2+ in salt-tolerant sunflower. Similar sensitivity to external Na + is known for animal KORs, such as Kv2.1 and related to Na + reaction with the high and low affinity Na + binding sites in Kv2.1 channel (Kiss et al., 1998). Potassium outwardly-directed conductances mediated by KORs in salttolerant T. halophila decreased 1.5-1.7 times after the addition of 100 mM external Na + (Volkov et al., 2004;Volkov and Amtmann, 2006). In salt-sensitive species A. thaliana, this blockade was 1.3-1.9 times both in root epidermis and leaf mesophyll cells (showing a tendency to increase with an increase in the concentration of extracellular Ca 2+ ; Shabala et al., 2006). From the present findings, we hypothesize that enhanced sensitivity of K + efflux system to Na + can play an important role for adaptation because this will decrease K + loss under salinity conditions. It fits well within the hypothesis that maintaining a high K + /Na + ratio in plant cells and prevention of K + efflux under salt stress are key mechanisms of salt tolerance in higher plants (Shabala and Cuin, 2012;Demidchik et al., 2014. Intriguingly, K + outwardly directed conductance in sunflower showed greater Na + sensitivity at higher extracellular CaCl 2 levels that can have a positive effect in conditions of salinity (as cells will lose less K + ; Figure 5). This can be explained by the influence of CaCl 2 on the Na + -induced blockade of KORs in the case of measurements which were carried out at 20 and 2 mM external Ca 2+ . In animal plasma membrane K + channels, Na + can compete with K + for binding sites within a pore region modulating channel characteristics and functions in Ca 2+ -dependent manner (Kiss et al., 1998;Sauer et al., 2013). In animals, Ca 2+ modifies the K + channel activity via action on the surface charge, reaction with the specific binding sites at extracellular loops, effect on the EF-hands and calmodulin binding sites at cytosolic side (Shah et al., 2006). We hypothesize that the elevated extracellular Ca 2+ controls the Na + block of the sunflower K + channel by increasing Na + sensitivity. Interestingly, Lemtiri-Chlieh et al. (2020) have recently reported that divalent cation Mg 2+ added to the pipette solution can change both the activity of leaf NSCCs and their sensitivity to Gd 3+ , suggesting sophisticated interactions of cations within the NSCC complex. Involvement of root KORs (potentially encoded by Shakertype GORK) to NaCl responses and salt stress adaptation have been demonstrated in a number of species (Adem et al., 2020). It is a redox-dependent phenomenon as GORK is additionally activated by ROS (Demidchik et al., 2010). Potassium loss via GORK triggered by depolarization and ROS can lead to ionic disequilibrium, induction of autophagy, and programmed cell death (Demidchik et al., 2010. Enhanced blockade of KOR by Na + will be the simplest and "economical" mechanism for preventing K + loss that will retain the greatest amount of metabolic energy for adaptation in salinity conditions. The cell's energy balance has recently been recognized as one of the main salt stress targets (Tyerman et al., 2019). Thus targeting KORs and their Na + sensitivity regions to save energy for reparation needs offers high hopes for generation of salt-tolerant varieties by molecular breeding techniques. In conclusion, the data presented here strongly suggest that the moderate resistance of sunflower to NaCl stress is programmed at potassium and non-selective channel level via the sensitivity of ion channels to Ca 2+ and Na + . DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS VD was responsible for research supervision, experimental design, management of experiments, data analysis, and writing the manuscript. SB and MY were involved in the preparation of plant material, research supervision, and design of experiments. PH, IN, YT, XH, and MK carried out electrophysiological experiments. VS and AV conducted hydroponics studies. AS and IS carried out routine cultivation of sunflower seedlings, maintained patch-clamp equipment, and participated in manuscript preparation. All authors contributed to the article and approved the submitted version. FUNDING This study was supported by joint Belarus-India Project 018/53 of the State Committee of Science and Technology of Belarus (to VD and SB), International Academic Exchange Research Project (China-Belarus; to VD, MY, and SH), Guangdong Province Pearl River Fellowship (to VD), and the Russian Science Foundation grant#15-14-30008 (VD).	v3-fos-license
2018-04-03T04:53:56.154Z	2016-08-06T00:00:00.000	14505773	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.1016/j.dib.2016.07.060", "pdf_hash": "d4997bf1dab7cfce6c382d16c5caa6bc09359634", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:751", "s2fieldsofstudy": [ "Biology" ], "sha1": "d4997bf1dab7cfce6c382d16c5caa6bc09359634", "year": 2016 }	pes2o/s2orc	Summary data of potency and parameter information from semi-mechanistic PKPD modeling of prolactin release following administration of the dopamine D2 receptor antagonists risperidone, paliperidone and remoxipride in rats We provide the reader with relevant data related to our recently published paper, comparing two mathematical models to describe prolactin turnover in rats following one or two doses of the dopamine D2 receptor antagonists risperidone, paliperidone and remoxipride, “A comparison of two semi-mechanistic models for prolactin release and prediction of receptor occupancy following administration of dopamine D2 receptor antagonists in rats” (Taneja et al., 2016) [1]. All information is tabulated. Summary level data on the in vitro potencies and the physicochemical properties is presented in Table 1. Model parameters required to explore the precursor pool model are presented in Table 2. In Table 3, estimated parameter comparisons for both models are presented, when separate potencies are estimated for risperidone and paliperidone, as compared to a common potency for both drugs. In Table 4, parameter estimates are compared when the drug effect is parameterized in terms of drug concentration or receptor occupancy. a b s t r a c t We provide the reader with relevant data related to our recently published paper, comparing two mathematical models to describe prolactin turnover in rats following one or two doses of the dopamine D 2 receptor antagonists risperidone, paliperidone and remoxipride, "A comparison of two semi-mechanistic models for prolactin release and prediction of receptor occupancy following administration of dopamine D 2 receptor antagonists in rats" (Taneja et al., 2016) [1]. All information is tabulated. Summary level data on the in vitro potencies and the physicochemical properties is presented in Table 1. Model parameters required to explore the precursor pool model are presented in Table 2. In Table 3, estimated parameter comparisons for both models are Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/dib presented, when separate potencies are estimated for risperidone and paliperidone, as compared to a common potency for both drugs. In Table 4 Plasma samples were collected for bioanalysis of risperidone, paliperidone, and remoxipride using an on-line solid phase extraction with liquid chromatographytandem mass spectrometry method. Serum prolactin levels were measured using an enzyme linked immunosorbent assay technique. Experimental features All animal procedures were performed at Leiden University, in accordance with Dutch laws governing animal experimentation. Male Wistar rats, received single intravenous doses of risperidone (2 mg/kg, n ¼16) or paliperidone (0.5 mg/kg, n ¼21). Plasma drug concentrations as well as plasma prolactin levels were measured at pre-dose and at serial intervals post-dose. In another study, remoxipride was administered to rats either as a single intravenous dose of 4, 8 or 16 mg/kg (n ¼10) remoxipride or two doses of 3.8 mg/kg at varying dosing intervals. Blood samples were serially collected. Plasma concentrations of the drugs as well as prolactin were assayed using validated analytical methods. Data source location Department of Pharmacology, Leiden Academic center for Drug Research, Leiden. Data accessibility The data is within this article. Data can be used To compare experimental findings in literature with our model-based approach. As prior information, especially when the available data is scarce. For exploratory modeling. For translation from rat to humans. Data The information is presented in 4 tables. Table 1 presents the in vitro inhibition constant (KI) values in rat and humans and physicochemical characteristics of the antipsychotics risperidone, paliperidone and remoxipride. Table 2 presents the pharmacokinetic-pharmacodynamic model Table 2 Model parameters used for the simulations in exploratory model analysis. Pharmacokinetic and pharmacodynamic parameters obtained from Kozielska et al. [3] and Stevens et al. [4], respectively. Parameter Estimate CL ¼ clearance from the central compartment, V1 ¼ volume of the central compartment, Q ¼ intercompartmental clearance, V2 ¼ volume of the peripheral compartment, F ¼ bioavailability, Ka ¼ absorption constant, C prl,0 ¼ plasma concentration of prolactin in the absence of antipsychotic drug, R form ¼ zero-order rate constant for prolactin synthesis, K base ¼ first-order rate constant of prolactin release from the pool, K out ¼ first-order rate constant of elimination of prolactin from plasma, E max ¼ maximum increase in the prolactin release from the pool, EC 50 ¼ drug concentration at half-maximal effect, γ ¼ slope parameter, E max_pf ¼ maximum prolactin feedback, EC 50_pf ¼ plasma prolactin concentration at half-maximal effect. n R form is calculated as the product of C prl,0 . K out (equation (5) of Taneja et al. [1]). parameters used to perform exploratory model simulations of the precursor pool model, as referred to in Section 3.2, Fig. 5 of Taneja et al. [1]. Table 3 presents the model parameters assuming equal or different potency of risperidone and paliperidone. Table 4 presents the model parameters obtained with different parameterizations, assuming either unbound drug concentration or dopamine D 2 receptor occupancy as the driving force for drug effect. Experimental design, materials and methods Details of the experimental procedures have been described previously [1,5,6]. R form ¼ zero-order rate constant for prolactin synthesis, K base ¼ first-order rate constant of prolactin release from the pool, K out ¼ first-order rate constant of elimination of prolactin from plasma, E max ¼ maximum increase in the prolactin release from the pool, EC u50 ¼ unbound drug concentration at half-maximal effect, γ ¼ slope parameter, IIV ¼ inter-individual variability, K in,0 ¼ basal prolactin release rate, K DA ¼ first-order turnover constant for hypothetical dopamine, DAs 0 ¼ hypothetical scaled dopamine concentration at baseline, KI ¼ drug potency parameter.	v3-fos-license
2018-04-03T03:50:25.841Z	2016-10-10T00:00:00.000	12870230	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://doi.org/10.1002/rcm.7725", "pdf_hash": "83e15e9e4b2b973da965889b3bb09ab6d8883b7f", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:939", "s2fieldsofstudy": [ "Chemistry", "Environmental Science" ], "sha1": "83e15e9e4b2b973da965889b3bb09ab6d8883b7f", "year": 2016 }	pes2o/s2orc	Laser ablation electrospray ionization high‐resolution mass spectrometry for regulatory screening of domoic acid in shellfish Rationale Domoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs. Methods We assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials. Results Domoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit. Conclusions These findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd. Domoic acid (DA; Fig. 1(A)) is a potent neurotoxin that is produced by marine diatoms and accumulates in shellfish. DA was first identified as the causative agent of amnesic shellfish poisoning (ASP) after a serious outbreak in 1987 that left 3 people dead after consuming contaminated blue mussels from Prince Edward Island, Canada. [1] Since then, regulatory analysis of DA in shellfish has been critical in safeguarding public health and multi-billion dollar shellfish industries worldwide, with the regulatory limit in Europe and North America set at 20 mg DA kg À1 edible tissue. [2,3] Testing has typically been carried out using 10 to 30 min liquid chromatography/ultraviolet (LC/UV) methods after extraction with aqueous methanol, which are robust and fit for purpose. [4,5] Routine LC/mass spectrometry (MS) methods are also used for DA analysis, either as a single analyte or as part of a multi-toxin analysis also capable of quantitating several classes of lipophilic toxins. [6,7] Routine LC/UV and dedicated LC/MS methods use external calibration with standards in neat solvent and are not significantly affected by matrix effects in electrospray ionization (ESI). More advanced LC/MS/MS methods are also available for DA, including sample preparation using strong anion-exchange solid-phase extraction (SPE) [5] and/or derivatization chemistry, [8,9] that combined have yielded limits of detection as low as 1 μg kg À1 . [10] The scope of DA analysis worldwide is large enough that increases in sample throughput would lead to significant cost and time savings, particularly for regulatory labs. For example, the Canadian Food Inspection Agency (CFIA) and the Marine Institute (MI) in Ireland currently run about 10,000 and 3000 shellfish samples for DA testing annually, respectively, the majority of which are negative. Efforts have already been made by regulatory labs to increase the throughput of DA analysis. The MI screens shellfish varieties other than scallops using a multi-toxin LC/MS method with a limit of detection of 0.5 mg kg À1 . Positive samples are then re-analyzed using the regulatory LC/UV method, [11] which is also used to analyze all scallop samples. The CFIA has developed rapid ultra-high performance LC/UV and LC/MS methods, similar to that reported previously. [12] These methods use a 2.5 min long chromatographic run and have been validated as fully quantitative with limits of detection (LODs) and limits of quantitation (LOQs) of 0.06 and 0.2 mg kg À1 , respectively, for LC/MS and 0.7 and 2 mg kg À1 , respectively, for LC/UV. Even compared with these more rapid methods, a screening method that does not require sample extraction or chromatographic separation would lead to a significant increase in sample throughput. Screening samples for the presence or absence of a regulated substance using a high-throughput technique followed by confirmatory analysis using a reference method has been recognized as an effective approach in many areas of food safety testing. The United States Department of Agriculture is currently moving towards LC/MS and gas chromatography (GC)/MS methods of high-throughput screening for its analysis of chemical residues in meat. [13] For algal toxins in shellfish, antibody-based test kits have been considered for screening of paralytic poisoning toxins in shellfish by several regulatory agencies. [14,15] This assay has been reported to give a false positive rate of up to 30%, but still represented a significant benefit to the testing lab when used alongside the mouse bioassay as a confirmatory method. [14] Recently, a comprehensive report compared the performance of five rapid test kits for qualitative and quantitative screening of DA in shellfish. [16] Overall good performance was observed, with lateral flow immunoassays giving the best results. However, all these approaches still require sample extraction prior to analysis, which represents the practical limit to their throughput. Laser ablation electrospray ionization (LAESI) is an ambient ionization technique for MS that uses a laser to produce a fine mist of neutral droplets of sample liquid followed by ionization of analytes by charge transfer from the plume of an electrosprayed buffer. [17] During application of the laser pulse, water functions as a matrix, absorbing the mid-infrared (λ = 2940 nm) laser energy by its O-H stretch mode. [18] The ionization specificity of LAESI is comparable with that of ESI rather than laser ablation techniques, making it more suitable for the analysis of polar, labile molecules. The majority of applications of LAESI have focused on mass spectrometry imaging of plant and animal tissues, [19] but it has also been used for direct targeted and non-targeted analysis in liquid samples and biological specimens. [20,21] To date, the capability of LAESI-MS for targeted quantitative analysis directly from complex samples has remained largely unexplored. [20,22] Recently, we proposed that LAESI-MS/MS could be used as a high-throughput method for quantitative DA analysis and showed detection of DA directly from shellfish tissue homogenates. [22] This preliminary proof of concept work considered only the blue mussel (Mytilus edulus) and only analyzed DA standards, spiked tissue and mussel tissue matrix reference materials. The LAESI-MS/MS conditions were relatively generic and it was proposed that further refinement could lead to improvements in analytical variability. Here, as a follow-up to our original communication, we investigate the suitability of LAESI-MS for analysis of DA in a wide range of real shellfish samples previously analyzed for DA in a regulatory setting. The first goal of this project was to refine the LAESI-MS method for DA in shellfish tissue homogenates compared with the previous work, [22] including transfer of the original method to a high-resolution mass spectrometer for improved selectivity and sensitivity. Various sample preparation approaches and LAESI-MS parameters were also optimized, with respect to the sensitivity of analysis, reproducibility and matrix effects. The second goal of this project was to evaluate the quantitative capabilities of the developed method for the analysis of real shellfish samples and to address the challenge of calibration using tissue homogenate standards. Finally, the quantitative results obtained by LAESI-MS were compared with those obtained by standard LC/UV and LC/MS methods for a large set of shellfish samples. EXPERIMENTAL Samples and standards A total of 189 shellfish samples analyzed previously as part of routine monitoring programs were acquired from the CFIA (Canada) and the MI (Ireland). The CFIA samples included 29 soft shell clam (Mya arenaria) samples ranging from 0.7 to 10 mg DA kg À1 wet tissue, five control (DA < LOD) clam samples and one blue mussel (Mytilus edulis) sample containing 14 mg DA kg À1 wet tissue. The MI samples included 13 king scallop (Pecten maximus) adductor muscle samples ranging from 0.8 to 16 mg DA kg À1 wet tissue and 13 control samples, 49 king scallop gonad samples ranging from 1.4 to 55 mg DA kg À1 wet tissue and five control samples, 23 king scallop remainder (viscera) samples with 0.6 to 222 mg DA kg À1 wet tissue and 38 blue mussels ranging from 2.8 to 21 mg DA kg À1 wet tissue, and 14 control samples. All samples were received as the homogenized tissue of several animals (≥100 g) from which sub-samples for regulatory analysis had previously been taken. NRC Certified Reference Materials (CRMs) for DA included a calibration solution (CRM-DA-f) and wet mussel matrix CRMs (CRM-ASP-Mus, CRM-PSP-Mus, CRM-FDMT, CRM-DSP-Mus, NRC-Zero-Mus). The optimization of MS and LAESI parameters was carried out using a standard curve and mussel tissue homogenate matrix-matched calibration standards prepared by spiking NRC CRM-Zero-Mus with a DA standard. The investigation of sample preparation approaches was then carried out by analyzing a sub-set of real samples and in-house reference standards prepared for this study. Matrix-matched standards were prepared by blending naturally contaminated tissue with control tissue of each matrix. First, the control tissue was blended 1:1 with water and analyzed by LC/UV before DA was added. High-level matrix-matched standards were then prepared for each matrix by blending control tissue with ≤5% by mass of a high-level, naturally contaminated, mussel tissue (>600 mg DA kg À1 tissue). A range of concentrations of matrix-matched standards was then prepared by blending these high-level samples with six different amounts of control tissue. Concentrations of DA were assigned to all these matrix reference materials using a validated LC/UV method. Optimized LAESI-MS screening method The test samples were diluted 1:1 with deionized water and further homogenized using an Omni TH handheld tissue homogenizer (Discovery Scientific, Vancouver, BC, Canada) to facilitate reproducible transfer of 20-μL aliquots to low-volume 96-well plates using an automatic volumetric pipette with wide-orifice 100-μL tips. A LAESI DP-1000 direct ionization system (Protea Biosciences Inc., Morgantown, WV, USA) was used to ablate samples with 50 pulses of a mid-IR (λ = 2940 nm) laser at 10 Hz with 700 μJ of energy and a dwell time of 2 s. A Q Exactive hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) was operated in in targeted positive ionization single ion monitoring (tSIM) mode with a 1 m/z unit window centred around m/z 312 (corresponding to the [M + H] + ion of DA), at a mass resolution setting of R = 140 000 and a C-trap fill time of 500 ms. The average MS peak height at m/z 312.144 across the LAESI peak was used to quantitate DA. Confirmatory LC/UV analysis A validated LC/UV method was used to quantitate DA in the matrix standard reference materials prepared for this study and to re-analyze some test samples after LAESI analysis. Sub-samples (4 g) of homogenized mussel tissue were accurately weighed into a centrifuge tube with 16 mL of 1:1 methanol/water. These were vortex mixed for 3 min, centrifuged at 2600 g for 10 min and the supernatant was filtered to 0.45 μm prior to analysis. Determination of DA was carried out on an 1100 series LC system equipped with a diode array detector (Agilent, Palo Alto, CA, USA). Separation was carried out on a 3 μm Luna C18 stationary phase (150 x 4.6 mm column; Phenomenex, Torrance, CA, USA) with isocratic elution using 1:9 acetonitrile/water with 0.1% trifluoroacetic acid. The mobile phase flow rate was 0.9 mL min À1 , the injection volume was 10 μL, the autosampler temperature was 6°C and detection was carried out at 242 nm. LAESI-MS method optimization In our original communication, we used a linear ion trap (LIT) mass spectrometer to quantitate DA in MS/MS mode using the m/z 266 product ion obtained from the [M + H] + ion at m/z 312. [22] This method showed good selectivity and an LOD of 1 mg DA kg À1 in spiked wet mussel tissue homogenate. As reported previously, analysis of DA standards by LAESI-MS and LAESI-MS/MS gives spectra comparable with those generated by ESI. [22] During method transfer to the Q Exactive (QE) instrument, we first compared sensitivity and selectivity between MS/MS and HRMS at various MS resolution settings using a control mussel tissue homogenate (CRM-Zero-Mus) spiked with DA at five levels between 1 and 60 mg kg À1 . A product ion scan of the m/z 312 precursor showed excellent selectivity when mussel tissue homogenates containing DA were compared with the control homogenate (Fig. 1). Excellent mass accuracy of ±0.2 ppm for m/z 266 was also observed by MS/MS. However, only inconsistent detection of a few spectral counts was possible in the 1 mg kg À1 mussel matrix standard using MS/MS. The high selectivity of MS/MS resulted in no measurable background at the accurate mass of the m/z 266 product ion ( Fig. 1(B)). This made it difficult to measure signal-to-noise ratios to determine an LOD, although this was estimated at about 2 mg kg À1 . The drop in sensitivity in MS/MS mode observed between the LIT and the QE could be due to a combination of factors. The LAESI sampling capillary is longer on the QE than on the LIT by about 7.5 cm, which could lead to greater ion losses. In addition, the types of fragmentation techniques and ion detectors used on the two instruments are different, with the LIT using ion trap collision-induced dissociation with an electron multiplier and the QE using higher energy collisional dissociation and Fourier transform detection. These methods have previously been shown to generate different data, both qualitatively and in terms of ion abundance. [23] Improved sensitivity and LODs were obtained by HRMS using the tSIM scan mode. This scan mode selects all ions of a specified nominal mass using the quadrupole for Orbitrap analysis as a way of minimizing space-charge effects in the C-trap and Orbitrap. The effect of resolution setting on selectivity and sensitivity in HRMS was also investigated using spiked mussel matrix-matched calibration curves. The default setting of R = 35,000 gave a 10-fold higher sensitivity than observed in MS/MS mode ( Supplementary Fig. S1, Supporting Information). Only a modest drop in sensitivity of about 20% was observed when increasing resolution to R = 70 000, and no further decrease was observed using the highest resolution setting of R = 140 000. Increasing resolution showed a significant improvement in selectivity, as shown in Fig. 2. A single peak observed at m/z 312.1444 using R = 35 000 is partially resolved at R = 70 000, and completely resolved at R = 140 000, into a peak for DA at m/z 312.1443 and another for an interfering matrix species at m/z 312.1391. Using the instrument resolution setting of R = 140 000, the measured resolution for DA (m/z/FWHM) was 127 000 ± 2000 (N = 9) and the mass errors were ±1.3 ± 0.3 ppm (N = 15) in matrix samples (uncertainties given as standard deviation). This, along with the detection of significant intensities of this matrix interference in real mussel samples (discussed below), means that either a resolution setting of R = 140 000, used for the remainder of the current work, or MS/MS detection, used previously, [22] is required for the selective analysis of DA from mussel tissue using LAESI-MS techniques. Investigation of the matrix interference at m/z 312.1391 by LC/HRMS suggested that it was the M + 1 isotope peak of a matrix compound detected at m/z 311.1350. The matrix interference was not retained in reversed-phase chromatography on a C 18 stationary phase, and therefore does not represent a potential source of matrix interference in routine LC methods. The LAESI pulse and the MS scan rate were synchronized by increasing the C-trap fill time to 500 ms, which allowed for a larger number of laser pulses to be averaged in each MS scan and improved LAESI peak shape ( Supplementary Fig. S2, Supporting Information). In order to maintain the number of data points per well at above 10, the number of pulses per well was increased from 30 to 50, all at a frequency of 10 Hz. To partially compensate for this decrease in throughput, the dwell time between wells was reduced from 5 s to 2 s. After the experiments for the current study were complete, a follow-up experiment was carried out on a prototype LAESI system capable of operating at laser pulse frequencies up to 20 Hz. A comparison of linearity and variability for a mussel tissue calibration curve run in triplicate at 10 Hz and 20 Hz showed improved linearity and a decrease in average RSD of replicate wells to 25% at 20 Hz, compared with 37% at 10 Hz (Supplementary Fig. S3, Supporting Information). Early in method development, a trend in variable response with well position on the 96-well plate was detected that showed significantly suppressed response from columns 1 and 2 of the 96-well plate compared with other columns. This can be attributed to stage effects in the LAESI source: the disruption of the ion sampling due to air currents created by the moving stage. A larger impact of air currents on plume dynamics could be expected during the long stage movement from the last well of one row to the first well of the next (e.g. A12 to B1) compared with the short movements between wells in the same row (e.g. A11 to A12). At the time that the current experiments were being carried out, the LAESI stage was only able to move through wells in the sequence from left to right and return from column 12 to column 1 before beginning a new row (e.g. A1 to A12, B1 to B12, C1, etc.). In order to quickly eliminate this stage effect, columns 1 and 2 of each plate were not loaded with samples for the remainder wileyonlinelibrary.com/journal/rcm of this study. In the time since these experiments were carried out, additional flexibility in stage movements has been accommodated in updated control software so that long stage movements can be avoided. It is expected that this will both correct the observed stage effect and increase throughput by eliminating the time required to scan back to the first well of the next row. Sample preparation optimization Figure 3 shows a comparison of the sensitivity of analysis for DA in shellfish homogenates using different sample preparation approaches (Fig. 3(A)) and different tissue types (Fig. 3(B)). The normalized relative response plotted in Fig. 3 is defined as the MS spectral counts divided by the concentration of DA. This allowed the comparison of sensitivities between analyses of real samples in Fig. 3(A) and matrix standard curves in Fig. 3(B). The low-volume 96-well plate format used for LAESI analysis requires reproducible dispensing of homogeneous 20-μL aliquots of shellfish tissue homogenate. Additional sample homogenization was therefore required compared with what is typically required for LC analysis, where 2-5 g sub-samples from larger batches of homogenized tissue are extracted with a 4:1 ratio of solvent/sample. To help with this additional homogenization and pipetting, the samples were diluted 1:1 with deionized water at this stage. For comparison, five samples from each tissue type were also analyzed without any further homogenization or dilution. Since it was not possible to pipette the crude tissue, small chunks were placed in each well using a spatula. Despite this practical limitation, DA was still successfully detected in positive samples, but with lower relative response than after dilution and further homogenization (Fig. 3(A)). It should be noted that the additional 1:1 dilution step required for LAESI-MS analysis represents minimal additional work once incorporated into existing regulatory workflows, which already require some homogenization of several animals to account for biological variability. Centrifugation of homogenates and analysis of crude supernatants by LAESI-MS was also considered as a possible approach for sample preparation. In practice, this offered the ability to more reproducibly deliver 20-μL aliquots into the low-volume 96-well plates. Analysis of supernatant by LAESI-MS also represents a possible solution to the problem of storage and sub-sampling of tissue homogenate standards needed to calibrate the LAESI-MS method. It may not be practical to store a homogenate standard in the freezer, thawing it periodically to take a sub-sample for LAESI calibration. By comparison, storage and sub-sampling an aqueous supernatant is more feasible and similar matrix effects could be expected. Analysis of homogenate supernatants from the same sub-set of samples as above showed a similar response and variability to the analysis of the diluted and blended samples (Fig. 3(A)). Since dilution and blending were found to be effective, centrifugation was not pursued further as an approach to sample preparation of the test samples, but it may still represent a reasonable solution to calibrating the method in the future. Figure 3(A) also confirms the significant ionization suppression reported in the preliminary work, when the signal intensity from real samples was compared with that from neat solvent standards and samples fully extracted and cleaned-up using strong anion-exchange (SAX) solid-phase extraction. [5] While effective at improving sensitivity and minimizing matrix effects, the SAX cleanup is not compatible with a high-throughput workflow and was only examined for comparison. Surprisingly, the response from NRC wet mussel tissue matrix CRMs was significantly higher than that of the raw shellfish samples, although the relative response between the CRMs was equivalent (Supplementary Fig. S4, Supporting Information). These materials have previously always been analyzed with methods requiring extraction and have excellent commutability with real samples with respect to matrix interference and ionization suppression when used in this way. Differences in processing between these CRMs and the real samples analyzed here include heat stabilization (cooking) and more rigorous homogenization, both of which could lead to differences in relative response in direct analysis by LAESI-MS. Differences in DA response and reproducibility, and matrix effects between the different shellfish tissue matrices were investigated and are shown in Fig. 3(B). The relative response between the different matrix calibration standards did not differ significantly, which is promising for use of a single matrix standard for calibration. In general, differences in matrix effects varied more between different sample preparation approaches than between the types of shellfish tissue being analyzed. Quantitative capabilities of the LAESI-HRMS method The quantitative capabilities of the developed LAESI-HRMS method using the tSIM scan mode were assessed by investigating selectivity, linearity, LODs, precision and trueness. The reference materials included six-point tissue homogenate matrix-matched standards for each tissue type, with DA concentrations assigned by LC/UV. These were analyzed in at least triplicate, and the 5 mg kg À1 levels were analyzed at least 12 times throughout the run as quality control (QC) samples. The samples included 189 real shellfish samples previously analyzed by LC/UV or LC/MS as part of routine toxin monitoring programs. Samples and standards of each tissue type were analyzed together on 96-well plates, with each sample spotted in triplicate. Figure 4(A) shows an example of the data generated from the analysis of a plate of mussel tissue homogenate samples. The selectivity was assessed by confirming the absence of a signal at m/z 312.144 in 20 samples that tested negative for DA using LC reference methods. To investigate the relevance of the potentially interfering matrix species, discussed above and shown in Fig. 2, to the detection of DA in real samples, the accurate mass of the matrix interference at m/z 312.139 was determined from sequences of real shellfish samples. This matrix peak was absent in clam and scallop tissue samples, but was present at variable levels in mussels up to signal intensities equal to those of 10 mg kg À1 DA (Fig. 4(B)). Sample carryover was assessed by reviewing data from the analysis of real samples where low-level samples followed high-level samples. In one case ( Supplementary Fig. S5(A), Supporting Information), significant sample carryover was observed after the analysis of a 0.5 mg kg À1 scallop adductor muscle sample immediately following a 40 mg kg À1 matrix standard. However, in nearly all other cases no carryover was observed. Supplementary Fig. S5(B) (Supporting Information) shows a near baseline signal for a 0.6 mg kg À1 scallop remainder sample when it was bracketed by the two highest level samples in the study: scallop remainder at 210 and 220 mg kg À1 . The reason for the isolated carryover observed in Supplementary Fig. S5(A) (Supporting Information) is not clear, but care should be taken in manual review of data following high-level detection. The linearity was assessed from replicate analyses of six-point matrix standards for each tissue type ranging from 1 to 40 mg kg À1 . Considering the magnitude of matrix effects (Fig. 3), excellent linearity was observed for matrix-matched calibration standards (Table 1), with R 2 values ranging between 0.98 and 0.9992 (see also Supplementary Fig. S5, Supporting Information). The upper limit to the linear range was not investigated here but is not relevant to the use of LAESI-MS in regulatory screening. Limits of detection of LAESI-HRMS were determined through replicate analyses of low-level (1-5 mg DA kg À1 wet tissue) matrix standards of each tissue type. This was done by calculating the signal-to-noise (S/N) as the ratio of the MS peak intensity at m/z 312.144 in low-level matrix standards to that in control samples. These S/N values were then extrapolated to S/N = 3, doubled to account for the 1:1 dilution in sample preparation and used to determine LODs (Table 1). Similar values were obtained from the standard deviations of measurements on control samples, but an insufficient number wileyonlinelibrary.com/journal/rcm of control samples for each tissue were available to allow rigorous calculation of LOD by this method. In all cases, samples close to the LOD as well as control samples were run to verify detection at these levels, which are similar to LODs of the LC/UV methods currently used for routine DA quantitation. [4,5,12] Compared with other tissues, scallop gonad samples exhibited higher background and poorer LAESI peak shape, and these are reflected in a higher LOD. The precision was assessed through replicate analysis of 5 mg kg À1 matrix reference standards of each tissue type run as QC samples throughout each sequence of real samples. These values (Table 1) are relatively high for quantitative analysis but generally acceptable for a screening method. They did, however, confirm that samples needed to be analyzed in at least triplicate to achieve good accuracy. Even with triplicate analyses, the run times were still about 30 s per sample. The accuracy of the method was evaluated by comparing results from LAESI-HRMS with those from LC/UV and LC/MS reference methods. In addition, two different matrix-matched calibration approaches were assessed using either single-point calibration at 5 mg kg À1 or six-point calibration from 1 to 40 mg kg À1 . In all cases, the matrix-matched standards consisted of shellfish tissue homogenates whose DA concentration was established using validated LC/UV methods. The results using the full calibration range were used to evaluate the performance of LAESI-HRMS for primary quantitation. Single-point calibration was used to evaluate its performance as a screening method. Two samples of scallop remainder with DA > 100 mg kg À1 showed saturation of detection and they were not considered when examining overall correlations. Both calibration approaches gave equal overall correlation coefficients (R 2 ) of 0.89 for the linear regression of LAESI vs LC results. Six-point calibration gave better overall agreement with a regression slope of 1.2 compared to 1.8 for single-point calibration. This difference was primarily influenced by the highest level samples, which showed a significant positive bias using one-point calibration. On the other hand, one-point calibration gave better results for screening of samples as being either above or below 5 mg kg À1 (Fig. 5). Using this screening approach, LAESI-HRMS was able to identify all 20 of the samples above the regulatory limit of 20 mg kg À1 . [2,3] A false positive rate of 4% was observed, with eight samples being incorrectly identified as containing DA above 5 mg kg À1 . Only one sample above 5 mg kg À1 was not identified by LAESI-MS, but this sample was still found to contain less than half of the regulatory limit for DA, as determined by LC/UV. The LAESI-MS system was very robust and the experiments carried out for this study can be considered a significant stress test of the instrument. Approximately 2500 wells of shellfish tissue were analyzed over 2 days, which greatly exceeds sample volumes expected for routine use. It was found that the MS extension tube required cleaning after approximately 500 wells of tissue homogenate. This process involved sonicating the tube in a formic acid solution for 20 min and could be expected to have minimal impact on throughput as long as two tubes are available. The primary indication that extension tube cleaning was required was a drop in sensitivity, particularly for higher level samples, resulting in a loss of dynamic range of the method. CONCLUSIONS LAESI-HRMS performed well as a high-throughput screening method for DA in a variety of shellfish matrices and was successful at identifying samples with [DA] >5 mg kg À1 . This value corresponds to one-quarter of the regulatory limit, and was chosen to allow for early detection of the beginning of an algal bloom event, to ensure no false negatives above 20 mg kg À1 and to minimize the number of false positives requiring confirmatory analysis by LC. No sample extraction or cleanup was required after the tissue had been homogenized. The analysis time, including replicates and standards required to obtain good trueness, was under 40 s/sample. Because of the high variability of the method, confirmatory testing of positive samples using an LC reference method is currently recommended. However, the availability of an isotopically labelled standard for DA in the future may allow LAESI-HRMS to be used for direct quantitation without confirmatory testing. Currently, the best approach for the calibration of LAESI-MS is to prepare a secondary standard of homogenized, naturally contaminated, shellfish tissue, and to characterize it using a validated LC method. Remaining challenges include how to store and aliquot the shellfish homogenate standards required for LAESI-MS calibration. Supernatants showed similar response to homogenates and could be useful as matrix-matched standards in the future. Use of this technique could result in significant cost and time savings for routine testing labs and expand their capacity during periods of unusually high sample volume, such as the Pseudo-nitzschia bloom on the west coast of North America in 2015. [24] The next stage of development should involve implementation of LAESI-MS alongside current methodology in a regulatory testing lab where an ongoing comparison of the techniques could inform further method refinements and validation.	v3-fos-license
2019-04-09T13:06:31.466Z	2017-10-25T00:00:00.000	104266470	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10404-017-2005-5.pdf", "pdf_hash": "1f8955c806a561a041e04ee008679e40b7b4a91e", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:964", "s2fieldsofstudy": [ "Materials Science" ], "sha1": "8d9c9e9fd1e99e11aaac28b3ce47461a2f6d1776", "year": 2017 }	pes2o/s2orc	Precision moulding of biomimetic disposable chips for droplet-based applications In this study, we present a biomimetic approach to improve the stability and reproducibility of droplet generation processes and to reduce the adhesion of aqueous droplets to channel surfaces of microfluidic polymer chips. The hierarchical structure of the lotus leaf was used as a template for a partial laser structuring of the moulds that were used for casting the polymer chips. The hydrophobic wax layer of the lotus leaf was technologically replicated by coating the polymer chips using a plasma deposition process. The resulting microfluidic polymer chip surfaces reveal a topography and a surface free energy similar to those of the lotus leaf. Subsequent droplet-based microfluidic experiments were performed using a 2D flow focussing set-up. Droplets from both, serum-supplemented cell culture medium and anticoagulated human whole blood, could be generated stably and reproducibly using a fluorocarbon as continuous phase. The presented results illustrate the application potential of the lotus-leaf-like polymer chips in life sciences, e.g. in the field of personalised medicine. Introduction Microfluidic chips are used in droplet-based microfluidics to prepare serially arranged micro-reactors based on the immiscibility of at least two fluids (Chong et al. 2016). An increasing number of research groups use such micro-reactors for biological applications, e.g. for screening of microorganisms regarding their potential to produce pharmaceuticals (Zang et al. 2013) and to discover novel enzymes (Beneyton et al. 2016) or for investigating their resistance against heavy metals (Cao et al. 2013). Other groups reported about the cultivation of single cells in microfluidic droplets to detect their protein expression quantitatively (Huebner et al. 2007) or about screening and development studies on multicellular spheroids (McMillan et al. 2016) and even on embryos of multicellular organisms like the zebrafish (Funfak et al. 2007). Our group has recently reported about the dropletbased cultivation of embryoid bodies (EBs) formed from murine embryonic stem cells [mESCs (Lemke et al. 2015)]. These mESCs experiments were performed using the modularly constructed technological platform "pipe based bioreactors" (pbb) which has the potential to serve as long-term cultivation system for 3D cell cultures in the volume scale from 100 nL up to 10 µL (Spitkovsky et al. 2016). Furthermore, droplet-based processes can be used to encapsulate pancreatic islets (Wiedemeier et al. 2011) and to analyse the quality of food (Schemberg et al. 2009(Schemberg et al. , 2010. An important prerequisite for the establishment of droplet-based applications is the availability of special devices for manipulating the droplets. One example is devices for the rapid and targeted sorting of droplets (Xi et al. 2017). Furthermore, new droplet generation concepts using different pressure conditions allow varying droplet sizes (Teo et al. 2017). However, despite the huge potential of droplet-based microfluidics and the continuously growing number of publications in this field (Dressler et al. 2014), most studies only present proof-of-concepts (Casadevall i Solvas and deMello 2011; Volpatti and Yetisen 2014). One reason for this limited transferability to commercial/industrial applications is the restricted availability of disposable polymer chips that allow for a stable and reproducible droplet generation of challenging biologically relevant fluids with a high protein content (Shembekar et al. 2016). Cyclic olefin copolymer (COC) (Mair et al. 2006) and polycarbonate (PC) (Sun et al. 2005) are well-established thermoplastic materials for microfluidic applications since they are optically transparent, sterilisable, easy to handle and economically priced. However, micro-channel surfaces of fluidic chips moulded from these polymers by standard injection moulding processes do not support a stable and reproducible droplet generation process. Especially in the case of generating droplets from biologically relevant fluids with a high protein concentration like cell culture media supplemented with serum, an adhesion of the droplets to the micro-channel surface and consequently droplet pinning resulting in cross-contamination are frequently observed. To avoid droplet pinning effects, micro-channel surfaces have to be modified to achieve superhydrophobic properties. There are a lot of papers describing surface modifications to achieve superhydrophobic properties on polymers, e.g. (Shirtcliffe et al. 2011;Xue et al. 2010). Usually, the surface will be microstructured and hydrophobically coated. Well-established procedures are hot embossing (Wang et al. 2017), sol-gel coating (Wu et al. 2016) and deposition of soot (Esmeryan et al. 2017) or nanoparticles (Saarikoski et al. 2009). Furthermore, laser ablation procedures are used to structure the surfaces (Bachus et al. 2017;Rowthu et al. 2015). However, all these procedures are more expensive than producing microstructured chips by moulding or not suitable for droplet-based microfluidic applications. The final hydrophobisation is usually carried out by means of a wet chemical or plasma-supported coating (Jankowski et al. 2011). In this study, we present a biomimetic approach to prepare anti-adhesive micro-channel surfaces on PC-and COC-chips with superhydrophobic lotus-leaf-like properties (Barthlott and Neinhuis 1997) for droplet-based microfluidic applications. The lotus effect is supposed to result from the combination of a hierarchical microtopography (i.e. papillae with a defined microstructure and nanostructure) and a hydrophobic epicuticular wax layer (Andreas et al. 2007;Darmanin and Guittard 2015;Quéré 2008). In order to replicate the lotus structure (Gornik 2004;Kim et al. 2013;Oh et al. 2011;Tuvshindorj et al. 2014), the surfaces of the moulds were equipped with a microtopography (Fig. 1) employing femtosecond pulsed laser ablation as described by Groenendijk (Groenendijk 2008). After injection moulding, the chips were treated with a plasma coating procedure. The resulting surfaces were characterised topographically as well as physico-chemically. In order to analyse these microstructured and plasma-coated fluidic chips regarding their behaviour during and after droplet generation from aqueous media with a high protein concentration, fluidic experiments were performed. The fluid micro systems (FMS chips) described in this paper were developed to handle cells and 3D cell structures like spheroids with diameters up to 500 µm. For this reason, the diameters of the droplet guiding channels were determined to be 1000 µm resulting in droplet volumes not less than 523 nL to guarantee droplet contact to the circular channel walls. Droplet contact to the walls prevents merging of droplets. The smallest channel diameter for a reasonable manufacture by milling is proven to be 200 µm resulting in droplet volumes of about 4 nL. However, using moulding techniques, smaller channel diameters could be realised. Preparation of microstructured moulds by laser ablation To investigate the chips surface properties on the one hand and their microfluidic properties on the other hand, two different chip types were moulded: (1) test elements (MST chips) without microfluidic channels but with a microstructured planar surface that were primarily employed for topographical and physico-chemical analyses (Fig. 1a) and (2) FMS chips with microstructured half-channels for droplet generation experiments (Fig. 4b, c). For these experiments, two of the half-channel possessing chips have to be assembled face-to-face resulting in circular cross sections of the microfluidic channels. For the characterisation of unstructured surfaces, the reverse surfaces of the MST chips were used. The FMS chips were designed to perform 2D flow focussing experiments, i.e. each face-to-face assembled microfluidic chip possesses one main channel (diameter: 1000 µm) and two side channels (diameter: 300 µm), perpendicularly arranged to the main channel, see Fig. 4b. The size of a FMS chip is 24 × 24 × 4 mm 3 . The respective moulds were manufactured from hot working steel (1.2343). The semi-circular negative structures for moulding the half-channels were manufactured by electrical discharge machining. Subsequently, surface microstructuring was performed by laser ablation using an ultrashort pulse laser (Hyper Rapid, Coherent GmbH, Germany) equipped with a Galvanometer scanner. The pulse width was 8 ps at a wavelength of 355 nm. The laser power was 0.5 W. For the mould of the MST chips, the microstructured area was 20 × 20 mm 2 . For the FMS chip mould, only the negative structure for moulding the main channel and also the parts of the side channel that are in close proximity to the droplet generation zone were microstructured. A home-made tilting stage (ifw Jena, Germany) was employed to rotate the mould during the laser ablation process in order to obtain an angle of incidence of close to 90° with respect to the curved surface of the semicircular negative structures of the mould. Both the tilting and the positioning of the samples demanded a high degree of precision (µm scale). Inspired by the microstructure of the anti-adhesive lotus leaves, micro-cavities with a depth of 10-15 µm and a spacing of 10-15 µm were created by laser ablation (Groenendijk and Meijer 2006). The diameter of the laser focus was 10 µm. The depths of the micro-cavities were adjusted by varying the density of the laser pulses per area (repetitions or pulse count). To achieve a high surface density, the microcavities were arranged in a hexagonal pattern (Andreas et al. 2007;Feng et al. 2008). Injection moulding and coating COC (COC615) and two PC materials (PC2400 and PC2805) with different viscosities were used as chip moulding polymers. The viscosity increased from PC2400 < PC2805 < COC615. The chips were manufactured by injection moulding (Allrounder 320 s, Arburg, Germany) with the following process parameters (Table 1). After injection moulding, the chips were cleaned by treatment with isopropanol for 15 min in an ultrasonic bath (Sonorex super RK 100 H, Bandelin electronic GmbH & Co. KG, Germany) and successive rinsing with ethanol [80%(v/v)] and deionised water. Scheme of the three-phase system: microstructured, plasma-coated channel surface/hydrophobic phase (perfluorodecalin)/hydrophilic phase (droplet) and c scheme of the Cassie-Baxter wetting regime Subsequently, the surfaces of the chips were coated with a hydrophobic layer employing a low-pressure plasma system (Pico 110265, Type F, Diener electronic GmbH + Co. KG, Germany). After evacuating the reaction chamber down to a pressure of 9 Pa, the octafluorocyclobutane precursor (C 4 F 8 , Air Liquide, Germany) was injected with 15 sccm, and the plasma was initiated at 13.56 MHz. Plasma coating was performed for 30 min. The coated chips were stored until use in a closed chamber. Scanning electron microscopy (SEM) After sputter coating with an approx. 9 nm gold layer (K550X Sputter Coater, Quorum Technologies Ltd, UK), the microstructured surface topography of the MST chips was characterised by means of stereo scanning electron microscopy (Evo LS10, Carl Zeiss Microscopy GmbH, Germany). SEM images from three tilt angels (0°-7°-15°) were recorded with a 250 fold magnification, 9 mm working distance and 1024 pixel × 768 pixel resolution. From these SEM images, a three-dimensional, digital surface model (3D-DSM) was composed employing the MeX ® software (Alicona Imaging GmbH, Austria). The analysis tool of the same software was used to determine the depths of the micro-cavities. Atomic force microscopy (AFM) AFM investigations on MST chips were performed using the atomic force microscope Nanowizard ® equipped with a 100 µm z-scan module CellHesion ® (both jpk instruments AG, Germany). All scans were performed in contact mode at ambient conditions with cantilevers ARROW-NC (NanoWorld AG, Switzerland) having a nominal spring constant of 42 N/m and a tip radius less than 20 nm. Due to the high aspect ratio of the micro-cavities, the scans were performed with a low scan rate of 0.1 Hz. In addition to the analyses of the microstructured chips, the hierarchical topography of a lotus leave (Nelumbo nucifera) was investigated, as well. The lotus leaf was stabilised with glycerol to avoid drying artefacts during the scans. Post-processing and 3D images of data were realised using the background correction feature and a Gaussian smoothing filter of the software SPIP™ (Image Metrology A/S, Denmark). Physico-chemical characterisation Surface free energy determinations (including polar and dispersive components) were performed on the microstructured MST chips before and after the plasma coating procedure according the OWRK approach (Owens, Wendt, Rabel und Kaelble). For this approach, the contact angles of deionised water, formamide, ethylene glycol (predominantly polar) and diiodomethane (dispersive) were recorded using the OCA System (sessile drop, 3 droplets of 3 µL, DataPhysics Instruments GmbH, Germany). Fluidic experiments Fluidic experiments were performed with a highly versatile microfluidic platform comprising the following functional modules: • Droplet generation FMS, • Detection module (equipped with two optical sensors), • Tube storage disc, • Stirring unit (only for experiments with blood), • Syringe pump system (neMESYS, cetoni GmbH, Germany). The droplet generation FMS is composed of two plasmacoated chip halves assembled reversibly face-to-face and pressed together with screws guaranteeing leak tightness . Pins and drillings serve as positioning elements to guarantee for a reproducible assembly and disassembly, e.g. for refreshing of the chip coating. After its assembling, the FMS is mounted into a frame that serves for the fluidic connections between the tubes and the FMS (Fig. 4a). All functional modules were connected with tubes made from polytetrafluoroethylene (PTFE) and fluorinated ethylene propylene (FEP), respectively. For both, the transport of droplets as well as the mutual separation of the droplets, perfluorodecalin (PFD) served as continuous phase. Additionally, reference experiments with Novec 7500 and Pico-Surf TM 2 as continuous phase were performed. Two aqueous fluids with a high biotechnological and biomedical relevancy were used for the experiments performed in this study: (1) cell culture medium Dulbecco's Modified Eagle's Medium (DMEM), product number D5523 (Sigma-Aldrich Chemie GmbH, Germany) supplemented with 4.5 g/L d-glucose, 2 mmol/l l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10%v/v foetal calf serum and 0.01%w/v phenol red and (2) anticoagulated human blood (supplemented with trisodium citrate 0.106 mol/L in S-Monovette ® , order number 04.1955.100, Sarstedt AG & Co., Germany). For droplet-based microfluidic experiments, those fluids are often challenging due to their high concentration of surface active compounds like proteins. In order to characterise the influence of the FMS surface microstructure and coating on the droplet generation process, most experiments were performed without surfactants. However, as reference to usual droplet-based applications, also experiments with surfactants were performed. Prior to the experiments, the droplet generation FMS and the tubes were intensively rinsed and filled with PFD. For each parameter, five experiments were performed with a PFD flow rate (Q c ) of 500 µL/min, an aqueous fluid flow rate (Q d ) of 100 µL/min and a droplet generation time of 6 min. To evaluate the droplet generation reproducibility, the volume of each droplet and its standard deviation were assessed. Each droplet was considered as a sphere (sphere diameter ≤ main channel diameter of the droplet generation FMS) or as cylinder with hemispherical ends. In the latter case, the cylinder diameter corresponds to the main channel diameter of the droplet generation FMS. For the cylinder volume calculation, the droplet length was measured photometric at 525 nm by recording the absorption shift between PFD and the droplet. These measurements were realised using a detection chip with two optical sensors. The droplet volume calculation bases on the precondition of an idealised droplet shape. However, depending on the droplet velocity, deviations from these idealised shapes were observed. Compared to a non-moving (nondeformed) droplet, the shape deviation became more significant with increasing velocity. Taking these shape deviations into account, a correction factor was introduced. This factor T s/s represents the ratio of the droplet length average s D,SP , calculated from the sample volume delivered by the syringes pump (SP) divided by the number of generated droplets and the droplet length average s D,OS , determined by the optical sensors (OS). Subsequently, the volume of each droplet was calculated employing this correction factor. To evaluate the droplet generation reproducibility, the following parameters were determined for each experiment: (1) the mean droplet volume, (2) the standard deviation and (3) the coefficient of variation (CV). For the final analysis of the droplet generation stability, two further experiments were performed. Within the scope of these experiments, interactions of the sample medium with the FMS channel surface should be investigated. For this, droplets were continuously generated for 6 h using the FMS chips to be examined. The evaluation was based on the pure optical observation of the droplet generation process. The experiment was stopped when the droplets extensively adhered to the channel surface, and the stability of droplet generation process was significantly disturbed. Influence of the microstructure on the physico-chemical properties With the applied injection-moulding process, papilla-like structures could successfully be prepared on different polymers surfaces (Fig. 2a-c). The use of different polymers resulted in different morphologies of the papilla-like structures. The flattest papillae were observed on the surface of MST chips made from COC (Fig. 2c), whereas the PC2805 surfaces were characterised by rather sharp papillae (Fig. 2b). The different polymers also caused different heights of the papillae; i.e. heights of ~ 5 µm for PC2805 (Fig. 2b) and COC6017 (Fig. 2c) and heights of ~ 6 µm for PC2400 (Fig. 2a). These differences are caused by the different viscosities of the molten polymers. A lower viscosity of the melt improves its intrusion into the mould cavities and consequently increases the height of the papillae. The polymer PC2400 with the lowest viscosity (PC2400 < PC2805 < COC6017) resulted in the maximum papillae height. Compared to unstructured and uncoated polymer MST chip surfaces, the contact angles of uncoated microstructured MST chip surfaces increased from ~ 90° to ~ 120° (Fig. 2d). The highest water contact angle on uncoated but microstructured surfaces was measured with ~ 130° on the COC6017 MST surface. This also connotes that microstructured COC6017 FMS should be well suited for droplet-based applications even without plasma coating. The surface free energy of the unstructured and uncoated MST surfaces was ~ 40 mN/m (Fig. 2e). Caused by the microstructure, the surface free energy was increased by ~ 6 mN/m for the MST chips with smaller papillae height (PC2805 and COC6017) and by ~ 20 mN/m for the MST chips with maximum papillae height (PC2400, Fig. 2e). In the case of unstructured surfaces of MST chips made from the polymers PC2400 and PC2805, ~ 95% of the surface free energy was contributed by the dispersive component. For COC6017, the dispersive component was ~ 89%. For the microstructured surface of MST chips, the dispersive component was increased by ~ 3% for PC2400 and PC2805 and by ~ 10% for COC6017. In summary, the uncoated microstructure of the MST chip surfaces increases both the water wettability and the surface free energy. This phenomenon was described by Wenzel (Wenzel 1949): for a homogeneous wetting scenario where microstructures intensify the surface properties of the bulk material (hydrophobic solid materials become more hydrophobic and vice versa). Influence of the plasma coating on the physico-chemical properties Plasma coating resulted in a significant increase of the water contact angle for both the unstructured and the microstructured MST surfaces (Fig. 2d, coated surfaces). The surface of an unstructured MST chips moulded from PC2805 revealed a water contact angle of 88.1° ± 3.4° and a surface free energy of 41.74 ± 1.0 mN/m (dispersive: 41.56 ± 1.1 mN/m, polar: 1.9 ± 0.3 mN/m). After plasma coating with C 4 F 8 , the wettability was significantly affected. The water contact angle increased to 119.4° ± 0.7°, and the surface free energy was reduced to 8.98 ± 0.2 mN/m (dispersive: 8.44 ± 0.3 mN/m, polar: 0.54 ± 0.2 mN/m). Interestingly, the wettability was not only reduced for aqueous test fluids but also for non-fluorinated oils like tetradecane. However, the coated surfaces were completely wettable with fluorinated oils like PFD (contact angle < 20°). From AFM measurements, the thickness of the C 4 F 8 -coating was estimated to be ~ 140 nm. For this, a glass slide was partially coated using the above-described coating procedure. For the microstructured surfaces, the water contact angle increased from ~ 110° to ~ 130° up to ~ 160° after plasma coating. Furthermore, the standard deviation of the water contact angles was reduced for all coated MST chips. After plasma coating the wettability of the three polymers were comparable even though there were significant differences in the wettability prior to the coating process (Fig. 2d). The polymer with the highest contact angle before coating (130° for microstructured COC6017) displayed the lowest contact angle after plasma coating (154°). Since the structure heights of PC2805 and COC6017 (Fig. 2d) are comparable, we assume that the rounded morphology of the COC6017 microstructure elements (Fig. 2c) causes an increase of the contact area to the polar phase resulting in a slightly increased wettability. In addition to the reduction of the water wettability, a significant reduction of the surface free energy was observed after coating both microstructured and not microstructured MST surfaces (Fig. 2e). For the unstructured surfaces, the surface free energy was reduced by more than 30 mN/m to values around 9 mN/m. An extremely low surface free energy of ~ 2 mN/m was observed for the microstructured surfaces after plasma coating. This reduction is predominantly due to a decrease of the dispersive part of the surface free energy. While the coating procedure does not significantly affect the polar component for unstructured surfaces (~ 1 mN/m), it is increased to ~ 4 mN/m for uncoated microstructured samples. When these microstructured surfaces are plasma coated, the polar part is reduced to values lower than 0.2 mN/m. In general, polar components account for less than 10% of the surface free energy irrespective of the coating. The high water contact angles additionally indicate a low contribution of the polar part of the surface free energy, especially since the coated microstructured polymer surfaces display an extremely low polar contribution. Comparison of the coated microstructured polymer surfaces with the lotus leaf surface In order to compare the technically prepared biomimetic surfaces with the leaves of the lotus flower, AFM analyses were performed on microstructured and plasma-coated PC2400 MST chips and Nelumbo nucifera leaves. Comparative AFM analyses reveal that both surfaces are characterised by a hierarchical micro-and nanotopography (Fig. 3). The morphology of the individual microstructure elements of the injection-moulded polymer surface reveals a striking similarity to the papillae of the lotus leaf (Fig. 3b, e). They also display comparable heights, diameters and spacing. The arrangement of the microstructure elements is characterised by a lower degree of variability (in terms of height and spacing of individual microstructure elements, Fig. 3a, d). Furthermore, both surfaces display a similar nanotopography on top of the microstructure elements (Fig. 3c, f). Both surfaces reveal an extremely low water wettability (~ 160° for the lotus leaf (Barthlott and Neinhuis 1997) as well as for the microstructured, plasma-coated polymer surface). These superhydrophobic properties indicate a heterogeneous wetting according to the Cassie-Baxter theory (Cassie and Baxter 1944;Hüger et al. 2009;Wagner et al. 2003). Microfluidic experiments The FMS microstructure was created on the surface of the main channel which is in direct contact with the test fluids. SEM and stereoSEM images of the main channel surface reveal that the microstructure that was created on the mould Table 2) could be successfully transferred onto the FMS chip elements (Fig. 4c, d). For the microfluidic experiments with DMEM and blood, the microstructured and plasma-coated FMS chips made from the above-mentioned polymers were investigated (Table 2,(1)(2)(3)(4)(5). Additionally, reference FMS chips ( Table 2, No. 6-7) were investigated to estimate the effect of a missing microstructure (unstructured PC2805) as well as of a missing C 4 F 8 coat (uncoated COC6017). Irrespective of the type of polymer, no significant adhesion of the cell culture medium to the channel surface was detected during the 30 min of droplet generation employing coated microstructured FMS (Table 2,(2)(3)(4)(5). However, severely reduced performance times of ~ 10 min for the unstructured but plasma-coated PC2805 reference FMS ( Table 2, No. 6) and ~ 15 min for the microstructured but uncoated COC6017 reference FMS ( Table 2, No. 7) were observed. This indicates that a combination of both the microstructure and the plasma coating is necessary to guarantee for a stable droplet generation process. In addition to these effects, the surface modification also affects both the droplet volume and the reproducibility of the droplet generation process (Fig. 4f). When the droplet generation was performed with microstructured and coated FMS, a consistent droplet volume of ~ 800 nL was obtained. For all microstructured and coated FMS investigated with DMEM ( Table 2, [3][4][5], there is a slight tendency of an increasing droplet volume for a decreasing water contact angle (and a decreasing surface free energy), compare (Table 2, No. 7). The droplet volumes increased to ~ 1100 nL (~+ 35%) when uncoated COC6017 FMS were employed (Fig. 4e, f). Each of these droplet volume increases correlates with a higher wettability of the surfaces (~ 30° lower water contact angles and higher surface free energy, Fig. 4e, f). The higher wettability of the FMS channel surface causes more pronounced interactions of DMEM with the channel surface, resulting in a delayed droplet break-up and thus in an increase of the droplet volume. For all microstructured and plasma-coated FMS, the CV values were smaller than 2.5%, indicating that the droplet generation process was highly reproducible. The lowest CV value of 1.45%, and thus, the highest reproducibility was observed for the microstructured and plasma-coated COC6017 FMS. For droplet generation from anticoagulated human whole blood, a coated microstructured PC2400 FMS was used ( Table 2, No. 1). Droplets could stably be generated during the whole process, lasting 30 min maximally. In contrast to the experiments with DMEM, droplets from blood could not be generated with microstructured but uncoated PC FMS. The blood droplet volumes and their CV values were higher than the respective data for droplets generated from DMEM. This may be due to the higher protein concentration of whole blood that supports interactions with the uncoated channel surface (Pham et al. 2016). Compared to the experiments without surfactants ( Finally, the experiments with DMEM and whole blood were continued with the four structured and coated FMS ( Table 2, No. 1-5) to study the long-term stability of the droplet generation. For this purpose, droplets were continuously generated on two successive days each for 6 h using the corresponding FMS as well as the test fluids DMEM and whole blood. Using three different, microstructured and coated FMS (Table 2, No. 2-5), droplets could be generated without disturbance over the complete test period of 12 h with DMEM. In the case of droplet generation from whole blood using the microstructured and coated PC2400 MST (Table 2, No. 1), punctiform adhesions of the blood on the channel surface were observed after approx. 10 h. However, these adhesions could be eliminated by increasing the flow rates and did not appear again. These experiments proved that injection-moulded, microstructured and plasma-coated FMS support a stable and long-term droplet generation process. In summary, both the introduced microstructure and the hydrophobic plasma coating lower the surface free energy of the FMS channel surfaces. Consequently, the tendency of biologically relevant fluids to adhere to the channel surface is lowered. According to Cassie's law (Cassie and Baxter 1945;Cassie 1948), the contact area between the droplets and the channel surface could be significantly reduced. Particularly (Absolom et al. 1987), this reduced contact area should also reduce the capability for protein adhesion (Fig. 1b, c). A combination of the microstructure and the hydrophobic plasma coating significantly reduces the adhesion of biologically relevant fluids to the channel surface. The resulting disposable FMS chips support a long-term stable droplet generation process with a highly reproducible droplet volume. Conclusions The results presented in this study demonstrate that microfluidic chips with biomimetic anti-adhesive channel surfaces can be prepared by combining injection moulding and plasma coating. Injection moulding was performed in moulds that were microstructured via laser ablation. The resulting superhydrophobic surfaces reveal a hierarchical microtopography and physico-chemical properties similar to the lotus leaf. Dropletbased microfluidic experiments reveal that only the combination of the microtopography and the anti-adhesive C 4 F 8 -plasma coating supports a stable long-term droplet generation process. Droplets could even be prepared from challenging fluids like whole blood with a high protein concentration in a stable and highly reproducible fashion. In combination with the modular platform (iba 2005; Lemke et al. 2015), the FMS chips possess a high potential for commercial, droplet-based applications in life sciences since they support a stable and reproducible droplet generation process and since they can be prepared as sterilisable disposables. The advantage of the approach for increasing the hydrophobicity of polymer surfaces proposed in this paper is the direct microstructure transfer from the mould to the chip, followed by a plain plasma coating. Compared to the manufacture of ordinary moulds, only one additional manufacturing step is necessary. After milling and electroerosion of the mould, the microstructures were realised by laser ablation. During the moulding process, these microstructures were transferred onto the FMS chips. After coating the FMS chips with plasma, two chip halves were mounted face-to-face to achieve round channels. After mounting the FMS chips, they can be used for the experiments. Laser ablation is the only additional manufacture step.	v3-fos-license
2018-04-27T04:56:13.281Z	2017-12-18T00:00:00.000	4942782	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2018/sc/c7sc05094a", "pdf_hash": "c3e6f48adc28713c27a27344f53ec4761e5d2457", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1000", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "ad56aede2310d4ca0d0ac8028d1c7d45defd6b5d", "year": 2017 }	pes2o/s2orc	Guiding a divergent reaction by photochemical control: bichromatic selective access to levulinates and butenolides Allylic and acrylic substrates may be efficiently transformed by a sequential bichromatic photochemical process into derivatives of levulinates or butenolides with high selectivity when phenanthrene is used as a regulator. Introduction Chemistry with light provides a range of creative synthetic possibilities. 1 Many chemical transformations promoted by light, such as photosynthesis, the cis-trans photoisomerization of the protonated Schiff base of 11-cis-retinal and the synthesis of vitamin D are vital for the existence of life on Earth. 2 Conversely, photocycloadditions caused by UV radiation, may result in DNA damage leading to cancer. 3 To safeguard us from this, perhaps mimicking organisms that evolved UV protecting molecules, 4 chemists have developed sunscreens, 5 which deect or absorb these harmful radiations. Inspired by the way evolution has selected molecules to block certain UV wavelengths to follow specic biochemical pathways, 6 selective photochemical syntheses of organic frameworks may be imposed depending on whether molecular light lters are present or not. Indeed, photochemical outcomes of divergent organic reactions may be controlled by subtle changes in the substrate, reaction conditions or the use of sensitizers. 7 Recently, we employed UV light absorbing organic molecules in a selective deprotection of photolabile protecting groups and photo-induced crossmetathesis. 8 Moreover, the selective use of light of different frequencies to promote distinct chemical reactions, i.e. chromatic orthogonality, 9 has been applied in synthetic procedures, including the synthesis of a natural peptide product. 10 During our studies on chromatically orthogonal metathesis reactions using photo-switchable catalysts, 11 the consecutive irradiation at 350 nm and 254 nm UV light of a solution of avinylbenzyl alcohol and methyl acrylate resulted in the nonselective synthesis of two products, a butenolide and a levulinate. 12 Levulinates 13 and butenolides 14 are exceptionally ubiquitous, and some prominent natural products and derivatives containing these motifs are highlighted in Fig. 1. 15 Indeed, the development of efficient synthetic routes for their preparation continues to be an active area of interest to synthetic chemists and many methodologies have been put forth recently. 13,15a,c-e, 16 The ability to obtain different products utilizing the same starting materials and intermediates, especially by photochemical processes under mild conditions, may pose an important synthetic and industrial advantage. 17 Moreover; photochemistry possesses the inherent property of being able to be remotely controlled. 18 Herein, we report selective divergent all-photochemical syntheses of levulinates and butanolide derivatives using simple starting materials. Following a UV-A photoinduced cross-metathesis reaction, UV-C irradiation generates the desired products. Mechanistic and computational studies elucidate the role of phenanthrene as a UV-C modulator: the presence of phenanthrene in the reaction suppresses the 1,5-H shi pathway leading to high selectivity in the formation of butenolides, while excluding the phenanthrene allows the opposite selectivity (Scheme 1). Several examples are shown highlighting the scope and broad implications of this method, including the selective synthesis of a natural product molecule in a very efficient and convenient manner. Results and discussion The rst step of the desired sequence is a photoinduced CM reaction between an allylic (1) and an acrylic (2) substrate. 19 For this, photolatent S-chelated ruthenium benzylidenes were used. 20 Even though the CM reaction may also be efficiently promoted by typical ruthenium metathesis catalysts, the use of photolatent precatalysts allows for an all-photochemical method that can be both spatially and temporally controlled. 10 Thus, UV-A induced CM between 1a and 2a or 2b was readily promoted by the robust catalyst, cis-Ru I in good yields (Table 1, entries 1 and 2). Evaporation of the excess acrylate, without the need for further purication, set up the stage for the second photochemical step. In more complex CM reactions, for example when acrylates 2c-2e or allyl alcohols 1c and 1d were used ( Table 1, entries 3-9), cis-Ru I proved to be a less efficient CM catalyst. To our satisfaction, a faster initiating photolatent catalyst (cis-Ru II) 21 could perform better in these difficult cases, highlighting the advantages of the versatility of the S-chelated family of catalysts. Because ruthenium by-products are known to catalyze nonmetathetic processes, 22 the possible role of the ruthenium species in the second part of the sequence was examined. Two samples of E-3a, one made by the aforementioned CM route and the other by a metal-free route, 23 were subjected to irradiation at 254 nm under identical conditions. Similar results were obtained in both cases; demonstrating that the conversion to 4a and 5a are sheer photochemical processes not assisted by ruthenium. We then surmised that the transesterication 24 could be stied by running the reaction at lower temperature or by the use of a bulky acrylate. Indeed, decreasing the reaction temperature by about 10 C reduced lactonization and shied the reaction towards the levulinate compound (see ESI for details †). Better still, higher than 90% selectivity for 5b was achieved when bulky 2b was used as the CM partner in step 1 ( Table 1, entry 2). Next, we tested whether the addition of a bulky alcohol could help provide the necessary steric barrier to Table 1 Sequential photochemical reactions a a CM was carried out at 0.05 M, unless otherwise noted. Isomerization reactions were conducted at 0.01 M concentration in DCM at 25-30 C. 10 equiv. of acrylate was used in the CM reaction. The reported yields are isolated yields. Product ratios in parentheses are based on 1 H NMR analyses. See ESI for further details. b For entries 1 and 2, cis-Ru I (3 mol%) was used. For entries 3-7, 3 mol% while for entries 8 and 9, 6 mol% cis-Ru II was used. c For entries 1-5, 2.1 equiv. and for entries 6-9, 0.3 equiv. of phenanthrene was used. The product ratios in the parentheses are 4 : 5. d For entries 2, 8 and 9, DCM and for entries 1 and 3-7, t BuOH/DCM (1 : 4) was used as the solvent. The product ratios in the parentheses are 5 : 4. e 0.1 mM aq. solution of adenosine was used as an external UV-C lter to minimize side reactions (vide infra). inhibit the lactonization reaction by hydrogen bonding to 3. This could be quite practical if less sterically demanding acrylates are desired as the CM partners, especially because methyl and butyl acrylates are amongst the most important acrylic esters, with great demand as commodity chemicals. In addition, the possibility to apply different acrylates expands the scope of levulinate products that may be directly obtained by the divergent sequence. To our satisfaction, high selectivities for the 1,5-H shi were achieved when one equivalent of t BuOH was added to the reaction solution during irradiation of E-3a with 254 nm light. Further optimization of reaction conditions showed that a mixture of t BuOH and DCM (1 : 4) gave the best selectivity for 5a (Table 1, entry 1). Notably, reactions with 2a and 2b were very convenient given that excess acrylate could be easily removed by evaporation aer the CM reactions, allowing sequential reactions without purication of the intermediate. To promote the opposite selectivity, we envisioned applying the molecular 'UV-C lter' protocol 8 to prevent the photochemically more demanding 1,5-H shi (vide infra). Indeed, aer screening several UV-C lters and reaction conditions, the irradiation of a DCM solution of E-3a with phenanthrene afforded 71% isolated yield of the butenolide ( Table 1, entry 1). The amount of phenanthrene required for selective lactonization was found to be dependent on the molar absorptivity of the substrate. Thus, for allylic alcohols bearing aromatic substituents, about 2 equiv. of phenanthrene were required to achieve optimal selectivity; while substoichiometric (0.3 equiv.) amounts of phenanthrene were sufficient for the allyl alcohols with aliphatic substituents ( Table 1). The type of acrylate used also had a signicant effect on the selectivity of the lactonization process; thus, lactonization with n-butyl acrylates was somewhat more sluggish and led to lower selectivity (Table 1, entry 3), while the increased leaving group ability of 4-t-phenyl acrylate ( Table 1, entries 5 and 7) resulted in butanolide formation even without the addition of phenanthrene (albeit in lower yields due to detrimental decomposition). The functional group tolerance and selectivity of the reaction sequence was also probed with more challenging starting materials that could, in principle, form different ring-size lactones. With this in mind, 3h was irradiated at 254 nm in the presence of phenanthrene (Table 1, entry 8). Hydroxymethyl butyrolactone (4c) 24,25 was selectively obtained with full retention of chirality, as expected. In the same manner, optically pure 4d was obtained from 3i (Table 1, entry 9). High selectivities, and decent overall isolated yields, were also obtained for the levulinates 5h and 5i, the only difference in the procedure being that the UV absorbing phenanthrene was not added to the reaction mixture. To better understand this remarkable photochemical divergent process, we decided to investigate possible reaction mechanisms. It is reasonable to assume that formation of the lactone (4a) occurred through a typical diabatic photochemical E-Z isomerization of the olen, 26 followed by trans-esterication. Likewise, ketone 5a may be formed through a 1,5-H shi, generating a dienol intermediate (i5), which may tautomerize to afford the nal product (Scheme 1). 27 Supporting this hypothesis, Z-3a was appreciably observed by 1 H NMR monitoring during UV-C irradiation of E-3a. Wavelengths of 350 nm, 380 nm or even thermal stimuli, e.g. heating to 140 C, did not lead to isomerization; however, upon irradiation with 300 nm light some Z compound was observed (see ESI for details †). The generation of the dienol intermediate (i5) was validated by deuterium labelling studies (Scheme 2A). The photochemical reaction in the presence of D 2 O/CD 2 Cl 2 showed the incorporation of deuterium at both the aand bpositions in product 5a-D. Also, UV-C irradiation of deuterium labelled intermediate 3b-D afforded a product without deuterium (Scheme 2A). As a further control experiment, a reaction of the corresponding TMS-protected E-3a was performed in CD 2 Cl 2 , forming the expected TMS-enolate intermediate (see ESI for details †). These experiments strongly support a 1,5hydrogen shi mechanism to produce the dienol intermediate (i5) and refute the occurrence of a plausible photochemically induced 1,3-hydrogen shi. The effect of phenanthrene on cyclization was probed by UV-C irradiation of 1e, a substrate that can only cyclize (Scheme 2B) both with and without phenanthrene. In both cases cyclization to the lactone was observed; however, the reaction was slower when phenanthrene was present. This experiment clearly suggests that the phenanthrene is not sensitizing the cyclization reaction, but is indeed inhibiting the 1,5-H shi. Quantum chemical calculations were used to further study the reaction mechanisms involving these processes. The brightest excited state was found to be S 2 for E-3j (Scheme 1, R ¼ R 0 ¼ Me) with 189 nm and S 3 for Z-3j with 192 nm. The excitation energies of the E and Z isomers are very close (DE ¼ 0.09 eV), and therefore both can be excited by irradiation of UV light of the same wavelength. The S 9 state of phenanthrene lies at 193 nm (Table S5 †), overlapping with the main absorption of both isomers of 3j. It is therefore expected that addition of phenanthrene should decrease the rate of the E-Z interconversion. As well noted in the literature, 28 the computed vertical excitation energies are shied from the experimentally observed absorption maxima due to lack of solvent effects, vibrational effects and accuracy of the quantum chemical methods. Hence, we need to consider the excitation wavelength of 254 nm to be blue shied in the theoretical context. To assess the effect of the solvent on the vertical excitation energies, quantum chemical calculations were carried out using an implicit solvent model (see Table S5 †). In solution phase the spectra show a red shi in the range of 2 to 16 nm. Hence, inclusion of the solvent model shis the vertical excitation energies towards the experimental values. In order to rationalize the effect of phenanthrene on the photochemical reactions, intermediates of the two divergent reaction pathways were analyzed theoretically. The intermediate for the levulinate formation pathway is a dienol (Scheme 1, i5j), and the intermediate for the cyclization pathway is i4e. Excited state energies reveal the brightest state (S 2 ) of dienol intermediate i5j at 237 nm which is nearly coinciding with the wavelength of a high-lying excited state S 4 of the phenanthrene (240 nm). Although both states are around 14-17 nm (0.29-0.35 eV) away from the experimental excitation wavelength (energy), considering (i) the blue shi of the computed vertical excitation energies with respect to the experimental absorption maxima, (ii) the broadness of the excitation source (ca. 10 nm) and (iii) the deformation of the geometry due to thermal energy, the excitation in this energy range is feasible. This is supported by the experimental nding that also excitation at 300 nm leads to levulinate formation. In case of the cyclization reaction, i4e (S 4 ) is found to absorb at 155 nm which overlaps with the absorption of phenanthrene at 153 nm (S 31 ). However, the energy difference to the excitation source is signicantly higher considering the blue-shied calculated energies (at least 1.5 eV compared to 3j). In addition, the cyclic geometry is more rigid than that of the dienol. The higher rigidity would lead to smaller distortion from the minimum structure and a more narrow absorption band. Hence, the probability of light activation of cyclization reaction is negligible. Therefore, we conclude that the major role of the phenanthrene is to lter the wavelength that triggers 1,5-H shi and to suppress the pathway of levulinate formation while the cyclization is expected to proceed largely unaffected. The scope of the sequential chromatic divergent process was scrutinized with several other substituted allyl alcohols and the results are displayed in Table 2. Both aromatic and aliphatic substrates underwent the expected photochemical transformations resulting in moderate to good isolated yields of the corresponding products. Notably, aryl bromides (3l and 3m) underwent radical dehalogenation 29 during the isomerization to ketone (to afford 5a), while the aryl chloride was stable under these conditions (3n). However, in the presence of phenanthrene, the radical dehalogenation is avoided, yielding the corresponding bromoaryl lactones (4f and 4g); and underlining the versatility of the UV-C lter method in yet another photochemical reaction. CM of 2a with methyl 4-hydroxyhex-5-enoate (1e) gave rise to a diester (3q) which in principle could form two different cyclic esters in the presence of phenanthrene: the saturated or unsaturated lactone. Interestingly, compound 4k was the major product when phenanthrene was added, verifying that the trans-cis isomerization process is faster than simple cyclization. On the other hand, when 3q was irradiated in the absence of phenanthrene (with added t BuOH) the symmetric ketodiester 5o was exclusively produced. Unfortunately, tertiary allyl alcohol CM products (3r and 3s) did not isomerize to the ketoester (no alkyl shi) by UV-C irradiation, but did follow the lactone pathway when protected by phenanthrene (4l, 4m). Further prospects for this selective photochemical transformation were investigated by probing the reaction sequence on amines and amides. Due to the known susceptibility of metathesis catalysts for amine containing substrates, 30 N-Boc protected allyl amine (1n) was used as the CM partner with methyl acrylate (2a), which upon N-Boc-deprotection and irradiation with 254 nm light in the presence of phenanthrene, afforded the desired lactam 4n. 31 On the other hand, g-ketoamides (5p and 5q) were obtained in decent yields by the irradiation of CM products of a-vinyl benzyl alcohol (1a) and Table 2 Reaction scope with allylic alcohols a a Reported yields are isolated yields aer purication and for the reaction times, see ESI. b For 3j, 3k, 3q and 3s, 0.3 equiv., for 3l-3p, 2.1 equiv., and for 3r, 2.3 equiv. of phenanthrene was used as internal UV-C lter during lactonization. c cis-Ru II (3 mol%) was used for metathesis reaction. d Around 21% of the saturated lactone was obtained. acrylamides in the presence of t BuOH, as expected for the irradiation without UV protection (Scheme 3). Moreover, the UV-C irradiation of 3t without phenanthrene resulted in a complex mixture; highlighting the special role that phenanthrene has in these reactions. Finally, the applicability of this divergent tandem photochemical process was rigorously tested in the total synthesis of a member of the anti-allergenic cladospolide family, isocladospolide B, 15a and the enantiomer precursor (5r) of another natural product isolated from the endophytic fungal strain Cladosporium tenuissimum of Maytenus hookeri 32 (Scheme 4). Thus, R-(+)-propylene oxide (7) was opened with a Grignard reagent prepared from 8-iodoocta-1,3-diene 33 in the presence of CuI. The diene intermediate obtained was selectively dihydroxylated using AD-mix-a to afford the novel allyl alcohol 1o with high enantioselectivity (see ESI for details †). Irradiation of 1o at 350 nm wavelength with 2b in the presence of cis-Ru II, furnished 3w in good yields, notwithstanding the presence of three hydroxyl groups in the molecule. 19a,b Due to the relative instability of the intermediates, further irradiation with 254 nm light in the absence of UV-C lter gave a more complex mixture than usual. The use of a dilute adenosine aqueous solution following the external UV-C lter protocol effectively inhibited the side reactions and CM product 3w underwent a smooth and selective conversion to ketone 5r, a valuable intermediate towards a biologically relevant twelve-membered macrocycle and complex levulinate containing compounds. 15e The alternative pathway, a photochemical tour de force, was achieved by irradiation with 254 nm light in the presence of phenanthrene as internal UV-C lter, following the isomerizationcyclization path to give iso-cladospolide B (4o), 15a a natural product isolated from marine fungi; in four protecting group free steps, the nal two of which are photochemical transformations and with a total overall yield of 26%. 34 Conclusions In summary, we have shown that by addition of phenanthrene as a UV regulator, commonly available starting materials may be selectively transformed into complex organic molecules by two consecutive light induced reactions. Quantum chemical calculations and experimental studies revealed important mechanistic aspects of this photochemical process, highlighting the fundamental role of phenanthrene in guiding the divergent reaction towards a selective product by hindering a 1,5-H shi. The scope of the presented sequential reactions and its limitations were exposed by the use of several allylic and acrylic substrates as CM partners to produce a wide variety of nal products; including a natural product in a very efficient manner. This method presents an original divergent synthetic pathway and may inspire other types of controlled photochemistry by adaptation of this protocol. Conflicts of interest There are no conicts to declare.	v3-fos-license
2016-05-04T20:20:58.661Z	2015-06-05T00:00:00.000	13958867	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0127165&type=printable", "pdf_hash": "603b469ba58080696908008af58d80b27259e62c", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1082", "s2fieldsofstudy": [ "Environmental Science", "Medicine" ], "sha1": "603b469ba58080696908008af58d80b27259e62c", "year": 2015 }	pes2o/s2orc	Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions. Introduction Arthritic diseases are characterized by pain, stiffness, and joint inflammation, which eventually lead to articular cartilage (AC) destruction and disability. Osteoarthritis (OA) and rheumatoid arthritis (RA) are the most debilitating forms of arthritis [1]. AC is the highly specialized connective tissue responsible for frictionless movement between the articulating joint surfaces and the transmission of loads with a low frictional coefficient [2]. AC lacks blood vessels and lymphatic supply, has a limited capacity for intrinsic healing and repair, and has structural arrangements that are challenging for repair and restoration [3]. Chondrocytes of AC are embedded in a matrix comprising type II collagen (CII) proteoglycans and water [4]. Water comprises 60-80% of the wet weight of cartilage. Biomechanical properties of collagen and proteoglycan provide tensile and cushioning properties of AC, respectively [5]. The destruction of the AC is associated with reduced synthesis of the matrix components by articular chondrocytes and the enhanced breakdown of the matrix by disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs) and matrix metalloproteinases (MMPs) [6]. The degradation of proteoglycan is an early and reversible process, whereas the breakdown of the collagen network in AC by collagenases results in the irreversible destruction of the fibrillar network [4,7]. The treatment of arthritis involves different combinations of drugs offered at different stages of the disease to control inflammation and swelling by blocking the prime inflammatory processes [8]. To date, no pharmacological intervention offers protection or treatment from destruction of AC in arthritic conditions [9,10]. Polyphenols, many of which are well known for their antioxidant and anti-inflammatory activities, are consumed as micronutrients in the human diet, with an average consumption of 1g/day [11,12]. Polyphenols taken orally are extensively metabolized in the intestinal and hepatic systems, and the metabolites in the plasma differ in their biological activities [12]. Polyphenols are also an integral part of traditional medicines for the treatment of arthritis in many countries [11]. Epigallocatechin gallate (EGCG), quercetin (QUE), and catechin (CAT) are the major polyphenols in preclinical research for the treatment of cancer [13,14], arthritis [15], diabetes [16,17], cardiovascular diseases [18], and other inflammatory diseases [16]. Tannic acid (TA) extracted from oak trees also has beneficial biological activities in cancer and diabetes [19][20][21]. Previous findings relating to the role of polyphenols in arthritis mostly elucidate the mechanisms of inhibiting inflammatory cytokines or MMPs [15,[22][23][24][25][26][27][28]. The process of vegetable tanning dates back to ancient times. In the process, the conversion of skin/hide (type I collagen) matrix into leather is done through the crosslinking of plant polyphenols (tannin) with the type I collagen matrix. Polyphenols interact with collagen through hydrophobic association and hydrogen bonding. The multiple hydroxyls functional groups present in the polyphenols will have the ability to have hydrogen bonding with the side functional groups and peptide backbone of collagen triple helices [29,30]. Thus, crosslinked collagen matrices attain stability against enzymatic degradation [31,32]. Based on conventional wisdom of vegetable tanning, we hypothesize the binding of polyphenols with type II collagen (CII) in AC and prevention of cartilage degradation. In this study, we demonstrate the binding of polyphenols (EGCG, QUE, CAT, and TA) to collagen in bovine AC explants, leading to stability against collagenases. The bioavailability of polyphenols (including those in the synovial space) through the oral route is probably nonexistent. Therefore, we attempted to study the effect of intra-articular injections of plant polyphenols on cartilage protection through an in vivo model of collagen-induced inflammatory arthritis (CIA). Ethics statement Animal experiments were carried out in strict accordance with the norms of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) in the institutional animal house. The animals were fed a standard commercial diet with water ad libitum. The protocol was approved by our Institutional Animal Ethical Committee (IAEC) of the CSIR-Central Leather Research Institute (IAEC No: 03/02/2011b). All the rats were purchased from the National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad, India. Articular cartilage explants Fresh bovine tibio femoral joints were collected from a slaughterhouse. The cartilage surfaces were visually inspected for the absence of degeneration. Cartilage slices in thicknesses ranging 1.2-2.4 mm were dissected from the surfaces (lateral and medial condyles) using a scalpel. The cartilage slices were punched to obtain samples of uniform size and weight. The samples were washed in cold saline, transferred to sterile cold water, and stored at -40°C until use. Collagen, glycosaminoglycan, and water content estimation of AC explants The cartilage samples were grouped and placed in centrifuge tubes containing PBS. They were then utilized for the estimation of water content, collagen, and glycosaminoglycans (GAG). The water content was determined by the difference between the wet and dry weights of the cartilage. The amount of GAG present in the cartilage explants was determined after papain digestion using dimethylmethylene blue (DMMB) dye [33]. Initially, the cartilage explants were incubated overnight at 65°C in 2 mL of papain digest solution (i.e., papain solution [1%], which was prepared by dissolving 1 g of papain in 100-mL PBE buffer [PBE buffer: 100-mM Na 2 HPO 4 , 10-mM EDTA, 5-mM cysteine, 500-mL deionized water, final pH 6.5) for complete cartilage digestion. The digested sample was then centrifuged at 6,000 rpm to remove any insoluble components, and the supernatant was stored at -4°C [33,34]. Chondroitin sulfate obtained from bovine trachea was used as a standard to estimate the amount of GAG. The total amount of GAG was estimated by adding 200 μL of DMMB solution to the volume of 50 μL of papain-digested supernatant and read on a microplate reader at 525 nm. The collagen content was determined using the method reported by Woessner [35]. By estimating the hydroxyproline content of the hydrolyzed cartilage (treated with 6N HCl at 118°C, for 12 h, in sealed hydrolysis tubes) using dimethylaminobenzaldehyde and chloramine T, a standard curve was generated with standard hydroxyproline. The amount of collagen = amount of hydroxyproline determined × 7.4 (conversion factor). Thermal stability of AC explants Differential scanning calorimeter (DSC) analysis is widely used to determine the physicochemical transformations that occur during thermal degradation [36,37]. The thermal stability of cartilage depends on the distribution of therapeutic (polyphenol) molecules inside the matrix and their interactions with the collagen fibrils of AC. AC explants were treated with polyphenols (200 μM), at 37°C for 48 h, in a shaking incubator. Stock solutions of polyphenols were prepared in PBS except QUE, which was prepared using DMSO as co-solvent at DMSO:PBS of 1:3. AC explants incubated only with PBS and stored at 4°C were used as controls to prevent autolytic degradation of AC at 37°C. The cartilage samples were washed within 24 h of incubation in PBS. After washing, the cartilage samples (with and without polyphenol treatment) were placed on tissue paper to remove excess surface buffer (the moisture of AC is~65%). They were then weighed and placed in an aluminum pan for calorimetric analysis using TA Instruments Model DSC Q200/NETZSCH DSC 204 F1. The samples were analyzed in the temperature range 25-150°C at a heating rate of 5°C/min [38]. Enzymatic stability of AC explants To study the effect of polyphenol treatment on the enzymatic stability of AC, the explants were treated with polyphenols dissolved in PBS (CAT, QUE, EGCG, and TA) at 200 μM for 48 h, in a shaking incubator, at 37°C. AC explants incubated only with PBS were used as controls. The treated explants were again incubated for 24 h in PBS to wash out free polyphenols, as their presence can inhibit collagenase. The explants were then incubated in collagenase at 37°C for 96 h. The ratio of collagen (in cartilage explants) to collagenase was maintained at 50:1 (w/w), and the reaction was buffered at pH 7.4 with 0.1-M Tris-HCl and 0.05-M CaCl 2 . After 96 h, the reaction was stopped, and the mixture was centrifuged for 15 min at 10,000 rpm. The supernatant was analyzed for soluble collagen and GAGs using the Woessner method [35] and DMMB dye [33,34] respectively, and the percentage release thereof was calculated using the Eqs 1 and 2 given below. Physical stability of AC explants To study the effects of the polyphenols on physical properties of cartilage, the compression strength of the cartilage with and without incubation in polyphenols was determined using a Brookfield CT3 10K Texture Analyzer (USA). AC explants 5 mm in diameter within a narrow weight range (18-22 mg) were obtained. The cartilage samples were separated into six groups (three samples per group). Five cartilage groups were incubated with or without 200 μM polyphenols (CAT, QUE, EGCG, and TA, respectively) at 37°C for 96 h. Another additional control group was stored at 4°C. The load required for 50% compression (i.e., half of its original thickness) of the incubated cartilage samples was determined. Prior to compression measurement, the cartilage samples were equilibrated with PBS pH 7.4 for 4 h at room temperature (25°C) and then placed directly below the probe in a stainless steel plate with a thin layer of PBS on the circular plate of the instrument. The probe was allowed to compress the cartilage with the target of 50% compression at a defined speed (0.1 mm/sec) to examine the unconfined compression properties [23,39]. In vivo analysis of the effect of polyphenol in protecting AC in collageninduced arthritic rats Forty-two female Wistar rats, 6-8 weeks old and weighing 130-180 g, were used to evaluate the effect of the intra-articular injection of polyphenols (EGCG and TA) in protecting AC under arthritic conditions. The animals were grouped for both prophylactic and therapeutic treatment conditions as shown in Table 1. The EGCG and TA solutions were prepared fresh each injection day by dissolving the desired weights in sterile saline under aseptic conditions. For each leg, a dose of 300 μg (i.e., 25 μl from 12 mg/ml stock solution) of EGCG or TA was injected intra-articularly in the tibiofemoral joints of the rats of the respective groups. Positive control (PPC & TPC) and NC were injected with saline. Collagen-induced arthritis CIA was induced in all rats except the NCs (i.e., without disease) by the intradermal injection of an antigenic mixture in the back (dorsal part) and rear feet. Type II collagen (CII) from bovine AC was prepared and purified; the final concentration of 2 mg/ml was obtained by suitable dilution using 0.05 M acetic acid and was then stored at 4°C. Heat-killed M. tuberculosis (HKMtb) collected from the Tuberculosis Research Centre (TRC-Chennai, India) was processed as described previously [40]. On the day of CIA induction, HKMtb was emulsified in an ice bath with an equal volume of CII solution and CFA to obtain a final concentration of 2 mg/ ml. Finally, the antigenic mixture (300 μl in six divided doses) was injected over four sites in the rats' backs and two at each foot on day 0 (induction injection) and similarly on day 7 (booster injection). The volumes of the rats' hind paws were examined periodically for arthritis development using a plethysmometer. Prophylactic treatment and induction of CIA Through prophylactic treatment, we sought to ensure that the binding of polyphenols took place before the induction of arthritis; therefore, any significant protection of cartilage Analysis of the articular cartilage Both the prophylactic and therapeutic groups were sacrificed on day 43 of immunization, and the tibiofemoral joints of 3 rats in each group were fixed in 10% formalin. They were then decalcified and dehydrated and spliced sagitally into two halves (S1 Fig). Midsections of 3-μm thickness were obtained from embedded tissues on spliced side and stained with hematoxylin and eosin (H&E) and masson's trichrome [24]. Degradation (blind) scoring of both tibial and femoral end cartilage was carried out in six histological sections (i.e. six joints from 3 rats). Quantitative histomorphological scoring of cartilage degradation was as follows: Score 0 (normal cartilage), Score 1 (minimal damage), Score 2 (moderate damage), and Score 3 (maximum damage) [41]. An independent expert pathologist, without knowledge of the experimental groups did the scoring. Statistical analysis The statistical analysis was performed with GraphPad Prism version 5. Statistical significance was defined as P-value 0.05. Data were analyzed by one-way ANOVA, RM-ANOVA, or the unpaired Student's t-test, as appropriate. Collagen, GAG, and water content In RA and OA, the degradation of proteoglycans and collagen on the cartilage surface allows more water to penetrate and loosen the matrix, thereby affecting the load-bearing abilities of the cartilage. The presence of water, proteoglycans, and collagen is important in maintaining the physical properties of AC. The loss of proteoglycans will cause a change in the water content and subsequent loss of elasticity and resilience. The cartilage explants were analyzed for water content, which was in line with previous reports, at 70.21± 2.41% [42]. The total collagen content of the wet cartilage was 14.03 ± 0.25%, about 47% of the dry weight. The GAG content of the explants was found to be 5.24 ± 0.81% (on a wet-weight basis). Thermal stability of polyphenol-treated cartilage Relatively little information is available on the thermal properties of mammalian hyaline cartilage, particularly the effect of drugs on the thermo-physiochemical properties of AC. Here, for the first time, bovine AC has been studied using DSC to evaluate the effect of polyphenols on the thermal stability of articular cartilage. DSC thermograms of native and polyphenol-treated AC are shown in Fig 1, and the denaturation temperature of cartilage is presented in Table 2. The polyphenol-treated cartilage showed increases in the thermal stability of collagen from 8-16°C with reference to native cartilage; EGCG and TA showed the maximum increases in the thermal stability of cartilage (12 and 16°C respectively), whereas QUE and CAT exhibited only 8 and 10°C increases in thermal stability respectively, with reference to native untreated cartilage. Enhancement in thermal stability indicates the binding of polyphenols with the collagen in the cartilage matrix, as well as collagen crosslinking. The aromatic rings of polyphenols could also be involved in hydrophobic association with aromatic side-chain functional groups of collagen type II [30,43]. Enzymatic stability of cartilage explants Polyphenol binding and crosslinking render cartilage resistant to enzymatic degradation. The percentages of collagen and GAG released from the cartilage explants after the enzymatic treatment are presented in Table 3. The untreated cartilage showed collagen degradation of about 72%, whereas the polyphenol-treated cartilage samples showed a significant level of protection from enzymatic collagen degradation. The EGCG, TA, and CAT treatments were more protective (i.e., statistically significant), showing only 24, 29, and 32% collagen degradation, Effect of intra-articular injections of polyphenol (EGCG or TA) in AC protection of CIA rats In vitro studies showed that the treatment of cartilage with polyphenols stabilizes the cartilage enzymatically and thermally, and does not significantly alter the mechanical properties. Because of the poor bioavailability of polyphenols in circulation, particularly in synovial fluids, it is likely that limited observations have been made concerning its salutary effect on AC. We hypothesize that polyphenols injected intra-articularly will interact with type II collagen of AC and stabilize it against degradation by MMPs. Furthermore, the more efficient (in the thermal and enzymatic stabilization of AC) polyphenols (EGCG and TA) were selected for the in vivo studies of the protective effect on cartilage degradation using CIA rat models. We evaluated two strategies: prophylactic and therapeutic intra-articular injection in a CIA rat model. Prophylactic treatment In the prophylactic treatment group, intra-articular injections of EGCG, TA, and PBS in five doses were completed before arthritis induction. The development of CIA was monitored by paw volume measurements. As shown in Fig 3A, paw volume was unchanged until day 8 in immunized rats. On day 15, we observed a significant (P<0.001, n = 6) increase in paw volume in PPC, PT, and PE compared to NC, indicating the onset of CIA in immunized rats. There was no significant difference in paw volume at the onset of CIA between the PPC and the PT or PE groups. Beginning on day 22, the PE rats showed significant increases in paw volume until day 36 compared to the PPC rats, but this was not observed in the PT groups. A significant maximum increase in paw volume was observed on day 29 (i.e., increase to 2.78 ml versus 2.21 ml in the positive controls). These observations indicate that the prophylactic treatments of polyphenols EGCG and TA did not alleviate inflammation in the paws of immunized rats. The evaluation of histological sections of joints from various groups was done blindly for cartilage damage and synovial inflammation. The synovial membrane and cartilage in the NC group was normal. PPC exhibited synovial adhesions and fibrous fatty tissue, and the synovial membrane was inflamed and hyperplasic (Fig 4A), confirming the induction of CIA. The synovial membranes of PT showed more inflammation than PE, whereas the latter group had few eosinophilic infiltrations. The dark blue stain (by Masson's trichrome staining) in the histological sections at the articulating surface indicates the type II collagen matrix of AC (Fig 4B). In the NC, PT, and PE sections, the collagen was intact (green arrows), whereas the PPC showed irregular collagen matrix loss (black arrows). Histomorphological scoring levels of the cartilage degradation are presented in Fig 3B. As shown, the combined scoring (of three joints) for cartilage degradation in PT and PE was significantly lower than in PPC (P<0.05). In addition, AC in PT showed some superficial surface irregularities and rice bodies in the joint space (Fig 4A and 4B). Therapeutic treatment In the therapeutic groups, intra-articular injections of EGCG, TA, and PBS in five doses were completed after arthritis induction. As shown in Fig 5A, the paw volume (indicator of CIA onset) began to change on day 17, confirming significant induction of CIA (P<0.001, n = 6) in the TPC, TT, and TE groups compared to NC. Early on, paw volume was not significantly different among these groups. In the case of the therapeutic EGCG treatment, paw volume significantly increased on day 22 (P<0.05) but on day 43 (final) it had dropped significantly (P<0.01) to a volume of 2.00 ml compared to positive controls (2.29 ml). On day 43, the TT group also showed a significant (P<0.001) decrease in paw volume compared to TPC (i.e., 1.93 ml for TA and 2.29 ml for positive controls) (Fig 5A). There was no significant difference between TA and EGCG in reduction of paw inflammation. These observations indicate that therapeutic treatment with polyphenols EGCG and TA did not result in any significant alteration of paw inflammation. The combined histomorphological cartilage damage scores (of six joints) in the therapeutic groups of TA-treated (TT) and EGCG-treated (TE) cartilage were significantly lower (P<0.05) than that of TPC (Fig 5B). The cartilage damage scoring of TE was slightly lower than that of TT but did not reach statistical significance. As shown in H&E histological sections, the cartilage damage was very mild to negligible in most TT and TE samples (Fig 6A and 6B). Upon further observation using Masson's trichrome, the cartilage damage was minimal and the cartilage appeared intact in the stained sections (Fig 6B) of the therapeutically treated joints in groups TT and TE. However, the cartilage in group TPC (as shown) was profusely degraded (black arrows). Cartilage in normal controls was intact (Fig 6B, green arrows). Synovial inflammation was found in both the TA-and EGCG-treated therapeutic groups. The severity of synovial inflammation in both TT and TE was lower than in TPC. Discussion Supra-molecular assemblies of type II collagen (CII) are similar to those of type I, forming fibrillar structures. AC, composed predominantly of CII, is a porous matrix that facilitates the diffusion of small molecules through the porous cartilage matrix, which could crosslink with the side-chain functional groups of collagen, as shown in Fig 7. We show for the first time that treatment with polyphenols increases the thermal stability of AC. Of the various polyphenols tested, EGCG and TA had greater effects on thermal stability. Collagen is an inside-out protein where the side-chain functional groups are projected outward and these can react with multiple hydroxyl functional groups of polyphenols resulting in multiple hydrogen-bonded interactions with collagen, thereby conferring stability to the cartilage matrix ( Fig 7B). Plant polyphenolic molecules can stabilize type I collagenous matrices through hydrogen bonding and hydrophobic interactions [44,45]. Schlebusch and Kern [46] studied the possible stabilizing effects of catechin on collagen for vascular tissue stabilization. Similarly various studies reports stabilization of collagenous tissues through natural crosslinking agents. Cardiovascular tissues [47][48][49][50][51][52], intestinal mucosa [53], corneas [54,55], tendons [56], dentin [57], cartilage [58] and other collagenous scaffolds [59] has been studied with crosslinkers like glutaraldehyde, tannic acid, pentagalloyl glucose, genipin, procyanidins and lysyl oxidase etc. Plant polyphenols are found to be equivalent and advantageous in stabilizing the tissues compared to the chemical crosslinking agent glutaraldehyde which are found to be irreversible, cytotoxic and produces calcification [50,51]. With the evidence that polyphenols bind to AC, we studied their efficacy in protecting cartilage from enzymatic degradation. EGCG and TA treatments showed greater effects in protecting the cartilage from enzymatic damage, whereas QUE and CAT had lesser effects (Table 3). This is consistent with increases in the thermal stability of AC caused by EGCG and TA. In addition to the collagenolytic degradation of AC, there was a significant release of GAGs from the enzyme-treated AC, which must have been due to the release of GAGs associated with the fragments of collagen fibrils digested from the cartilage. Interestingly, both TA and EGCG treatment exhibited higher efficacy in stabilizing cartilage for collagen degradation and GAG release than did either QUE or CAT. This could be due to the ability of EGCG and TA to form better crosslinking with collagen and other matrix components. Recently, a study to improve collagen-based biomaterial used for skin anti-aging was conducted using polyphenols, revealing that they block the site at which collagenase acts upon collagen [60]. Previous studies have also reported the collagenase inhibitory effects of polyphenols [60,61]. In our studies, the polyphenol-treated cartilage was thoroughly washed to leach out any free or weakly bound polyphenols and hence eliminating the possibility of having free polyphenols to inhibit collagenase. Therefore, the resistance to collagenolytic degradation was predominantly due to increased crosslinks between polyphenols and CII in the cartilage. The presence of water, proteoglycans, and collagen is important in maintaining the compressive properties of AC. The loss of proteoglycans will cause a change in the water content and subsequent loss of elasticity and resilience, and the cushioning property will be lost if the collagen and GAG are degraded in cartilage. Therefore, it was important to study the effect of polyphenols on the compressive strength of cartilage. As shown, polyphenol-treated cartilage explants showed less deterioration in load-bearing ability than controls (Fig 2). The change in load after polyphenol treatment was not more than 10% compared to the change in untreated cartilage (controls). This shows that polyphenolic treatment did not alter the mechanical (compression) properties of cartilage compared to the untreated cartilage explants. Similarly, Sung HW et al reported that genipin and glutaraldehyde treatment to porcine aortic valves did not seem to have significant difference in altering the mechanical properties. Glutaraldehyde treatment showed an increased denaturation temperature (~22°C) than that of Genipin (~15°C) compared to that of untreated tissues [62]. Recently, Satyam et al reported, that treatment of genipin with collagen scaffold was found to improve enzymatic and thermal stability (by2 4°C) than that of control and produces significant change in the mechanical properties [59]. Polyphenols protecting articular cartilage To enable the polyphenols to be efficient, the intra articular route of injection was selected, which will provide the advantage of direct drug administration but can be subjected to rapid clearance (t 1/2 of 0.1-6 h) [63]. Additional advantage is that biochemically intact polyphenols without being subjected to metabolic changes in the intestinal or hepatic systems would be delivered in the joints. We hypothesize that polyphenols injected intra-articularly will interact with type II collagen and stabilize cartilage against degradation, as illustrated in Fig 7B. The more efficient polyphenols (in the enzymatic stabilization of AC), EGCG and TA were selected for the in vivo studies using CIA rat models. The onset of CIA was clearly established with increases in paw volumes in both the prophylactic and therapeutic groups (Figs 3A and 4A). The lack of change in paw volume in the prophylactic group (Fig 3A) is a clear indication that polyphenols have little influence on the time of onset of arthritis or the maintenance of the inflammation level as measured by paw volume; whereas, in the case of the therapeutic groups, there was again no delay in the onset of arthritis or the maintenance of inflammation, except for some reduction in paw volume at the end of the experiment (day 43) (Fig 4A) compared to the positive controls. In all cases, the positive controls (immunized) had significant inflammation compared to the un-immunized (PBS only) controls. Against this backdrop, the prophylactic intra-articular administration of EGCG or TA showed significant (P<0.05) protective effects on the degree of cartilage damage as measured by histomorphological scoring compared to the positive controls (Figs 3B and 5B). This indicates that AC in prophylactic polyphenol-injected joints becomes resistant to the subsequent induction of inflammation-induced degradation. In the therapeutic polyphenol group, there was some reduction in paw volume, indicating a decrease in the degree of inflammation but only during the latter part of CIA, as compared to paw volume in positive controls. This may be consistent with the known anti-inflammatory effects of polyphenols. The therapeutic groups also showed the significant benefit of intra-articular injections of polyphenols in protecting cartilage from degradation during CIA (Figs 4B and6B). These observations are statistically significant (P<0.05). EGCG showed a better cartilage-protecting effect than TA. Together this indicates that AC in therapeutic polyphenol-injected joints becomes resistant to concurrent inflammation-induced degradation compared to subsequent inflammation in the prophylactic groups. Previously, catechin (20 μM) was found to inhibit the degradation of bovine nasal and AC explants by inhibiting the chondrocyte catabolic response [15]. In another study, EGCG (100 and 200 μM) was effective in inhibiting the IL-1β-induced production of matrix-degrading enzymes [34]. Recently, curcumin (1-25 μM) and quercetin (10-50 μM) also inhibited the matrix-degrading enzymes [64]. It is possible the protective effect on cartilage in the therapeutic groups could have been caused by the direct inhibition of MMPs. However, polyphenols in synovial fluid would have been rapidly cleared and metabolized, and little would have remained to inhibit the gradual degrading process of MMPs on the cartilage. Another explanation for the protective effect on cartilage could be a reduction in the degree of inflammation in the joints, but this was observed only in the latter part of CIA and was not confirmed by histopathological observation of inflammation in the synovium. Based on our in vitro data and the observations in the prophylactic polyphenol treatment groups, we predominantly credit the binding of polyphenols to AC as the likely protective factor against cartilage degradation in the therapeutic groups. Human collagen type II in the AC matrix is a long-lived protein with an estimated half-life of >117 years [65]. Collagen maturation involves extensive crosslinking, a strategy used for cartilage maintenance throughout the lifespan of most humans. Based on conventional wisdom of vegetable tanning, we have hypothesized that polyphenols could be used to crosslink cartilage collagen type II, rendering it resistant to degradation, as shown in schematic form in Fig 7. Intra-articular injections of polyphenols prevented cartilage degradation amid the milieu of inflamed joints. Further research is warranted to study the effects of polyphenols injections into the joint cavity and also establishing its role in an osteoarthritis models. In summary, we suggest a unique novel role for intra-articular injections of polyphenols in the therapeutics of cartilage degradation.	v3-fos-license
2021-05-05T00:08:32.729Z	2021-03-22T00:00:00.000	233708770	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1996-1073/14/6/1768/pdf", "pdf_hash": "eac613ae090734edbc3baa123a6cd242ce88dcf8", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1083", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "a81da7ece2c9f418f85014dbd8607d82a19cd6ce", "year": 2021 }	pes2o/s2orc	Conversion of Slaughterhouse Wastes to Solid Fuel Using Hydrothermal Carbonization : In this study, cattle and pig slaughterhouse wastes (SHWs) were hydrothermally carbonized at 150–300 ◦ C, and the properties of SHW-derived hydrochar were evaluated for its use as a solid fuel. The results demonstrated that increasing the hydrothermal carbonization (HTC) treatment temperature improved the energy-related properties (i.e., fuel ratio, higher heating value, and coaliﬁcation degree) of both the cattle and pig SHW-derived hydrochars. However, the improvements of cattle SHW-derived hydrochars were not as dramatic as that of pig SHW-derived hydrochars, due to the lipid-rich components that do not participate in the HTC reaction. In this regard, there was no merit of using HTC treatment on cattle SHW for the production of hydrochar or using the hydrochar as a solid fuel in terms of energy retention efﬁciency. On the other hand, a mild HTC treatment at approximately 200 ◦ C was deemed suitable for converting pig SHW to value-added solid fuel. The ﬁndings of this study suggest that the conversion of SHWs to hydrochar using HTC can provide an environmentally benign method for waste treatment and energy recovery from abandoned biomass. However, the efﬁciency of energy recovery varies depending on the chemical composition of the raw feedstock. Introduction The increase in the consumption of meat and meat products has led to the expansion of the slaughtering industry and the consequent increase in biological waste production [1]. Slaughterhouse waste (SHW) is the animal product remaining after the manufacture of the principal commodity in slaughterhouses and formally consists of inedible offal and fats [2]. Most SHW is used as a raw material in the rendering industry for the production of pet and animal feed. However, outbreaks of livestock infectious diseases, such as foot-and-mouth disease, mad cow disease, and African swine fever, hinder the use of the SHW in pet and animal feed production, and even in application as fertilizers [3]. Thus, significant amounts of SHW are underutilized and are discarded via incineration or landfills. The disposal methods not only have a negative impact on the environment by generating secondary pollutants (e.g., odor and leachate), but also lead to an economic burden on the meat industry. Recent advances in the SHW utilization pathways include its application as a source of industrial proteins, enzymes, and lipids [4,5]. Despite being an organic-rich source for industrial raw materials, SHW has process-applicable limitations and is its use is considered challenging for functional applications due to its heterogeneous composition (e.g., nonorganic compounds) and poor solubility [1,6]. More recently, SHW has received considerable attention as a feedstock for bioenergy production [7,8], particularly due to its high organic content. Increasing environmental concerns (e.g., fossil fuel depletion, carbon emissions, and waste management) necessitate efficient methods for energy recovery and waste management. Hydrothermal carbonization (HTC) is a thermochemical reaction-based method which occurs in a relatively low temperature range (150-300 • C) with moisture and autogenous pressure [9,10]. The relatively low energy input and mild treatment conditions of HTC have caused it to receive attention as an alternative biomass treatment technology rather than conventional thermal treatment technologies (e.g., pyrolysis, combustion) [11,12]. During the HTC reaction, various types of organic matter are converted into a valuable carbon-rich material (i.e., hydrochar). The operating conditions of the HTC (e.g., treatment temperature) affect the yield, physicochemical properties, and the functionality of hydrochar [13]. Hydrochar is a multifunctional carbonaceous material which can aid environmental remediation, soil amendment, and carbon sequestration, as well as serve as an alternative solid fuel [14]. Recently, the potential use of hydrochar as a solid fuel has received considerable attention owing to its enhanced heating value, thermal stability, and material structure, especially when compared to untreated biomass [15]. HTC boosts the energy-related properties of hydrochar, making it a sole and/or auxiliary fuel source for combustion facilities [16]. The production of hydrochar using waste biomass and its application as a solid fuel can double its profits, mitigate the environmental burden of SHW management, and help find the key to sustainable development. For the above reason, considerable studies on HTC reactions for hydrochar production and its use as a solid fuel have gained significant interest in this research area. Li et al. and Roy et al. reported on potential routes for solid fuel production using hydrochar derived from red jujube branch and peat moss, respectively [12,17]. Not only experimental research but also process efficiency modeling and cost analysis studies on HTC reactions have been published. Lucian and Fiori provide are a useful reference to evaluate the HTC process in terms of the environment and economics [18]. Herein, we focused on the conversion of cow and pig SHWs into hydrochar. The properties of the resultant hydrochars were evaluated for their use as solid fuels. We investigated the effects of the chemical feedstock composition on the hydrochar yield and properties. Finally, we determined optimal HTC treatment conditions for the tested SHWs to recover energy more efficiently. Feedstock Cattle and pig SHWs were obtained from a domestic meat processing company and were used as feedstocks for hydrochar production by HTC. The SHWs were moved to the laboratory immediately after collection, and the impurities (e.g., horn, feather, toenail) were removed. The cattle SHW used in this study was mainly composed of fat, kidney, and genitals, whereas pig SHW consisted of fat, liver, lung, and brain tissues. The components of each SHW type were ground using a cryogenic grinder (SPEX 6875D Freezer/Mill, SPEX SamplePrep, Metuchen, NJ, USA) and homogenized by blending. The sample pulverizing was conducted following manufacturer's instruction, but grinding time and rate were modified referring to our previous work. The sample (~2 g) was continuously cooled until −196 • C, and the constant temperature was maintained during the entire grinding process. The grinding time was 60 s at a rate of 10 cycles per second. To avoid sample spoilage, the prepared SHWs were frozen (<−20 • C) until further use. Hydrothermal Carbonization (HTC) SHWs were hydrothermally carbonized using a batch-type laboratory-scale reactor at four different temperatures (150, 200, 250, and 300 • C). The hydrothermal carbonization at each temperature was conducted in duplicate, and the products were mixed and analyzed together. The Teflon-lined stainless-steel reactor body with a total inner volume of 1 L was connected to a heating control system and a steam condenser. The HTC reaction was performed for 30 min at the preset temperature, and the pressure of the reactor was not regulated (autogenous pressure atmosphere was maintained during the HTC reaction). For each treatment, 300 mL of cattle or pig SHW was loaded with the same volume of deionized water into the reactor vessel and nitrogen gas was purged for 5 min to create anaerobic conditions, and it was then sealed. A mechanical agitator was operated at a speed of 200 rpm, and the contents in the reactor were continuously mixed during the reaction. After the HTC reaction, the residual steam was discharged, and the inner temperature and pressure of the reactor were lowered until they reached room temperature and atmospheric pressure, respectively. Both solid and liquid products of the HTC reaction were collected and oven-dried overnight at 105 • C. Finally, the hydrochar was obtained as dry matter and analyzed. The simple layout of the experimental setup is shown in Figure 1. Energy densification = HHV of hydrochar/HHV of feedstock where HHV is the higher heating value (2) Energy retention efficiency = Product yield × Energy densification (3) Analytical Methods Proximate analyses of ash and volatile matter (VM) contents in the samples were conducted according to the ASTM D3174 and D3175 standard test methods, respectively. The fixed carbon (FC) content in the samples was analyzed based on the differences between the ash and VM contents. ASTM international standard test method E1758-01, AOAC international method 2001.11, and the procedure recommended by Bligh and Dyer (1959) [19] were followed to determine the total carbohydrate, protein, and lipid contents in the test samples, respectively. A high performance liquid chromatography (HPLC) system consisting of a Waters 2695 Separations Module (Waters, Milford, MA, USA) associated with a refractive index detector (Waters 2414, Waters, USA) and Sugar-Pak 1 column (Waters, USA) was employed for determination of total carbohydrate. Deionized waster was used as the mobile phase and the flow rate was 0.6 mL min −1 . The analytical grade for HPLC standard solution was used (Sigma-Aldrich). For the instrumental analyses, the dried raw SHW and hydrochars were ground into fine particles and sieved to particle sizes of less than 250 µm. An elemental analyzer (Flash1112, Thermo Fisher Scientific, Bremen, Germany) was employed to perform the final analyses. The HHVs of the test samples were evaluated using a bomb calorimeter (Parr6400, Parr Instrument, Moline, IL, USA), followed by a standard method for calorimetric analysis (US EPA 5050). The functional group changes during the HTC reaction were analyzed using Fourier transform infrared (FTIR) spectroscopy (Vertex70, Bruker, Karlsruhe, Germany). The absorbance values of the test samples were in the range of 4000-400 cm −1 . All analyses were replicated three times for precision, and the average values of the obtained variables were used. A one-way analysis of variance (ANOVA) test using Microsoft Office Excel 2013 was conducted to evaluate a significant difference between the analysis results, where the significance level was determined at p < 0.05. Properties of SHW and Hydrochar The raw SHWs were converted into hydrochar during the HTC reaction. The hydrochar properties at different treatment temperatures are shown in Table 1. Higher HTC temperatures led to lower VM and higher ash contents in the hydrochars due to the accelerated hydrolysis rate and dehydration of raw SHW [20]. The hydrochar ash content affects pollutant emissions and HHVs, thereby determining the suitability of the hydrochar for use as a fuel source [21]. Many countries regulate the maximum permissible level of ash content in bio-solid refuse fuel (SRF), and the domestic regulations in Korea allow a maximum ash dry weight of 15% in bio-SRF. The FC content in the hydrochar gradually increased with increasing HTC temperatures. The FC content in the cattle and pig SHWderived hydrochars increased from 0.10 to 0.55 wt.% (dry) and from 0.27 to 0.76 wt.% (dry), respectively. Higher FC content in fuels help maintain the stable state of the flame during the combustion process. The elemental composition of the obtained hydrochar is presented in Table 1. A gradual increase in carbon content was observed in both the cattle and pig SHW-derived hydrochars with an increase in the HTC temperature. The carbon content is known to be closely associated with energy capacity of combustible material [22,23]. The carbon contents in the cattle and pig SHW-derived hydrochars increased from 65.52 to 71.94 wt.% (dry) and from 50.91 to 71.24 wt.% (dry), respectively. Meanwhile, a drastic decline in nitrogen content in the cattle SHW-derived hydrochar was observed with increasing HTC temperatures. The observation was attributed to the devolatilization of volatile nitrogen in the raw cattle SHW and the elimination of devolatilized nitrogen into the gas or liquid phase [24,25]. The deterioration of nitrogen content in feedstock during HTC can lower NOx emissions during the combustion process if the hydrochar is used as a solid fuel. Furthermore, negligible sulfur levels in both the cattle and pig SHWs revealed the potential use of the hydrochar as a clean energy source without any risk of SOx emissions. Generally, the variable components in the feedstock undergo complex reactions during HTC and affect the hydrochar combustion characteristics [26]. The three main feedstock components (i.e., carbohydrates, proteins, and lipids) are associated with prominent changes in the physicochemical properties of hydrochar [27]. In the cattle SHW, ultrahigh lipid content was observed (74.75 wt.%, dry), whereas carbohydrates and proteins constituted only 1.45 and 1.00 wt.% (dry), respectively. However, these components were distributed more evenly in the pig SHW (the content of carbohydrates, proteins, and lipids equaled 13.86, 13.13, and 33.25 wt.% (dry), respectively). During HTC, carbohydrates tend to contribute to the formation of hydrochar, while proteins help develop N-heterocyclic functional groups; lipids do not participate in the formation of carbonization products [26]. Therefore, each component in the raw SHW can cause changes in the energy-related properties and functional groups of the SHW-derived hydrochar. This issue is addressed in more detail in the following sections. With an increase in the HTC temperature, the fuel ratios of both the cattle and pig SHWs gradually increased. However, there was no significant increase in the fuel ratio of the pig SHW above 200 • C. The higher the fuel ratio, the better the produced solid fuel. Because the FC and VM contents in a combustible material are correlated with the combustion atmosphere by flame violence and heat flow balance, the fuel ratio is essential for determining fuel source potential [16]. Furthermore, the HHVs of the SHW-derived hydrochars were investigated to evaluate the energy potential of the hydrochars. As the HTC treatment temperature increased, all SHW-derived hydrochars had enhanced HHVs. The HHV of the pig SHW-derived hydrochar markedly increased from 4674 to 8804 kcal kg −1 , while the equivalent value of the cattle SHW-derived hydrochar increased only by~1600 kcal kg −1 under the same HTC conditions. This is attributed to the aforementioned differences in the chemical compositions of the cattle and pig SHWs. Although higher lipid content in the cattle SHW led to the higher initial HHV of the raw feedstock, the increase was not dramatic because the lipids were nonreactive during the HTC reaction. Even so, the hydrolyzed lipids were probably adsorbed onto the hydrochar surface [26], and the highest HHV of the cattle SHW-derived hydrochar was comparable to that of the pig SHW-derived hydrochar. Improvements in the SHW Properties The lower H/C and O/C ratios represent a greater coalification degree and advanced energy potential [13]. Both the cattle and pig SHWs underwent a combination of complex chemical reactions, especially dehydration and decarboxylation, during HTC. The coalification degree of the SHW-derived hydrochars gradually increased with an increase in the HTC temperature. Thus, it can be concluded that the economic value of raw SHWs for solid fuel use is improved by eliminating oxygen and hydrogen and proportionally increasing the carbon content in the product. In this study, the raw SHWs followed the trend of biomass coalification. However, due to the lipid-rich characteristics of the raw SHW, the SHW-derived hydrochars are positioned differently to the biomass-derived coals (i.e., in the upper left corner of Figure 3). The shift in the coalification degree during HTC was evaluated using the van Krevelen diagram, which compares the aromaticity (atomic H/C ratio) and the polarity (atomic O/C ratio) of the samples (Figure 3). Figure 4 shows the energy-related properties (i.e., energy retention efficiency, energy densification, and product yield) of the cattle and pig SHW-derived hydrochars. The ED of the SHW-derived hydrochars increased with an increase in the HTC temperature, and the ED value was improved to the raw SHW feedstocks (the ED of the raw SHW equaled 1.0). Owing to the aforementioned effects of chemical composition on hydrochar formation, a more substantial increase in ED was observed with the pig SHW-derived hydrochars. Meanwhile, the product yields of both the cattle and pig SHW-derived hydrochars was lowered by the continuous loss of VM and organic matter with the augmented severity in the HTC reaction. The product yield did not decline significantly, and ERE showed a similar trend to ED. When considering ERE and energy consumption at higher HTC temperatures, HTC for cattle SHW is not recommended, whereas a mild HTC temperature of~200 • C is optimal for converting pig SHW into value-added solid fuel. Changes of Functional Groups during the HTC Treatment The changes of functional groups on the SHW and hydrochar surfaces were analyzed using FTIR spectra analysis ( Figure 5). Some noticeable peaks were determined based on different peak intensities at various HTC treatment temperatures. The peak at 3000-2800 cm −1 is associated with the aliphatic C-H structure of hydrochars [28]. It represents the miscibility between lipids and water that increases when hydrogen bonding between water molecules weakens during HTC [29]. The lipid solubility in both the cattle and pig SHWs increased after HTC, and the lipid content in the raw feedstock probably became completely miscible under the supercritical conditions. The dissolved lipids were then adsorbed onto the hydrochar surface or ejected with steam after the HTC reaction. Thus, the relative peak intensity slightly decreased. The peak observed at 1650 cm −1 is related to the C=O bond of the carboxylic group [30], indicating that decarboxylation occurred in both SHW-derived hydrochars during the hydrothermal reaction. The reaction was more intense at higher HTC temperatures. The observation of the corresponding peak in hydrochars confirmed the results of the van Krevelen diagram and the H/C and O/C ratios obtained herein. The changes in peak intensity at 1130 cm −1 with respect to the C-O bond represent the carbohydrate component in the SHW [31]. The peak collapse is observed between HTC temperatures of 200 and 250 • C, revealing the thermal degradation of carbohydrates [32]. In summary, the FTIR spectra analysis demonstrated that the studied SHWs were well carbonized and converted into hydrochar after HTC. The differences in the hydrochar functional groups were attributed to the inherent properties of the cattle and pig SHWs. Conclusions This study focused on the hydrothermal treatment of cattle and pig SHWs and the potential use of SHW-derived hydrochars as a solid fuel to reduce the environmental and economic burden on the meat industry. Both the cattle and pig SHW-derived hydrochars demonstrated enhanced energy-related properties. However, the cattle SHW treated with HTC was not effective in terms of energy retention due to the lipid-rich characteristics of raw cattle SHW. A relatively low HTC treatment temperature (approximately 200 • C) was found to be suitable for pig SHW. Thus, the HTC of cattle and pig SHWs may prove effective for waste management and the production of solid fuel. The HTC treatment efficiency should be considered as it changes based on the chemical composition of the raw feedstock. Conflicts of Interest: The authors declare no conflict of interest.	v3-fos-license
2018-05-11T16:40:29.095Z	2018-05-08T00:00:00.000	13700546	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/293/27/10457.full.pdf", "pdf_hash": "b5836485dbefdab6dbdea3eb06c1345d6ca50ec1", "pdf_src": "Highwire", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1094", "s2fieldsofstudy": [ "Biology", "Chemistry", "Environmental Science" ], "sha1": "e4f2d116284b636ddc3cbeae4c0055a7d17dfb09", "year": 2018 }	pes2o/s2orc	From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus (Mcc.) capsulatus (Bath) and Methylomicrobium (Mm.) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function. Methanotrophic bacteria convert methane, the second most abundant greenhouse gas, to methanol in the first step of their metabolic pathway (1,2). As the main methane sink in nature, these microorganisms are promising biological tools for methane remediation and biofuel production (3)(4)(5)(6). Methanotrophs activate a 105 kcal/mol C-H bond in methane using metalloenzymes called methane monooxygenases (MMOs), 3 which are classified as soluble or membrane-bound (particulate, pMMO) (7). pMMO is the predominant methane oxidation catalyst in nature but is less well-characterized (8). A detailed understanding of methane oxidation by pMMO has the potential to guide synthetic catalyst design and facilitate methanotroph engineering. pMMO is a complex integral membrane enzyme that requires copper for activity (9 -11). Crystal structures of pMMO from four different methanotrophs reveal a 300-kDa ␣ 3 ␤ 3 ␥ 3 trimer composed of the subunits PmoA, PmoB, and PmoC (11)(12)(13)(14). PmoA and PmoC are integral membrane subunits, whereas PmoB consists of two periplasmic domains linked by two transmembrane helices. Present in all of these structures is a copper site at the N terminus of PmoB, with the N-terminal histidine of PmoB and two histidines from an HXH motif as ligands. This copper center, assigned as the active site (10), has been modeled with either one or two copper ions in the different structures. An additional PmoB monocopper site is found only in the structure of pMMO from Methylococcus (Mcc.) capsulatus (Bath) (12). The PmoC subunit houses a variable metal binding site that can be occupied by copper or zinc, depending on the crystallization conditions. The presence of multiple subunits with variable metal content has complicated efforts to determine the nuclearity of the copper active site and the roles of the other observed metal centers. Moreover, all studies of pMMO have been hindered by significant decreases in enzymatic activity upon isolation from the membranes and solubilization with detergents (7). In some cases, activity appears to be completely abolished upon removal from the membranes. As a result, the physiological relevance of structural and spectroscopic studies of purified pMMO has been questioned, and alternative hypotheses for the active site have been proposed, largely based on the assumption that solubilization and purification of pMMO removes catalytically essential copper ions (15,16). An alternative possibility is that removal of pMMO from the membranes, rather than loss of copper, has deleterious effects on activity. Detergent micelles are frequently used for mem-brane protein characterization because of ease of use and compatibility with many experimental methods (17,18). However, detergent micelles lack the structure and pressure provided by lipid bilayers and can cause instability and loss of function (19,20). Membrane mimetics provide a way to study membrane proteins in more native-like environments. In many cases, addition of lipids or use of these mimetics has restored functional activity to purified membrane proteins (21)(22)(23)(24). In particular, bicelles, discoidal lipid bilayers surrounded by detergent, have been used to characterize and crystallize a range of membrane proteins (25)(26)(27)(28). To address the hypothesis that pMMO inactivation upon solubilization is due to removal from the native membranes, we have reconstituted purified pMMO from Methylomicrobium (Mm.) alcaliphilum 20Z (20Z-pMMO) and Mcc. capsulatus (Bath) (Bath-pMMO) into bicelles. Bicelle reconstitution recovers the methane oxidation activity of both pMMOs without addition of exogenous copper ions or substantial alteration in the copper sites, as observed by electron paramagnetic spectroscopy (EPR). A crystal structure of 20Z-pMMO provides some insight into how solubilization might affect protein stability. Finally, extended X-ray absorption fine structure (EXAFS) analysis of 20Z-pMMO does not indicate the presence of the short copper-copper interaction observed in previous samples, prompting further investigation of the active site nuclearity. Recovery of [ 13 C]methane oxidation activity by bicelle reconstitution To systematically investigate loss of pMMO activity, methane oxidation activity was measured for as-isolated, solubilized, purified, and bicelle-reconstituted pMMO samples. pMMO activity assays are typically performed using either NADH or duroquinol as a reductant. Duroquinol can directly reduce pMMO, whereas a type 2 NADH dehydrogenase (NDH-2) likely oxidizes NADH and reduces quinones for subsequent electron transfer to pMMO (9). Solubilization with the detergent dodecyl maltoside (DDM) separates pMMO from the membranes (solubilized pMMO), which abrogates NADHdriven activity (Fig. 1). Solubilized pMMO was then reconstituted in bicelles to mimic the lipid bilayer and to investigate membrane-dependent activity loss. Methane oxidation activity was measured for as-isolated membranes, solubilized and purified pMMO in detergent (DDM), and bicelle (3% (w/v) DMPC-CHAPSO) reconstituted pMMO using both reductants (Fig. 1). Because of [ 12 C]methanol contamination in many buffers and reagents, a new activity assay was developed in which conversion of [ 13 C]methane to [ 13 C]methanol is detected via GC-MS. Methane oxidation activities for Bath-pMMO and 20Z-pMMO were measured at 30°C after 5 min because of solidification of bicelles at higher temperatures and longer incubation times. NADH-driven activity (41.1 Ϯ 1.7 and 14.5 Ϯ 1.2 nmol [ 13 C]methanol mg Ϫ1 protein for Bath-pMMO and 20Z-pMMO, respectively) is abolished upon solubilization and purification ( Fig. 1 and Table S1). For membrane-bound and solubilized samples, the activity measured using duroquinol was significantly lower than the NADH-driven activity for Bath-pMMO and not detected for 20Z-pMMO ( Fig. 1). For both pMMOs, reconstitution into bicelles recovers the methane oxidation activity of solubilized and purified samples using duroquinol as a reductant ( Fig. 1 and Table S1). However, NADHdriven activity is only restored for Bath-pMMO. It may be that an NDH-2 or other components of the electron transport chain responsible for NADH-dependent methane oxidation are not properly reassembled after solubilization and reconstitution of 20Z-pMMO. Notably, for both pMMOs, duroquinol-driven activity is significantly higher for bicelle-reconstituted samples than for asisolated membranes and is comparable with NADH-driven activity in membranes ( Fig. 1 and Table S1). The different properties of phosphatidylcholine (PC), the main lipid in DMPC bicelles, and phosphatidylethanolamine (PE), the predominant phospholipid found in these methanotrophs (29 -31), provide a possible explanation for this observation. The amine head group of PC is less polar than that of PE and may increase the solubility and access of duroquinol as well as O 2 and methane. Additionally, DMPC is composed of saturated 14:0 PC, whereas methanotroph PEs are primarily composed of a saturated and unsaturated mixture of 16:0 and 16:1 PE. The various head groups and acyl chain compositions can affect lipid packing, membrane fluidity, and even the structure of membrane pro- pMMO from micelles to bicelles teins (32). Finally, in as-isolated membranes, it is possible that the native quinones occupy the binding site duroquinol needs to access to reduce pMMO. Taken together, these results indicate that solubilized pMMOs are not irreversibly inactivated. Interestingly, solubilized or purified pMMO samples were reconstituted in bicelles without the addition of copper, suggesting that bicelles alone are responsible for the recovered activity. Effect of bicelle reconstitution on pMMO copper centers To further investigate the relationship between bicelle reconstitution and the pMMO copper sites, the copper concentrations of pMMO samples in as-isolated membranes, detergent, and bicelles were measured using inductively coupled plasma optical emission spectroscopy. The presence of approximately three copper ions per protomer in purified Bath-pMMO ( Fig. 2 and Table S2) is consistent with previous studies (12). Purified 20Z-pMMO contains ϳ2.7 eq of copper per protomer (Table S2). The copper contents of the native membranes and solubilized pMMOs are batch-dependent, accounting for the variability in copper stoichiometry values for these samples. Loss of some adventitiously bound copper is also typically observed during solubilization and purification (11). The copper stoichiometry does not change between pMMO samples in detergent and in bicelles ( Fig. 2 and Table S2). This observation, in conjunction with the recovered activity, indicates that the catalytically essential copper ions are still present in detergent-solubilized pMMO samples. The differences in activity between as-isolated membranes, detergent-solubilized pMMO, and bicelle-reconstituted pMMO therefore cannot be attributed to changes in copper content. Consequently, the membrane, and not copper depletion, is a crucial factor contributing to activity loss upon solubilization. To directly assess the Cu 2ϩ electronic and geometric structure through the bicelle reconstitution process, we collected EPR spectra of Bath-pMMO and 20Z-pMMO before and after bicelle reconstitution (Fig. 3). A previous EPR analysis of purified Bath-pMMO revealed two distinct type 2 Cu 2ϩ signatures (33). The bicelle-reconstituted Bath-pMMO exhibits the same Cu 2ϩ EPR spectrum as the purified Bath-pMMO and is simulated with the same parameters as reported previously. However, the bicelle-reconstituted enzyme contains slightly more Cu 2ϩ per protomer than the purified sample. Consequently, some of the Cu 2ϩ observed in the bicelle-reconstituted sample is Cu 1ϩ in the purified sample and oxidizes to Cu 2ϩ during the reconstitution procedure. The purified 20Z-pMMO EPR spectrum exhibits the Cu 2ϩ spectrum seen in both forms of Bath-pMMO as well as a small contribution from additional Cu 2ϩ resonance ( Fig. 3B and Table S2), suggesting adventitious Cu 2ϩ binding to 20Z-pMMO in a site either unoccupied or containing Cu 1ϩ in Bath-pMMO. Similar to Bath-pMMO, incorporation of 20Z-pMMO into bicelles oxidizes some Cu 1ϩ to Cu 2ϩ , as evidenced by the slightly altered g Ќ region and increased amount of Cu 2ϩ per protomer (Table S2), but the signal is otherwise the same as observed for the purified sample. Importantly, the EPR spectra of both pMMOs show that the Cu 2ϩ ligation is not substantially altered by the bicelle incorporation procedure. Therefore, the appreciable recovery of pMMO activity upon insertion of Bath-pMMO into the bicelle pMMO from micelles to bicelles is not due to differences in the active site copper structure, consistent with the notion that the membrane environment plays a critical role in modulating activity. Crystal structure of 20Z-pMMO To further characterize 20Z-pMMO, a crystal structure was determined to 2.7 Å resolution ( Table 1). The protein was purified in the presence of DDM, exchanged into the detergent Cymal-5, and then crystallized with ammonium sulfate as the precipitant. Varying the concentration of this precipitant was crucial for obtaining well-diffracting crystals. The 20Z-pMMO structure exhibits a similar overall architecture to Bath-pMMO, with an ␣ 3 ␤ 3 ␥ 3 trimeric structure. Unlike previous pMMO structures (11)(12)(13)(14), there is a single protomer in the asymmetric unit (Fig. 4A). Despite the overall structural similarity, the PmoC subunit of 20Z-pMMO is significantly disordered compared with the previous structures (11)(12)(13)(14) (Fig. 4B). Electron density is not observed for 60% of the PmoC subunit, including residues 1-89, 123-156, and 193-218. These disordered regions include the variable metal binding site (Asp-128, His-132, and His-145) and surrounding residues. This significant disorder may result from destabilization of PmoC in detergent and could be related to the complete loss of activity upon detergent solubilization and purification (Fig. 1B). PmoC, at least in 20Z-pMMO, is thus more flexible than suggested by previous structures. The metal binding sites of 20Z-pMMO also differ from those observed in previous pMMO structures (11)(12)(13)(14). In the Bath-pMMO PmoB subunit, there is a monocopper site coordinated by His-48 and His-72 (12). Although both residues are conserved in 20Z-pMMO, electron density attributable to copper or any other metal ion is not present (Fig. 4C). It is unclear why this site is only occupied in Bath-pMMO, but the metal binding residues are not conserved in all pMMOs, with His-48 substituted by Asn and Gln in type II methanotrophs, indicating that this metal center is not essential for methane oxidation. The PmoB subunit also contains a bound copper that is coordinated by residues His-33, His-137, and His-139 and has been assigned as the active site. In some pMMO structures, this site has been modeled with two copper ions, including Bath-pMMO (12)(13)(14). The dicopper site model is based on EXAFS data that consistently indicate the presence of a short copper-copper distance as well as the measured copper stoichiometry upon purification (10, 11,13,14,34). However, in other structures, the site has been modeled with a single copper ion (11,14). In the 20Z-pMMO structure, this PmoB site is also best modeled with one copper ion (Fig. 4D). The site is square planar with copper-nitrogen distances of 2.1 Å for the His-137 ␦N, 2.1 Å for the His-139 ⑀N, 2.5 Å for the His-33 ␦N, and 1.9 Å for the N-terminal nitrogen of His-33. The electron density for His-33 is not as well-defined as that for other two histidine residues. Interestingly, very strong additional electron density is observed for PmoB residue Lys-155 in PmoB appended to the side-chain N atom. We could not conclusively model this density, but it could potentially arise from posttranslational modification of this residue. XANES and EXAFS analysis of 20Z-pMMO The copper X-ray absorption near edge structure (XANES) spectra measured for 20Z-pMMO indicate a mixed Cu(I) and Cu(II) metal environment. A subtle transition, observed at 8978.8 eV (Fig. 5A) is consistent with the forbidden 1s 3 3d transition for Cu(II) (35). Additional edge transitions, observed at 8983 and 8986.3 eV and illustrated in the first derivative of the edge at 8982.3 and 8985.5 eV in Fig. 5A, inset, are characteristic of the 1s 3 4p transitions often observed for systems containing a mixture of Cu(I) and Cu(II) (35). Analysis of the copper EXAFS spectra for 20Z-pMMO suggest a mononuclear copper ligand environment constructed by only oxygen and nitrogen within the first ligand sphere (Fig. 5B). Simulations of copper-oxygen/nitrogen nearest neighbor pMMO from micelles to bicelles ligand scattering suggest a disordered ligand environment composed of approximately 2.5 to 3.5 oxygen/nitrogen ligands at an average bond length of 1.96 Å ( Table 2). Inclusion of a direct copper-copper scattering vector was not justified in our simulations. Long-range scattering could be simulated using low Z (carbon/nitrogen) scattering at bond lengths of 2.97, 3.36, and 3.97 Å, reminiscent of patterns observed because of imidazole scattering interactions from coordinated histidines (36). In support of imidazole scattering, the pronounced camelback feature at 4 Å Ϫ1 , characteristic of metal-histidine ligation (37), is also observed. Discussion The recovery of methane oxidation activity upon pMMO reconstitution into bicelles underscores the importance of studying membrane proteins in native-like environments. Although studying membrane proteins in a membrane context seems obvious, detergent micelles are still typically used instead. Besides their amphipathic nature, detergent micelles lack important lipid bilayer characteristics that provide structural support (19). Reconstitution of pMMO into bicelles restores the methane oxidation activity of inactive detergent-solubilized pMMOs close to levels measured for membrane-bound pMMO (Fig. 1). The copper stoichiometries and EPR spectroscopic features are nearly identical for inactive detergent-solubilized and active bicelle-reconstituted pMMO samples and are consistent with previous observations (12,33). These data indicate that the copper centers detected in detergent-solubilized pMMO are functionally relevant. In previous pMMO crystal structures, one to three copper ions were modeled per protomer, found only in the PmoB and PmoC metal centers, and only the PmoB site coordinated by His-33, His-137, and His-139 consistently houses copper ions (11)(12)(13)(14). Chan and Yu (15) and Chan and co-workers (16) have proposed that Bath-pMMO instead contains ϳ15 copper ions, including a tricopper active site in PmoA and six to seven Cu 1ϩ ions bound to the C terminus of PmoB, and have suggested that copper loss from these sites upon membrane solubilization is responsible for the reduced activity of purified Bath-pMMO. However, the recovered activity of bicellereconstituted pMMO samples indicates that large numbers of essential copper ions are not lost during isolation from the membranes. The crystal structure of 20Z-pMMO provides some insight into how removal from the membrane could affect activity. PmoC is largely disordered, suggesting destabilization upon solubilization and resultant activity loss. PmoB only contains two transmembrane helices, and PmoA is sandwiched between PmoB and PmoC, features that may contribute to their structural stability in detergent micelles. In contrast, only the PmoC helices near PmoA are ordered (Fig. 4A), whereas the disordered regions are exposed to the lipid membrane and perhaps more susceptible to perturbations upon reconstitution into detergent micelles. Without lateral pressure or specific lipid binding, PmoC may be structurally less stable in micelles. PmoC is positioned directly adjacent to the proposed PmoB active site and could be involved in stabilization of the active site or copper binding that may be essential for activity. In addition, for a hydrocarbon monooxygenase homolog of pMMO, mutation of the PmoC metal binding residues reduces activity, suggesting an important functional role (38). Previous efforts have mainly focused on characterizing perturbations in PmoB to explain activity loss. Some of this attention should be shifted to understanding how the transmembrane subunits, particularly PmoC, play an essential role in methane oxidation. Finally, a mononuclear copper active site remains a viable possibility (6,8). The 20Z-pMMO PmoB site is best modeled as monocopper (Fig. 4). Additionally, the short copper-copper distance detected in the EXAFS analysis of other pMMOs (11,14,35) is not present in 20Z-pMMO (Fig. 5). Its absence in Figure 5. XANES and EXAFS analysis of 20Z-pMMO. A, copper XANES spectra for 20Z-pMMO. Inset, the first derivative of near edge and edge features is displayed to more clearly highlight the features. B, raw copper EXAFS for 20Z-pMMO. Simulations were fit using a standard conservative approach that follows rules governing both spectral resolution relative to acceptable intraligand scattering interaction bond lengths and acceptable bond lengths (55). C, Fourier transform of the EXAFS. Raw unfiltered data are shown in black, and the best fit simulations are shown in gray. EXAFS were fit over a k range of 1.0 -12.85 Å Ϫ1 . pMMO from micelles to bicelles 20Z-pMMO could be due to lower protein concentrations, a heterogeneous distribution of copper-copper vectors in the samples that cancel out the overall signal, or even the reduced presence of other copper contaminant proteins that could contribute to the observed feature. This result is consistent with a recent quantum refinement of the Bath-pMMO PmoB copper site (39). Most relevant to a pMMO monocopper active site are the lytic polysaccharide monooxygenases (LPMOs), which utilize a monocopper active site for oxidative cleavage of glyosidic bonds. Both the PmoB copper site and the LPMO active site contain a histidine brace metal-binding motif. However, LPMOs lack a third histidine ligand and additional metal binding sites. In addition, some contain a methylated histidine ligand. Moreover, the substrates of pMMO and LPMO are drastically different (40 -42). Overall, studying pMMO in a membrane-bound context has validated past characterizations and provides new insights into the importance of the PmoC subunit and the nature of the active site. It will be important to continue this approach in future studies of pMMO activity and mechanism. Methanotroph cell growth Mm. alcaliphilum 20Z was cultured as described previously (43,44). Briefly, cells were grown in 1ϫ modified nitrate mineral salts medium, 0.5 M NaCl, 2.3 mM phosphate buffer, and 50 mM carbonate buffer (pH 9.5) supplemented with 40 M CuSO 4 ⅐H 2 O and trace elements solution under a 1:3 methaneto-air ratio in 12 liters bioreactors. Mcc. capsulatus (Bath) cells were grown in 1ϫ nitrate mineral salts medium and 3.9 mM phosphate buffer (pH 6.8) supplemented with 50 M CuSO 4 ⅐H 2 O, 40 M iron NaFe(III)EDTA, and trace element solution under a 1:4 methane-to-air ration in 12 liters of bioreactors (12). All bioreactor cell growths were harvested at an OD 600 of 8 -10 and centrifuged at 8,000 ϫ g for 30 min. Cell pellets were flash-frozen in liquid nitrogen and stored at Ϫ80°C for future use. Membrane isolation Mm. alcaliphilum 20Z cell pellets (10 g) were resuspended in 100 ml of 25 mM PIPES and 500 mM NaCl (pH 7) supplemented with EDTA-free protease inhibitor tablets (Roche). The cells were manually stirred for resuspension on ice. The cell resuspension was sonicated at 4°C for 1.5 min with an on/off interval of 1 s/3 s at 25% amplitude and centrifuged at 8,000 ϫ g for 1 h to remove cell debris. The supernatant was centrifuged at 100,000 ϫ g for 1 h to isolate the pelleted membranes containing pMMO. The membrane pellet was washed twice with a Dounce homogenizer in 25 mM PIPES and 250 mM NaCl (pH 7). 1-ml aliquots of pMMO-containing membranes at total protein concentrations of 10 mg/ml (measured by Bio-Rad DC assay using BSA as a standard) were flash-frozen in liquid nitrogen and stored at Ϫ80°C. Mcc. capsulatus (Bath) membranes were isolated as described previously (45). pMMO purification and bicelle reconstitution Membranes were solubilized using 1.2 mg of DDM (Anatrace) per 1 mg of crude protein at 4°C for 1 h. The solubilized protein was centrifuged at 100,000 ϫ g for 1 h, and the supernatant was collected for purification. Solubilized 20Z-pMMO was buffer-exchanged into 25 mM PIPES, 50 mM NaCl (pH 7), and 0.02% (w/v) DDM using a 100-kDa molecular mass cutoff Amicon (Millipore). 20Z-pMMO was purified using a 15Q anion exchange column (GE Healthcare) and eluted using a 50 -800 mM NaCl gradient (Figs. S1 and S2). Solubilized Bath-pMMO was concentrated to 1 ml using a 100-kDa molecular mass cutoff Amicon and loaded onto a 120-ml Superdex 200 size exclusion column (Fig. S3). All eluted pMMOs were concentrated using a 100-kDa molecular mass cutoff Amicon to 10 mg/ml in 25 mM PIPES, 250 mM NaCl (pH 7), and 0.02% (w/v) DDM. Freshly solubilized or purified pMMO at 10 mg/ml was reconstituted with a 30% (w/v) DMPC:CHAPSO 2.8:1 bicelle solution (Molecular Dimensions) using a 4:1 volumetric ratio and incubated on ice for at least 30 min to prepare pMMO samples at 8 mg/ml reconstituted in 6% (w/v) bicelles. The copper concentration was measured by inductively coupled plasma optical emission spectroscopy at the Quantitative Bio-element Imaging Center at Northwestern University. [ 13 C]Methane oxidation activity assay To measure the methane oxidation activity of membranebound, solubilized, purified, and 6% (w/v) bicelle-reconstituted pMMOs, samples were diluted to 4 mg/ml (or 3% (w/v) bicelles) in 100-l reactions consisting of reductant (280 M NADH (Sigma-Aldrich) or excess duroquinol) in 2-ml screw-top vials with septa tops (Agilent). A 1-ml volume of headspace gas was withdrawn and replaced with 1.5 ml of [ 13 C]methane (Sigma-Aldrich). All reactions were performed at 30°C (bicelle samples solidify at 45°C, the temperature typically used for Bath-pMMO activity assays). pMMO reconstituted in 3% or 1.5% (w/v) bicelles gave the highest activity, which decreased with lower bicelle concentrations (Fig. S4). Reactions were incubated at 30°C and 200 rpm for 5 min, put on ice for 5 min, and then quenched with 500 l of chloroform spiked with 1 mM dichloromethane. The reaction was vortexed at 2,000 rpm for 10 min and centrifuged at 2,000 ϫ g for 30 min to separate precipitate from the chloroform mixture. 2.5 l of sample was injected onto a PoraBOND Q column (25 m ϫ 250 m ϫ 3 m) on an Agilent 7890B/5977A MSD GC/MS instrument with a split ratio of 10:1. The GC was maintained under a constant flow of 1.2 ml/min of helium gas. The initial oven temperature was maintained at 80°C for 3.5 min, followed by an increase of 50°C/min to 150°C and held for 1.5 min. A second ramp rate of 15°C/min was used to reach the final temperature of 300°C, held for 1 min. The mass spectrometer was maintained under an ion source temperature of 230°C, quad temperature of 150°C, 70 eV, and a detector voltage of 2,999 V. Masses 31, 33, and 49 were monitored for detection of [ 12 C]methanol, [ 13 C]methanol and dichloromethane (dwell times of 10 ms, 100 ms, and 10 ms, respectively). The [ 13 C]methanol peak area was integrated, quantified from a standard calibration curve, and normalized to the concentration of the internal standard dichloromethane. The lower limit of detection was determined to be 10 M [ 13 C]metha-pMMO from micelles to bicelles nol, and a stringent cutoff for minimum concentration was set at 30 M. Methane oxidation activity values using [ 13 C]methanol detection by GC-MS compared with [ 12 C]methanol detection using the GC-flame ionization detector (FID) are shown in Table S3. EPR spectroscopy EPR samples were prepared by aliquoting 100 M (DDM samples) or 80 M pMMO (bicelle samples) in 25 mM PIPES, 250 mM NaCl (pH 7), and 0.02% (w/v) DDM or 6% (w/v) bicelles into Wilmad quartz EPR tubes (Sigma-Aldrich). Measurements were collected on a continuous wave X-band Bruker ESP-300 spectrometer using a liquid helium flow Oxford Instruments ESR-900 cryostat. Spectra were corrected for background resonance by subtraction of a spectrum of 50 mM Tris (pH 8.0), 150 mM NaCl collected under the same conditions. Cu 2ϩ spin quantitation was performed by double integral area comparison of pMMO spectra to Cu 2ϩ -EDTA standards containing 25-500 M Cu 2ϩ . All EPR simulations were performed using EasySpin (46). X-ray absorption spectroscopy Purified 20Z-pMMO samples were concentrated to 385 M using a 100-kDa molecular mass cutoff Amicon centrifugal concentrator and resuspended in 30% (v/v) glycerol. The copper concentration of the 20Z pMMO samples was 732 M. These samples were loaded into Lucite XAS cells wrapped with Kapton tape, flash-frozen in liquid nitrogen, and stored at Ϫ80°C. XAS data were collected at the Stanford Synchrotron Radiation Lightsource on beamline 9-3, equipped with a Si[220] double-crystal monochromator that contains an upstream mirror used for focusing and harmonic rejection. Fluorescence spectra were collected using a 100-element Ge solid-state Can-berra detector. During data collection, the Oxford Instruments continuous-flow liquid helium cryostat was stabilized at 10 K. Copper excitation data were collected using a 6-m nickel Lytle filter and solar slits placed between cryostat and detector to reduce scattering fluorescence. XAS spectra were measured using 5 eV steps in the pre-edge region (8,750 -8,960 eV), 0.25 eV steps in the edge region (8,986 -9,050 eV), and 0.05 Å Ϫ1 increments in the EXAFS region (to k ϭ 13.3 Å Ϫ1 ), integrating from 1 to 25 s in a k 3 weighted manner for a total scan length of~40 min. A copper foil spectrum was collected simultaneously with each protein spectrum for real-time spectral energy calibration, with an assigned first inflection point for the copper foil spectrum at 8,980.3 eV. Spectra were closely monitored for any photodamage, and slight photoreduction was observed. To diminish the extent and impact of photoreduction, six individual spectra were collected at unique positions on the sample surface, following a matrix positioning grid to ensure a new radiation exposure surface, and only the initial exposure spectrum at each position was used during overall data analysis. Spectra were collected on duplicate independent samples, and data presented in this report represent the average of six scans. XAS spectra were processed and analyzed using the EXAFSPAK program suite written for Macintosh OS-X (52) 4 integrated with the Feff v8 software (53) for theoretical model generation. EXAFS fitting analysis was performed on raw/unfiltered data. Single scattering models were calculated for oxygen, nitrogen, sulfur, copper, and carbon coordination to simulate possible copper ligand environments, with values for the scale factors (Sc) and E 0 calibrated by previous fittings of characterized Cu(I)/Cu(II) crystallographic copper model compounds (35). Standard criteria for judging the best-fit EXAFS simulations included a reasonable Debye-Waller factor for the fit ( 2 Ͻ 0.006 Å 2 ) (54); the spectral resolution of the data, calculated based on the energy range extent of usable data (55); and the lowest mean square deviation between data and fit width, corrected for the number of degrees of freedom (FЈ) (55). During the standard criteria simulations, only the bond length and Debye-Waller factor were allowed to vary for each ligand environment. A dimensionless Sc ϭ 1 and E 0 values of Ϫ12, Ϫ14, and Ϫ16 eV were used for Cu(I,II)-C/N/O, -S, and -Cu theoretical model calibrations, respectively, during simulations (35).	v3-fos-license
2021-05-08T00:03:31.809Z	2021-02-19T00:00:00.000	233898520	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://translational-medicine.biomedcentral.com/track/pdf/10.1186/s12967-021-02879-2", "pdf_hash": "16d520ce678ba9604e53da37ff48f5d7a3aa709b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1108", "s2fieldsofstudy": [ "Biology", "Medicine" ], "sha1": "2464bd7f5befeb406c209c182ac92bc861d3d0c9", "year": 2021 }	pes2o/s2orc	METTL13 inhibits progression of clear cell renal cell carcinoma with repression on PI3K/AKT/mTOR/HIF-1α pathway and c-Myc expression Background Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive type of renal malignancy. Methyltransferase like 13 (METTL13) functions as an oncogene in most of human cancers, but its function and mechanism in ccRCC remains unreported. Methods qRT-PCR, western blotting and immunohistochemistry were used to detect METTL13’s expression in tissues. The effects of METTL13 on ccRCC cells’ growth and metastasis were determined by both functional experiments and animal experiments. Weighted gene co-expression network analysis (WGCNA) was performed to annotate METTL13’s functions and co-immunoprecipitation (co-IP) was used to determine the interaction between METTL13 and c-Myc. Results METTL13 was underexpressed in ccRCC tissues compared to normal kidney tissues and its low expression predicted poor prognosis for ccRCC patients. The in vitro studies showed that knockdown and overexpression of METTL13 respectively led to increase and decrease in ccRCC cells’ proliferation, viability, migratory ability and invasiveness as well as epithelial-mesenchymal transition (EMT). The in vivo experiment demonstrated the inhibitory effect that METTL13 had on ccRCC cells’ growth and metastasis. Bioinformatic analyses showed various biological functions and pathways METTL13 was involved in. In ccRCC cells, we observed that METTL13 could negatively regulate PI3K/AKT/mTOR/HIF-1α pathway and that it combined to c-Myc and inhibited c-Myc protein expression. Conclusions In general, our finding suggests that high expression of METTL13 is associated with favorable prognosis of ccRCC patients. Meanwhile, METTL13 can inhibit growth and metastasis of ccRCC cells with participation in multiple potential molecular mechanisms. Therefore, we suggest METTL13 can be a new diagnostic and therapeutic target for ccRCC in the future. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-021-02879-2. still the major and the most effective therapeutic method for localized RCC, while metastasis occurs in approximate 25% cases, which makes it difficult for patients to undergo surgery [3]. Among all the histological and molecular subtypes of RCC, clear cell RCC (ccRCC) is the most common subtype, accounting for about 75% [1,4]. Besides, ccRCC is resistant to radiotherapy and chemotherapy, which makes patients' prognosis far from satisfying [5,6]. Thus, it's urgent and important to identify crucial biomarkers for ccRCC and to have comprehensive insights into deep molecular mechanisms playing in this neoplasm with the aim to provide molecular bases and inspirations for the diagnosis, monitoring and treatment. Protein methyltransferase like 13 (METTL13), also called FEAT, is coded by gene METTL13, which is located at chromosome 1q24.3. Purified from rat livers in 2011 by a Japanese researcher, METTL13 was found to be abnormally overexpressed in several human cancers including lung cancer and to drive tumorigenesis in transgenic mice [7][8][9][10]. A study indicated that METTL13 inhibits apoptosis of lung, breast and liver cancer cells and miR-16 can repress the expression of METTL13 by binding to its 3′-untranslated region [8]. In 2016, researchers detected high expression levels of METTL13 in the plasma of patients with breast, ovarian and lung cancer [9], following which it was identified as a recurrence predictor for breast cancer [10]. As a methyltransferase protein, it can specifically methylate the Lys55 of eEF1A, resulting in the increase of its translational output and tumorigenesis of lung and pancreatic cancer [11,12]. In 2019, scholars demonstrated that expression of METTL13 is positively regulated at transcriptional level by HN1L and METTL13 can enhance hepatocellular carcinoma development by up-regulating TCF3 and ZEB1 [13]. Despite the studies above proving the oncogenicity role of METTL13, an article elucidated its downregulated expression in bladder cancer and its tumor-suppressing functions [14]. However, studies targeted at METTL13 are still very scarce and unintegrated with uncertainty of its roles in various cancers; meanwhile, its expression and biological functions in ccRCC remain unknown, which are worth further exploration. In this study, it was revealed that METTL13 was underexpressed in ccRCC tissues compared to normal kidney and its low expression was associated with unfavorable prognosis. METTL13 was detected by us to have significant inhibitory effect on growth and metastasis of ccRCC cells. Besides, METTL13 could repress PI3K/ AKT/mTOR/HIF-1α signaling pathway as well as c-Myc expression and might participate in other potential mechanisms, including metabolism regulation and cell adhesion alteration. These findings may provide insights into better understanding of METTL13's molecular functions in ccRCC as well as inspiration for ccRCC diagnosis and therapy. Bioinformatic analyses Website UALCAN (http:// ualcan. path. uab. edu/) was used to obtain gene expressions in different sample types (533 ccRCC tissues and 72 normal kidney tissues), different pathological grades and clinical stages of ccRCC with transcriptome data provided by The Cancer Genome Atlas (TCGA) database. METTL13 expressions in 72 pairs of ccRCC tissues and adjacent normal tissues were also acquired from a dataset (GSE53757) of the Gene Expression Omnibus (GEO) database (http:// www. ncbi. nlm. nih. gov/ geo/) [15]. Website GEPIA (http:// gepia. cancer-pku. cn/) directly produced survival curves of ccRCC patients with high and low METTL13 expression levels based on an appropriate expression threshold. Transcriptome data of kidney renal clear cell carcinoma (KIRC) cohort was downloaded from TCGA database and differentially expressed genes (DEGs) was filtered out with \|logFC\|> 1 and false discovery rate (FDR) < 0.05 as the criteria. We performed weighted gene co-expression network analysis (WGCNA) with the WGCNA package in R (The WGCNA package in R). ClusterProfiler R package was used to determine gene modules' gene ontology and KEGG pathway enrichments regarding FDR < 0.05 as threshold. Patient samples All the ccRCC tissues and their corresponding adjacent normal tissues were obtained from the urology surgery department of the first hospital of China Medical University (Shenyang, China). 50 pairs of ccRCC tissues and corresponding adjacent normal kidney tissues were respectively analyzed by qRT-PCR and 13 pairs were analyzed by western blotting assay. 48 ccRCC tissues were used for immunohistochemistry analysis. This study was approved by Research Ethics Committee of China Medical University (No: AF-SOP-07-1. 1-01, Additional file 1) and all the patients had supplied the written informed consent. Cell transfection Two strands of small interfering RNA (siRNA) targeting at METTL13 were designed and purchased from JTS-BIO Co. (China), sequences of which were as following: si-METTL13#1 (sense: GCG GGG UGC UAC AUA AAU ATT; anti-sense: UAU UUA UGU AGC ACC CCG CTT), si-METTL13#2 (sense: GCU CUG CCC UUC AGA UCU UTT; anti-sense: AAG AUC UGA AGG GCA GAG CTT). Usage of LipofectamineTM3000 (Invitrogen, USA) was involved in siRNA transfection with the protocol provided by manufacturer's guidelines. METTL13 overexpression plasmid was purchased from GeneChem (Shanghai, China) and transfection was performed according to manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from tissue samples and cells with the use of RNAiso Plus (Takara Biotechnology, Dalian, China) according to manufacturer's instructions and subsequently the Prime Script RT Master Mix (Takara Biotechnology, Dalian, China) was utilized to conduct reverse transcription to synthesize cDNA. qRT-PCR was performed by Sybr Premix Ex Taq TMKit (Takara Biotechnology, Dalian, China) and LightCy-clerTM 480 II system (Roche, Basel, Switzerland), after which the 2 −ΔΔCt method was used to calculate the relative expression level of each sample referring to internal β-actin or GAPDH expression. Information of primer sequences is listed in Additional file 2: Table S1. Western blotting assay Total protein from patient samples or cells was obtained by RIPA lysis buffer with 1% Phenylmethylsulfonyl fluoride (PMSF) contained and protein concentrations were detected by bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). Equal mass of proteins (30 μg/lane) were used for electrophoresis in 10% SDSpolyacrylamide, followed by PVDF membrane (0.2 μm) transfer, membrane blocking by 5% non-fat milk, primary and secondary antibody incubation as well as image capture by an EasySee Western Blot kit (Beijing Transgen Biotech, Beijing, China) and a chemiluminescence system (Bio-Rad, CA, USA). Information of primary antibodies is listed in Additional file 3: Table S2. 5-Ethynyl-2′-deoxyuridine (EdU) assay EdU assay was performed with the usage of an EdU kit (BeyoClickTM, EDU-488, China). Cells were co-cultured with EdU working solution (1:1000) at 37 °C in a humidified 5% CO2 atmosphere for 2-4 h, followed by fixation with 4% paraformaldehyde for 30 min and treatment with 0.3% Triton X-100 for 30 min at room temperature. Then, according to the manufacture's protocol, cells were coincubated with click reaction solution for 30 min at room temperature in a dark environment, after which cells were treated with Hoechst solution for 10 min. We used a fluorescence microscope (Olympus Corporation, Japan) to capture images with a magnification of 200× and cell counting was conducted by ImageJ software. Cell viability assay (CCK-8 assay) Counting Kit-8 (CCK-8) solution (Bimake, USA) was added to each well of 96-well plates to the concentration of 0.5 mg/ml, where 2.0 × 10 3 cells had been initially loaded. After incubation for one hour at 37 °C with 5% CO 2 , absorbance at 450 nm was measured by plate reader (Model 680; Bio-Rad Laboratories). Wound-healing assay When the density of cells in 6-well plates reached above 90%, we used a 1-mL pipette tip to vertically scratch an artificial wound in the middle of the wells. Then cells were washed with PBS and new FBS-free medium was added. Images were obtained with the help of an inverted microscope (EVOS XL system, AMEX1200; Life Technologies Corp, Bothell, WA, USA) at 40 X magnification. After cultured for 48 h, cell images were re-obtained. Cell migration and invasion assay 8-μm-pore transwell chambers in 24-well plates (Corning Costar, Corning, NY, USA) were used. Chambers coated with Matrigel (BD, San Diego, CA, USA) were for cell invasion detection, while those without Matrigel coating were used to determine cell migration. 600 μl 10%-FBS containing medium was placed into each bottom chamber, while equal number of suspended cells (1.0-1.5 × 10 4 cells for migration assay, 3.0-4.0 × 10 4 cells for invasion assay) in 200 μl medium without FBS were imbedded onto each upper chamber. After cultured at 37 °C with 5% CO2 for 48 h, suspended cells in the upper chamber were washed out, while cells adhering to the bottom membrane were stained by crystal violet. Images were obtained by using the inverted microscope described previously at 100X magnification and cell counting was performed by software Image J. Co-immunoprecipitation (Co-IP) assay Cells were lysed by RIPA lysis buffer containing 1% PMSF and 1% protease inhibitor. A certain amount of cell lysate was isolated as input, while 5 μg primary antibody (METTL13, abcam, ab186002; c-Myc, Santa Cruz, sc-40) or homologous IgG (Santa Cruz Biotechnology) was co-incubated with remaining lysate at 4 °C overnight. Then, 30-μl protein A/G-beads was co-incubated with the lysis solution at 4 °C for an hour, after which beads was extracted and washed by washing buffer three times. Next, proteins were isolated by using beads into 2x protein loading buffer after co-incubation at 100 °C for 15 min and western blot was finally conducted. Immunohistochemistry assay Tissues previously formalin-fixed and paraffin-embedded were cut into 4-μm slices and they were treated according to procedures previously described [16], which involved use of rabbit anti-METTL13 antibody (GTX120626, GeneTex, USA) and an UltraSensitiveTM SP (Mouse/ Rabbit) IHC kit (Maxin-Bio, Fuzhou, Fujian, China) according to the manufacture's guidance. Images were captured by the inverted microscope with magnifications of 200× and 400×. Animal experiments Experiments with animals involved were approved by China Medical University Ethics Committee of Medical Experimental Animal Welfare and were conducted following the institute's guidelines. 14 female BALB/c-nude mice of 4-6 weeks old, purchased from Beijing Vital River Experimental Animal Technology Co. Ltd., were housed in a pathogen-free environment at Experimental Animal Department of China Medical University. As for the tumorigenicity study, 1.0 × 10 6 OS-RC-2 cells (empty vector or METTL13 overexpression) in 150 μl serumfree 1640 medium containing 40% Matrigel were injected subcutaneously into flank of each mouse, 30 days after which mice were euthanized and tumors were excised. Each group included 4 mice. Primary tumors were measured for their size and weight. As for the metastasis study, 1.0 × 10 5 OS-RC-2 cells (empty vector or METTL13 overexpression) in 150 μl pathogen-free PBS were injected into per mouse via its lateral tail vein. After 45 days, lungs were separated and metastatic tumors were counted. Each group included 3 mice. Hematoxylin and eosin staining was applied in observing serial histological sections of the lungs. Statistical analysis Each experiment was performed independently for at least 3 times and data were expressed as the mean ± standard deviation (SD). Software GraphPad Prism of version 8.0 (La Jolla, CA, USA) was used to perform all the statistical analysis. Differences between two groups were evaluated by Student's t test. Differences in expression for samples with paired measures were analyzed by Wilcoxon signed rank test. Survival status was analysed by Kaplan-Meier/Logrank methods. As for all the data, P < 0.05 was regarded as statistically significant. * indicates P < 0.05; indicates P < 0.01, * indicates P < 0.001. Low expression of METTL13 is associated with poor outcome of ccRCC Despite the oncogenic role of METTL13 in most of human cancers, not only TCGA database but also GEO dataset showed that METTL13 was underexpressed at transcriptional level in ccRCC tissues (Fig. 1a, b). In addition, by using the website UALCAN, we detected that METTL13 expression levels were significantly and negatively correlated to tumor grades and to cancer stages of ccRCC (Fig. 1c, d). According to Kaplan-Meier survival curves provided by GEPIA on the basis of TCGA database ( Fig. 1e), it was found that ccRCC patients with higher METTL13 expression levels were more likely to have better prognosis (P = 0.01). By analyzing 50 pairs of samples using qRT-PCR assay, we observed significant decrease of METTL13 mRNA expression in ccRCC tissues compared to that in adjacent normal tissues (Fig. 1f, g). The result of western blotting also indicated the underexpression of METTL13 in ccRCC tissues at protein level (Fig. 1h). Immunohistochemistry results suggested that METTL13 protein expression declined with increase of tumor grade (Fig. 1i). Therefore, we could suggest that low expression of METTL13 was significantly associated with ccRCC occurrence and unfavorable prognosis of ccRCC patients. Knockdown of METTL13 promotes ccRCC cells' proliferation, migration and invasion Results of METTL13 protein expression obtained from 5 ccRCC cell lines (OS-RC-2, 760-P, ACHN, Caki-1 and 786-O) and a normal renal proximal tubule epithelial cell line (HK-2) showed that METTL13 was significantly underexpressed in most of the cancer cell lines (Fig. 2a). According to the result, two cell lines, 786-O and Caki-1, in which METTL13 was expressed relatively high, were selected to perform functional experiments with by knocking down METTL13 expression. Two strands of siRNA targeted at METTL13 were designed and their knockdown efficiencies were validated by immunoblotting assay (Fig. 2b). After transfecting the siRNA, we found that proliferation and viability of the two cell lines were significantly enhanced (Fig. 2c, d). In addition, knockdown of METTL13 respectively led to increase in wound healing rates of 786-O cells and Caki-1 cells (Fig. 2e), demonstrating its promotion of cell migration, furtherly validated by migration assay (Fig. 2f ). Meanwhile, silencing METTL13 expression significantly improved cells' invasiveness (Fig. 2f ). Western blotting results revealed that N-cadherin expression was upregulated in METTL13-silenced ccRCC cells, whereas E-cadherin was distinctly decreased (Fig. 2g). In general, inhibition of METTL13 expression facilitated proliferation, viability, migration and invasion of ccRCC cells as well as epithelial-mesenchymal transition. METTL13 inhibits ccRCC cells' proliferation, migration and invasion Then by using lentiviral vector expressing METTL13, we respectively constructed METTL13 stable-overexpressed OS-RC-2 and ACHN cell lines (Fig. 3a). Results of EdU assay suggested significant decrease in proliferating cells' portion after overexpressing METTL13 expression (Fig. 3b). In accordance with our expectation, METTL13 overexpression gave rise to not only cell viability inhibition (Fig. 3c) but also to restraint on cell migration and invasiveness (Fig. 3d, e). After overexpressing METTL13, the alterations in expressions of EMT-related proteins, N-cadherin and E-cadherin, were shown in (Fig. 3f ), which were opposite to what resulted from siRNA disposals, indicating METTL13's role in inhibiting EMT of ccRCC cells. These data suggested upregulation of METTL13 inhibited growth, metastasis and EMT of ccRCC cells. Functional correlations of METTL13 The next step to investigate METTL13's potential molecular mechanisms in ccRCC was performed by bioinformatic analyses with data obtained from TCGA database. After standardization, we inserted all the differently expression genes (DEGs) in ccRCC including METTL13 into WGCNA. The merged dynamic result showed that genes were divided into eight modules, the black, blue, green, magenta, pink, purple, red and turquoise ones c-e Cell proliferation, viability and migration were measured by EdU assay (c), CCK-8 assay (d) and wound-healing assay (e), respectively. f Cell migration assay and invasion assay were respectively used to detect cells' migratory ability and invasiveness. g Expression levels of EMT-related proteins (N-cadherin, E-cadherin) from cells were detected via western blotting. Each experiment was conducted independently at least three times. Student's t-test (two-tailed) was used for statistical analysis, P < 0.05, P < 0.01, *P < 0.001. Bar graphs: mean ± S.D., n = 3 . 4a). METTL13 and genes with similar expression mode were located in the turquoise module (Fig. 4b). Functional enrichment analysis of the turquoise module shown in Fig. 4c suggested that METTL13 had tremendous potential to participate in metabolism regulations, including metabolism-related pathways like the HIF-1 signaling pathway. Furthermore, by extracting nodes in METTL13's secondary connection and drawing network, we noticed that despite location in turquoise module, METTL13 directly connected to many genes situated in the blue module, which share high correlation with METTL13 (Fig. 5a). Meanwhile, the interactions between METTL13 and other modules were mainly dependent on the blue module. As for genes directly linked to METTL13, results of functional enrichment analyses were shown in Fig. 5b, based on which we predict that METTL13 affects tumor metastasis not only via EMT regulation but also by modulating cell adhesion. Taken together, METTL13 might regulate various biological functions as well as signaling pathways in ccRCC. METTL13 inhibits PI3K/AKT/mTOR/ HIF-1α signaling pathway in ccRCC With the guidance of bioinformatic analyses, we noticed that METTL13 participated in regulation of HIF-1 signaling pathway. As a core factor participating in HIF-1 signaling pathway, hypoxia-inducible factor-1α (HIF-1α) has been reported to affect multiple biological behaviors of renal cell carcinoma cells [17,18]. Then, we tried to figure out the impact that METTL13 had on HIF-1α. Results showed that silencing METTL13 resulted in significantly increase in HIF-1α protein levels in Caki-1 cells and on the contrary in OS-RC-2 cells, the overexpression of METTL13 led to an opposite effect (Fig. 6a). However, HIF-1α mRNA expressions were not influenced by METTL13 expression alterations (Fig. 6b), Cell proliferation was detected by EdU assay. c CCK-8 assay was performed to measure cell viability. d Cell migration rate was observed via wound-healing assay. e Migratory ability and invasiveness of ccRCC cells were detected by migration assay and invasion assay, respectively. f EMT-related protein levels were detected by performing western blotting. Each experiment was conducted independently triplicate. Student's t-test (two-tailed) was used for statistical analysis, P < 0.05, P < 0.01, P < 0.001. Bar graphs: mean ± S.D., n = 3 which suggested METTL13 might regulate HIF-1α in a post-transcriptional manner. It's known that PI3K/AKT/ mTOR signaling pathway importantly participates in HIF-1α protein translation [17] and after we respectively silenced and overexpressed METTL13, we detected that METTL13 could negatively regulate the phosphorylation levels of PI3K, AKT and mTOR without obvious impact on the total protein expressions (Fig. 6c, d). Taken together, we suggested that METTL13 could inactivate the PI3K/AKT/mTOR/HIF-1α pathway in ccRCC cells. METTL13 binds to c-Myc and inhibits c-Myc expression Meanwhile, METTL13 has been confirmed to have properties of protein binding and protein modification [11,12]. Out of great interest, we surveyed IntAct [19], a protein-protein interaction database and we displayed a number of protein interactions of METTL13, which was visualized by software Cytoscape [20] and shown in Fig. 7a. According to this result, we selected c-Myc, the most classic member of Myc family, as a target interacting with METTL13 to investigate because of its critical role in tumorigenesis and metabolism. By performing co-immunoprecipitation assay, we were convinced that METTL13 could physically bind to c-Myc (Fig. 7b). Furthermore, results showed that silencing METTL13 resulted in increase in c-Myc protein levels in Caki-1 cells and on the contrary in OS-RC-2 cells, the overexpression of METTL13 led to an opposite phenomenon (Fig. 7c), which indicated an inhibitory effect that METT13 had on c-Myc protein expression. Similarly, after alterations of METTL13 expression, no significant impact on c-Myc mRNA expression levels was observed (Fig. 7d), suggesting a post-transcriptional modification of c-Myc by METTL13. In conclusion, METTL13 seems to physically interact with c-Myc and to negatively regulate c-Myc protein expression in ccRCC, while many other potential interactors of METTL13 are worthy of further studies. METTL13 inhibits tumor growth and metastasis in vivo To further investigate the biological functions of METTL13 in vivo, OS-RC-2 cells stably overexpressing METTL13 were subcutaneously injected into BALB/c nude mice while OS-RC-2 cells transfected with empty vector was processed in the same way as the negative control. After 4 weeks, tumors from the METTL13 overexpression group showed significantly smaller sizes and lower weights by comparison to tumors from the negative control group (Fig. 8a-c). After extracting proteins from the tumors and performing western blotting assay, we detected significantly lower protein expressions of HIF-1α and c-Myc in the METTL13 overexpression group (Fig. 8d), which was accordant to what we previously observed by experiments in vitro. Next, teil vein injection was performed. METTL13-overexpressed and empty vector-expressed OS-RC-2 cells were respectively injected into BALB/c nude mice, 45 days after which lung colonization was analyzed. The result showed that lungs excised from mice in the METTL13 overexpression group were observed with presence of fewer metastatic tumors (Fig. 8e, f ), suggesting METTL13's inhibitory effect on metastatic ability of ccRCC cells. Each experiment was carried out at least for three times independently. Student's t-test (two-tailed) was used for statistical analysis, P < 0.05, P < 0.01, P < 0.001. Bar graphs: mean ± S.D., n = 3 Discussion Despite the oncogenic role played by METTL13's enhanced expression in many types of tumors, this molecule is underexpressed in ccRCC and its low expression is associated with poor prognosis according to public datasets, which aroused our interest in further research. By using qRT-PCR and western blotting assay, we detected significant decreases in mRNA and protein levels of METTL13 in ccRCC tissues compared to normal adjacent tissues as well as a negative relevance between METTL13 expression and malignancy grades of ccRCC via immunohistochemistry. In vitro and in vivo studies confirmed the inhibitory effect that METTL13 had on ccRCC cells' proliferation and metastasis. Furthermore, the alteration of ccRCC cells' metastatic ability by METTL13 might result from the regulation of epithelial-mesenchymal transition (EMT). Based on these, we identify METTL13 as a tumor suppressor gene in clear cell renal cell carcinoma, affecting a variety of biological behaviors of cancer cells and contrary to its role in most other cancers. Therefore, METTL13 is likely to act as a new biomarker for ccRCC in the future. By performing bioinformatic analyses with transcriptome data provided by TCGA datasheet, we found that METTL13 had a great potential to participate in metabolism regulation, including glycolysis, gluconeogenesis, TCA cycle, fructose and mannose metabolism, which had been proved to be closely associated with occurrence and development of ccRCC [21][22][23][24]. What appealed to us was that HIF-1 signaling pathway was involved and it's also been reported to play an important role in cancer metabolism [25,26]. HIF-1α, one of the most important molecules in the HIF-1 signaling pathway, has brought about scholars' interest with its crucial roles in various diseases, including malignancies [27][28][29][30]. Previous studies have stated HIF-1α's abnormal overexpression in ccRCC tissues and have demonstrated its tumorigenic functions as well as multiple molecular mechanisms [31][32][33][34]. According to our experiments, the expression of HIF-1α was observed to be negatively regulated by METTL13 at post-transcriptional level. Additionally, the PI3K/AKT/mTOR signaling pathway, which has been confirmed to promote HIF-1α protein translation, was observed to be inactivated by METTL13. However, it still The interaction between METTL13 and c-Myc was confirmed by co-immunoprecipitation (Co-IP). c, d Protein levels (c) and mRNA levels (d) of c-Myc were respectively detected by western blot and qRT-PCR after silencing or overexpressing METTL13. Each experiment was performed independently triplicate. Student's t-test (two-tailed) was used for statistical analysis, P < 0.05, P < 0.01, *P < 0.001. Bar graphs: mean ± S.D., n = 3 requires further efforts to determine whether METTL13 regulates HIF-1α expression via other mechanisms or not and how METTL13 specifically participates in the whole HIF-1 signaling pathway. Besides, the roles that it plays in other biological behaviors and processes of ccRCC, especially metabolism, are left to discover. According to the WGCNA result, we also suppose that METTL13 is able to regulate cell adhesion in ccRCC by possibly connecting to other molecules. Thus, inhibition of ccRCC metastasis by METTL13 may result from other mechanisms besides the EMT alteration. Furthermore, the database IntAct provided us with more than 20 proteins potentially combining with METTL13. Among these interactors, TRAF2 influences mitochondrial apoptosis of ccRCC [35]; KLK6's expression is negatively associated with renal carcinoma grades [36]; higher HLA-E mRNA level predicts better prognosis of ccRCC patients [37]; FAS can be potentially regarded as a biomarker for predicting survival of renal cancer patients who have received nephrectomy [38]. The result also involved Myc, a family of proto-oncogenes, which extensively functions in cancer formation and development [39]. c-Myc is the most classic and important member of the Myc family and it has been reported to control various biological behaviors of ccRCC cells with abundant mechanisms including metabolism regulation [40][41][42][43]. Via our experiments, we evidenced not only the physical interaction between METTL13 and c-Myc but also the restraining effect that METTL13 had on c-Myc protein expression. However, the specific mechanism still remains a mystery. Based on our findings, METTL13 is of great potential to act as a new biomarker for ccRCC diagnosis and therapy. On the one hand, the expression level of METTL13 in ccRCC is likely to be considered a potential molecular indicator in the future, which may assist pathologists in diagnosing clinicopathological characters of the tumors and predicting patients' prognosis; on the other hand, we could propose new methods using METTL13 agonists, taking advantage of METTL13's tumor suppressing role in ccRCC for renal cancer therapy. Besides, with an increasing number of studies aimed at metabolism regulation in renal carcinoma [21][22][23][24], we are looking forward to a new therapeutic strategy for ccRCC by utilizing METTL13's potential participation in metabolism, including METTL13's inhibitory impact on expressions of metabolism-related genes, HIF-1α and c-Myc. Conclusion Collectively, our research demonstrated METTL13's tumor suppressing role in clear cell renal cell carcinoma for the first time, as featured by inhibiting growth and metastasis of cancer cells. In addition, we summarized a variety of biological processes and molecular mechanisms that METTL13 might be involved in, according to which we validated its inhibitory effect on PI3K/ AKT/mTOR/HIF-1α pathway. Moreover, we confirmed that METTL13 could bind to c-Myc and restrain its expression. On the basis of these, METTL13 has a great potential to act as a new diagnostic biomarker and therapeutic target for ccRCC in the future, while its molecular mechanisms are worthy of further validation and investigation.	v3-fos-license
2020-12-10T09:06:14.955Z	2020-12-05T00:00:00.000	230639674	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/fsn3.2041", "pdf_hash": "6f1cc2c92de33ea69b1f663aa9adb105c17f42e5", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1112", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "sha1": "1ea4ce74e313aa3538c52633c62c804c1b392b30", "year": 2020 }	pes2o/s2orc	Optimization of fortified sponge cake by nettle leaves and milk thistle seed powder using mixture design approach Abstract Powdered nettle leaf and milk thistle (MT) seed were added to the cake batter with certain percentages selected by the Design‐Expert v. 10 software (0–25, 25–0, 18.75–6.25, 6.25–18.75, and 12.5–12.5). Addition of nettle and MT seeds to the cake reduced the moisture content, volume, and springiness and increased hardness of the samples. 12.5% nettle‐12.5% MT seed had the least hardness and the highest amount of springiness and cohesiveness. The highest BI, WI, SI, L, a, and b* and the lowest ΔE were observed in 12.5% nettle‐12.5% MT seed and 25% MT seed samples, respectively. Antioxidant activity and antimicrobial properties were increased in all samples compared to the control sample, so that 6.25% nettle‐18.75% MT exhibited the highest antioxidant activity and antimicrobial properties. The highest levels of quercetin and silymarin were observed in 25% nettle and 25% MT seeds, respectively. In the sensory evaluation, 12.5% nettle‐12.5% MT seed took the best scores regarding flavor, texture, color, and overall acceptance. Based on the lowest hardness, 13.65% nettle‐11.34% MT seed was determined as optimized points by the software, which was equivalent to desirability of 0.72. The optimum sample contained 62.90 mg quercetin and 886.70 mg silymarin. According to the HPLC analysis results, consumption of 10 optimal cakes daily could theoretically decrease the blood sugar level, which requires further studies. The remaining amount of quercetin and silymarin in 2.5 g of nettle leaves and 2.5 g of MT seeds after heating was 11 and 19 mg, respectively. In other words, heat did not have much effect on the destruction of quercetin and silymarin. Milk thistle (MT) is an annual or biennial flowering plant growing all over the world. MT is recommended for the treatment of varicose veins, menstrual disorders, splenic congestion, kidney, and liver in the early 19th century (Abenavoli et al., 2018). Silymarin, extracted from MT seeds and then dried in standard conditions, contains mostly flavonolignans (up to 80% w/w) in addition to polymeric and oxidized polyphenolic compounds. Silymarin is also reported to have positive effects on the blood sugar control and reduction (Luminita et al., 2016). Silymarin is composed mainly of silybinin, silychristin, silydianin, and isosilybinin with approximate percentages of 60, 20, 10 and 5%, respectively. Furthermore, diastereoisomers of the mentioned compounds, including silybinin A, silybinin B, isosilybin A, isosilybin B are found in addition to taxifolin. The use of nettle and MT in cereal products such as cakes and the resistance of silymarin and quercetin to the heat process can produce functional foods that have medicinal properties, texture quality and consumer acceptance, and lead to an increase in human health (Tayoub et al., 2018). Ataei and Hojjatoleslamy (2017) evaluated the amount of oleuropein remaining in the cake. They reported that oleuropein remained more in the olive leaf powder than its extract did. Therefore, in this study, nettle leaves and MT seeds were added to the cake in powder form. Moreover, the amount of silymarin and quercetin, which is effective in reducing blood sugar, was evaluated (Ataei & Hojjatoleslamy, 2017). In addition, the mixture design method was used, which contributes to the reduction of the number of treatments and repeat tests, so that results are obtained faster. \| Sponge cake preparation We prepared the sponge cake according to the recipe introduced by the authors in an earlier study with some modifications. Table 1 shows the ingredients based on 100 g of flour. Nettle leaf and MT seed powder was added to the formulation in the last step of batter preparation. After cooling, the baked cakes were packed in poly propylene bags at room temperature and kept for future physicochemical and sensory evaluation analyses (Ataei & Hojjatoleslamy, 2017). \| Optimization of mixing ratio The mixture Design of Experiment method was used to find the optimum mixture ratio of the nettle-MT powder (p < .05). The Design-Expert (V. 10) software, Stat-Ease Inc., USA, was used in this research. Nettle leaf and MT seed were assumed to be independent variables with a total proportion of 25% in a 100-g sponge cake. The software then suggested the amount of nettle and MT seed for different treatments (Table 1). The best model expressing the behavior of the treatments was chosen according to the obtained p-value. Moreover, more parameters, including R 2 and Adjusted R 2 , were studied where the lack of fit test was not significant. Finally, the response and the dependent variable were considered desirable based on the independent variables as: (Mohammadi et al., 2019). where; D, is total desirability, d, is desirability of each response, and n, is response number. \| Chemical properties of sponge cake Chemical properties of the cake samples were measured using AACC approved methods as: moisture content, crude protein, ash content and crude fat by AACC 46-40, AACC 46-11A, AACC 08-01, and AACC 30-10, respectively. According to the method AACC 46-11A, the crude protein content was assessed using the nitrogen conversion factor of 6.25 (Ataei & Hojjatoleslamy, 2017). \| Physical properties of sponge cake The cake volume and density were measured according to Prokopov et al. using rapeseed displacement (Prokopov et al., 2015). Samples with dimensions of 20 × 20 × 20 mm were taken from the midsection of the cakes on the first and after thirty days of storage for the texture profile analysis (TPA) using a texture analyzer (Brookfield Springiness quantifies the elasticity of the cake by measuring the distance recovered between the first and second compressions. Cohesiveness indicates the extent at which the product texture resists to the second deformation relative to the first one (Salehi et al., 2016). The Hunter Lab system was used to determine the color characteristics of the crumb of cake samples using a ColorFlex Ez, USA colorimeter. The L, a and b* values were determined. Where the L* value indicates the lightness from dark (0) to light (100), the a* value indicates the degree of the green to red color, the higher positive the value the more reddish. The value b* represents the yellow to blue color, the higher positive the value the more yellow. ΔE, which shows total color difference from the reference color, was obtained from Equation 2 (Hafez, 2012). \| Quercetin and Silymarin measurement To evaluate the quercetin and silymarin retention in baked cakes, the amount of quercetin and silymarin was measured. The measurement was performed according to the method proposed by Haghi and Hatami (2010) and Bourgeois et al., 2016), using the HPLC technique. The HPLC system (Azura, Knauer Co) with a k-1001 pump and a UV-Visible detector k-260 was used for this purpose. The Teknokrom C18 column was chosen for the analysis, and the UVvisible detector was set at 370 nm and 150 nm for quercetin and silymarin, respectively. The mobile phase to measure quercetin was acetonitrile (7%) (A) and phosphoric acid (B). Injection volume was 20 μl, and the column temperature was ambient temperature. The elution protocols applied for quercetin were: 0-9 min, 75% B; 9-19 min, 75%-25% B; and 19-24 min, 25% B (Bourgeois et al., 2016). \| Microbial load determination The microbial load of cake samples was determined according to the International Commission on Microbiological Specification of Foods (ICMSF, 2009). For this purpose, a unit weight of a cake sample was taken and mixed with 9 ml of sterile distilled water in a test tube. It was then serially diluted until the desirable dilutions were obtained. A volume of 0.01 ml aliquot from each diluent was aseptically transferred into different previously autoclaved plates poured with already sterilized molten media. The plates were then incubated at 28°C for 72 hr. The plates in the incubator were daily checked for growth. The number of grown colonies on each plate was counted using a colony counter. The number of colonies was reported in term of the number of the colony forming unit (CFU). \| Determination of antioxidant activity Free radical scavenging activity, DPPH radical elimination, was de- where absorbance control is the absorbance of DPPH solution without extract. \| Sensory evaluation The hedonic scale as a unique and commonly used scale to measure food product liking and preference yields reliable results. Hence, a 5-point hedonic scale, according to the method employed by the authors in a previous study was conducted to determine the degree of overall liking of the sponge cakes (Ataei & Hojjatoleslamy, 2017). A total of 20 semi-trained panelists were selected, each panelist received five cake slices, cut from the midsection of the cakes maintained at ambient temperature, and was asked to score each sample based on the degree of liking on a fivepoint hedonic scale (one: dislike very much, two: dislike, three: neither dislike nor like, four: like, five: like very much). The panelists received samples. \| Physicochemical characteristics of sponge cake reported that addition of green tea to the sponge cake had no significant effect on the moisture content of the cake. % inhibition of DPPH = 100 × (Absorbance control − Absorbance sample) Absorbance control Table 2, addition of the nettle leaf and MT seed powders caused to decrease the cake volume and to increase its density, compared to the control sample. The cake volume is generally influenced by the characteristics of the fibers added, via both the nature and amount. Fiber addition can disturb the gluten network, and eventually the gluten protein is diluted. A weak gluten network might result in letting gases such as carbon dioxide and water vapor to escape from the cake pores and consequently decrease the cakes volume (Kim et al., 2012;Tsong-Ming lu et al., 2010). It appears that it occurred more considerably in the sample containing the 25% MT, so that it had the lowest volume, while the 12.5% nettle-12.5% MT seed had the highest volume among the cake samples. These findings were consistent with those reported by Aydogdu et al. (2018) in one study of the rheological properties of cake batter and quality of the final product as affected by addition of different fibers. It was stated that a significant correlation was found between the consistency index and the specific volume. As the batter consistency index was increased, cakes with lower specific volume were obtained. The type of fiber is highly important. The consistency index of the batter containing lemon and apple was higher than that with oat and pea. Particularly, addition of lemon fiber resulted in the highest consistency index of batter and the lowest specific volume of cakes. Gómez et al. (2003) reported that the lowest volume of bread was obtained, when the coffee fiber with higher soluble fiber content was added to bread. Furthermore, higher elastic and viscous modulus values inhibited cake development, leading to lower volume. and their texture hardness. The 25% MT seed sample had the lowest moisture content and the highest hardness, while the 12.5% nettle-12.5% MT seed sample revealed the highest moisture content and the lowest hardness. Furthermore, a predictable reduction of the moisture content of the samples occurred during storage time, being concurrent with the hardness increase of the samples. Figure 3 These results correspond to those obtained by Seo et al. (2010) in the study of the sponge cake containing turmeric. Addition of turmeric to the cake increased the cake hardness, gumminess, and chewiness, and thus the softness of the cake was decreased due to its effect on the gluten network. By addition of 129.5% sugar, 0.5% turmeric powder, and 10.0% oil, linear models were selected for the hardness, gumminess, and chewiness (Seo et al., 2010). the storage period of the cake, this network gradually is weakened, the internal strength of the cake structure is decreased, and consequently the cohesiveness and springiness of the cake are decreased (Kim et al., 2012). In this study, the results revealed that the weakening of the gluten network and eventually reduction of cohesiveness and springiness occurred largely when nettle and MT seed were simultaneously added to the cake compared to the control. However, addition of either nettle or MT seed alone led to further disruption of the gluten network and further reduction of the cake cohesiveness and springiness. The results were in agreement with the findings obtained by Tsong-Ming lu et al., 2010) for the cakes added with the green tea powder. They replaced different percentages (0%, 10%, 20%, and 30%) of the cake flour by the green tea powder and reported that the viscosity of the cake batter was higher than that of the control sample, but the cake volumes exhibited a reverse trend. It was attributed to the increased replacement of flour with cellulose and the weakened gluten matrix. Thus, the cohesiveness and springiness were decreased with addition of the green tea powder. Kim et al. (2012) reported that hardness and gumminess were reduced, and cohesiveness and springiness were increased with addition of the cactus Opuntia humifusa powder. This was due to the mucilaginous properties of the cactus pectin. Gums displaying viscous properties in the solution state were added to cake batters in order to increase the moisture retention during baking. Pectin preserves the moisture, increases the volume, springiness and cohesiveness of cake, and decreases its hardness. \| Antioxidant activity Antioxidants serve as preventatives from the destructive and detrimental influence of free radicals, also known as oxidants, on the cells and the resulting diseases. Therefore, assessment of antioxidant and free radical scavenging characteristics of different natural products and additives has been the aim of many research studies. Polyphenols, including phenolic acids, and flavonoids are the major and most popular compounds with antioxidant properties (Kamkar & Khodabakhshiyan, 2017;Viktorova et al., 2019). According to the literature, antiradical properties and inhibitory potential of nettle are greater than those of many synthetic antioxidants (Zeipina et al., 2015). Antioxidant activity of nettle leaves was reported to range from 17.31% to 80.77%. Zeipina et al. attributed this variation to the fertility of soil, the clone and the plant age. (Zeipina et al., 2015). Saa (2017) studied the antioxidant properties of silymarin and reported that silymarin had higher antioxidant properties than BHT had. As different concentrations of silymarin were added to the sunflower oil, the antioxidant activity of the sunflower oil was increased compared to BHT. Table 2 presents the results of the antioxidant activity measurement. The quartic model was selected by the software to fit the data. According to our findings, the highest and lowest DPPH scavenging elimination was observed in the 6.25% nettle-18.75% MT seed sample and the 25% nettle cake. It appears that the combination of nettle and MT seeds used in the cake could exhibit higher antioxidant effect than when used alone. so that as the amount of antioxidants in food increases so do antimicrobial properties (Fazelinasab et al., 2017). Both nettle and \| Antimicrobial activity MT have antimicrobial and antioxidant properties owing to their phenolic and flavonoids content (Hasanloo et al., 2014;Moradi & Amini, 2017). The results of this study showed that the growth of mold in all treatments was lower than that of the control sample. It was due to the presence of nettle and MT seed in the formulations. spectively. The quercetin content ranged from 0 to 116mg, and the silymarin content ranged from 0 to 2,025 mg in the cake samples. \| Quercetin and Silymarin content The linear model was selected as the best model fitting to experimentally measured quercetin and silymarin values. As nettle and MT seed were increased, the quercetin and silymarin content was increased in the samples. Table 4 presents the results of the sensory evaluation performed based on the 5-point hedonic scale. Flavor, texture, and overall acceptance values were fitted by a quadratic model, while odor and color were predicted by linear and cubic models in the best way, respectively. Among all treatments, the cake with 12.5% nettle leaf and 12.5% MT seed gained the highest overall acceptance score. \| Optimization The optimized combination of the nettle leaf powder and the MT seed powder to be used in the cake was determined by the Design-Expert software. Independent variables were assumed to be different amounts of nettle and MT seed, up to 25%, according to the minimum hardness value and the highest sensory test scores, and then optimization was performed by the software. According to The results demonstrated that approximately 10 optimal cakes could theoretically decrease the blood sugar level, which needs further studies. The amount of quercetin and silymarin in 2.5 g of nettle leaves and 2.5 g of MT seed was 0.44% w/w equal to 11 mg and 0.87% w/w equal to 11 mg, respectively. After heating, their amount was 11 mg and 19 mg, respectively. In other words, heat did not have considerable effect on the destruction of quercetin and silymarin, and the substances could resist the thermal processes of the food industry. It is recommended that nettle and MT seeds be used in various food industries, particularly in the cereal industry in order to have acceptable physical and chemical properties and to use its medicinal properties like lowering the blood sugar. In this respect, further studies are required. ACK N OWLED G M ENTS This work was supported by Azad University, Shahrekord branch DATA AVA I L A B I L I T Y S TAT E M E N T The data that support the findings of this study are available from the Shahrekord Azad University upon reasonable request.	v3-fos-license
2021-05-05T00:09:57.105Z	2021-03-09T00:00:00.000	233622552	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.3390/m1195", "pdf_hash": "901d761601148d8deaefe587a6e4a813bcf0c6d7", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1140", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "sha1": "6f66af30f76f4195b935f7d7924fa7045b5740a1", "year": 2021 }	pes2o/s2orc	2-Hydroxybenzoate : A new hybrid compound of chalcone-salicylate (title compound) has been successfully synthesized using a linker mode approach under reﬂux condition. The structure of the title compound has been established by spectroscopic analysis including UV-Vis, FT-IR, HRMS, 1D, and 2D NMR. Then, computational approach was also applied in this study through molecular docking and MD simulation to explore its potency against breast cancer. The results of the molecular docking study showed that the title compound exhibited more negative value of binding free energy ( − 8.15 kcal/mol) than tamoxifen ( − 7.00 kcal/mol). In addition, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. Based on the studies, it can be predicted that the title compound has a cytotoxic activity potency against breast cancer through ER α inhibition and it presumably can be developed as anticancer agent candidate. Introduction Chalcones (1,3-diphenyl-prop-2-en-1-ones) are natural products that are found in several plant species and they have been reported for broad spectrum of biological activities. Their analogues and derivatives have been widely synthesized to explore their potential uses [1][2][3], especially in anticancer drug discovery researches [4]. Some researchers have reported that several hydroxylated and methoxylated chalcones exhibited potent cytotoxic activity against the MCF-7 cell line [4,5]. Besides chalcones, some salicylic acid derivatives have also been reported as potent cytotoxic agents against several cancer cell lines, including MCF-7 [6]. Based on the report, the IC 50 values of some tested compounds are close to the IC 50 of 5-fluorouracil. In addition, some hybrid compounds such as β-carbolinesalicylates have also exhibited potent cytotoxic activity against various cell lines [7]. A hybrid of 4-farnesyltiosalicylate-salicylate also possessed the higher cytotoxic activity against MCF-7 cell line than a withdrawn drug, sorafenib (Nexavar) [8]. Therefore, designing and synthesizing a new compound through the molecular hybridization approach is an interesting option in order to search for new anticancer agent candidates. Some workers have reported that this approach has been proven to be an effective way to develop new multifunctional compounds from hybridization or conjugation of two or more molecules that are expected to have a better biological activity than their parent compounds [9]. In this work, we report the synthesis of a new hybrid compound, (E)-4-(3-(3-(4methoxyphenyl)acryloyl)phenoxy)butyl 2-hydroxybenzoate (title compound) using a linker mode approach. The potency of cytotoxic activity of the title compound against breast cancer was studied in silico using computational approaches (molecular docking and MD simulation). Results and Discussions 2.1. Synthesis of Title Compound (2) In this work, we have successfully synthesized a new hybrid compound of chalconesalicylate (2) by combining both structures of a substituted chalcone analog (1) and salicylic acid, using linker mode approach [9]. The linker was used because the steric hindrance from both molecules that will be linked usually became a problem in molecular hybridization [10,11]. Application of a linker is a way to minimize the steric hindrance [11,12]. In this work, we used the 1,4-dibromobutane as a linker. The reaction was performed under reflux condition in acetonitrile with the presence of potassium carbonate as catalyst, as described in Figure 1. more molecules that are expected to have a better biological activity than their parent compounds [9]. In this work, we report the synthesis of a new hybrid compound, (E)-4-(3-(3-(4-methoxyphenyl)acryloyl)phenoxy)butyl 2-hydroxybenzoate (title compound) using a linker mode approach. The potency of cytotoxic activity of the title compound against breast cancer was studied in silico using computational approaches (molecular docking and MD simulation). (2) In this work, we have successfully synthesized a new hybrid compound of chalconesalicylate (2) by combining both structures of a substituted chalcone analog (1) and salicylic acid, using linker mode approach [9]. The linker was used because the steric hindrance from both molecules that will be linked usually became a problem in molecular hybridization [10,11]. Application of a linker is a way to minimize the steric hindrance [11,12]. In this work, we used the 1,4-dibromobutane as a linker. The reaction was performed under reflux condition in acetonitrile with the presence of potassium carbonate as catalyst, as described in Figure 1. The title compound in pure form was obtained as clear yellow crystal in 33.13% yield, with melting point of 55-57 °C. The purity of the synthesized product was determined by TLC and HPLC analysis, as shown in Supplementary Material. The structure of the title compound has been confirmed by spectroscopic analysis including UV, FT-IR, HRMS, 1D, and 2D NMR. The FT-IR analysis was performed to ensure that the functional groups present in the synthesized compound match the functional groups present in target molecule. Based on the FT-IR spectra, the title compound showed the broad absorption band at 3112 cm −1 . This absorbance showed the presence of hydroxyl (O-H group) in ortho position bonded to carbonyl group of salicylate through intramolecular hydrogen bond formation. The absorption bands at 3076 cm −1 and 1594-1449 cm −1 showed the presence of aromatic C-H and C=C bonds in three aromatic rings of title compound, while the absorption band at 2953 and 2868 cm −1 showed the symmetrical and asymmetrical stretching of C-H in methoxy group of title compound. The presence of carbonyl group of ester and ketone is shown by the two vibration bands at 1672 and 1657 cm −1 , respectively. In addition, the absorption bands around 1298-1028 cm −1 showed the presence of C-O bonds in ether and ester groups of title compound. The other types of vibrations can be seen in Supplementary Material. In addition, the HRMS analysis was also performed to determine the molecular weight of the synthesized compound. Based on the mass spectral analysis, the synthesized compound has a molecular weight corresponding to the molecular weight of the title compound. The molecular ion peak [M + Na + ] of the title compound is found at m/z 469.1616 with intensity of 100%, while the calculated mass is 469.1627, as shown in Supplementary Material. The title compound in pure form was obtained as clear yellow crystal in 33.13% yield, with melting point of 55-57 • C. The purity of the synthesized product was determined by TLC and HPLC analysis, as shown in Supplementary Material. The structure of the title compound has been confirmed by spectroscopic analysis including UV, FT-IR, HRMS, 1D, and 2D NMR. The FT-IR analysis was performed to ensure that the functional groups present in the synthesized compound match the functional groups present in target molecule. Based on the FT-IR spectra, the title compound showed the broad absorption band at 3112 cm −1 . This absorbance showed the presence of hydroxyl (O-H group) in ortho position bonded to carbonyl group of salicylate through intramolecular hydrogen bond formation. The absorption bands at 3076 cm −1 and 1594-1449 cm −1 showed the presence of aromatic C-H and C=C bonds in three aromatic rings of title compound, while the absorption band at 2953 and 2868 cm −1 showed the symmetrical and asymmetrical stretching of C-H in methoxy group of title compound. The presence of carbonyl group of ester and ketone is shown by the two vibration bands at 1672 and 1657 cm −1 , respectively. In addition, the absorption bands around 1298-1028 cm −1 showed the presence of C-O bonds in ether and ester groups of title compound. The other types of vibrations can be seen in Supplementary Material. In addition, the HRMS analysis was also performed to determine the molecular weight of the synthesized compound. Based on the mass spectral analysis, the synthesized compound has a molecular weight corresponding to the molecular weight of the title compound. The molecular ion peak [M + Na + ] of the title compound is found at m/z 469.1616 with intensity of 100%, while the calculated mass is 469.1627, as shown in Supplementary Material. Synthesis of Title Compound The 1D NMR spectra was measured to confirm the structure of the title compound based on the number and chemical environment of protons and carbons. The 1 H NMR spectra of the title compound in CDCl 3 showed a singlet peak (1H) at δ 10.82 ppm due to the presence of hydroxyl group in the aromatic ring of the salicylate moiety. In addition, the aromatic proton signals around δ 7.84-6.87 ppm (12 H) were assigned as aromatic protons in three substituted aromatic rings of the title compound, whereas the signals around δ 4.46-2.01 ppm (11 H) were also assigned as aliphatic protons. Then, the two doublet signals at δ 7.80 ppm (1H) and 7.40 ppm (1H) were assigned as Hβ and Hα protons, respectively. Based on the calculation, the coupling constants (J) of both doublet signals are 15.5 Hz. The coupling constants showed that both protons are in trans (E) position. Based on the interpretation of 1 H NMR spectral data, the synthesized compound has 26 protons, corresponds to the number of protons present in the expected structure. The presence of a methoxy group was observed by the appearance of a singlet signal at δ 3.87 ppm. Then, the appearance of two triplet signals and a multiplet signal in the aliphatic proton region were assigned as four methylene groups (-CH 2 -) 4 in the title compound, as described clearly in Supplementary Material and Table 1. The 13 C NMR spectra of the title compound in CDCl 3 showed that two specific signals at δ 190.19 and 170.15 ppm were assigned as carbonyl signals of ketone and ester, respectively. In addition, the carbon signals around δ 161.70-112.46 ppm were assigned as aromatic carbon signals on three aromatic rings of the title compound including three oxyaryl carbons C2 (161.67), C1 (159.13), and C4 (161.70). Then, five carbon signals around δ 67.42-25.44 ppm were assigned as aliphatic carbon signals of a methoxy group and four methylene carbons in the title compound, as described in Supplementary Material and Table 1. Based on the interpretation of 13 C NMR spectral data, the synthesized compound has 27 carbons, and it is matched with the number of carbon in the expected structure. The assignments of 1 H and 13 C NMR spectra of the title compound were supported by 1 D-TOCSY, 13 C-DEPT, COSY, HSQC, and HMBC spectra, as described clearly in Supplementary Material. The 1 H-1 H correlations in HMBC spectra ensured that the linker is bound to carboxyl group, as depicted in Figure 2. The interpretation result of COSY, HSQC, and HMBC spectra showed that the 1 H-1 H and 1 H-13 C correlations in the title compound are matched with the expected structure. δ 67.42-25.44 ppm were assigned as aliphatic carbon signals of a methoxy group and four methylene carbons in the title compound, as described in Supplementary Material and Table 1. Based on the interpretation of 13 C NMR spectral data, the synthesized compound has 27 carbons, and it is matched with the number of carbon in the expected structure. The assignments of 1 H and 13 C NMR spectra of the title compound were supported by 1 D-TOCSY, 13 C-DEPT, COSY, HSQC, and HMBC spectra, as described clearly in Supplementary Material. The 1 H-1 H correlations in HMBC spectra ensured that the linker is bound to carboxyl group, as depicted in Figure 2. The interpretation result of COSY, HSQC, and HMBC spectra showed that the 1 H-1 H and 1 H-13 C correlations in the title compound are matched with the expected structure. Molecular Docking Study and MD Simulation The molecular docking study was performed to predict the ability of the title compound to bind with the estrogen receptor. This receptor is classified into two subtypes, ERα and ERβ. Both receptors are present in the mammary gland [13]. ERα plays an important role in cell proliferation [14] and pathogenesis of breast cancers [15]. Approximately 75% of breast cancers have positive expression of this specific type of hormonal receptor [16]. Therefore, targeting this receptor is an attractive option for finding a new anticancer agent for breast cancer. In this work, the molecular docking study was performed for the starting material (chalcone analogue), title compound (hybrid of chalcone-salicylate), and tamoxifen as a reference drug to treat breast cancer. The crystal structure of ERα was downloaded from rcsb.org with PDB ID of 3ERT. This crystal structure is bound to a co-crystalized ligand, 4-OHT, one of the major active metabolites of tamoxifen [17]. The docking study was performed in several steps, as described in the procedure section. After the protein and ligands were prepared, a validation of docking protocol is required to ensure the accuracy of the docking result [18][19][20]. The validation of docking protocol can be performed by redocking the co-crystalized ligand (4-OHT) to the prepared receptor. The validation result of docking protocol is presented in Supplementary Material. The result showed that the similarity of binding poses between co-crystalized ligand and re-docking ligand is 81.48% with RMSD value less than 2. These results showed that the docking protocol was valid. The 3D and 2D binding poses of re-docked ligand (4-OHT) is depicted in Supplementary Molecular Docking Study and MD Simulation The molecular docking study was performed to predict the ability of the title compound to bind with the estrogen receptor. This receptor is classified into two subtypes, ERα and ERβ. Both receptors are present in the mammary gland [13]. ERα plays an important role in cell proliferation [14] and pathogenesis of breast cancers [15]. Approximately 75% of breast cancers have positive expression of this specific type of hormonal receptor [16]. Therefore, targeting this receptor is an attractive option for finding a new anticancer agent for breast cancer. In this work, the molecular docking study was performed for the starting material (chalcone analogue), title compound (hybrid of chalcone-salicylate), and tamoxifen as a reference drug to treat breast cancer. The crystal structure of ERα was downloaded from rcsb.org with PDB ID of 3ERT. This crystal structure is bound to a co-crystalized ligand, 4-OHT, one of the major active metabolites of tamoxifen [17]. The docking study was performed in several steps, as described in the procedure section. After the protein and ligands were prepared, a validation of docking protocol is required to ensure the accuracy of the docking result [18][19][20]. The validation of docking protocol can be performed by re-docking the co-crystalized ligand (4-OHT) to the prepared receptor. The validation result of docking protocol is presented in Supplementary Material. The result showed that the similarity of binding poses between co-crystalized ligand and re-docking ligand is 81.48% with RMSD value less than 2. These results showed that the docking protocol was valid. The 3D and 2D binding poses of re-docked ligand (4-OHT) is depicted in Supplementary Material and the comparison of binding poses of co-crystalized (yellow color) and re-docked ligand (atomic coloring) are depicted in Figure 3. Based on the figure, we can observe that they are superimposed. Based on the docking result, the chalcone analogue, title compound, show similar interactions to 4-OHT. Based on the Table 2, we observed tha pound had more negative binding free energy (−8.15 kcal/mol) when co Based on the docking result, the chalcone analogue, title compound, and tamoxifen show similar interactions to 4-OHT. Based on the Table 2, we observed that the title compound had more negative binding free energy (−8.15 kcal/mol) when compared to the chalcone analogue (−6.32 kcal/mol) and tamoxifen (−7.00 kcal/mol). More negative binding free energy indicates that the title compound is more easily bound to the ERα [18]. In addition, we also observed that the binding free energy of the title compound was very close with the binding free energy of 4-OHT as native ligand (−9.02 kcal/mol). The chalcone analogue is able to form one hydrogen bond to Glu353 residue in the active site of ERα. This interaction was also observed between 4-OHT and ERα. However, 4-OHT has more van der Walls and other hydrophobic interactions that might cause 4-OHT to have more negative value of binding free energy than chalcone analogue. In addition, the title compound is also able to form hydrogen bond, but with different amino acid residue (Cys530). This interaction is not observed for other docked compounds. However, there are 18 similar interactions between the title compound and tamoxifen, and there are 19 similar interactions between the title compound and 4-OHT, as depicted in Table 2 MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between ketone carbonyl group with Thr347 (2.02 Å). Based on the literature [20], the hydrophilic interactions with both residues are playing important role in antagonist activity of 4-OHT to ERα. The MD simulation results are presented in Table 3 and depicted in Supplementary Material. A comparison of binding poses of the title compound before and after MD simulation is depicted in Figure 6. Notably, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. In addition, they are still superimposed. ketone carbonyl group with Thr347 (2.02 Å). Based on the literature [20], the hydrophilic interactions with both residues are playing important role in antagonist activity of 4-OHT to ERα. The MD simulation results are presented in Table 3 and depicted in Supplementary Material. A comparison of binding poses of the title compound before and after MD simulation is depicted in Figure 6. Notably, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. In addition, they are still superimposed. Instrumentations The instruments used in synthesis, purification, and structure characterization of the title compound are a set of reflux apparatus, vacuum rotary evaporator (Buchi ® , Flawil, Figure 6. The 3D visualization of superimposition of binding poses of the title compound before and after MD simulation. Binding pose before MD simulation is presented in blue and after MD is presented in yellow. The amino acid residue with hydrogen bond is presented in green and the other maintained interactions during MD simulation are presented in red. Synthesis of (E)-4-(3-(3-(4-Methoxyphenyl)acryloyl)phenoxy)butyl 2-Hydroxybenzoate As much as 9 mmol (1.9431 g) of 1,4-dibromobutane was diluted in 50 mL of acetonitrile in a round bottom flask (mixture 1), 3 mmol (0.762 g) of chalcone analogue was dissolved in 15 mL acetonitrile (mixture 2), and 6 mmol (0.8292 g) of potassium carbonate was added into mixture 2. Then, the mixture 2 was poured into mixture 1. The reaction mixture was refluxed and stirred on the oil bath at 80-85 • C until the reaction was completed (27 h). After the reaction was completed (observed by TLC analysis), the intermediate solution was used immediately without further purification. A mixture of 6 mmol (0.828 g) of salicylic acid and 4 mmol (0.5528 g) of potassium carbonate in 7.5 mL acetonitrile was added to the intermediate solution and then refluxed and stirred at 80 • C until the reaction was completed (48 h). After the reaction was completed (observed by TLC analysis), the mixture of the product was poured in a separatory funnel and washed by distilled water (3 × 15 mL). Then, the solvent was evaporated using vacuum rotary evaporator to afford the crude product. This crude product was purified through a SiO 2 column chromatography with a mixture of n-hexane and ethyl acetate (8:2) as mobile phase to get pure product of chalcone-salicylate. The purity of hybrid compound was confirmed by TLC and HPLC analysis. Then, the structure of hybrid compound was confirmed by spectroscopic analysis including FT-IR, HRMS, 1D NMR ( 1 H NMR, 13 13 Molecular Docking Study The structure of ligands were drawn using ChemDraw Professional 15.0 and a database of ligand was created using MOE 2019.0101 software package (Chemical Computing Group, Tokyo, Japan). The crystal structure of ERα with PDB ID 3ERT was downloaded from http://www.rcsb.org [18][19][20] and was prepared in Discovery Studio 2020 Client to remove the water, co-crystalized ligand, and then, it was further prepared using MOE to minimize the energy. Before running the docking, the placement and refinement method were set as alpha triangle and rigid receptor, respectively. The placement and refinement scores were set as Afinity dG. Then, the placement and refinement poses were set as 100 and 30, respectively. The other parameters were set as default. The 4-OHT as native ligand was re-docked to the prepared protein to validate this docking protocol. After the protocol was validated, the other ligands were docked with same method. The best poses of docking results were selected based on some parameters such as binding free energy, RMSD, and the similarity of interactions compared to the 4-OHT as native ligand and to tamoxifen as breast cancer drug. The best poses of the docked ligands were presented in 2D and 3D visualization using Discovery Studio 2020 Client. MD Simulation The MD simulation was also performed using MOE. Before run the simulation, the algorithm was set as NPA. The start time, checkpoint, and sample time were set as 0, 300, and 0.01, respectively. Then, the forcefield was set as CHARMM27, and the other parameters were set as default. The result of MD simulation was presented in 2D and 3D visualization using Discovery Studio 2020 Client. Then, the protein-ligand interactions before and after MD simulation were compared. Conclusions In this work, we have successfully synthesized a new hybrid compound of chalconesalicylate. Based on spectroscopy analysis, the structure of the synthesized compound was matched with the expected structure. The molecular docking and MD simulation results predicted that the title compound has cytotoxic potency against breast cancer through binding with ERα and it presumably can be developed as a new anticancer agent candidate. However, the in vitro and in vivo evaluations are required to ensure its cytotoxic potency. Supplementary Materials: The following are available online, Figure S1: the clear yellow crystal of the title compound (hybrid compound), Figure S2: TLC chromatogram of hybrid compound (HC) compared to chalcone analog (CA) and salicylic acid (SA) as starting materials under UV lamp, 254 nm. H = n-hexane and E = ethyl acetate, Figure S3: the result of TLC analysis of hybrid compound using various mobile phases: (a) n-hexane: ethyl acetate (9:1), (b) DCM: n-hexane (8:2), (c) D100% = DCM 100%, Figure S4: HPLC chromatogram of hybrid compound, analysis was performed using reverse phase column Shim-Pack VP-ODS (150 × 4.6 mm) with gradient elusion method using water and acetonitrile (HPLC grade) as mobile phase for 20 minutes with flow rate 1 ml/minute, Figure S5: the FT-IR spectra of hybrid compound, Figure S6: The HRMS spectra of hybrid compound, Figure S7: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), Figure S8: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aromatic region, Figure S9: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aliphatic region, Figure S10: the 1D TOCSY spectra of aromatic region of hybrid compound in CDCl 3 (500 MHz), Figure S11: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), Figure S12: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R aromatic region, Figure S13: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R" aromatic region, Figure S14: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R"' aromatic region, Figure S15: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aliphatic region, Figure S16: the 13 C NMR spectra of hybrid compound in CDCl 3 (125 MHz), Figure S17: the overlay of 13 C NMR and DEPT-135 spectra of hybrid compound in CDCl 3 (125 MHz), Figure S18: the overlay of 13 C NMR and DEPT-135 spectra of hybrid compound in CDCl 3 (125 MHz), expansion in aromatic region, Figure S19: the HSQC spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aromatic region, Figure S20: the 1 H-13 C correlations of R aromatic region in HSQC spectra of hybrid compound in CDCl 3 , Figure S21: the 1 H-13 C correlations of R" aromatic region in HSQC spectra of hybrid compound (title compound) in CDCl 3 , Figure S22: the 1 H-13 C correlations of R"' aromatic region in HSQC spectra of hybrid compound in CDCl 3 , Figure S23: the 1 H-13 C correlations of aliphatic region in HSQC spectra of hybrid compound in CDCl 3 (500 MHz), Figure S24: the HMBC spectra of hybrid compound in CDCl 3 , Figure S25: the important 1 H-13 C correlations of aliphatic protons in HMBC spectra of hybrid compound in CDCl 3 , Figure S26: the important 1 H-13 C correlations of hydroxyl group in HMBC spectra of hybrid compound in CDCl 3 , Figure S27: the important 1 H-13 C correlations of R" and R"' aromatic protons and α, β protons in HMBC spectra of hybrid compound in CDCl 3 (a) correlation with C190.19-C159.13, (b) correlation with C144.47-C127.60, Figure S28: 3D and 2D binding poses of re-docked native ligand (4-OHT) to ERα, Figure S29: the 3D and 2D visualization of binding modes of docked compounds to ERα, (a) chalcone analogue, (b) title compound, (c) Tamoxifen, Figure S30: the visualization of MD simulation result of the title compound using Discovery Studio 2020 Client (a) 3D visualization (b) 2D visualization, Table S1: mobile phase composition for HPLC analysis of hybrid compound, Table S2: the result of docking protocol validation.	v3-fos-license
2019-07-31T13:03:55.982Z	2019-01-01T00:00:00.000	198982893	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/10717544.2019.1642420?needAccess=true", "pdf_hash": "3b2b0094ca486038ec5f559646ca6bd169ffc6b9", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1191", "s2fieldsofstudy": [ "Biology", "Engineering", "Chemistry" ], "sha1": "0f79ab592768f31d7ca21f5a34a00a95f1e335c9", "year": 2019 }	pes2o/s2orc	Lipid-chitosan hybrid nanoparticles for controlled delivery of cisplatin Abstract Lipid-polymer hybrid nanoparticles (LPHNP) are delivery systems for controlled drug delivery at tumor sites. The superior biocompatible properties of lipids and structural advantages of polymers can be obtained using this system for controlled drug delivery. In this study, cisplatin-loaded lipid-chitosan hybrid nanoparticles were formulated by the single step ionic gelation method based on ionic interaction of positively charged chitosan and negatively charged lipid. Formulations with various chitosan to lipid ratios were investigated to obtain the optimal particle size, encapsulation efficiency, and controlled release pattern. Transmission electron microscope and dynamic light scattering analysis demonstrated a size range of 181–245 nm and a zeta potential range of 20–30 mV. The stability of the formulation was demonstrated by thermal studies. Cytotoxicity and cellular interaction of cisplatin-loaded LPHNP were investigated using in vitro cell-based assays using the A2780 ovarian carcinoma cell line. The pharmacokinetics study in rabbits supported a controlled delivery of cisplatin with enhanced mean residence time and half-life. These studies suggest that cisplatin loaded LPHNP have promise as a platform for controlled delivery of cisplatin in cancer therapy. Introduction Nanoparticles have shown superiority to other drug delivery systems due to greater solubility for hydrophobic drugs, enhanced circulation time, and targeted drug delivery (Fang et al., 2010). The size gap between endothelial cells in leaky tumor vascular is 100-600 nm, so nanoparticles can reside the tumor site for specific delivery of drugs (Cho et al., 2008). By exploiting the small size of nanoparticles and the unique pathophysiological abnormality of the tumor's vasculature, the nanoparticles more readily extravasate through the relatively leaky vasculature and release drug at the tumor site. The typical poor lymphatic drainage of the tumor contributes to enhanced retention and accumulation of drug at the tumor site (Acharya & Sahoo, 2011). A novel lipid-polymer hybrid drug delivery system has been developed to mitigate disadvantages related to liposomal and polymeric nanoparticles systems (Mandal et al., 2013). Hydrophilic biocompatible polymers, integrated with hydrophobic lipid moieties, can self-assemble into nanoparticles. Phospholipids are nonimmunogenic and broadly applied in formulating biocompatible drug delivery systems such as liposomes. However, lipids have stability problem such as degradation at elevated temperature (Robinson, 1996). Combining the polymer and lipid into a single system ensures more effective drug delivery (Elsabahy & Wooley, 2012). Chitosan is natural cationic polymer with an exceptionally low immunogenicity and greater absorption profile. It also exhibits a facilitated drug release profile in a tumor microenvironment at a low pH (Kumar, 2000). The pH-responsive manner of chitosan makes it suitable as a tumor targeting delivery vector for cancer therapy (Prabaharan, 2015). The lipid component consists mainly of phosphatidylcholine, a natural component of biological membranes, is biocompatible and used in many nanoparticle formulations (Kelmann et al., 2007). The lipid used is LIPOID S75 V R that contains 74% phosphatidylcholine and is suitable for delivery of drug. Studies have shown an 18% increase in the AUC of drug delivered with such nanoparticles (Fricker et al., 2010). Lipidchitosan hybrid nanoparticles have the advantage of both polymeric and liposomal drug delivery systems, nanoparticles can be obtained by interaction of positively charged chitosan and negatively charged lipid using an ionic gelation method (see supplementary data) (Barbieri et al., 2013). Chemotherapy is frequently used for treatment of various forms of cancer. To explore a possible solution to the major drawbacks of chemotherapeutics, including nonspecific and uncontrolled drug delivery, lipid-polymer hybrid nanoparticles were prepared (Tahir et al., 2017). Cisplatin is a first line agent for the treatment of testicular and ovarian cancer and is also used for a number of other malignancies such as lung and esophageal cancer (Comis, 1994). However, the poor solubility of cisplatin in water and oil phases limits the development of nanoparticles with high drug loading and encapsulation (Hamelers & De Kroon, 2007). Conventional intravenous administration of cisplatin results in high toxicity in normal tissues particularly liver and kidney. Encapsulation of cisplatin into a lipid-polymer system has resulted in safer delivery to tumors . Intravenous administration of the cisplatin also leads to rapid clearance due to its low molecular weight (Li et al., 2008a). Application of a lipidpolymer controlled release system can overcome problems associated with low retention time and contribute to improved antitumor efficacy. This approach for the delivery of cisplatin loaded polymer-lipid hybrid nanoparticles achieved a significantly higher cellular internalization and an enhanced cytotoxic effect compared cisplatin alone. In this study, we employed lipidchitosan hybrid nanoparticles with high drug entrapment for controlled delivery of cisplatin to tumor cells. Materials Low molecular weight chitosan was obtained from Sigma Aldrich (Chememie, Germany). Lipid (Lipoid S75) was obtained from Lipoid AG (GmbH, Nattermannallee 1, D-50829 K€ oln, Germany) as a gift sample. Cisplatin was kindly provided by Pharmedic Laboratories PVT (Ltd.) Pakistan. Ethanol and acetic acid were obtained from Fisher Scientific. Cell TiterBlue V R was purchased from Promega V R (WI, USA). The doxorubicin resistant ovarian cell line A2780 was purchased from Sigma Aldrich. Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), and penicillin-streptomycin solution were obtained from CellGro (VA, USA). Hoechst 33342 was purchased from Molecular Probes Inc. (Eugene, OR). Paraformaldehyde was from Electron Microscopy Sciences (Hatfield, PA, USA). Trypan blue solution was obtained from Hyclone (Logan, UT, USA). Preparation of nanoparticles Nanoparticles were prepared as described by Sonvico et al. (2006). Briefly, 10 mg of chitosan was dissolved in 92 mL of 0.1% acetic acid in deionized water. Cisplatin was dissolved in the same solution with continuous stirring. Lipid was dissolved in pure ethanol (25 mg/mL). The ethanolic solution was then added drop wise to the drug solution. The nanoparticles formed via ionic gelation. The nanoparticles with lipid and chitosan at various ratios were centrifuged at 10,000 rpm for 30 min followed by lyophilization. Six different formulations with lipid: chitosan ratios ranging from 5:1 to 60:1 were characterized for their size, surface charge, entrapment efficiency, and drug loading. In case of cell uptake studies that require incorporation of fluorescent dyes, rhodamine 123 and rhodamine-PE were dissolved in ethanol along with lipid and then ethanolic solution was added drop wise to chitosan solution. Nanoparticles size and surface charge The prepared cisplatin-loaded formulations were analyzed for the size, polydispersity index (PdI), and surface charge using a Zeta Sizer (Malvern Ver.7.11 Royston, UK). Dynamic light scattering was used for the determination of size and surface charge. The measurement was performed at 25 C and a 90 scattering angle in triplicate for each sample. Determination of drug contents and entrapment efficiency The entrapment efficiency was calculated by an indirect method by measuring the amount of unentrapped drug in the supernatant after centrifugation (Xu et al., 2006). The drug contents were determined using UV Spectrophotometry (Spectrum Scientific). The absorbance of standard and samples were measured at 210 nm. The determination was performed in triplicate. Transmission electron microscopy (TEM) Surface morphology of the cisplatin loaded hybrid nanoparticles was determined by transmission electron microscopy (JEOL USA, Inc.). Sample of LPHNPS with lipid to chitosan ratio 20:1 were applied directly on the grid. The excess of sample was removed from the grid, and suitable images were taken at different magnifications. Differential scanning calorimeter (DSC) DSC analysis was performed to evaluate the physical form of cisplatin within the nanoparticles (Alam et al., 2014). The calibration was done using indium for the temperature and heat flow. Samples were placed on one pan and another aluminum pan was used as a reference. The samples were heated over the temperature range of 25-400 C. Thermogravimetric analysis (TGA) Thermogravimetric analysis was performed to measure the change of mass of LPHNPs over a range of temperatures. The dried formulation of chitosan-lipid nanoparticles was used. The LPHNP formulation (38.1 mg) was put on the gravimetric analyzer and the temperature varied from 25 to 500 C. The change in the mass with the change of temperature was recorded for each second till the completion of run time. In vitro drug release The drug release study was conducted using a dialysis bag method (Avgoustakis et al., 2002) . The dialysis membrane of MWCO 10-12 kDa was used. The drug release study was performed for all six formulations and the release of the drug from LPHNPs was compared with the pure drug solution. The LPHNPs formulation was suspended in PBS (pH 7.4) in dialysis bags at 37 ± 0.5 C with constant stirring at 100 rpm. All the formulations were loaded with 5 mg of cisplatin. Samples were withdrawn at predetermined time intervals. Kinetic modeling was applied using zero-order, firstorder, Higuchi and Korsmeyer-Peppas models. Cell viability Cell viability studies were performed on A2780 cells. Cells (5000) were seeded in each well of 96-well plates. After 24 hours incubation, cells were treated with cisplatin-loaded LPHNP and cisplatin solution at a cisplatin concentration range of 1.25 to 50 mg/mL to check the effect at different concentrations (Wang et al., 2014). Formulations were washed out after four hours treatment and replaced with fresh RMPI media. The effect on cytotoxicity was observed at 20 and 44 hours using a Cell TiterBlue V R assay measuring fluorescence from cells on plate reader (BioTek). Fluorescence microscopy Cells (100,000) were seeded on microscope cover glasses in a 12-well plate. After incubation, cells were treated with the cisplatin loaded LPHNP formulation labeled with Rh-PE and a control blank formulation for 4 h. After four hours, cells were washed with PBS, pH 7.4, and fixed with PBS pH 7.4 containing 2% paraformaldehyde (PFA) for 30 min at room temperature. Cells were then washed with PBS, pH 7.4, three times and stained with 10 mg/mL Hoechst 33342 in PBS pH 7.4 for 15 min. Cells were washed again with PBS, pH 7.4, and mounted on Fisherbrand Superfrost V R microscope slides with Fluoromount G V R mounting buffer (SouthernBiotech, AL, USA) for analysis by fluorescence microscopy using KEYENCE(BZ-X710) fluorescence microscope. Cellular uptake The cellular association of cisplatin loaded LPHNP was evaluated using flow cytometry (Beckton Dickinson FACS Calibur TM , NJ, USA). Cells (500,000) were seeded in each well of six-well plates. After overnight incubation, cells were treated with Rh-123 containing formulations (0.5 mol%) for four hours in serum complete media. After that, cells were detached using trypsin and washed with PBS, pH 7.4, three times and centrifuged at 1000 rpm for 5 min and the resuspended in 300 mL of PBS. The fluorescence signal was obtained using a 488 nm laser and the emission was recorded using a 530/30 nm wavelength filter. A total of 10,000 gated live cell events were collected. In vivo pharmacokinetics Twelve healthy rabbits were selected for the pharmacokinetics studies (average weight, 2.4 ± 0.4 kg) after taking approval from the research and ethics committee on animals of Faculty of Pharmacy and Alternative Medicines, The Islamia University of Bahawalpur (PhD-IUB-Ph-13). Rabbits were obtained from the animal house of Faculty of Pharmacy and Alternative Medicines, The Islamia University of Bahawalpur. Studies support rabbits as an animal model to study pharmacokinetic comparable to human (Nair et al., 1981). Rabbits were divided into two group of six. One group was administered 4 mg/kg of the cisplatin drug solution via an intravenous route as a reference while the other group received 4 mg/kg of the cisplatin loaded lecithin-chitosan hybrid nanoparticles (Huo et al., 2005). Rabbits were fasted overnight before the start of the study. Blood samples were collected at defined time intervals over a period of 24 hours and centrifuged immediately to separate plasma. The plasma samples were treated with nitric acid: perchloric acid to remove plasma proteins (Navolotskii et al., 2015). Plasma was stored at -20 C. The samples were treated to separate protein and 20 mL samples were used to determine drug concentration using HPLC. HPLC was performed using C18 column with flow rate 1.5 mL/min and UV detector at 250 nm, while acetonitrile/methanol/water was used as a mobile phase in concentration of 30/40/30. Particle size and surface charge Size and polydispersity index have effects on the loading and release of drug from the nanoparticle formulation (Souza et al., 2014). The particle size and zeta potential of all formulations are shown in (Table 1). The particle size of all developed formulations varied between 181 nm and 245 nm, which is suitable for passive targeting of tumor via an EPR effect (Cho et al., 2008). The surface charge at the 5:1 ratio of lipid-chitosan ratio to a 30:1 ratio remained positive due to the electrostatic interaction of chitosan and lipid. The cationic charge of chitosan prevails. However, as the concentration of lipid was increased, the zeta potential value shifted from positive to negative. At a ratio of 20:1 a monodispersity and a better stability profile were observed because of an interaction of negative and positive charges in appropriate concentration (Sonvico et al., 2006). Morphology Morphology of the lipid-chitosan nanoparticles was studied using transmission electron microscopy. The TEM images showed the spherical shaped nanoparticles ($200 nm) with the lipoplex structure of the lipid and polymer entangled with each other through positive and negative charge in the hybrid nanoparticles (Figure 1). The images exhibited a lipoplex morphology with some lipid covering which prevented diffusion of drug and water penetration into the system the shell of chitosan-lipid provides long circulating characteristics (Mandal et al., 2013). Entrapment efficiency and drug loading Entrapment of drug in the nanoparticles was calculated by an indirect method. Parameters were evaluated at different concentrations of lipid and chitosan while keeping the concentration of drug constant. All the formulations showed more than 70% encapsulation efficiency. Lipid-polymer nanoparticles at a ratio 20:1 showed the highest encapsulation efficiency (89.2%). The hybrid nanoparticles demonstrated significantly increased encapsulation and drug loading as compared to polymeric nanoparticles, owing to the presence of a lipid layer . The presence of a lipid coat on the outer surface of the polymer increased drug encapsulation, and the lipid layer provided structural integrity and prevented leakage of hydrophilic drugs (Cheow & Hadinoto, 2011). Differential scanning calorimetry The DSC analysis was performed to check the crystalline/ amorphous nature of the drug, polymer and lipid in the formulations (Figure 2(A)). The formulation of cisplatin loaded lipid-chitosan hybrid nanoparticles demonstrated a typical dehydration peak at 102 C. This is due to the evaporation of water associated with the drug. The cisplatin pure drug showed a dehydration peak at around 100 C due to the water associated with it and then an endothermic peak at 270 C, which relates to the melting point of the drug (Dixit et al., 2015). However, there is no sharp endothermic peak of cisplatin in the nanoparticle formulation, attributable to a loss of its crystalline nature in the hybrid nanoparticle formulation, as reported in a previous study (Dixit et al., 2015). The curve of chitosan typically showed a broad endothermic peak over the temperature range of 70-150 C, corresponding to the loss of water of crystallization and melting point of the chitosan (Cervera et al., 2011).Thermal decomposition of chitosan starts at 300 C, which is an exothermic process and its peak is seen at around 320 C (Drebushchak et al., 2006). This behavior of chitosan is also seen in the formulation as an endothermic peak starting at 70 C and broader endothermic peak in the formulation is due to the presence of chitosan and lipid and cisplatin conversion into an amorphous form. Thermogravimetric analysis TGA was performed to measure the percentage of weight loss of LPHNPs over time over a certain range of temperatures (Figure 2(B)). TGA of cisplatin, chitosan, and lipid formulation were also performed. The results show a weight loss by cisplatin occurs at 270 C, which corresponds to the melting point of cisplatin (Prodana et al., 2014). Chitosan exhibited a slight weight loss at 70 C and at around 290 C a gradual weight loss (Javaid et al., 2018). Weight loss in the lipid mixture began at about 250 C. The melting points of all three components of the formulation were different from each other and showed a peak of weight loss in their respective range confirming the absence of any physical interaction between components and thermal stability of the LPHNPs formulation. In vitro drug dissolution Drug release studies were performed in phosphate buffer saline pH 7.4 at 37 C at 100 rpm using the dialysis membrane method (Jeong et al., 2008). None of the formulations demonstrated burst release of cisplatin after immediate immersion in the medium, this is in accordance with previous studies (Spenlehauer et al., 1986). The release pattern of the polymerlipid hybrid system also showed an absence of immediate burst release and suggested that drug can be released in a controlled manner. There was a sustained release over 24 hours from the lipid-polymer complex (See supplementary data) (Mandal et al., 2016). The results indicated that best fit model for the formulation is a Korsmeyer-Peppas model which is usually followed in lipid-polymer hybrid systems (Tran et al., 2015). The controlled release of drug was attributed to the lipid layer and a minor contribution of polymer and a change in the concentration of lipid effect on the rate of release of the drug from formulations (Chan et al., 2009). The lipid-polymer hybrid nanoparticles with drug distributed inside the polymer showed a better controlled release profile. The release of drug from the polymer matrix depends on diffusion. Further lipid layering prolongs the drug release (Li et al., 2008). The value of release component (n) suggests that the drug follows a super case II transport mechanism in most of the formulations (Sonawane et al., 2016). Formulation with 20:1 ratio was selected for further studies. Cell viability The results were observed using Cell Titer Blue. Six different concentrations of drugs were used 50, 25,12.5, 6.25, 3.12, 1.6 mg/mL. The concentrations with more significant difference are shown in Figure 3. The results showed that blank lipidpolymer hybrid nanoparticles have no effect on cell cytotoxicity and indicate the biocompatibility of the formulation (Figure 3). After 24 hours of incubation the drug solution had greater cytotoxicity to A2780 ovarian cell lines as compared to cisplatin loaded lipid-polymer hybrid nanoparticles. This is due to the drug solution's easy access to the cell. The drug is in the lipid and polymer layer and takes time to release. After 48 hours, the cisplatin loaded lipid polymer hybrid formulation had more cytotoxic effect on A2780 ovarian cancer cell lines compared to drug solution, which showed that the release of drug from the formulation could be controlled and produce a greater effect with passage of time. Kim et al. also proved that sustained release of cisplatin from glycol chitosan nanoparticles had higher toxicity compared to cisplatin solution after 48 hours . Hence the cytotoxic effect of cisplatin is time and concentration dependent. Optimum activity after 48 hours is in agreement with the previous studies (Zhang et al., 2017). It is also important that after 48 hours, the cisplatin loaded lipidpolymer hybrid nanoparticles showed a 20-30% increased cytotoxicity at same concentration as compared to 24 hours and up to 50% of cell death at the lowest concentration. These results are in agreement with the controlled release formulation of cisplatin observed by Reardon et al. (2017). The controlled release of formulation is also evident from the in vitro dissolution and in vivo pharmacokinetics studies. Cellular uptake studies A cell uptake study is an important tool for evaluation of delivery potential of the nanoparticle system. Cell uptake was studied with flow cytometry and cell association by fluorescence microscopy. The results of flow cytometry indicated that there was an eightfold increase in the uptake of cisplatin LPHNPs loaded with fluorescence dye Rh-123 as compared to the control (Figure 4(A)). The cell association of the nanoparticles was also observed by staining with the fluorescent dyes. Hoechst produces a blue color after interaction with DNA of the cell. Rhodamine PE produces the red fluorescence in cells. The images that nanoparticle show internalization in A2780 ovarian cancer cells. Lipid present in the LPHNP may facilitate the internalization by interacting with the lipid layer at the surface of the cell membrane (Guo et al., 2015). Yue et al. showed that chitosan nanoparticles can deliver drug to the perinuclear space and produce fluorescence (Yue et al., 2011). The cells exhibited an association with LPHNPs and produced fluorescence (Figure 4(B)). In vivo pharmacokinetics To determine the effect of loading cisplatin in lipid-polymer hybrid nanoparticles, analysis of drug was performed by injecting the same amount of drug in two groups of six rabbits. The concentration versus time relationship graph was drawn to evaluate the pharmacokinetics parameters ( Figure 5). These studies suggest that combination of polymer and phospholipid can strongly influence the pharmacokinetics properties and could serve as a vehicle for controlled delivery (Feng et al., 2011). The time to reach maximum concentration was 1 ± 0.05 hours in the drug solution group and 6 ± 0.15 hours in the lipid-chitosan hybrid formulation group. Maximum serum concentration was observed at 1.01 mg/mL compared to 4.07 mg/mL in the drug solution group. A lower peak concentration is considered effective for prolonged exposure and reduced side effects of chemotherapeutic drugs (Cheng et al., 2015). The half-life of the cisplatin loaded lipid-chitosan formulation was 14.0 ± 0.4 hours which was much greater than 1.25 ± 0.04 hours for the drug solution group, which indicates a prolonged release of drug in controlled manner. Mean residence time (MRT) was 20.8 ± 0.3 hours compared to 6.0 ± 0.5 hours in cisplatin drug solution, indicating that the lipid-polymer hybrid formulation provides a controlled release of cisplatin. The lipid layer has been observed to protect drug from protein binding and improve absorption (Mei et al., 2013). A similar increase in MRT was observed for a cisplatin formulation by Nakano et al. (1997). The AUC with the lipid-chitosan formulation was 9.83 ± 0.3 mg h/mL as compared to drug solution at 18.4 ± 0.5 mg h/mL. Similarly, the lipid-polymer formulation group shows a 4.6-fold increase in volume of distribution of cisplatin as compared to the cisplatin solution. Kai et al also showed a 4.2-fold increase in volume of distribution of cisplatin (Kai et al., 2015). Overall, the pharmacokinetics parameters of cisplatin were vastly improved with the lipid-polymer hybrid nanoparticle system. Conclusion Cisplatin loaded lipid-chitosan hybrid nanoparticles were successfully fabricated and characterized for various physicochemical parameters including particle size, entrapment efficiency, drug loading and thermal behavior, compatibility of excipients and crystalline behavior and in vitro drug release profile. The best formulation with a suitable combination of lipid and chitosan showed monodispersity, small size, and a controlled release profile. Further release of drug was affected by an increase in the lipid concentration. The rate of release of drug was attributable to both polymer and lipid. The release of drug is controlled by the polymer matrix and further by a lipid layer that prevents leakage of drugs. There was an absence of burst release of cisplatin due to its entrapment in the inner polymer layer and outer lipid covering. Cell viability studies confirmed the cytotoxic effect on the A2780 ovarian cancer cell line over a 48-hour period. Cell uptake studies showed increased cellular uptake of LPHNPs. In vivo pharmacokinetics studies in rabbits showed a controlled release behavior. Toxicity studies in rats provided safety profile of the LPHNPs. Based on the characterization and invitro release profile, the lipid-chitosan hybrid nanoparticles can provide controlled delivery of cisplatin and act as a useful platform for the potential delivery of cisplatin to tumors. Further studies in tumor animal models should be undertaken to examine the effectiveness of treatment of tumors with LPHNPs.	v3-fos-license
2021-10-20T15:59:53.744Z	2021-09-01T00:00:00.000	240527592	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2073-4344/11/9/1103/pdf?version=1631960820", "pdf_hash": "1831527272b4e31e136f1bb02e6dcb20a17edb4f", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1210", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "e2dbe379ac606d3322d15aed69e48fc095a12a74", "year": 2021 }	pes2o/s2orc	Evaluation of 3,3′-Triazolyl Biisoquinoline N,N′-Dioxide Catalysts for Asymmetric Hydrosilylation of Hydrazones with Trichlorosilane A new class of axial-chiral biisoquinoline N,N′-dioxides was evaluated as catalysts for the enantioselective hydrosilylation of acyl hydrazones with trichlorosilane. While these catalysts provided poor to moderate reactivity and enantioselectivity, this study represents the first example of the organocatalytic asymmetric reduction of acyl hydrazones. In addition, the structures and energies of two possible diastereomeric catalyst–trichlorosilane complexes (2a–HSiCl3) were analyzed using density functional theory calculations. Results and Discussion We recently developed the modular method to synthesize axial-chiral 3,3′-triazolyl biisoquinoline N,N′-dioxides from readily available triazoles and optically pure 3,3′dibromo-biisoquinoline N,N′-dioxide [57] as part of our longstanding interests in developing new chiral Lewis-bases [58][59][60][61][62]. Since this new class of catalysts was found capable of activating trichlorosilane at relatively low temperatures, we envisioned that they might be able to catalyze the reduction of acyl hydrazones under conditions where no background reaction would take place. We set out on our investigation by employing benzoyl hydrazone 1a as a model substrate (Scheme 2). To our delight, the background reaction was found negligible at −40 °C, and catalyst 2a provided hydrazine (R)-3a in 48% yield with 53% ee (entries 1 and 2). Next, we looked at several solvents that are commonly used for trichlorosilane-mediated reactions. Chloroform provided the product with a lower yield but with a slightly higher enantioselectivity (34% yield, 66% ee). Acetonitrile gave 3a in a comparable yield but with a lower ee of 32%. The reaction in tetrahydrofuran afforded the opposite enantiomer (S)-3a with a much lower yield and selectivity (entry 5). Overall, dichloromethane was found optimum. We tested with twice as much solvent since benzoyl hydrazone 1a was not fully dissolved under the reaction conditions (entry 6). However, it did not improve the result. Previously, we found that 4 Å molecular sieve was an effective acid scavenger for adventitious HCl in trichlorosilane [63], but its use did not positively impact the outcome in the present case (entry 7). The use of 3.0 equivalent of trichlorosilane did not improve the yield, either (entry 8). As the protecting groups on hydrazones are known to influence their reactivities and enantioselectivities in many cases (e.g., see; [19]), we evaluated the Boc and Cbz protected hydrazones (1b and 1c in entries 9 and 10, respectively). While enantiomeric excesses of the corresponding products were slightly higher than that of the benzoyl counterpart, both Boc and Cbz protecting groups adversely affected the yields. Since the C=O unit of Boc or Cbz group is more Lewis basic than that of the benzoyl counterpart, we tested a less Lewis basic hydrazone (1d). However, the yield decreased to 14% albeit with a slightly higher enantioselectivity (entry 11). Overall, the present method was found to be quite sensitive to reaction solvents and the hydrazone protecting groups. Next, we evaluated different triazolyl groups on the biisoquinoline that are expected to play important roles on the catalyst's reactivity and selectivity (Scheme 3, entries 1-4). Catalyst 2a was clearly superior to the other three catalysts (2b-d) in terms of their reactivity. Catalyst 2c was more enantioselective than others, albeit with a low yield. These results indicate that the reactivity and selectivity of this new class of catalysts can be tuned by changing the triazolyl groups. We also compared these triazolyl catalysts to conventional 3,3′-substituted biisoquinoline N,N′-dioxides (entries 5 and 6). To our surprise, neither 2e nor 2f promoted the reaction although 2f was as reactive as 2b-d for the hydrosilylation of an N-phenyl ketimine with trichlorosilane [57]. Nonetheless, these results clearly demonstrated that this new class of axial-chiral biisoquinolines is indeed complementary to the existing Lewis-base catalysts and bode well for the development of their applications. As we determined the basic reaction parameters, we proceeded to evaluate the extent to which the present catalytic system could enantioselectively promote the hydrosilylation of various benzoyl hydrazones with trichlorosilane (Scheme 4). To our surprise, a paramethyl substitution (3e)-which is a minimal change from the model substrate (3a)-had a detrimental effect on the chemical yield while the corresponding meta-substitution (3f) did not. An ortho-methyl substitution (that is known to push the aromatic ring out of conjugation with a C=N bond) completely shut down the reaction (3g). Eventually, it was gleaned that the para-substitutions have adverse effects on the reactivity but not much on the enantioselectivity regardless of their electronic nature (3e, 3h-m) (these enantioselectivities are approximately the same). A heteroaromatic hydrazone was moderately less reactive and selective than the model substrate (3n). Although an ethyl group at the C=N bond is in general expected to lead to an increased steric demand in the TS, it did not affect the reactivity in the present case (3o). It is noteworthy that a cyclohexyl counterpart provided the opposite sense of enantioselection to the model substrate (3p). Differentiation of the two similar alkyl groups franking the C=N bond was difficult by the present catalytic system (3q). Unreacted hydrazones and corresponding ketones were the major components of the crude reaction mixtures besides the desired products, and no significant amounts of by-products were observed for 1a-1q. An α,β-unsaturated hydrazone was not a viable substrate for this method as the conjugate reduction took place (3r) [64]. We also tested a 1.0 mmol scale reaction with the model substrate. To our delight, it provided essentially the same result (Scheme 5), demonstrating a potential robustness of the method. Furthermore, catalyst 2a was quantitatively recovered after a flash column chromatography on silica gel (see Supplementary Materials for details). The recovered catalyst promoted the model reaction (1a on 0.25 mmol scale) with no loss in reactivity and enantioselectivity. The structure of the active reducing species generated from a chiral catalyst and HSiCl 3 is considered to play a central role for the enantioselectivity of a reaction. Even with notable advances made in this area (selected references; [35][36][37][38][39][40][41][42][43][44][45]), it remains largely elusive and significantly challenging to control the relative populations and reactivities of diastereomeric reducing species that are reversibly produced from a chiral Lewis-base and trichlorosilane. C 2 -symmetric 2a and HSiCl 3 can give rise to two diastereomeric complexes that are expected to have different enantioselectivities (as long as 2a acts as a bidentate Lewis-base). Therefore, the binding geometry of 2a to HSiCl 3 was investigated computationally with the aim of shedding some light on the structure of the active reducing species. To our delight, 2a was found to bind to HSiCl 3 through its two oxygen atoms (i.e., a C 2 -symmetric bidentate ligand), generating two diastereomeric complexes ( Figure 1). Complex 1 was found to be 1.91 kcal/mol lower in energy than the complex 2. The analysis of their electrostatic potentials revealed an anion-π-type interaction between the hydrogen atom in complex 1 or one of the chlorine atoms in complex 2 and the phenyl ring. It should be mentioned that a pileup of electron density occurs at the peripheral atoms of a hypervalent silicon complex of this kind [59,65,66]. This anion-π-type interaction appears to effectively lock the conformation of the benzyl group at least at the ground state, leading to a welldefined chiral pocket around the hypervalent silicon atom. This computationally identified non-covalent attractive interaction could offer a possible basis to rationalize why 2a (benzyl) was as enantioselective as 2d (1-adamantyl), and why 2c (benzhydryl) was substantially more enantioselective than 2d (53% ee, 54% ee and 74% ee, respectively; Scheme 3). Conclusions Axial-chiral 3,3′-triazolyl biisoquinoline N,N′-dioxides offer potential for functioning as effective catalysts for the asymmetric hydrosilylation of acyl hydrazones with trichlorosilane. Since catalyst's triazolyl units indeed tuned the reactivity and enantioselectivity of the reaction and our modular synthesis allows ready access to a variety of 3,3′-triazolyl biisoquinoline N,N′-dioxides, potential for the identification of more effective catalysts than those presented herein clearly exits. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Funding: Support is currently provided by the National Institute of Health (1R15 GM139087-01) and the Florida Institute of Technology. Data Availability Statement: Data is contained within the article and Supporting Information. Computed structures of the two lowest energy minima for the 2a-HSiCl 3 complex (i.e., two diastereomeric complexes) calculated with PBEh-3c//C-PCM (DCM). Both are shown with balls-and-sticks (left) and space filling (right) models. Molecular electrostatic potentials are also shown in the space filling models. Complex 1 (top) is 1.91 kcal/mol lower in energy than complex 2 (bottom).	v3-fos-license
2018-04-03T01:23:03.491Z	2014-02-20T00:00:00.000	13888768	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://downloads.hindawi.com/journals/tswj/2014/581737.pdf", "pdf_hash": "90481d560658fc509071ff8c1bc25cb95b3cec17", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1245", "s2fieldsofstudy": [ "Chemistry", "Biology" ], "sha1": "bd459d1cfe228752f2877924f43534f4495ae5e8", "year": 2014 }	pes2o/s2orc	A Mini Library of Novel Triazolothiadiazepinylindole Analogues: Synthesis, Antioxidant and Antimicrobial Evaluations A new series of novel triazolothiadiazepinylindole analogues were synthesized with an aim to examine possible antioxidant and antimicrobial activities. The titled compounds (3a–z) were obtained in good yield by reacting 5-(5-substituted-3-phenyl-1H-indol-2-yl)-4-amino-4H-1,2,4-triazole-3-thiols 1a–c with 3-(2,5-disubstituted-1H-indol-3-yl)-1(4-substituted phenyl)prop-2-en-1-ones 2a–i. All the newly synthesized compounds were characterized by IR, 1H NMR, mass spectroscopic and analytical data. The synthesized analogues were tested for antioxidant and antimicrobial potency. Among the tested compounds 3a–c and 3j–l have shown very promising free radical scavenging activity and total antioxidant capacity. Compounds 3d–f, 3m–o, and 3s–z have shown excellent ferric reducing antioxidant activity. An outstanding antimicrobial activity is observed with compounds 3a–c and 3j–l. Introduction Antioxidants [1][2][3] act as "free radical scavengers" hence to prevent or slow the damage done by the free radicals [4][5][6]. Free-radical-induced oxidative stress associated with several cellular toxic processes including oxidative damage to protein, and DNA, membrane lipid oxidation, enzyme inactivation, and gene mutation leads to carcinogenesis [7]. Antioxidants are involved in processes such as immunity, protection against tissue damage, and reproduction and prevent growth or development caused by free radicals [8][9][10]. Antioxidants are useful in the prevention and treatment of Parkinson's and Alzheimer's disease [11][12][13]. Heterocycles constitute one of the major areas of organic chemistry and play important roles in drug discovery. Many of the best selling drugs currently in use contain one or more heterocyclic rings. Several fused heterocycles as well as biheterocycles are referred to as privileged structures [14]. Among them, sulfur-and nitrogen-containing heterocyclic compounds have maintained the interest of researchers and their unique structures led to several applications in different areas [15]. Triazoles and their derivatives constitute an important class of heterocyclic compounds and their analogues have been reported to possess various biological activities such as antimicrobial [16], anti-inflammatory [17], antihypertensive, anti-HIV [18], anticancer, and antitumor [19,20]. Several compounds containing 1,2,4-triazole rings known as drugs like fluconazole, posaconazole, alprazolam, [21] and triazolothiadiazepine analogues represent a wellknown class of drug substances at different stages of research, which possess antiviral [22] and antimicrobial properties [23]. Indole is a heterocycle of great importance in biological systems [24,25]. The indole moiety is present in a number 2 The Scientific World Journal of drugs currently [26] in the market; in our previous approaches, we have described some new indole analogues with highly potent antioxidant, DNA cleavage and antimicrobial activities [27][28][29][30]. Interestingly, we have developed a new green protocol for the synthesis of rapid and clean synthetic route towards mini library of triazolothiadiazepinylindole analogues, which showed in vitro antioxidant and antimicrobial activities. Materials and Methods 2.1. Chemistry. All chemicals used in this investigation were of analytical grade and were purified whenever necessary. Melting points of the synthesized compounds were measured in open capillaries and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F 254 aluminium sheets (MERCK). Iodine vapour was used as detecting agent. IR spectra were recorded in KBr on PerkinElmer and FTIR spectrophotometer (] max in cm −1 ). 1 H NMR and 13 C NMR spectra on BRUKER AVENCE II 400-MHz NMR spectrometer and the chemical shifts were expressed in ppm ( scale) downfield from TMS as an internal reference. The mass spectra were recorded on LC-MSD-Trap-SL instrument. The elemental analysis was performed by using FLASH EA 1112 SERIES instrument. General Procedure for the Synthesis of Compounds 3a-z (1) Conventional Method. To a solution of substituted indolyltriazole 1a-c (0.01 mol) in acetic acid substituted chalcones 2a-i (0.01 mol) were added. The reaction mixture was refluxed 3-4 hrs. The completion of the reaction was monitored by TLC. After the completion, the reaction mixture was poured to a beaker containing 100 mL of ice-cold water. The crude products thus separated were filtered and recrystallized from ethanol to yield target compounds 3a-z. (2) Microwave Oven Method. A mixture of substituted indolyl triazole 1a-c (0.01 mol) and substituted chalcones 2ai (0.01 mol) was powdered, mixed, and introduced to borosil sample crucible containing few drops of acetic acid. This was subjected to microwave irradiation for 10 minutes with 70% microwave power. After the completion (TLC), reaction mixture was brought to room temperature, washed with ethanol, and recrystallized to get the title compounds 3a-z which were found to be in good purity (TLC) and excellent yield. Antioxidant Activities (1) Free Radical Scavenging Activity. Free radical scavenging activity was done by DPPH method [32]. Different concentrations (25 g, 50 g, and 100 g) of samples and butylated hydroxy anisole (BHA) were taken in different test tubes. The volume was adjusted to 100 L by adding MeOH. Five milliliters of 0.1 mM methanolic solution of DPPH was added to these tubes and shaken vigorously. The tubes were allowed to stand at 27 ∘ C for 20 min. The control was prepared as above without any samples. The absorbances of samples were measured at 517 nm. Radical scavenging activity was calculated using the following formula: (2) Total Antioxidant Capacity. Various concentrations of samples (25 g, 50 g, and 100 g) were taken in a series of test tubes. To this, 1.9 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) was added. The tubes were incubated at 95 ∘ C for 90 min and allowed to cool. The absorbance of each aqueous solution was measured at 695 nm against a blank. Antioxidant capacities are expressed as equivalents of ascorbic acid. Ascorbic acid equivalents were calculated using standard graph of ascorbic acid. The values are expressed as ascorbic acid equivalents in g per mg of samples. (3) Ferric Reducing Antioxidant Power. Various concentrations of samples (25 g, 50 g, and 100 g) were mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 ∘ C for 20 min. Next, 2.5 mL of 10% trichloroacetic acid (w/v) was added. From this solution, 5 mL was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride and absorbance was measured spectrophotometrically at 700 nm. BHA was used as standard. Candida tropicalis ATCC 8302 and Candida albicans ATCC 60193by applying the agar plate diffusion technique [33]. Dilution process was adopted at 25 g, 50 g, and 100 g/mL concentrations, respectively. The activity is compared with reference drugs gentamycin for antibacterial and fluconazole for antifungal activity. The zone of inhibition after 24 hr of incubation at 37 ∘ C in case of antibacterial activity and 48 hr in case of antifungal activity was compared with that of standards. to thiadiazepine C=N stretching, respectively. The 1 H NMR spectrum of 3a has exhibited a singlet at 12.47 ppm due to indole NH and peak at 11.63 ppm is due to indole NH which is also D 2 O exchangeable. A multiplet between 7.31-8.47 ppm corresponds to twenty aromatic protons present in the molecule and a peak at 5.65 ppm is assigned for the -CH= of thiadiazepine ring proton. The 13 C NMR spectrum of compound 3a has shown peaks at 108, 111, 113, 117, 118, 118, 118, 120, 123, 125, 126, 126, 126, 128, 128, 128, 128, 129, 129, 129, 130, 132, 133, 134, 135, 138, 138, 144, 145, and 166. The mass spectrum of compound 3a has shown molecular ion peak at m/z 712 [M] +• which is corresponding to molecular weight of the compound. The above spectral data supports the formation of compound 3a. Results and Discussion Various new triazolothiadiazepinylindole analogues synthesized during the present investigation are listed in (Table 1). Biological Activities. The compounds 3a-z were screened for their antioxidant (free radical scavenging, total antioxidant capacity, and ferric reducing antioxidant power) and antimicrobial activities. Antioxidant Activities (1) Free Radical Scavenging Activity. The target compounds were screened for free radical scavenging activity by DPPH method [32]. The samples were prepared at concentrations of 25, 50, and 100 g/100 L and butylated hydroxy anisole (BHA) was taken as standard. DPPH is a stable free radical in a methanolic solution. Because of the unpaired electron of DPPH, it gives a strong absorption maxima at 517 nm in the visible region (purple color). In addition, the unpaired electron of the radical becomes paired in the presence of a hydrogen donor (a free radical scavenging antioxidant), decreasing the absorption. Among the compounds tested 3ac and 3j-l have shown very promising free radical scavenging activity. The increased activity is due to the existence of halogen substitution at the five positions of both indoles. The hydrogen of indole NH could be donated to the DPPH to form DPPH free radical; by the presence of phenyl ring at the third position of indole, the DPPH free radical will be stabilized by the resonance. Compounds 3d-f, 3mo, and 3s-x containing halogen atom at five positions of indole and a methyl group at another indole ring have shown moderate activity, whereas compounds 3g-i, 3pr, and 3y-z have shown the least activity compared with the standard. The bar graph representation of percentage of free radical scavenging activity is displayed in Figures 1 and 2. 6 The Scientific World Journal (2) Total Antioxidant Capacity. Total antioxidant activity was performed to all the newly synthesized compounds [34]. analogues were screened for in vitro antibacterial activity against ( Table 2) gram-negative bacteria Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) and gram-positive bacteria Staphylococcus aureus (S. aureus) at 25 g/mL, 50 g/mL, and 100 g/mL concentrations, respectively. Gentamycin was used as standard. The zone of inhibitions was measured in mm for each concentration. Most of the screened compounds were found to have significant antibacterial activity. Compounds 3a-c and 3j-l have shown very good activity against all the three bacterial strains. Compounds 3d-f, 3m-o, and 3s-x have shown moderate activity and compounds 3g-i, 3p-r, and 3y-z have shown the least activity. Antifungal screening of the compounds was carried out in vitro against two fungi strains Candida tropicalis and Candida albicans at 25 g/mL, 50 g/mL, and 100 g/mL concentrations using fluconazole as standard. Among the tested indole analogues the majority of compounds exhibited moderate to significant antifungal activity. Conclusions We have synthesized titled compounds 3a-z by economic, better yield, and safer methods through the formation of compounds 1a-c and 2a-i under thermal and microwave condition. The compounds 3a-z were subjected for their antioxidant and antimicrobial screening. Very potent antimicrobial, scavenging and antioxidant activity was observed with compounds containing halogens at the fifth position of indoles. Excellent ferric reducing activity was observed with compounds containing electron donor group at five positions The Scientific World Journal 9 of one/both indoles. Therefore, the findings will provide a great impact on chemists and biochemists for further investigations in the indole field in search of molecules possessing potent antioxidant and antimicrobial activities.	v3-fos-license
2019-04-13T13:02:44.594Z	2019-04-11T00:00:00.000	109939333	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://nottingham-repository.worktribe.com/preview/1828317/Nick%20Besley%20Probing%20Elusive%20Cations.pdf", "pdf_hash": "0d75e314d6403a3f051a3dc5603593cd1e76c212", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1302", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "343f91d50d89b3fc628570b89cd1a48c7fb7f22f", "year": 2019 }	pes2o/s2orc	Probing Elusive Cations: Infrared Spectroscopy of Protonated Acetic Acid Protonated carboxylic acids, (RCOOH)H + , are the initial intermediates in acid-catalyzed (Fischer) esterification reactions. However the identity of the isomeric form is under debate. Surprisingly, no optical spectra have been reported for any isomer of the protonated carboxylic acid monomer, despite it being a fundamental organic cation. Here, we address these issues by using a new approach to prepare cold He-tagged cations of protonated acetic acid (AA), which entails electron ionization of helium nanodroplets containing metastable dimers of AA. The protonated species is subsequently probed using infrared photodissociation spectroscopy and, following a comparison with calculations, we identify the two isomers whose roles are debated in Fischer esterification. These are the carbonyl-protonated E , Z isomer and the metastable hydroxyl-protonated isomer. Our technique provides a novel approach that can be applied to other elusive ionic species. 3 The mechanism of the acid-catalyzed (Fischer) esterification of carboxylic acids was first explored in detail in the 1930s. 1,2 This early mechanistic work suggested that initial protonation occurs at the hydroxyl oxygen atom. In the case of an acetic acid monomer (AA), this leads to formation of the structure labelled as prot-OH in Figure 1. However, NMR studies of the protonation of simple carboxylic acids carried out in the 1960s indicated that protonation occurs exclusively at the carbonyl oxygen. 3,4 The NMR work was performed under aggressive conditions with essentially 1:1 concentrations of magic acid (FSO3H-SbF5) and carboxylic acid. At -60 °C a double peak structure was observed, indicating that a single isomer, the E,Z isomer, is formed ( Figure 1). It is now a standard assumption in modern organic chemistry textbooks that protonation occurs at the carbonyl oxygen in Fischer esterification. 5 However, a recent study combining DFT calculations with electrospray ionization mass spectrometry reached a different conclusion. 6 This work suggests that carbonyl protonation 4 produces an intermediate which is too stable to react with an alcohol to yield an ester. Instead, they found that only hydroxyl protonation can lead to subsequent reaction and this occurs because of the production of the reactive acylium ion (cf. prot-OH structure in Figure 1). This proposal agrees with the original mechanistic predictions from the 1930s, 1,2 creating new uncertainty about the reaction mechanism. The protonated monomer of AA has previously been studied using ab initio calculations and mass spectrometry. [7][8][9][10][11][12] However, despite being a rather fundamental organic cation, no optical spectra have been reported for it or for the monomer of other simple protonated carboxylic acids, (RCOOH)H + . Inokuchi and Nishi have measured infrared (IR) photodissociation spectra in the OH stretching region for protonated formic acid cluster ions, (HCOOH)nH + , and their AA analogues, for n ³ 2. 13 However, this technique could not access the protonated monomers because the IR photon has insufficient energy to induce fragmentation. In a separate study, Hu et al. reported IR photodissociation spectra of protonated AA cluster ions. 14 However, instead of seeing a reduction in ion signal at the mass of the protonated monomer, m/z 61, an enhancement was observed that was attributed to IR absorption by a larger ion, (CH3COOH)COOH + , which subsequently fragmented to give (CH3COOH)H + and CO2. A similar problem was encountered in the case of protonated propanoic acid. 15 We introduce an experimental technique for measuring IR spectra of He-tagged cations, which utilizes electron ionization of neutral clusters embedded in helium nanodroplets. Mass spectrometry has been used extensively to study electron and multiphoton ionization of doped droplets, 16 but there have been few IR spectroscopic studies of the ions produced in such processes. 17 In Figure 2b and c, we present IR depletion spectra for He-tagged protonated AA, HeN-(CH3COOH)H + , where N = 1 (m/z 65) and 3 (m/z 73). A spectrum for the untagged ion (N = 5 0 at m/z 61) is shown in the Supporting Information ( Figure S1) rather than here because it contains broad spectral features due to hot bands, as well as congestion arising from competing photofragmentation pathways. In contrast, spectra for the He-tagged species show relatively narrow and well resolved features. Also shown in Figure 2 is an IR spectrum recorded at m/z 57. Because this corresponds to a mass of one helium atom below that of an isolated protonated AA monomer, the observed depletion in ion signal cannot be attributed to IR absorption by protonated AA. However it is possible that spectral features from a smaller ion could appear in mass channels 65 and 73 6 through the addition of more helium atoms. The spectrum at m/z 57 therefore acts as a background spectrum and the two weak features at ~3430 and ~3505 cm -1 are assigned to He3-HOCO + . 18 Consequently, peaks observed at ~3430 cm -1 in the m/z 65 and 73 spectra are assigned to He5-HOCO + and He7-HOCO + , respectively. We now turn our attention to the new features observed at m/z 65 and 73, which are not present at m/z 57, and are therefore assignable to IR absorption by He-tagged protonated AA. Most notable are the two strongest peaks at ~3480 and ~3550 cm -1 . The shape of the lower frequency band clearly changes with the number of attached helium atoms (from N = 1 to 3) and this is attributed to frequency shifts caused by helium solvation effects. Band shifts induced by helium solvation have also been reported for a different cation, C60 + , although that study used electronic rather than vibrational spectroscopy. 19 However, the N-dependent behavior is a separate topic and will be the subject of future work. From here onwards, we choose to focus on identifying the structural isomers of protonated AA that are formed. To assist with the spectral assignments, we have performed ab initio calculations at the MP2/aug-cc-pVTZ level of theory for the four isomers of protonated AA illustrated in Figure 1. For each isomer considered, the ZPE-corrected relative energy, as well as the harmonic vibrational frequencies and intensities, were calculated. We summarize the key findings in Table 1 and present further results in Figure S2 and Table S1. To account for anharmonicity, the calculated OH stretching frequencies have been scaled by an empirical factor of 0.955, derived from a comparison of the computed OH stretching vibrational frequency for the AA neutral monomer and the known experimental gas phase value of 3583 cm -1 . 20 Before comparing our calculated spectra for the untagged isomers of protonated AA (N = 0) with the experimental spectrum measured at m/z 65 (N = 1), we consider whether the helium atom causes a significant shift in the OH stretching frequency. For this purpose, we performed calculations for each isomer with an attached helium atom in various locations relative to the ion, which led to the identification of 18 stable structures ( Figures S3 and S4, Table S2). The addition of a single helium atom is found to shift the OH stretching frequencies by < 2 cm -1 in all but one case: the exception was a He-tagged Z,Z structure, where a shift of 5 cm -1 was calculated. These small shifts allow us to simply compare the four predicted spectra for the bare ions with the Hetagged spectrum recorded here. In Figure 3, the calculated IR spectra for the untagged isomers are plotted below the spectrum measured at m/z 65 for He- Next we consider whether the Z,Z and E,E conformers could be responsible for any of the experimental features in Figure 3a. The weak band at 3533 cm -1 could potentially be assigned to the Z,Z conformer, or alternatively to a combination band of the E,Z conformer that gains intensity via coupling to the strong E,Z band at 3550 cm -1 . Furthermore, the E,E conformer could provide a minor contribution to the intensity of the 3550 cm -1 band, but again the evidence is circumstantial. In summary, the formation of isomers E,E and Z,Z can't be ruled out, but if they are present then the populations are likely to be small and therefore we will not discuss them further. We now turn to the two weak, higher frequency bands centered at 3629 and 3698 cm -1 ( Figure 3a). These features are absent from the spectrum at m/z 57 ( Figure 2a) and are therefore assigned to He-tagged protonated AA. However, we can immediately exclude the OH stretching fundamentals for isomers E,E, E,Z and Z,Z because the calculated frequencies are at least 70 cm -1 to the red of both experimental bands (Figure 3). We have also considered assignments to overtone and combination bands for the E,Z conformer, both for the bare and He-tagged ions. For this purpose, anharmonic vibrational frequency calculations were performed. The calculated IR intensities for all overtone and combination bands between 3300 and 4000 cm -1 were found to be < 5 km mol -1 , which is very weak compared with the OH stretching fundamental transitions of the E,Z conformer (> 250 km mol -1 ). Further reasons for discounting these bands are provided in the Supporting Information. A more viable assignment is to the OH stretching fundamentals of isomer prot-OH, for which the calculated and experimental band frequencies agree to within 18 cm -1 and this close agreement is illustrated in Figure 3. Furthermore, the calculated band intensities of >65 km mol -1 (Table 1) are at least an order of magnitude higher than those predicted for overtone and combination bands in this spectral region, which also supports this assignment. The calculated structure for isomer prot-OH (Figure 3d) indicates that hydroxyl protonation results in a substantial lengthening of one of the C-O bond lengths to 2.437 Å, such that we are left essentially with the acylium ion, CH3CO + , forming a weakly bound complex with H2O. Indeed, the experimental bands centered at 3629 and 3698 cm -1 lie fairly close to the known OH stretching frequencies for a gas-phase water monomer but are red-shifted, as expected when complexation occurs. Another facet of these experiments is that they enable the measurement of spectra not only for the He-tagged cation, but also the neutral precursor. Knowledge of the precursor structure can then be used to predict which cation structures are likely to form following electron ionization. Our technique has the further benefit of rapid cooling by the surrounding helium, enabling access to metastable structures. For example, for AA we have previously shown that two metastable dimers of neutral AA are formed inside helium nanodroplets rather than the lowest energy cyclic dimer. 21 The two dimers are structurally similar but have distinct hydrogen bonding motifs, a feature that can be exploited to generate different protonated AA conformers. The calculated dimer structures are shown in Figures 4a and b, whilst the superimposed blue boxes indicate potential structures for protonated AA that would require minimal structural rearrangement. Remarkably, the identified structures match those assigned in the current study. Therefore, these calculations provide an insight into likely mechanisms for generating the observed protonated AA structures using electron ionization of metastable AA dimers (Figures 4a and b). Furthermore, the proven capability to create protonated species suggests that this technique has potential applications of biological, as well as chemical, importance. Given our assignments, the ability to prepare the E,Z and prot-OH isomers from metastable AA dimers opens the possibility to investigate their potential roles as intermediates in Fischer esterification. 6 If these isomers were prepared inside helium nanodroplets, just one molecule per droplet, then subsequent pickup and reaction with an alcohol could allow the first stage of the esterification reaction to be studied. Energy to initiate the reaction would be provided by photoabsorption and any subsequent depletion of reactants would be monitored using IR spectroscopy, thus allowing the relative reactivity of the two isomers to be determined. In this way, the helium nanodroplets would act as nanocryostats allowing the isolation and investigation of specific reaction pathways. In Experimental Methods Protonated AA was created by electron ionization of liquid helium nanodroplets doped with neutral clusters of AA, a process which generates cations that may be tagged with one or more 13 helium atoms. The tagged ions, which are intrinsically cold because weakly bound helium atoms will readily detach from hot ions, were probed by the beam from a tunable IR laser. Spectra were recorded as a function of IR wavelength by monitoring any reduction in ion signal induced by resonant photoabsorption. These measurements were made mass-selectively using a quadrupole mass spectrometer and our technique is a variant of IR photodissociation spectroscopy. The instrument has been described previously [21][22][23][24] but this is the first time it has been used to measure IR spectra for He-tagged ions rather than neutral molecules or clusters inside helium nanodroplets. Further details can be found in the Supporting Information. Supporting Information Experimental details; IR spectra showing fragmentation pathways; computational method; calculated structures, energies and frequencies for bare and He-tagged protonated acetic acid	v3-fos-license
2018-05-21T22:38:44.931Z	2018-03-06T00:00:00.000	21693334	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.iucr.org/e/issues/2018/04/00/eb2005/eb2005.pdf", "pdf_hash": "c1d34318f15db22e4ffb971de2707eb1a244637a", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1311", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "c1d34318f15db22e4ffb971de2707eb1a244637a", "year": 2018 }	pes2o/s2orc	Crystal structure of aquachlorido(nitrato-κ2 O,O′)[1-(pyridin-2-yl-κN)-2-(pyridin-2-ylmethylidene-κN)hydrazine-κN 2]manganase(II) The asymmetric unit comprises a discrete molecule in which the cation MnII is heptacoordinated. The environment around the cation is an almost perfect pentagonal bipyramid. In the crystal, extensive hydrogen bonding leads to a three-dimensional framework. The search for novel manganese(II) compounds having interesting magnetic properties, using 1-(pyridin-2-ylmethylidene)-2-(pyridin-2-yl)hydrazine (HL) as a tridendate ligand, led to the preparation of the title mononuclear material, [MnCl(NO 3 )(C 11 H 10 N 4 )(H 2 O)], and the determination of its structure by XRD. The asymmetric unit comprises a discrete molecule in which the cation Mn II is heptacoordinated. The environment around the cation is an almost perfect pentagonal bipyramid. The base is defined by the two N atoms of the pyridine rings, the N atom of the imino function of the ligand and the two O atoms of the chelating bidentate nitrate ligand. The apical positions are occupied by a Cl atom and a water molecule. In the crystal, there are numerous hydrogen bonds of the types Ow-HÁ Á ÁONO 2 and C-HÁ Á ÁONO 2 , which generate layers parallel to the bc plane in which the ligands in the axial positions point into the interlayer space. These axial ligands give rise to hydrogen bonds of the types Ow-HÁ Á ÁONO 2 , Ow-HÁ Á ÁCl, N-HÁ Á ÁCl and C-HÁ Á ÁCl, leading to a threedimensional framework. The chain bridging the two pyridine rings is disordered over two sets of sites in a 0.53 (2):0.47 (2) ratio. Chemical context Although very much studied, the coordination chemistry of manganese remains very interesting as this metal can have several degrees of oxidation and its complexes can display different coordination numbers and geometries that are not always easily predicted (Chiswell et al., 1987;Baldeau et al., 2004;Mikuriya et al., 1997). Although the coordination numbers four and six are the most common in the coordination chemistry of manganese, the coordination numbers five, seven and eight are also observed (Louloudi et al., 1999). As a result of the multiple degrees of oxidation of this metal, interest in the coordination chemistry of manganese complexes is considerable. The involvement of manganese in various important biological processes such as oxidation of water by photosynthetic enzymes (Whittaker & Whittaker, 1997), hydrogen peroxide disproportionation by catalase (Meier et al., 1996), superoxide dismutase (SOD) (Schwartz et al., 2000), ribonucleotide reductase and lipoxygenase (Baffert et al., 2003) increases the interest of scientists in this metal. These examples from nature inspire chemists to search for biomimetic catalysts of these metalloenzymes that are highly selective and cause little damage to the environment ISSN 2056-9890 (Krishnan & Vancheesan, 1999). Manganese complexes are also used as catalysts in many processes such as epoxidation of alkene (Castaman et al., 2009), oxidation (Wegermann et al., 2014) and hydrogenation of ketones (Bruneau-Voisine et al., 2017). The involvement of the metal center in these processes depends as much on its degree of oxidation as on its coordination number in the complex. The synthetic procedures adopted are essential for yielding complexes with specific properties. In this context, for the synthesis of the heptacoordinated Mn II title complex, we use a one-pot synthesis method, which is an efficient approach to prepare a large variety of coordination compounds (Oyaizu et al., 2000). Manganese dichloride tetrahydrate is mixed with the synthesized organic ligand (HL), which provides three soft nitrogenbinding sites in the presence of nitrate anions that can act with hard oxygen-binding sites to yield a mononuclear heptacoordinated manganese(II) complex. Structural commentary The structure of the title complex is shown in Fig. 1. The asymmetric unit comprises a discrete molecule in which the cation Mn II is heptacoordinated. The coordination polyhedron of the Mn II center is best described as a distorted pentagonal bipyramid with an MnN 3 O 3 Cl chromophore. The basal plane is occupied by two nitrogen atoms from the pyridine rings, one nitrogen atom from the imino function and two oxygen atoms from the chelating bidentate nitrate group. The metal-bound ligand nitrogen atoms exhibit angles of 69.85 (7) (N1-Mn1-N2) and 69.62 (7) (N2-Mn1-N4) which are slightly different from the ideal angle for a regular pentagon (72 (14) Å ]. The two pyridine rings are connected by a disordered chain C-CH=N-NH-C in which the bond lengths are slightly different from those observed in similar complexes; this may be related to the observed disorder. Two intramolecular hydrogen bonds, C1-H1Á Á ÁO2 and C11-H11Á Á ÁO3, are also observed in the structure ( Figure 1 An ORTEP view of the title compound, showing the atom-numbering scheme and intramolecular hydrogen bonds as dashed lines. Displacement ellipsoids are plotted at the 50% probability level. Supramolecular features In the crystal, the complex molecules are linked by hydrogen bonds, giving rise to a three-dimensional network (Fig. 2, Table 2). The structure is built up from pentagonal bipyramids around the Mn II atom, which are assembled in layers parallel to the bc plane. These layers are interconnected by hydrogen bonds. The coordinating axial water molecule points into the interlayer space and act as a hydrogen-bond donor towards oxygen atom O2-NO 2 and chlorine atom Cl1 (Fig. 2) via the hydrogen bonds O1W-H1WBÁ Á ÁO2 ii and O1W-H1WAÁ Á ÁCl1 i , [symmetry codes: (i) x + 1, y, z; (ii) Àx + 2, Ày + 1, Àz + 2]. The axial Cl1 atom points also in the interlayer space and acts as a hydrogen-bond acceptor toward N3-H3NÁ Á ÁCl1 iii and C6-H6Á Á ÁCl1 iii [symmetry code: (iii) Àx + 1, Ày + 1, Àz + 1]. The combined hydrogen bonds link the layers into a three-dimensional framework. Within a layer, the molecules are interconnected by hydrogen bonds of the type C-HÁ Á ÁONO 2 [C8-H8Á Á ÁO4 iv -NO 2 ; symmetry code: (iv) x, y, z À 1]. , Cu II (Mesa et al., , 1989Rojo et al., 1988;Ainscough et al., 1996;Chowdhury et al., 2009;Mukherjee et al., 2010;Chang et al., 2011), Co II (Gerloch et al., 1966), Ni II (Chiumia et al., 1999) and Zn II (Dumitru et al., 2005;. In all cases, the ligand behaves as a tridentate ligand acting through the soft nitrogen donor atoms from the two pyridine rings and the imino function. The hard protonated nitrogen atom remains uncoordinated in all complexes. Synthesis and crystallization A mixture of 2-hydrazinopyridine (1 mmol) and 2-pyridinecarbaldehyde (1 mmol) in ethanol (10 mL) was stirred under reflux for 30 min. A mixture of ammonium nitrate (3 mmol) and MnCl 2 Á4H 2 O (1 mmol) in ethanol (10 mL) was added to the solution. The mixture was stirred for 30 min and the resulting yellow solution was filtered and the filtrate was kept at 298 K. A yellow powder appeared after one day and was collected by filtration, yield 65%. Analysis calculated for [C 11 H 12 Refinement Crystal data, data collection and structure refinement details are summarized in Table 3. All H atoms ( CH, NH and OH 2 groups) were optimized geometrically (C-H = 0.93, N-H = 0.86 and O-H = 0.87-0.91 Å ) and refined as riding with U iso (H) = 1.2U eq (C) or 1.5U eq (O). The chain bridging the two pyridine rings is disordered. This disorder may be explained by the fact that the sequence of atoms C(Py)-CH N-NH-C(py) overlaps with the sequence C(py)-NH-N CH-C(py), meaning two orientations of the ligand. In such a case, for the refinement it was assumed that the C atom of the CH group from one chain and the NH atoms from the second chain occupy the same position. The same relates inversely. The occupancy factor refined to 0.53 (2):0.47 (2). Aquachlorido(nitrato-κ 2 O,O′)[1-(pyridin-2-yl-κN)-2-(pyridin-2-ylmethylidene-κN)hydrazine-κN 2 ]manganase(II) Crystal data Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.	v3-fos-license
2019-04-03T13:09:14.433Z	2018-12-07T00:00:00.000	92113091	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=89652", "pdf_hash": "33dde01f7922aba8293c67e93ad758b12b730759", "pdf_src": "ScienceParseMerged", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1334", "s2fieldsofstudy": [ "Medicine", "Agricultural and Food Sciences" ], "sha1": "702e79136e23526d3fd778817816e5ccf4935f82", "year": 2018 }	pes2o/s2orc	Dietary Effect of Squalene on Lipid Metabolism of Obese / Diabetes KK-Ay Mice and Wild-Type C 57 BL / 6 J Mice The purpose of this study was to evaluate the influence of squalene (SQ) on plasma and hepatic lipid levels of obese/diabetic KK-Ay mice and wild-type C57BL/6J mice. SQ supplementation significantly increased the HDL cholesterol of KK-Ay mice, which was paralleled with no significant difference in the total and non-HDL cholesterol levels. The increase in HDL cholesterol was also found in the plasma of normal C57BL/6J mice, but the difference was not significant. SQ administration significantly increased neutral lipids (NL) in the liver of KK-Ay mice, while no significant difference was observed in the polar lipids and the total cholesterol levels. The increase in NL was primarily due to the increase in TAG. However, the cholesterol level significantly increased due to SQ intake in the liver of C57BL/6J mice, while no significant difference was found in other lipid levels. The present study suggests that SQ may effectively increase HDL cholesterol level, an important anti-atherosclerotic factor, especially in subjects with metabolic disorders. Introduction Squalene (SQ), a naturally occurring triterpenic hydrocarbon, is widespread in nature, especially among shark liver oil and olive oils.The shark Centrophorus squamosus has been reported to contain approximately 14% liver oil, mainly composed of SQ (nearly 80% of the oil) [1].Although shark liver oil remains the richest natural source of SQ, its use is limited by persistent organic pollutants and shark resource protection [1].SQ is also found in many vegetable oils in va-S.Liu rying concentrations.Among vegetable oils, oil from Amaranthus sp. is known to have the highest concentration of SQ (up to 73.0 g/kg oil) [2] [3].The SQ content in olive oil is also high (5.64 g/kg oil) compared to other vegetable oils such as that derived from hazelnuts (0.28 g/kg oil), peanuts (0.27 g/kg oil), corn (0.27 g/kg oil) and soybean (0.10 g/kg oil) [4]. Olive oil intake has shown beneficial health effects [5] [6] [7] [8] and these effects have been recognized to partially derive from olive oil minor compounds, mainly phenolic compounds.In addition, due to the relatively high content in olive oil compared with other vegetable oils, SQ is also regarded as a contributing factor in the reduced risk of diseases associated with olive oil intake.To date, SQ has been reported to show anticancer, anti-inflammatory, antioxidant, skin protection, liver protection, and neuroprotective activities [9] [10] [11] [12].In particular, many studies have been conducted on the relationship between the reduced risk of cancer due to olive oil intake and the role of SQ as a vital dietary cancer chemopreventive agent [9] [10] [11] [12] [13]. Furthermore, the higher intake of olive oil in Mediterranean countries compared to northern European countries is related to the low incidence of cardiovascular disease (CVD) [10].CVD is the leading cause of morbidity and mortality worldwide.In many cases, CVD is caused by atherosclerosis, a chronic vascular disease that generally occurs in the aorta and muscular-type arteries, such as coronary arteries, brain arteries, renal arteries and carotid arteries [14]. Although the exact cause of atherosclerosis is still unknown, modification and deposition of lipids in the vascular wall can induce this event.Among types of lipid deposition, low density lipoprotein (LDL) cholesterol deposition, especially oxidized LDL, is regarded as a main cause.Thus, cholesterolemia is known as a major inducer of atherosclerosis, and much attention has been paid to the hypocholesterolemic activity of olive oil components, such as oleic acid and other minor compounds including SQ [10]. SQ is known as an important intermediate for the biosynthesis of phytosterol or cholesterol in plants, animals and humans [11], and its endogenous synthesis begins with the conversion of acetyl coenzyme A to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), followed by the reduction of HMG-CoA to mevalonate, mediating HMG-CoA reductase.Thus, the involvement of SQ in cholesterol synthesis is easily expected, and the possibility has been demonstrated for the use of SQ as a biomarker to evaluate endogenous cholesterol biosynthesis (Brown et al., 2014).Indeed, numerous studies from this viewpoint have explored this possibility of using several experimental approaches in both animals and humans [11]. Many studies on the effect of dietary SQ on blood cholesterol levels have been [16].The varied effects of SQ are hypothesized to be due to differences in the experimental approaches and sexes [10] [15] [17]. The controversial effect of SQ on blood cholesterol level has been replicated in animal models.Several researchers have reported a reduction of blood lipid levels, including cholesterol in rats after SQ intake [18] [19] [20], while other researchers have found increases in the cholesterol levels in normal animals by SQ feeding [21] [22].In addition, Chmelik et al. [23] found the increase in high density lipoprotein (HDL) cholesterol levels of C57Bl/6J SPF mice fed SQ, while the total cholesterol and LDL cholesterol levels decrease with SQ feeding.This specific increase in HDL cholesterol by SQ intake was also found in three different mouse models (wild-type, Apoa1-and Apoe-deficient) [17]. Many animal and human studies have revealed SQ as a promising agent in CVD prevention [10] [11] [15].The effect of SQ has been explained via several different mechanisms, including the elimination of cholesterol as fecal bile acids [16], inhibition of HMG CoA reductase by dietary SQ due to negative feed-back regulation [24], inhibition of oxidized LDL uptake by macrophages [25], stimulation of reverse cholesterol transport [26], inhibition of isoprenaline-induced lipid peroxidation [18], and attenuation of homocysteine-induced endothelial dysfunction [11].These mechanisms are basically dependent on the antioxidant activity of SQ and the involvement of SQ in cholesterol metabolism [1] [10] [11] [15]. However, SQ's role in plasma lipids is not yet clear, although hyperlipidemia, especially hypocholesterolemia, is regarded as a major risk factor for CVD.In the present study, we assessed the effect of SQ on the plasma lipid content of animal models.For animals, we used C57BL/6J genetic background mice.These mice have been widely used due to their higher predisposition to atherosclerosis development [27].Furthermore, we compared the effect of SQ in normal and obese/diabetes mice.Obesity and diabetes are a major risk factor for CVD [28] [29].Therefore, the comparison may help to elucidate the effect of SQ on the reduction of CVD risk. Animals and Diets Obese/diabetic KK-A y mice (male, four weeks old) and wild-type C57BL/6J mice (male, four weeks old) were obtained from the Japan CREA Co., Tokyo, Japan. The mice were housed individually in an air-conditioned room (23˚C ± 1˚C and 50% humidity) with a 12 h light/12 h dark cycle.After acclimation feeding of a normal rodent diet MF (Oriental Yeast Co., Ltd, Tokyo, Japan) for 1 week, the mice were randomly divided into 3 groups of seven and were fed experimental diets for four weeks (Table 1).The body weight, diet and water intake of each mouse was recorded daily. Ethics The research project was approved by the Ethical Committee at Hokkaido University, and all procedures for the use and care of animals for this research were performed under approval by the Ethical Committee of Experimental Animal Care at Hokkaido University. Sample Collection Mice were sacrificed under diethyl ether anesthesia after 12 h fasting on day 28.Blood samples were taken from the caudal vena cava of the mice.A portion of blood was used for blood glucose analysis, while the remaining part was stored for lipid analysis.Blood glucose was measured using a blood glucose monitor, namely, the Glutest Neo Sensor (Sanwa Kagaku Kenkyusyo Co. Ltd., Aichi, Japan).This sensor is an amperometric sensor with flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase and ( ) Blood Lipid Analysis The blood plasma analysis was performed by the Analytical Center of Hakodate Medical Association (Hakodate, Japan).The analysis included measurement of the following parameters: total cholesterol, HDL cholesterol, non-HDL cholesterol, triacyglycerols (TAG) and phospholipids. Hepatic Lipid Analysis Total lipids (TL) was extracted from the liver with chloroform/methanol (2:1, v/v) [30].The TL (ca.20 mg) was further separated on a Sep-Pak Silica cartridge (Waters Japan, Tokyo, Japan) by elution with chloroform (70 mL) and methanol (50 mL).The neutral lipids (NL) and polar lipids fractions were eluted with chloroform and methanol, respectively.Both lipid contents in the liver (mg/g liver) were calculated from the TL level per liver weight.Total cholesterol and TAG were measured using enzymatic kits (Cholesterol E-test and Triglyceride E-test) obtained from Wako Pure Chemical Industries, Osaka, Japan. The fatty acid composition of the TL was determined by gas chromatography (GC) after conversion of fatty acyl groups in the lipid to their methyl esters.The fatty acid methyl esters (FAME) were prepared according to the method of Prevot and Mordret [31].Briefly, 1 mL of n-hexane and 0.2 mL of 2 N NaOH in methanol were added to an aliquot of total lipid (ca. 10 mg), vortexed and incubated at 50˚C for 30 min.After incubation, 0. In a preliminary experiment, we found that SQ and docosahexaenoic acid (22:6n-3, DHA) could not be separated clearly on the chromatogram using the Omegawax-320 capillary column.Therefore, the prepared FAME sample was Quantitative Real-Time PCR Total RNA was extracted from the livers of mice using RNeasy Lipid Tissue Mini Kits (Qiagen, Tokyo, Japan) according to the manufacturer's protocol.The cDNA was synthesized from total RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems Japan Ltd., Tokyo, Japan).Quantitative real-time PCR analyses of individual cDNA were performed with ABI Prism 7500 (Applied Biosystems Japan Ltd., Tokyo, Japan) using TaqMan Gene Expression Assays (Applied Biosystems Japan Ltd., Tokyo, Japan).Gene expression was normalized to the reference gene GAPDH. The mRNA analyses were performed on genes associated with lipid metabolism, which included liver X receptor (LXR), sterol regulatory ele- HMG-CoA Reductase Analysis Activity of HMG-CoA reductase, a key enzyme of hepatic cholesterol synthesis, was measured according to the procedure described by Rao and Ramakrlshnan Statistical Analysis Results are expressed as mean ± SEM.Statistical significances between groups were evaluated by one-way ANOVA with post hoc comparisons (Scheffe's F-test).Differences with P < 0.05 were considered to be statistically significant. Results 3.1.Food Intake, Water Intake, Weight Gain, Tissue Weights, Blood Glucose Levels, Serum and Hepatic Lipid Parameters The weights of major tissues were not significantly different, except for a significant increase in the liver weight of KK-A y mice fed SQ (1% and 2%) (Table 2).Significant increases in weight gain were also found in KK-A y mice fed SQ (2%) (Table 2).Although a tendency in the decrease in plasma non-HDL cholesterol was found in KK-A y mice, other lipid parameters increased (Table 3).A significant increase in HDL cholesterol was found.However, all plasma cholesterols levels (total cholesterol, HDL cholesterol and non-HDL cholesterol) increased in C57BL/6J mice, but the difference was not significant (Table 3).SQ intake significantly increased TL, NL and TAG in the liver of KK-A y mice, while no significance was observed in the hepatic lipid levels of C57BL/6J mice, except for total cholesterol (Table 4).Total cholesterol level in the liver from C57BL/6J mice significantly increased due to SQ (2%) feeding, but levels in KK-A y mice fed SQ (1% and 2%) were lower than those fed control diet (Table 4). Fatty Acid Levels of Liver Lipids SQ (1% and 2%) supplementation significantly all fatty acid contents in the liver from KK-A y mice except for 20:4n − 6, resulting in a significant increase in the total fatty acid contents (Table 5).The increase in total fatty acids presented in Table 5 was consistent with the result in Table 4 showing the increasing effect of SQ on liver TL and NL.However, there was little difference in the fatty acid content in the liver of C57BL/6J mice (Table 5).This was also expected due to the TL and NL levels in the liver of C57BL/6J mice (Table 4).Different letters (a, b) show significant differences at P < 0.05. Gene Expression Related to Lipid Metabolism and HMG-CoA Reductase Activity To determine the effect of dietary lipids on liver lipid metabolism, the related gene expressions were analyzed using real-time PCR.Although the analysis showed no significant effect of SQ on the gene expression of C57BL/6J mice (Figure 1), a significant difference was found in the HMGCR and CYP7A1 genes in the liver from KK-A y mice fed SQ compared with the control (Figure 2).Furthermore, SQ (2%) supplementation significantly increased HMG-CoA reductase activity in the liver from KK-A y mice, but not C57BL/6J mice. Discussion Epidemiological studies have revealed an inverse correlation between HDL cholesterol levels and the risk of cardiovascular disease and atherosclerosis.HDL cholesterol promotes reverse cholesterol transport and has several atheroprotective functions, such as anti-inflammation, anti-thrombosis, and antioxidation [32] [33].In prospective epidemiologic studies, every 1-mg/dL increase in HDL is associated with a 2% to 3% decrease in CVD risk, independent of LDL cholesterol and TAG levels [34].Furthermore, normal or high HDL levels appear to have anti-atherosclerotic, anti-inflammatory, antioxidant and anti-thrombotic properties, even in the presence of high LDL cholesterol [35].In the present study, we found a significant increase in the plasma HDL cholesterol of obese/diabetic KK-A y mice, with no significant difference in the total and non-HDL cholesterol levels (Table 3).The increase in HDL cholesterol was also results in the effect of SQ on plasma lipid levels, recent studies have demonstrated the effect of SQ on blood HDL cholesterol level as important factor in atherosclerosis protection [11].Administration of SQ for seven weeks (2.1 g/kg) to C57Bl/6J SPF mice showed a 60% increase in HDL cholesterol with no changes in total cholesterol [23].Likewise, SQ administration for 11 weeks at a dose of 1 g/kg caused a specific increase in HDL cholesterol levels in three male mouse models (wild-type, Apoa1and Apoe-deficient) with the C57BL/6J genetic background [17].In a rat model, specific increase in HDL cholesterol has also been reported [18].These studies have demonstrated that high HDL level would be independent of an anti-atherosclerotic factor [35].The present study confirmed that the increase in HDL cholesterol level is a major effects of SQ in atherosclerosis protection.HDL cholesterol biogenesis and its development are involved in various complex metabolic networks, such as upregulation of ATP-binding cassette transporter A1, apoA-I transcription and liver X receptor (LXR) [36].Therefore, the increasing effect of SQ on HDL cholesterol would be related to these events; however, the mechanisms by which SQ elevate plasma HDL-cholesterol levels remain unclear.SQ is known to show a broad repertoire of biological action based on its antioxidant activity [11].This effect could be exerted in HDL to prevent oxidative modifications of the apolipoprotein A-I (ApoA-I), other HDL proteins, and HDL lipids.The prevention by HDL of oxidative stress can make it more fluid and thus more functional.Further study will be needed. In humans, orally administered SQ is well absorbed (60% -85%).This, and the intestinal de novo synthesized SQ, are transported by chylomicrons into circulation and are rapidly taken up by the liver, where it is converted into cholesterol [17].A significant increase in total hepatic cholesterol found in normal C57BL/6J mice may be reflected by the conversion of SQ to cholesterol in the liver (Table 4).However, cholesterol level decreased in the liver of obese/diabetic KK-A y mice (Table 4).However, significant increase in NL by SQ intake was observed in KK-A y mice, while no significant difference was observed in polar lipids (Table 4).The increase in NL resulted in higher TL levels in the liver.SQ administration to KK-A y mice induced its accumulation in the liver.Moreover, SQ is eluted as the NL fraction in the separation of TL with the column chromatography used in the present study.However, the level of SQ measured was less than 20 mg/g liver in both groups.Therefore, the increase in NL found in Table 4 was mainly due to the increase in TAG.The TAG increase due to SQ intake was strongly related to the higher level of total fatty acids found in Table 5. As shown in Table 4 and Table 5, squalene administration to KK-A y mice induces TAG accumulation in the liver.To determine the effect of squalene, gene expression related to fatty acid (FASN and SCD1) and TAG (DGAT1 and DGAT2) synthesis was analyzed (Figure 1).However, no significant difference was found in these gene expressions, together with other kinds of genes related to lipid metabolism, except for HMGCR.It is difficult to explain the discrepancy found between gene expression and TAG content in the liver of KK-A y mice.One possibility is the involvement of TAG that originated from other tissues. HMGCR is a gene related to cholesterol metabolism.Its expression was significantly decreased by squalene (1%) intake, but no significance was observed with administration of squalene (2%) (Figure 1), while a significant increase in HMG-CoA reductase activity was found in the liver of KK-A y mice fed squalene (2%), but with no significance in squalene (1%) (Figure 2).Although HMG-CoA reductase activity and its gene expression, e.g., HMGCR, is known as a key factor in cholesterol synthesis, the changes in these factors would not greatly affect cholesterol concentrations in the liver (Table 4).This may be due to the compensation of other pathways of cholesterol metabolism and/or the effect of cholesterol supplementation from other tissues.CYP7A1 is known to catalyze the cholesterol catabolic pathway.This enzyme expression was decreased by squalene intake (Figure 1).Although the difference was not significant, this may affect the cholesterol level of the liver. Conclusion The present study showed that SQ supplementation to C57BL/6J background mice increased plasma HDL cholesterol level, which is an important and independent anti-atherosclerotic factor.It is noteworthy that this effect of SQ was found more clearly in an obese/diabetes mouse model compared with normal mice.Although more research is needed to clarify the effect of SQ on the lipid metabolism and dynamics related to atherosclerosis, the present study suggests the anti-atherosclerotic effect of SQ, especially for subjects with metabolic disorders. 2 mL of 2 N HCl in methanol solution was added to the solution and vortexed.The mixture was separated by centrifugation at 1000 g for 5 min.The upper hexane layer containing fatty acid methyl esters was recovered and subjected to GC. GC was performed on a Shimadzu GC-14B equipped with a flame-ionization detector and a capillary column [Omegawax 320 (30 m × 0.32 mm i.d.); Supelco, Bellefonte, PA].The injection port and flame ionization detector were set at 250˚C and 260˚C, respectively, and the column temperature was held at 200˚C.The carrier gas was helium at a flow rate of 50 kPa.Fatty acid content in the lipid samples was expressed as a weighted percentage of the total fatty acids.Each fatty acid level of liver tissue (1 g) was calculated by comparing the peak ratio to that of the internal standard (17:0) and the total lipid content. ( 1975).This procedure is based on the formation of hydroxymate from the reaction of HMG-CoA and mevalonate with hydroxylamine.The resulting hydroxymate can be quantitatively measured using the colorimetric assay.The HMG-CoA and mevalonate concentration in the liver homogenate were collected separately by changing pH to avoid interference by mevalonate in the HMG-CoA analysis.Therefore, liver homogenate was reacted with freshly prepared hydroxylamine reagent (alkaline hydroxylamine reagent in the case of HMG-CoA) at pH 5.5 for HMG-CoA and pH 2.1 for mevalonate, respectively.HMG-CoA reductase activity was calculated from the ratio of HMG-CoA concentration to mevalonate concentration.S. Liu et al.DOI: 10.4236/fns.2018.9121081504 Food and Nutrition Sciences Figure 1 . Figure 1.Effect of squalene administration on hepatic mRNA expression in KK-A y mice (a) and C57BL/6J mice (b). Figure 2 . Figure 2. Effect of squalene administration on HMG-CoA reductase activity in liver of KK-A y and C57BL/6J mice. Table 2 . Weight gain (g) and tissue weight (g per 100 g body weight). Different letters (a, b,c) show significant differences at P < 0.05. Table 5 . Major fatty acid content in liver (mg/g liver).	v3-fos-license
2017-10-30T17:06:35.676Z	2017-10-30T00:00:00.000	36790456	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fphar.2017.00741/pdf", "pdf_hash": "58de8751bf4df016786f8ede66fe1d00c9934b51", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1337", "s2fieldsofstudy": [ "Biology" ], "sha1": "58de8751bf4df016786f8ede66fe1d00c9934b51", "year": 2017 }	pes2o/s2orc	GYY4137, an H2S Slow-Releasing Donor, Prevents Nitrative Stress and α-Synuclein Nitration in an MPTP Mouse Model of Parkinson’s Disease The neuromodulator hydrogen sulfide (H2S) was shown to exert neuroprotection in different models of Parkinson’s disease (PD) via its anti-inflammatory and anti-apoptotic properties. In this study, we evaluated the effect of an H2S slow-releasing compound GYY4137 (GYY) on a mouse PD model induced by acute injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). GYY was intraperitoneally (i.p.) injected once daily into male C57BL/6J mice 3 days before and 2 weeks after MPTP (14 mg/kg, four times at 2-h intervals, i.p.) administration. Saline was given as a control. Behavioral tests (rotarod, balance beam, and grid walking) showed that 50 mg/kg GYY significantly ameliorated MPTP-caused motor impairments. At lower doses (12.5 and 25 mg/kg) GYY exhibited a less obvious effect. Consistent with this, immunohistochemistry and western blot analysis demonstrated that 50 mg/kg GYY attenuated the loss of tyrosine hydroxylase (TH) positive neurons in the substantia nigra and the decrease of TH expression in the striatum of MPTP-treated mice. Moreover, at this regimen GYY relieved the nitrative stress, as indicated by the decreases in nitric oxide (NO) generation and neuronal NO synthase (nNOS) upregulation elicited by MPTP in the striatum. The suppression of GYY on nNOS expression was verified in vitro, and the results further revealed that Akt activation may participate in the inhibition by GYY on nNOS upregulation. More important, GYY reduced the nitrated modification of α-synuclein, a PD-related protein, in MPTP-induced mice. Overall, our findings suggest that GYY attenuated dopaminergic neuron degeneration and reduced α-synuclein nitration in the midbrain, thus exerting neuroprotection in MPTP-induced mouse model of PD. INTRODUCTION Hydrogen sulfide is the third gasotransmitter next to NO and carbon monoxide. It regulates a variety of physiologic and pathologic processes in a wide range of biological systems. For example, the cse (a gene encoding the H 2 S synthase CSE) deficient mice developed hypertension early at 7 weeks after birth (Yang et al., 2008). H 2 S also exerted a beneficial role in atherosclerosis and related disorders (Wang et al., 2009). We previously demonstrated its anti-fibrotic property in chronic kidney disease (Song et al., 2014). Indeed, H 2 S was first proposed as an endogenous neuromodulator by Abe and Kimura (1996). However, it was until recently that its function in the central nervous system gains the attention of scientists. Increasing studies identify a potential role of H 2 S in neurodegeneration. For example, sodium hydrosulfide (NaHS, an H 2 S fast-releasing salt) reduced amyloid beta-peptideinduced neuronal injury and ameliorated the learning memory impairment in APP/PS1 transgenic mice (Fan et al., 2013;Liu et al., 2016). In addition, cse deficiency contributed to the neurodegeneration in Huntington's disease (Paul et al., 2014). Parkinson's disease is the second most common neurodegenerative disorder, affecting approximately 1.7% of the population over 65 years old. Pathologically, it is featured by dopaminergic neuron losses in the SN and formation of inclusion bodies that are composed of α-synuclein (α-syn). Its etiology remains elusive. Several pathogenic factors such as oxidative stress, mitochondrial dysfunction, protein misfolding and neuroinflammation, have been reported to be involved. In addition, higher level expressions of NO and its synthases were detected in the brains of PD patients and animal models (Eve et al., 1998;Dehmer et al., 2000). Inhibitors or depletion of NOS protected against dopaminergic neuron degeneration in MPTP-induced mouse models (Schulz et al., 1995;Liberatore et al., 1999). These studies strongly suggest a role of nitrative stress in PD progression. Inhaled H 2 S or NaHS has been shown to exhibit neuroprotection in neurotoxins-induced rodent models of PD (Hu et al., 2010;Kida et al., 2011;Lu et al., 2012). Of note, manipulation of H 2 S gas deals with a lot of safety issues in practice. NaHS is unstable in water solution. Moreover, NaHS or Na 2 S delivers a rapid bolus of H 2 S in aqueous solution within a quite short period (seconds) that does not accurately mimic the biological process of H 2 S release in vivo. These limit our understanding to the role of H 2 S in PD pathogenesis. As such, in this study we evaluated the effect of GYY, which slowly releases low but consistent amounts of H 2 S within several hours in aqueous solution at physiologic pH and temperature (Li et al., 2008), in an MPTP-induced mouse model of PD, and the underlying mechanism involved. Our findings clearly demonstrate a protective action of GYY against dopaminergic neuron losses caused by MPTP via its anti-nitrative stress property and inhibition on α-syn nitration, and thus consolidate a favorable role of H 2 S in PD development. Animals and MPTP Treatment Male C57BL/6J mice (8-10 weeks old; 23-28 g weight) were purchased from the SLRC Laboratory (Shanghai, China). The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University. All mice were housed in a SPF animal facility with a temperature at 21-25 • C and a controlled light-dark cycle. Parkinson's disease animal models were established by intraperitoneally (i.p.) injecting the mice with MPTP (14 mg/kg, Sigma-Aldrich, St. Louis, MO, United States) four times at 2-h intervals. Saline was given as controls. To evaluate the effect of GYY (Cayman Chemical, United States), the mice were administered with GYY at individual doses (12.5, 25, and 50 mg/kg, i.p.) once daily for 3 days prior to MPTP treatment, and continued throughout the experimental period. The animals were sacrificed 2 weeks after MPTP administration for biochemical studies. Behavioral Assessment Behavioral assessments were performed at 2 weeks after MPTP injection. The data were analyzed by independent observers blinded to the treatments. Rotarod test was performed to estimate motor balance and coordination. Before test, each mouse was trained three times on the rod with an accelerating speed at 5-20 rpm/min. During the test session, the rotational speed remained constant at 18 rpm/min. A trial was stopped if the mouse fell down or the latency to fall reached 5 min. Each mouse was subjected to three trials with an interval of 30 min during the test session, and the average value of the latency to stay on the rod was calculated and included for further analysis. Balance Beam Walking The mice were placed on a batten and tempted with fodder to cross a wooded balance beam (64 cm long, 1.5 cm wide, 15 cm high). The test was stopped and repeated again if the mouse fell off. The time that a mouse successfully transversed the balance beam was recorded. The test was repeated three times at 5 min intervals, and the mean value of the walking time was presented as the data for analysis. A longer latency to cross the beam indicated a poorer motor coordination. Grid walking test was applied to evaluate the sensorimotor coordination of hindlimbs in mice. The mice were placed on a 50 * 40 cm wire grid with 3 * 3 cm grid squares (iron wire at a diameter of 4 mm) in a quiet room with dim lighting, and allowed to walk for 30 s. The number of hindlimb slips was counted by an independent experimenter when the paw completely failed to hold a rung. The animal was put on the grid twice for habituation before test without pre-training. Each trial was repeated three times and the average of foot slips was used for analysis. Immunohistochemical Staining Following the behavioral tests, the animals were deeply anesthetized and perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde (PFA). Mouse brains were immediately removed from the skull and post-fixed in PFA solution overnight. After that, brains were dehydrated in a serial of alcohol solution and dimethylbenzene was applied to make the brains transparent. Brains were then embedded in paraffin wax. Next, 4 µm thickness sections were cut through SN compacta (SNpc), and every fourth slice was reserved. For staining, sections were deparaffinized and rehydrated, followed by epitope retrieval by boiling the sections in 0.01 M citrate buffer (pH 6.0) for 10-15 min and cooling at room temperature for 20-30 min. After that, sections were thoroughly washed in 0.01 M PBS three times. Endogenous peroxidase activity was blocked with 3% H 2 O 2 in 0.01 M PBS for 30 min. Next, the sections were incubated in 5% BSA with 0.1% Triton X-100 in PBS for 30 min, followed by incubation with mouse monoclonal anti-TH (1:1000, Sigma, St. Louis, MO, United States) at 4 • C overnight. Subsequently, the sections were washed in PBS, incubated with goat anti-mouse secondary antibody at room temperature for 1 h and stained using a DAB kit (Gene Tech, GK500705, China). The sections were observed and photographed using the Zeiss microscope (Carl Zeiss, 37081, Germany). Striatal MPP + Level Analysis Striatal MPP + levels were determined as previously described (Jackson-Lewis and Przedborski, 2007) with minor modifications. In brief, striatum and whole blood were collected 90 min after four injections of MPTP (14 mg/kg, i.p.) at 2-h intervals. The tissue samples were homogenized by sonication in 0.2 M chilled perchloric acid and then centrifuged at 15,000 g for 30 min at 4 • C. The whole blood samples were kept at room temperature for 30 min and subjected to centrifuged at 1000 g, 30 min. Serum was harvested from the resulting supernatant, and mixed with 0.2 M perchloric acid before subjected to the second centrifuged at 15,000 g for 30 min at 4 • C. The supernatants were transferred into a clean eppendorf tube for MPTP and MPP + analysis. MPTP/MPP + was analyzed by HPLC equipped with UV detectors (MPTP at 295 nm; MPP + at 245 nm). An aliquot (20 µl) of each supernatant was eluted through a C18 reverse phase column (5 µm, 250 mm × 4.6 mm, SHIMADZU VP-ODS) attached to the HPLC system (Waters, Milford, MA, United States) at a flow rate of 1.0 ml/min. The mobile phase consists of 15% acetonitrile, 50 mM potassium phosphate adjusted to pH 3.2 with ultrapure 18 M phosphoric acid. The detection limit is 3 ng/ml and this method provides good reproducibility. NO Production Determination The striatal NO content was evaluated by measuring the accumulation of nitrite and nitrate using the kit from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Briefly, striatum tissues were homogenized (1:10 m/v) in lysis buffer and centrifuged at 15,000 g for 30 min at 4 • C. Next, an aliquote (50 µl) of the resulting supernatant was mixed with the dilution buffer at an equal volume, followed by addition with 5 µl nicotinamide adenine dinucleotide phosphate (NADPH), 10 µl flavin adenine dinucleotide (FAD) and 5 µl nitrate reductase and incubation at 37 • C for 15 min. After that, 10 µl lactate dehydrogenase (LDH) was added and incubated for another 5 min. Subsequently, 50 µl Griess reagent I (0.1% naphthylethylene diaminedihydrochloride in 1% sulfanilamide) and 50 µl Griess reagent II (2.5% H 3 PO 4 ) were added and incubated for 10 min. The optical density of the mixture was measured at 540 nm using a microplate reader (Tecan Infinite M200 PRO, Switzerland). The results were calculated by a standard curve with sodium nitrite, and expressed as µmol per gram protein. The protein concentrations of striatal homogenates were determined using the BCA protein assay kit (Thermoscientific, United States). Cell Culture and Treatment PC12 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (China), and cultured in RPMI1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cultures were maintained in a incubator with 5% CO 2 /95% air at 37 • C. For experimentation, cells were seeded into a 12-well plate and cultured overnight before treatment. Statistical Analysis All data were presented as mean ± SEM of at least three independent experiments. All statistical analyses were performed with GraphPad Prism 5.0 software. The differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis. Statistical significance was set at P < 0.05. GYY Improves Motor Deficits Induced by MPTP in Mouse To study the ability of GYY to affect MPTP-induced impairment in motor function, we assessed the animals' motor coordination using rotarod, balance beam walking, and grid walking test at 2 weeks after MPTP injection. The results are shown in Figure 1. Compared to saline-treated group (control), the latency to fall off the rod significantly decreased in MPTP-injected mice ( Figure 1A). Co-treatment with GYY at 25 and 50 mg/kg prolonged the time of staying on the rod compared to MPTPonly group. Balance beam walking test also showed that 50 mg/kg GYY co-treatment improved the MPTP-induced motor deficits, as indicated by a shorter time period to cross the beam compared to MPTP only group (Figure 1B). Similarly, in grid walking test we found that MPTP injection increased the number of foot slips in mice, implying that the sensorimotor function was also impaired by MPTP. This was markedly attenuated by 50 mg/kg GYY co-treatment ( Figure 1C). And the treatment with GYY alone at 50 mg/kg, the maximal tested dose in this study, did not have any impact on the motor behavior. Striatal MPP + Levels Are not Affected by GYY Preventive Treatment As MPTP-caused neurotoxicity to dopaminergic neurons is dependent on the metabolism of MPTP to its active metabolite MPP + via monoamine oxidase (MAO) in the nigrostriatal system, the effect of GYY on striatal MPP + levels was determined in a parallel study. In this part, 10 male C57BL/6J mice (10 weeks old) were applied and randomly divided into two groups: MPTP and GYY + MPTP group (n = 5 per group). For MPTP group, the mice received four injections with MPTP (14 mg/kg, i.p., at 2 h intervals). For GYY + MPTP group, the mice were treated with GYY (50 mg/kg, i.p.) once daily for 3 days followed by MPTP administration at 1 h after the last injection with GYY. No significant changes were observed in the striatal MPP + level in MPTP-treated mice with and without GYY (50 mg/kg, i.p.) pretreatment (MPTP 3.90 ± 1.98 nmol/mg tissue; GYY + MPTP group, 3.96 ± 1.46 nmol/mg tissue; P > 0.05; Figure 2), which was measured 90 min after the last MPTP injection. Measurement of serum MPP + levels revealed no significant difference between the two tested groups either (MPTP 3.25 ± 0.39 µg/ml; GYY + MPTP group, 3.14 ± 0.33 µg/ml, P > 0.05). This indicates that the observed effects of GYY on MPTP-induced neurotoxicity are not caused by the reduction of MPTP conversion into MPP + . GYY Attenuates Dopaminergic Neuron Losses in the Substantia Nigra The dopaminergic neuron degeneration was examined by quantifying the number of TH immunoreactive neurons in the SNpc using immunohistochemistry study. The result showed that MPTP caused an approximate 40% decrease in the number of dopaminergic neurons in the SN compared to saline, and this was attenuated in the mice co-treated with 50 mg/kg GYY (Figures 3A,B). Treatment with GYY alone did not have any impact on TH-positive staining in the SN. Western blot analysis showed that 50 mg/kg GYY co-treatment prevented the reduction of striatal TH protein level caused by MPTP ( Figure 3C). These results indicate that co-treatment with GYY FIGURE 1 \| GYY ameliorated motor deficits in MPTP-induced mouse model of PD. Performance on the rotarod (A) and balance beam (B) was impaired by MPTP administration, as indicated by a shorter latency to stay on the rod (A) and a longer period to cross the beam (B) in MPTP-injected mice. The number of foot slips in the hindpaw (C) was also increased by MPTP injection compared to controls. GYY (12.5, 25, and 50 mg/kg/d, i.p.), administered 3 days before and 2 weeks after MPTP injection, alleviated the MPTP-induced motor dysfunction to a different extent, and the effect of 50 mg/kg GYY was consistently significant. Treatment with 50 mg/kg GYY or saline alone did not affect the motor behavior. Results are presented as mean ± SEM from 6 to 7 mice per group. * P < 0.05, * * P < 0.01, * * * P < 0.001; one-way ANOVA. Frontiers in Pharmacology \| www.frontiersin.org FIGURE 2 \| GYY did not affect MPTP metabolism. Striatal and serum MPP + levels quantification by HPLC in combination with UV detection. Panels show typical chromatographs of striatal (A) and serum (B) MPP + and MPTP. Typical retention times for MPP + at 9.3 min and MPTP at 17.5 min. MPTP levels, almost near the detection limit (3 ng/ml), were much lower than those of MPP + and thus were not included for analysis. Mice received four injections of MPTP (14 mg/kg, i.p., at 2 h intervals), with or without GYY (50 mg/kg, i.p.) pretreatment for 3 days (five mice per group). MPTP was given at 1 h after the last GYY injection. Striatum and whole blood were harvested 90 min after the last MPTP administration. GYY pretreatment did not affect the striatal and serum MPP + levels resulting from MPTP metabolism (C). Student's t-test. N = 5 for serum sample; n = 10 for striatal homogenates (both sides of the striatum from five mice per group). at a suitable dose was able to prevent dopaminergic neuron losses in the SN of MPTP-treated mice. GYY Mitigates MPTP-Induced NO Overproduction and Nitrated Modification of α-Synuclein in the Striatum Overproduction of NO and its associated nitrative stress participate in PD pathology (Jimenez-Jimenez et al., 2016). In line with previous studies (Dehmer et al., 2000), we observed an accumulation of nitrite, a metabolite of NO, in the striatum of the mice receiving MPTP administration ( Figure 4A). This increase of NO generation was abated in the mice co-treated with GYY at the dose of 25 and 50 mg/kg, respectively. Likewise, co-treatment with GYY at this regimen inhibited the MPTP-induced upregulation of nNOS expression in the striatum (Figure 4B). Treatment with GYY alone did not modulate NO or nNOS expression level. The result was verified by an in vitro study, which demonstrated that pre-treatment with 100 µM GYY prevented MPP + (100 µM, 24 h) elicited increase of nNOS expression in PC12 cells (Figure 4C). Excessive NO generation often results in peroxynitrite formation and tyrosine nitration in proteins. Human α-syn contains four tyrosine residues (Tyr39, Tyr125, Tyr133, and Tyr136) that can be nitrated (Schildknecht et al., 2013), which may play a role in protein aggregation in PD. Therefore, we further examined the effect of GYY on α-syn nitration by western blot using the specific antibody against nitrated α-syn (Tyr125, Tyr133). The result showed that although the total α-syn level was not altered in the striatum following MPTP injection, the nitrated α-syn level was markedly elevated in MPTP-treated mice, and this elevation was abolished by GYY co-treatment ( Figure 4D). Akt Signaling Mediates GYY-Induced Inhibition on nNOS Upregulation Last, we studied the signaling pathway that underlies the inhibition by GYY on nNOS expression. Treatment with 100 µM MPP + was found to reduce the phosphorylation of Akt Ser 473 in a time-dependent manner, which was detected early at 0.25 h and remained to be dramatically declined at 1 h after treatment in PC12 cells ( Figure 5A). Pretreatment with 100 µM GYY almost reversed the decline of Akt phosphorylation caused by MPP + exposure ( Figure 5B). However, co-treatment with Akt inhibitor perifosine (5 µM) was able to attenuate the GYY-mediated inhibition on nNOS expression in MPP + -treated cells ( Figure 5C). These results indicate that Akt signaling may be activated and thus contribute to the inhibition on nNOS expression by GYY. DISCUSSION In this study we demonstrate the potential of GYY as a neuroprotectant against dopaminergic neuron losses in a mouse model of PD based on the following findings. First, intraperitoneal injection with GYY at an appropriate dose (50 mg/kg) alleviated MPTP-induced motor impairments in mice. Second, this H 2 S slow-releasing compound attenuated dopaminergic neuron losses in the SNpc caused by MPTP. Last, GYY mitigated MPTP-induced nitrative stress and α-syn nitration in vivo. In addition, GYY preventive treatment did not affect MPTP metabolism, as serum and striatal MPP + levels in GYY + MPTP group did not differ from those in MPTP group. To our knowledge, this is the first study demonstrating the neuroprotection of an H 2 S slow-releasing compound in the MPTP-induced mouse model of PD. . The number of TH positive neurons in both sides was counted from six brain slices for each mouse, with 3-5 mouse brains per group. The counting was performed by the experimenters blind to the treatments. (C) Striatal TH expression in MPTP-intoxicated mice was markedly reduced. N = 6 per group. Co-treatment with 50 mg/kg GYY was able to attenuate the loss of TH neuron in the SNpc (A,B) and the decrease of TH expression (C) in the striatum. The molecular masses of the protein markers were indicated to the right of the blots. * P < 0.05, * * P < 0.01, * * * P < 0.001; one-way ANOVA. Hydrogen sulfide is linked to the pathogenesis of PD. Hu et al. (2010) first reported that NaHS exerted neuroprotection against dopaminergic neuron losses in 6-hydroxydopamine and rotenone-induced rat models of PD. This beneficial role of H 2 S in PD was subsequently confirmed by other studies. Two independent groups demonstrated that systemic administration with NaHS or inhaled H 2 S prevented neurodegeneration and movement dysfunction in the MPTP-induced mouse model of PD (Kida et al., 2011;Paul et al., 2014). Vandiver et al. (2013) demonstrated that sulfhydration, a post-translational modification afforded by H 2 S, mediated the neuroprotective actions of parkin, and this study again highlighted a potential role of H 2 S in PD. However, it should be noted that most studies utilized NaHS as an H 2 S donor, which often leads to a burst of H 2 S release in solution. This does not mimick the biological synthesis of H 2 S, which is tightly regulated in vivo. To fully understand the role of H 2 S in PD, it is essential to assess the effect of H 2 S slowreleasing compounds in PD models. GYY is a water-soluable compound that releases H 2 S slowly over a period of hours in vitro and in vivo, producing H 2 S-related beneficial effects in several pathological conditions (Lin et al., 2016;Weber et al., 2017). Thus, in this study we evaluated the impact of GYY on a classic mouse model of PD induced by acute MPTP injection. Functionally, administration of GYY improved the motor impairments. Pathologically, GYY cotreatment attenuated the loss of dopaminergic neurons in the SN. Moreover, GYY pretreatment did not alter the striatal MPP + level, which excluded the possibility that GYY inhibited the MPTP metabolism via MAO although sulfides was reported to be MAO inhibitors (Warenycia et al., 1989;Stewart et al., 2014). These findings suggest the protective effects of GYY in this PD model. In support of this, two recent articles published in Movement Disorders proposed that the action of coffee consumption and smoking in lowering the risk of PD may be linked to H 2 S. Coffee intake was shown to improve the Prevotella population, which served as an important source of H 2 S generation and was decreased in the gut of PD patients (Cakmak, 2015(Cakmak, , 2016Scheperjans et al., 2015). But, it remains to delineate whether the role of H 2 S in PD relates to the gut microbiota. Human α-syn contains four tyrosine residues, which can be nitrated by peroxynitrite when excessive NO is generated. 3-nitrotyrosine, as a footprint of tyrosine nitration in proteins, FIGURE 4 \| GYY mitigated MPTP-induced nitrative stress and α-syn nitration in the striatum. (A) Co-treatment with 50 mg/kg GYY abolished NO overproduction in the striatum of MPTP-injected mice. The nitrite level was normalized by the protein concentration of striatal homogenates, n = 3. (B) Western blot study showed an upregulation of striatal nNOS induced by MPTP compared to controls, and this was attenuated by GYY (25, 50 mg/kg) co-treatment. N = 5 per group. Treatment with 50 mg/kg GYY alone did not affect NO generation or nNOS expression. (C) Effect of GYY on nNOS expressions in PC12 cells treated with 100 µM MPP + or vehicle for 24 h. Cells were pretreated with 100 µM GYY for 1 h before MPP + being added. Relative values were obtained after normalization to loading controls (Tubulin/TUBB), n = 4. (D) Co-treatment with 50 mg/kg GYY led to a reduced nitrated modification of α-syn, but not the total α-syn in the striatum, indicated by a lower ratio of nitro-α-syn over total α-syn compared to MPTP-treated mice. Actin served as loading controls. Mean ± SEM, n = 5. * P < 0.05, * * P < 0.01, * * * P < 0.001; one-way ANOVA. was detected in both PD patients and neurotoxins-induced animal models of PD (Schulz et al., 1995;Good et al., 1998). Moreover, several studies demonstrate that lewy bodies, the pathologic hallmark of PD and other synucleinopathies, largely contain α-syn that is modified by nitration on tyrosine residues (Giasson et al., 2000). So far, the pathophysiological function of nitrated α-syn remains elusive. Some studies report that α-syn nitration may facilitate protein aggregates formation and thus contribute to the pathogenic processes of PD and other synucleinopathies. Nitrated α-syn was suggested to serve as a clinical biomarker for PD diagnosis (Fernandez et al., 2013). In this study, we found that MPTP caused an increase of nitrated but not the total α-syn level in the striatum, and this increase was attenuated by GYY co-treatment. This also indicates the inhibition by GYY on nitrative stress induced by MPTP. The inhibition of GYY pretreatment on nNOS expression was abolished in the presence of AKT inhibitor perifosine. PC12 cells were pretreated with perifosine (5 µM) for 1 h, followed by incubation with GYY for 1 h and subsequent exposure to MPP + for 24 h. N = 5. * P < 0.05, * * P < 0.01, * * * P < 0.001; one-way ANOVA. As a sibling of NO, H 2 S has been demonstrated to directly and indirectly interact with NO in biological systems. Moreover, H 2 S signaling, mediated by S-sulfhydration of its targeting proteins, often functions in an opposite manner to NO (Mustafa et al., 2009;Lu et al., 2013). In this study, GYY not only reduced the excessive generation of NO but also blocked the increase of nNOS expression induced by MPTP in the striatum. However, treatment with GYY did not have an obvious impact on NO or nNOS expression in control mice. This may be due to the relative lower level of NO under physiological conditions. Alternatively, the slower release of H 2 S from GYY which results in a consistent but lower level of H 2 S may also explain the lack of a direct effect of GYY on NO and nNOS expression, as previously reported (Li et al., 2008). Here, we demonstrated an indirect effect of GYY on nNOS expression, which may, at least in part, be mediated by Akt signaling, because GYY was found to enhance Akt activation. And in the presence of the Akt inhibitor perifosine, GYY-induced inhibition on nNOS expression was obviously abated. Therefore, activation of Akt signaling may participate in the suppression on nitrative stress and the neuroprotection by GYY on this mouse model. However, it should be cautious that a number of treatments have been shown to be efficacious in the animal models but finally failed in human clinical trials. Therefore, much more preclinical investigations need to be carried out to fully understand the role of H 2 S and its related strategies for treatment in PD. In summary, our data demonstrated that GYY, a novel H 2 S slow-releasing compound, was protective in an in vivo mouse model of PD via its inhibition on nitrative stress. The findings may have implications for the research and development of H 2 S-based therapies for PD. ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Laboratory Animal Care Guidelines of the Institutional Animal Care and Use Committee (IACUC) of Soochow University. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University. AUTHOR CONTRIBUTIONS XH: study design, data acquisition, data analysis and manuscript preparation. YY and YS: experiments design, sample collect and data analysis. BY, YW, and JZ: sample acquisition, data collection, statistical analysis. C-FL and XZ: study design and manuscript revision. L-FH determined the study theme, designed the experiments, ensured all the data and approved the final version to be published. All authors have contributed to this article.	v3-fos-license
2018-04-03T04:07:08.683Z	2017-11-01T00:00:00.000	4992130	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0187229&type=printable", "pdf_hash": "f3e3c76f9973df342842d7b97b4b20bf5b5793e9", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1365", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Environmental Science" ], "sha1": "f3e3c76f9973df342842d7b97b4b20bf5b5793e9", "year": 2017 }	pes2o/s2orc	Lactobacillus reuteri suppresses E. coli O157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy to improve food safety? The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants. Introduction Enterohaemorrhagic Escherichia coli (EHEC) are Shiga toxin-producing E. coli (STEC) responsible for severe human diseases such as haemolytic uraemic syndrome [1]. The majority of infections, commonly attributed to the consumption of contaminated food is caused by EHEC with serotype O157:H7 and the gut of ruminants, mainly cattle, is considered as the principal reservoir [2]. Therefore, it is important to develop nutritional or ecological strategies to reduce EHEC survival in the gastrointestinal tract (GIT) of cattle and to limit shedding and further contamination of food products. Several approaches have been proposed to reduce the prevalence of E. coli O157:H7 in cattle, including feeding of antagonistic microorganisms, vaccination or bacteriophage treatment. Lactobacilli, which are known to exhibit an inhibitory effect against various enteric pathogens, are widely used as probiotics or direct-fed microbials in humans and animals [3,4]. Lactobacilli display antimicrobial activities as a result of production of metabolites such as lactic acid, bacteriocins or other non-proteinaceous molecules [4]. Lactobacillus reuteri is used commercially as a probiotic microorganism and possesses antimicrobial properties against intestinal pathogens. It is well documented that specific L. reuteri can produce antimicrobial factors, such as hydroxypropionaldehyde (HPA) (also named reuterin), reutericyclin, reutericin, lactate or Mucus Adhesion-Promoting (MAP) protein [5,6]. HPA has been proven effective against bacteria, fungi and protozoa survival [7,8]. It has been postulated that HPA inhibits the activity of bacterial ribonucleotide reductase, a key enzyme catalysing the first step in DNA synthesis, which would explain the broad-spectrum activity of HPA [7,9]. HPA is produced by L. reuteri during a two-step anaerobic fermentation of glycerol: a glycerol dehydratase first catalyses the conversion of glycerol to HPA and HPA is then reduced to 1,3 propanediol (1,3-PD) [7,8]. In addition, L. reuteri is known to excrete HPA in high amounts when the level of fermentable carbohydrates is low [10]. In germfree mice, L. reuteri administration reduced both colonization and clinical signs due to EHEC infection and resulted in HPA production in the caecum [11]. In cattle, the terminal recto-anal junction has been referred as the major site of E. coli O157: H7 multiplication [12]. However, O157:H7 isolates are found throughout the GIT compartments (including the rumen) of experimentally infected calves and naturally shedding cattle [13,14]. In addition, O157:H7 isolates in the rectum are clonally similar to isolates found in the rumen and fecal shedding has been shown to be positively associated with the presence of E. coli O157:H7 in the rumen [15]. All these observations demonstrate that the rumen is a reservoir for the contamination of hindgut compartments. Cattle regurgitate digesta during the rumination process and E. coli O157:H7 in the rumen may directly participate to EHEC dissemination in environment and to animal-to-animal transmission. In addition, quorum sensing molecules homoserine lactones (AHLs) promote EHEC colonization of the bovine gut [16]. Interestingly, AHLs are produced by the rumen microbiota but are not found in other GIT compartments highlighting the role of the rumen in the successful EHEC colonization of the bovine gut [16]. For all these reasons, among strategies for decreasing EHEC burden in cattle GIT, it can be proposed to focus on reducing EHEC survival in the rumen. Because the rumen of cows is under strict anaerobiosis and contains a low level of easily fermentable carbohydrates, we speculated that L. reuteri could produce HPA at this site. In this study, we explored for the first time the potential growth inhibition exerted by L. reuteri towards EHEC in bovine rumen fluid. We tested several L. reuteri strains and demonstrated that L. reuteri LB1-7 isolated from raw bovine milk [17] produced HPA and could achieve a total suppression of EHEC in rumen fluid. In vitro experiments described in this study also showed that the exposure of EHEC to L. reuteri and glycerol in rumen fluid decreased drastically EHEC inoculation of lower bovine digestive segments. Materials and methods Bacterial strains and growth conditions L. reuteri and EHEC strains used in this study are listed in S1 DNA (oligonucleotides used in PCR amplification were described in S2 Table). Bacterial counts from freshly collected RF samples were performed under anaerobiosis using the Most Probable Number (MPN) method and revealed the presence of % 5 x 10 10 MPN/mL. At least two cows at three different days were used for each experiment with RF samples. After sampling, rectum contents were rapidly transferred to O 2 -free, CO 2 -saturated sterile flasks, diluted (1:1) in sterile reduced potassium phosphate buffer (50 mM potassium phosphate, resazurin 0.1%, 40 mM Na 2 CO 3 , 3 mM cysteine, pH 7) and used immediately. Three different cows were used for experiments with Rec samples. Small intestine and caecum contents were collected from three cows at slaughter as previously described [19], rapidly transported to the laboratory, pooled in equal proportions and stored at -20˚C until use. Agar spot test An agar spot test was performed to detect antimicrobial activity of L. reuteri strains as previously described [20]. The antimicrobial activity was recorded as growth-free inhibition zones (diameter > 1 mm) around the spots. In vitro HPA production by L. reuteri HPA was produced under anaerobiosis in PBS buffer (pH 7.4) and RF samples using a twostep fermentation protocol [20]. Briefly, RF samples were first centrifuged twice at 4100 g for 20 min and the supernatants were filter-sterilized through 0.45-μm nylon filter (Merck-Millipore, St Quentin en Yvelines). The filtrate was placed into glass tubes and left without stoppers in an anaerobic chamber (Jacomex, France) (100% CO 2 ) during three days to allow the rumen fluid filtrate to be under anaerobic conditions. The tubes were then recapped with butyl rubber stoppers and filtered again (0.45 μm) under CO 2 flux. L. reuteri strains were incubated in MRS broth without shaking under anaerobiosis for 24 hours at 37˚C. The cultures were then centrifuged (4100 g, 10 min), washed in sterile PBS buffer and adjusted to % 10 9 CFU/mL in O 2 -free, CO 2 -saturated PBS or filter-sterilized RF samples supplemented with 250 mM glycerol (Fisher-Aldrich). After incubation for 2 hours at 37˚C, the bacterial cultures were pelleted and HPA-containing supernatants were collected and filtered (0.22-μm) before storage at 4˚C. HPA quantification HPA production was quantified by a tryptophan-HCl colorimetric assay as previously described [10]. Briefly, the HPA-containing supernatants were diluted (10-fold) in sterile H 2 O, mixed with 10 mM tryptophan solution (0.01 M in 0.05 HCl, stabilized with a few drops of toluene) and 12 M HCl before incubation for 30 min at 37˚C. Optical density (OD 560 ) was measured with a spectrophotometer Spectronic BioMate TM 3 (Thermo Scientific). Acrolein (Fisher-Aldrich) was used as standard (the method is specific to both reuterin and acrolein quantification [10]). Standard curves were generated using an acrolein solution diluted to a concentration range of 0-20 mM in PBS or filter-sterilized RF. All the samples were filtered (0.45 μm) and diluted in PBS buffer if necessary. Co-incubation of EHEC and L. reuteri strains Pre-cultures of EHEC FCH6 Rif R or EDL933 Rif R inoculated from a single colony were incubated aerobically overnight at 37˚C in LB supplemented with rifampicin. Bacterial cultures were then centrifuged (4100 g, 10 min) and the pellets were resuspended in sterile PBS buffer. At the same time, L. reuteri strains were cultured in MRS broth for 24 hours (37˚C) without shaking. RF samples or LB broth (5 mL) were introduced into glass tubes equipped with butyl stoppers and screwed caps (Hungate tubes) under a 100% CO 2 atmosphere. Ground feed (25 mg) was then added to RF samples (80% meadow hay, 20% concentrate) to mimic a feeding cycle. RF samples or LB broth were inoculated with both L. reuteri (% 10 7 CFU/ml) and the EHEC Rif R (% 10 4 CFU/mL) strains. To mimic the physiological conditions encountered in the rumen, the cultures were incubated under strict anaerobiosis at 39˚C (bovine temperature) for 24 hours (% transit time of forage-rich digesta) with gentle shaking (mixing of digesta). The bacterial cultures were then 10-fold serially diluted in PBS and spotted (10 μL) in triplicate on LB plates containing rifampicin before incubation overnight at 37˚C. Each experiment was performed independently at least three times. The values presented are the log 10 mean number of CFU/mL. In vitro feedstuff degradation by the rumen microbiota The effect of L. reuteri on feedstuff degradation by the rumen microbiota was assessed using the in vitro Daisy II incubation system (ANKOM Technology Corporation, Fairport, NY, USA). This system allows evaluation of feed digestibility during simultaneous incubation of up to 4 glass vessels, which rotate in an insulated chamber maintained at 39.5˚C (% rumen temperature) [21][22][23]. RF samples were diluted (1:4, vol:vol) with Goering and Van Soest (GVS) anaerobic buffer [24] and dispensed under O 2 -free CO 2 atmosphere. Feedstuff, under the form of alfalfa hay (AH) or corn silage (CS) (0.25 g of each forage, ground to 1 mm particle size), was bagged (ANKOM filter bags F57, porosity 25 μm, 5.0 cm×5.5 cm) and incubated in the presence of diluted RF for 24 hours. Six glass beads (4 mm diameter) were added to each bag to improve bag immersion in the rumen fluid as previously described [22]. All filter bags were heat-sealed at 0.5 mm from the edge of the bags. One bag with only 6 glass beads was also placed in each jar as blank. Six replicates for each feedstuff were used, and the experiment was repeated three times with RF collected at one-week intervals. For each replicate, one vessel was used as control and contained only RF, buffer, AH and CS bags, and one was inoculated with % 10 7 CFU/mL of L. reuteri LB1-7 and 80 mM glycerol at the start of incubation. L. reuteri suspension was prepared from an overnight MRS culture at 37˚C. The culture was centrifuged and resuspended in GVS buffer. pH was monitored at the start and end of incubation in a 10 mL sample of the incubation mix. Samples were collected and stored (-20˚C) for fermentation end products analysis and microbial populations quantification by qPCR (see sections below). After 24 hours of incubation, bags were removed, washed with tap water and dried at 65˚C for at least 48 hours for determination of residual DM. Six bags of each feedstuff were not incubated in order to assess passive DM loss by tap water washing. It was then possible to calculate the disappearance of DM (%). Incubation of EHEC in rectum contents Survival of EHEC during its passage in different segments of the bovine GIT was simulated. To this, we performed a first incubation of EHEC in filter-sterilized RF samples (FS-RF) (0.22 μM) followed by a second incubation in Rec samples (RF samples were indeed filtered in order to retrieve only the inoculated bacteria and not the feed particles neither the rumen endogenous microbiota). Briefly, RF samples were centrifuged, filter-sterilized and placed into glass tubes in an anaerobic chamber as described above. The strain FCH6 Rif R (% 10 4 CFU/ mL) was incubated under strict anaerobiosis with gentle shaking in FS-RF samples (5 mL) inoculated or not with L. reuteri (% 10 7 CFU/mL) and supplemented with 80 mM glycerol. After 24 hours of incubation at 39˚C, the samples were centrifuged (4100 g, 10 min) and the bacterial pellet (containing potentially L. reuteri and/or EHEC) was suspended in sterile CO 2satured PBS (0.5 mL). The bacterial suspension was then inoculated into Rec samples freshly collected (containing its endogenous microbiota) (5 mL) and incubated at 39˚C under anaerobiosis. The concentration of EHEC Rif R was measured after 24h of incubation in FS-RF and after 6 and 24 hours of incubation in Rec samples. DNA extraction Genomic DNA from pure culture of L. reuteri was extracted using the Easy-DNA TM kit (Fisher Scientific, Illkirch, France). The L. reuteri strains were cultured aerobically for 24 hours in MRS broth at 37˚C without shaking. The next day, the bacterial cultures were centrifuged (10,000 g, 15 min) and the pellet was washed twice in sodium phosphate buffer (50 mM, pH 8). Initial cell lysis was performed using proteinase (Easy-DNA TM kit) for 5 min at 37˚C. Bacterial cells were then disrupted by bead beating for 3 min with 0.2 g of zirconia beads (0.1 mm diameter, Sigma-Aldrich). Subsequent genomic DNA extraction was performed according to the manufacturer's recommendations. Total DNA from the rumen and intestinal contents was extracted as previously described [27]. Briefly, bacterial cells were suspended in buffer (50 mM Tris buffer, 0.5 M NaCl, 50 mM EDTA, 4% SDS) (pH 8) and disrupted by bead beating for 3 min with zirconia beads. The mixture was incubated at 70˚C for 15 min and centrifuged (16,000 g for 2 min at 15˚C). The resulting supernatant was then precipitated, washed and resuspended as previously described [27]. Contaminating RNA was removed using a DNAse-free RNAse (10 mg/mL) and an additional incubation for 15 min at 37˚C with 15 μL of Proteinase K (20 mg/mL, Sigma-Aldrich) was also performed. Subsequent DNA purification was performed using the Qiamp Fast DNA Stool mini kit (Qiagen, Courtaboeuf, France). Oligonucleotide primer design and PCR screening L. reuteri genes were detected by PCR amplification using the primer pairs described in S2 Table. The primers used to detect Lactobacillus spp. target a 16S rRNA sequence conserved among Lactobacillus spp. but also Leuconostoc spp., Pediococcus spp. and Weissella spp. [28]. Therefore, in this study, the bacteria detected by these primers were designed as Lactobacillus group. DNA sequences from L. reuteri available in databases ( Table). Amplification was performed from genomic DNA extracted from L. reuteri strains as described above. Taq polymerase and enzyme buffer were from MP-Biomedicals (Illkirch, France). The PCR procedure was performed on a Mastercycler Personal apparatus (Eppendorf) using the following programme: initial denaturation at 94˚C for 4 min, 30 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 1 min, and a final elongation at 72˚C for 10 min. Bacterial enumeration in RF samples by quantitative-PCR Total bacteria, Fibrobacter succinogenes, Ruminococus flavefaciens, L. reuteri and the Lactobacillus group were quantified by quantitative PCR (q-PCR). The q-PCR quantification was performed as previously described [27]. The primer pairs targeting the rrs and hsp60 genes are described in S2 Table. The standard curves (10 8 to 10 3 rrs or hsp60 copies) targeting Lactobacillus group and L. reuteri, respectively, were established from genomic DNA extracted from L. plantarum and L. reuteri strains. DNA extracted from RF samples inoculated with LB1-7 and L. plantarum (% 10 6 CFU/mL) was used as positive and negative control respectively for L. reuteri quantification. The standard curves (10 8 to 10 3 rrs copies) targeting R. flavefaciens and F. succinogenes and total bacteria were established as previously described [27]. Statistical analysis Values are expressed as mean ± SEM. Statistical analyses were performed with the GraphPad Instat statistical software (La Jolla, CA, USA). Student's t-test was used to compare means and discuss the effect of L. reuteri. All tests were two-tailed paired and the level used to establish significance was P < 0.05. Survival of EHEC in bovine rumen fluid The fate of the spontaneous rifampicin mutants of EHEC FCH6 and EDL933 (S1 Table) was assessed in rumen fluid (RF) containing endogenous microbiota under in vitro culture conditions allowing the growth of the endogenous microbiota and mimicking the rumen physiological conditions (see the Materials and Methods section). A concentration of % 10 4 EHEC/mL was chosen because 3-6 log 10 colony-forming unit (CFU)/mL were enumerated from the rumen of steers experimentally infected with E. coli O157:H7 [29]. Results analysis showed that FCH6 Rif R survived well after incubation in RF samples (only 0.7 log 10 decrease in CFU/ mL after 24 hours of incubation) whereas the spontaneous Rif R mutant of the reference EHEC strain EDL933 (EDL933 Rif R ) showed higher cell death after anaerobic incubation in RF (1.9 log 10 decrease in CFU/mL) (S1 Fig). The strain FCH6 was thus selected for further studies. Ability of L. reuteri to produce HPA and to inhibit EHEC growth Five L. reuteri strains (S1 Table) were tested for their ability to inhibit the growth of EHEC FCH6 on soft BHI agar plates. Results showed that FCH6 was susceptible to L. reuteri strains LB1-7 and F275 under anaerobiosis when 2% glycerol (217 mM) was added whereas it was poorly or not inhibited by the remaining L. reuteri strains, with or without glycerol supplementation (S2 Fig). L. reuteri strains were also tested by PCR to detect the genes gldC and dhaT encoding glycerol dehydratase large subunit (EC 4.2.130) (required to convert glycerol to HPA) and 1,3-propanediol dehydrogenase (EC 1.1.1.202) (required to reduce HPA to 1,3-PD), respectively. Amplified products with the expected size for dhaT were obtained from all the L. reuteri strains tested, whereas the amplicon specific for gldC was only obtained for L. reuteri LB1-7, F275 and 65A (S3 Fig). In accordance with the results of agar spot assay, gldC was not amplified from L. reuteri F70 and 100-23 genomic DNA. The capacity of L. reuteri strains to produce HPA was then tested in PBS buffer supplemented with 250 mM glycerol. Results showed that HPA production at 37˚C under anaerobiosis was strain dependent since LB1-7, F275 and 65A produced 114, 84 and 19 mM HPA, respectively. As expected, HPA was not detected in the supernatant of L. reuteri strains 100-23 and F70. The production of HPA by L. reuteri was also tested in filter-sterilized RF samples supplemented with 250 mM glycerol: the strains LB1-7 and F275 produced 189 and 105 mM HPA, respectively, whereas HPA was not detected in the presence of L. reuteri strains 65A, 100-23 and F70. In view of these results, the L. reuteri strain LB1-7 was chosen to perform co-incubation experiments. The L. reuteri strain 100-23 was used as negative control. Persistence of L. reuteri in bovine intestinal fluids The quantification of L. reuteri and Lactobacillus group in intestinal contents was performed by qPCR amplification of the genes hsp60 (encoding heat shock protein) and rrs (encoding 16S ribosomal RNA), respectively. Lactobacillus group was detected in RF samples (7.5 log 10 rrs copy per μg of DNA ± 0.16), small intestine contents (7.8 log 10 rrs copy per μg of DNA ± 0.3), caecum contents (7.4 log 10 rrs copy per μg of DNA ± 0.09) and rectum contents (7.6 log 10 rrs copy per μg of DNA ± 0.05). In contrast, L. reuteri was undetectable in all GIT compartments tested, including the rumen (detection limit: 2 log 10 hsp60 copies). The capacity of L. reuteri LB1-7 to grow in RF samples was then analyzed. After 24 hours of incubation in RF at 39˚C under strict anaerobiosis, the spontaneous rifampicin-resistant mutants of LB1-7 (LB1-7 Rif R ) was enumerated at a concentration similar to the inoculation rate (% 10 7 CFU/mL), demonstrating the capacity of the strain to persist in RF. Antimicrobial activity of L. reuteri and antimicrobial potency of HPA against EHEC The growth of EHEC FCH6 Rif R (% 10 4 CFU/mL) was tested in the presence of different concentrations of L. reuteri LB1-7 (% 10 5 , 10 6 and 10 7 CFU/mL) and glycerol (20,40, 80 and 160 mM) in RF samples to determine the optimal conditions to decrease EHEC survival. The strain FCH6 Rif R survived well after 24 hours of anaerobic incubation in RF samples in the absence of glycerol or supplemented with 20 or 40 mM glycerol whatever the L. reuteri concentration (S3 Table). In contrast, no EHEC CFU was enumerated when LB1-7 (10 7 CFU/mL) was inoculated in RF supplemented with 80 or 160 mM glycerol (detection limit: 10 CFU/mL) (Fig 1). The inhibition of higher levels of EHEC (% 10 5 and 10 6 CFU/mL) were also tested. L. reuteri LB1-7 (% 10 7 CFU/mL) suppressed EHEC FCH6 in RF supplemented with 80 mM glycerol (RF-Glyc80) whatever the EHEC concentration. Similarly, co-incubation of L. reuteri LB1-7 (% 10 7 CFU/mL) and EDL933 Rif R (% 10 4 CFU/mL) in RF-Glyc80 also resulted in EHEC suppression after 24 hours of anaerobic incubation. As expected, L. reuteri 100-23 (% 10 7 CFU/ mL) had no effect on EHEC survival with or without glycerol (Fig 1). Results also showed that glycerol alone was not responsible for EHEC suppression since L. reuteri LB1-7 Rif R showed similar growth curves when incubated in RF and RF-Glyc80. This demonstrated the capacity of L. reuteri to persist in RF in the presence of glycerol. Different dilutions of HPA-containing supernatants were added to RF samples inoculated with FCH6 Rif R to test the antimicrobial potency of exogenous HPA. Addition of 25 mM HPA in RF samples (not inoculated with LB1-7) completely suppressed FCH6 Rif R after 2 hours of incubation. Production of HPA by L. reuteri in rumen fluids Kinetics of EHEC disappearance and HPA accumulation were monitored during co-incubation of FCH6 Rif R (% 10 4 CFU/mL) and LB1-7 (% 10 7 CFU/mL) in RF-Glyc80. As shown in Fig 2, a significant decrease in EHEC population was observed from 4 hours of co-incubation (P<0.05) and no EHEC colony could be detected after 6 hours of co-incubation. A total suppression of EHEC was observed when HPA concentration reached % 12.5 mM and a maximal HPA concentration (13.8 mM) was quantified after 8 hours of incubation (Fig 2). As expected, HPA and 1,3-PD accumulation correlated with glycerol disappearance and the glycerol concentration was only 51 mM and 34 mM in RF-Glyc80 after 6 and 24 hours of co-incubation, respectively. The concentrations of HPA and 1,3-PD accumulated in RF-Glyc80 were similar during the first 8 hours of incubation. Noteworthy, the level of 1,3-PD reached 34 mM after 24 hours whereas a decrease in HPA concentration was observed (7.3 mM) (Fig 2 and Table 1). These results suggested that HPA accumulation was transient probably because a part of HPA was reduced to 1,3-PD. As expected, HPA was not detected in the supernatant of RF-Glyc80 inoculated with L. reuteri 100-23 as well as in RF-Glyc80 samples not inoculated with L. reuteri. Because Schaefer et al. show that HPA production by L. reuteri is stimulated by interaction with different bacteria [8], we investigated the potential stimulating role of EHEC in HPA production. A similar maximal concentration of HPA (% 14 mM HPA) was quantified at 8 hours of incubation of RF-Glyc80 co-inoculated with LB1-7 and FCH6 Rif R or inoculated with LB1-7 alone demonstrating that the presence of EHEC in RF samples did not stimulate the production of HPA by L. reuteri under our experimental conditions. Production of HPA and antimicrobial activity of L. reuteri LB1-7 in LB broth HPA production and antimicrobial activity of L. reuteri LB1-7 against EHEC was also investigated during co-incubation in sterile laboratory medium. As shown in Fig 3A, 10 mM glycerol was required to completely suppress FCH6 Rif R co-inoculated with LB1-7 in LB broth after 6 hours of co-incubation. Up to 2.1 mM HPA was produced by L. reuteri LB1-7 after 3 hours of co-incubation with FCH6 Rif R in LB broth supplemented with 10 mM glycerol (Fig 3B). Taken together, the results showed that only 10 mM glycerol were required to completely suppress FCH6 Rif R in LB medium inoculated with L. reuteri LB1-7 while 80 mM glycerol were necessary in RF. Furthermore, it is noteworthy that the HPA concentration produced by L. reuteri and apparently sufficient to suppress EHEC was 2.1 and 12.5 mM in LB broth and RF samples, respectively. Glycerol metabolism by endogenous rumen microbiota Fermentation end products were analysed in RF samples. As expected, the three major shortchain fatty acids present in RF samples freshly collected were acetate, propionate and butyrate (RF t = 0, Table 1). The concentration of additional metabolites was below the detection limit: glucose (< 4 x 10 −3 mM), glycerol (< 10 −2 mM), 1,3-PD (< 2 x 10 −2 mM), isobutyrate (< 10 −2 mM), valerate (< 10 −2 mM), lactate (< 10 −2 mM) and ethanol (< 4 x 10 −2 mM). Acetate, propionate and butyrate concentration increased after 24 hours of anaerobic incubation due to fermentation by the endogenous rumen microbiota of simple and complex carbohydrates provided by the ground feed added to RF samples (see the Materials and Methods section) (RF t = 24h, Table 1). The glycerol concentration was still % 62 mM in RF-Glyc80 after 24 hours of incubation, indicating that the rumen microbiota assimilated % 24% of the glycerol. In addition, incubation of RF-Glyc80 resulted in an altered fermentation pattern when compared with RF samples not supplemented with glycerol: disappearance of glycerol was associated with an increase in propionate and butyrate concentration (P<0.05) ( Table 1). The 1,3-PD concentration was 2.6 mM in RF-Glyc80 after anaerobic incubation suggesting that the rumen microbiota was able to reduce glycerol to 1,3-PD to some extent. The HPA concentration in RF samples was below the detection limit (< 5 x 10 −2 mM). Impact of L. reuteri on rumen fermentation and glycerol metabolism The fermentation pattern of RF-Glyc80 samples inoculated with L. reuteri was analyzed (Table 1). After 24 hours of incubation under anaerobiosis, the glycerol concentration was 34.2 mM in RF-Glyc80 inoculated with LB1-7 (Table 1). Comparison with the level of glycerol recovered after incubation of non-inoculated RF-Glyc80 (61.9 mM) suggested that LB1-7 was able to metabolize glycerol, while the change in glycerol concentration in the presence of L. reuteri 100-23 was not significant. As expected, HPA was detected in RF-Glyc80 inoculated with LB1-7 but not with 100-23 (Table 1). Furthermore, as shown above, a relatively high concentration of 1,3-PD (34.3 mM) was quantified in RF-Glyc80 inoculated with L. reuteri LB1-7 whereas the level of 1,3-PD quantified in RF-Glyc80 inoculated with L. reuteri 100-23 was close to that obtained after incubation of RF-Glyc80 alone ( Table 1). This suggests that the slight 1,3-PD accumulation observed in RF-Glyc80 inoculated with 100-23 was essentially due to anaerobic reduction of glycerol by the endogenous rumen microbiota. Similar patterns were obtained when RF-Glyc80 was inoculated with L. reuteri alone or co-inoculated with both L. reuteri and EHEC FCH6 Rif R . In the absence of glycerol, incubation of RF with or without L. reuteri led to similar concentrations of acetate and butyrate, and a lower concentration of propionate (P<0.05) ( Table 1). In contrast, incubation of L. reuteri LB1-7 in RF-Glyc80 led to lower concentrations of acetate (P<0.05), butyrate (P<0.01), and propionate (P<0.001) ( Table 1). Noteworthy, lactate accumulation (28.7 mM; 0.13 mM undisociated from) was observed when LB1-7 was inoculated in RF-Glyc80 but not in RF. To analyze the role of lactate in EHEC inhibition, the strain FCH6 Rif R was incubated in RF-Glyc80 samples supplemented with 30 mM lactate. After 24 hours of incubation under anaerobiosis, a similar EHEC quantification (% 10 4 CFU/mL) was recovered in RF samples supplemented or not with 30 mM lactate suggesting that the LB1-7 bactericidal action was not due to lactate production. Impact of L. reuteri on the degrading activity of the endogenous rumen microbiota A potential inhibitory effect of L. reuteri on the feedstuff degradation capacity of the rumen microbiota was investigated. After 24 hours of incubation, 55% and 43% of corn silage and alfalfa hay dry matter (DM) respectively, were degraded by the rumen microbiota ( Fig 4A). Rumen microbiota activity was confirmed by the production of SCFAs after 24 hours of incubation (S4 Fig). Inoculation of L. reuteri LB1-7 and glycerol supplementation (80 mM) resulted in a significant decrease of DM degradation: 50.4% and 35.8% of corn silage and alfalfa hay DM were degraded, respectively (P<0.05) (Fig 4A), corresponding to inhibition decrease of 8.3 and 16.7%. A shift in SCFA profile was also measured (S4 Fig), indicating a disruption in the rumen microbiota. All incubation vessels had the same initial pH (7.10). After 24 hours of incubation, the pH was slightly decreased (6.70) in the vessels inoculated with L. reuteri compared with non-inoculated ones (pH 7.05). It is of interest to note that pH 6.70 was still a physiological value for rumen microbiota activity. The decrease in pH value and the fact that lactate and 1,3-PD were recovered in the vessels supplemented with glycerol after 24 hours of LB1-7 incubation (5.2 mM and 7.6 mM, for lactate and 1,3-PD respectively) (S4 Fig) suggested that L. reuteri remained metabolically active during the course of fermentation. In view of these results, a potential antimicrobial activity of L. reuteri against the total rumen bacterial population and two major rumen cellulolytic bacteria, Fibrobacter succinogenes and Ruminococcus flavefaciens, was investigated (Fig 4B). After 24 hours of incubation, the total bacterial population was slightly greater (P<0.05) with L. reuteri inoculation compared with control. R. flavefaciens population decreased during incubation (P<0.05) both in control vessels and in vessels inoculated with L. reuteri and supplemented with glycerol (decrease of 1 and 0.8 log 10 rss copy per μg of DNA, respectively) ( Fig 4B). The F. succinogenes population remained unchanged in the control vessels during the course of the fermentation whereas a significant decrease (P<0.001) was observed in the vessels inoculated with L. reuteri. This suggested that F. succinogenes was more affected by the presence of L. reuteri than R. flavefaciens, with on average a decrease of 0.5 log 10 rrs copy per μg of DNA compared to control vessels. Survival of EHEC in rectum contents after passage in RF-Glyc80 inoculated with L. reuteri Experiments were performed to mimic the passage of EHEC through different segments of the bovine GIT. To this aim, a first incubation of EHEC was performed in filter-sterilized RF (FS-RF) samples followed by a second incubation in Rec samples. The fate of FCH6 Rif R was evaluated after incubation in FS-RF-Glyc80 with L. reuteri LB1-7 and in following Rec samples. The EHEC strain was also incubated in FS-RF without L. reuteri and glycerol as a control. A bacterial concentration of 1.3 log 10 FCH6 Rif R CFU/mL was recovered after 24 hours of incubation in FS-RF-Glyc80 samples inoculated with L. reuteri LB1-7 (Rec t = 0/RF t = 24h, Fig 5). This concentration was markedly lower than that measured in control RF samples (5.1 log 10 CFU/mL, P < 0.05) (Rec t = 0/RF t = 24h, Fig 5). Note that, in contrast to incubation in RF samples containing its endogenous microbiota (Figs 1 and 2), L. reuteri LB1-7 strongly repressed but not totally suppressed the growth of EHEC in FS-RF-Glyc80, suggesting that absence of nutritional competition with the ruminal endogenous microbiota is responsible for the better survival of EHEC. In a second step, bacteria that survived passage through the rumen were inoculated in Rec samples containing its endogenous microbiota. Without prior exposure to L. reuteri and glycerol in RF, EHEC concentration in Rec samples reached 5.9 and 6.1 log 10 CFU/mL after 6 and 24 hours of incubation respectively (Fig 5). EHEC concentrations were much lower (2.3 and 2.7 log 10 CFU/mL, P < 0.001 and P <0.10 respectively) after 6 and 24 hours of incubation in Rec samples following preliminary exposure to LB1-7 in FS-RF-Glyc80 (Fig 5). The results demonstrated that the passage of EHEC in RF in the presence of L. reuteri and glycerol was efficient to drastically reduce in vitro EHEC survival in rectum content. Discussion Strategies can be proposed to reduce the carriage of E. coli O157:H7 in cattle i) during the animal growth period in order to limit pathogen shedding and thus environmental burden and ii) just before slaughter to limit the contamination of carcasses and thus the burden of E. coli O157:H7 entering the processing plant [30]. Many studies have tested the use of direct fed microbials (DFMs) as a pre-harvest strategy to reduce fecal shedding of E. coli O157:H7 in beef cattle [31]. Although results of field studies were variable, a recent meta-analysis showed that distribution of lactic acid bacteria appeared efficient in reducing the prevalence of E. coli O157:H7 fecal shedding [30]. To our knowledge, the use of L. reuteri to reduce EHEC survival in bovine gastrointestinal fluids is investigated for the first time. We have compared several L. reuteri strains and selected L. reuteri LB1-7 able to convert glycerol to HPA and to suppress EHEC in RF containing metabolically active endogenous microbiota. L. reuteri LB1-7 appeared to be of particular interest because of its resistance to simulated gastric and intestinal conditions [17], in addition, it can be potentially endowed with probiotic activities as already demonstrated for other lactobacilli isolated from raw milk [32]. Furthermore, in in vitro simulation of EHEC fate along the bovine digestive tract, we showed that when the EHEC strain are exposed to L. reuteri and glycerol in rumen fluid, its viable load sharply decreased, affecting also EHEC viability in a posterior digestive compartment (i.e. the rectum). The rumen environment should be appropriate for HPA production by L. reuteri because it is strictly anaerobic and the concentration of available sugars has been reported to be generally low (<1-2 mM post feeding [33]), a required condition for anaerobic glycerol fermentation by L. reuteri (a low glucose concentration favours HPA instead of 1,3-PD accumulation [34]). Indeed, the soluble carbohydrates resulting from polysaccharide breakdown by rumen microbiota are either readily fermented or very rapidly sequestered under the form of oligosaccharides into the cells, preventing soluble sugars to be released in RF [35]. The rumen microbiota is able to produce low levels of 1,3-PD in RF-Glyc80 suggesting transient HPA accumulation. This indicates that some bacteria of the rumen microbiota possess the metabolic pathways to convert glycerol to 1,3-PD via HPA reduction. Among 1,3-PD-producers, Clostridium butyricum and C. perfringens are the most commonly reported mammal intestinal species [36]. Although 1,3-PD production is not described in the rumen of cattle, we speculate that members of the Clostridium genus, which belong to the core ruminal bacterial community [37], could be responsible for HPA production in RF-Glyc80. However, RF-Glyc80 alone was not effective in preventing EHEC multiplication suggesting that transient HPA produced by the endogenous microbiota was insufficient to achieve an antimicrobial effect. As expected, the acetate:propionate ratio decreased in RF-Glyc80 after 24 hours of incubation indicating that part of the glycerol was fermented by the rumen microbiota. Several ruminal bacterial species such as Anaerovibrio lipolytica and Selenomonas ruminantium are able to ferment glycerol into propionate [38][39][40]. However, incubation of L. reuteri in RF-Glyc80 resulted in decrease in propionate concentration suggesting that part of the glycerol that could be fermented to propionate by the ruminal microbiota was more efficiently metabolized by L. reuteri. It can also be hypothesized that the HPA produced by L. reuteri in RF-Glyc80 resulted in an inhibition of the propionate producing population. One of the main functions of the rumen microbiota is the degradation and fermentation of plant biomass into short chain fatty acids providing energy for the animal [41]. Therefore, it is important to analyze the effects of LB1-7 inoculation on the degrading activity of the rumen microbiota. In our study, a significant decrease in corn silage and alfalfa hay degradation in the presence of L. reuteri LB1-7 and glycerol was observed. This decrease is probably due to an inhibitory effect of HPA on the rumen fibrolytic populations, as observed for F. succinogenes. However, qPCR assay targeting bacterial DNA, as well as DM degradation quantification could lead to underestimation of the effect of L. reuteri on the rumen bacteria, due to contribution of DNA and active enzymes released by dead bacterial cells. Fermentation end-products profiles also suggested that L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota in the presence of glycerol. In addition to the effect on fibrolytic populations, production of HPA by L. reuteri may lead to inhibition of lactate-utilizing and butyrate-and propionate-producing rumen bacteria, resulting in perturbed fermentations. Consequently, a potential supplementation of L. reuteri and glycerol during the animal growing period appears to be unsuitable. However, since EHEC carriage at the time of slaughter represents the potential entry of the pathogen into the meat production process, administration of L. reuteri and glycerol could be considered in view of application to finishing beef cattle, a few days before slaughter, when performance goals (decreased rate of gain-finished weight) are achieved. However, the pre-slaughter glycerol and L. reuteri administration should avoid additional stressing factors that may have a negative impact on meat quality [42]. Therefore, the approach we suggest for reducing E. coli shedding by cattle needs to be carefully evaluated to assess the stress responses in living animal and in post-mortem muscle metabolism, in addition to killing activity against EHEC. Accumulation of lactate was observed in RF-Glyc80 after incubation of L. reuteri under anaerobiosis. The efficiency of lactate in inhibiting E. coli O157:H7 multiplication in food products and cattle hides is well documented [43,44]. Furthermore, Ogawa et al. showed that the bactericidal activity of L. casei against E. coli O157:H7 was due to production of undissociated lactate, which permeates the bacterial membrane by diffusion and releases protons into the cell [26]. Our data clearly showed that lactate was not involved in EHEC suppression in rumen contents, probably because the weak level of the corresponding undissociated form was below the minimal inhibitory concentration [26]. Nonetheless, it is possible that the distribution of glycerol and L. reuteri to ruminants leads to EHEC inhibition due to HPA as well as lactate production in the rumen. In further steps, it will be important to analyze the impact of L. reuteri and glycerol association in vivo, in order to assess their effects on bovine gastrointestinal health due to lactate production and microbiota disruption. The potential use of L. reuteri as a feed supplement to reduce EHEC burden in cattle would necessarily be associated with glycerol administration. It is generally admitted that supplementing feed with a formulation containing freeze-dried micro-organisms is easy to implement, as far as strains stability issues are resolved. Introduction of L. reuteri as direct-fed microbial (DFM) would be of interest because the use of DFMs is widely accepted in ruminant nutrition and is perceived as a natural non-antibiotic way of improving animal performance and health [45]. The use of glycerol as cattle feed supplement has already been proposed to improve animal performance because of its cost-effective energetic value. Because the energy content of glycerol is close to that of corn, the replacement of corn by dry glycerol in cows daily supplementation has been considered [46]. Furthermore, the glycerol concentrations needed for EHEC inhibition by L. reuteri in vitro are close to those used in cattle production [46,47]. However, in vivo experiments will be needed to determine the glycerol concentration required for production of sufficient HPA to suppress EHEC in the rumen of cows, as part of the glycerol might be absorbed across the rumen wall [48]. In conclusion, L. reuteri LB1-7 appeared to resist the physical and microbiological conditions encountered in the rumen of cows and was effective in suppressing EHEC O157:H7 in RF in vitro. Data presented in this report define valuable information on the optimal antagonistic effect of a selected L. reuteri strain against EHEC (L. reuteri-EHEC concentration ratio, glycerol level, aeration conditions) and provide helpful data to set up more targeted in vivo trials to assess the efficiency of L. reuteri to decrease EHEC shedding by cattle. Future studies will be required to determine i) DFM supplementation parameters, ii) the effect of L. reuteri and glycerol on rumen microbiota and iii) the survival of EHEC strains in lower gastrointestinal tract. In vivo studies will also be necessary to explore the potential of HPA-producing L. reuteri alone or in combination with already commercialized DFMs in order to ultimately validate an efficient pre-harvest strategy in beef cattle with the aim to improve food safety. (LB1-7, F275, 65-A, F70 and 100-23) were first spotted onto the surface of Brain Heart Infusion (BHI) agar supplemented with 20 mM glucose and incubated anaerobically. The EHEC strain FCH6 was then inoculated in soft agar with or without glycerol (2%) and poured over the L. reuteri spots (in order to facilitate or not HPA production by spots containing L. reuteri). The plates were then incubated and the antimicrobial activity was recorded as growth-free inhibition zones around the spots as previously described [20].	v3-fos-license
2017-08-14T22:43:40.358Z	2012-04-30T00:00:00.000	30506728	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://revistas.inia.es/index.php/sjar/article/download/1948/1671", "pdf_hash": "08ef973ebd459dff711f849b32bbf104965c9f28", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1367", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "sha1": "a87772bbc890d220ea247eec0f3a8f91d126fc74", "year": 2012 }	pes2o/s2orc	Effect of extruded whole soybean dietary concentrate on conjugated linoleic acid concentration in milk in Jersey cows under pasture conditions Contradictory results has been found on the effects of soybean supplementation and conjugated linoleic acid (CLA) content in milk on feeding systems based on fresh forage The objective of the study was to evaluate the effect of a dietary supplement with different quantities of extruded whole soybean on the production and composition of milk, and CLA concentration or their isomers in Jersey cows under pasture conditions. Twenty-one Jersey cows were randomly assigned into 3 groups of 7 animals each. The cows were supplemented with a dietary concentrate (5 kg d –1 ), and each group received one of the three next treatments: control without soybean (0-SB), with extruded whole soybean at 0.5 kg d –1 (0.5-SB) or at 1 kg d –1 (1-SB). The basic diet was a pasture composed of Lolium perenne (70%), Trifolium repens (25%) and other species. The duration of the study was 75 d. Milk production ( p = 0.706) and protein production ( p = 0.926) were not affected by treatments. Fat ( p = 0.015) and protein ( p = 0.045) content as well as fat production ( p = 0.010) were lower in the 1-SB group. There was no effect of the inclusion of extruded soybean on total CLA content ( p = 0.290) or the content of cis -9, trans -11 ( p = 0.582), trans -10, cis -12 ( p = 0.136) and cis -10, cis -12 ( p = 0.288) isomers. However, concentrations of all isomers were affected by the nutritional quality of the pasture, with low values observed at greater maturity stages of pasture. . Although, it has been documented that concentration of CLA in milk from Jersey cows is 18% lower than that observed in Holstein cows (White et al., 2001), apparently few studies have been conducted regarding CLA content in Jersey cows. Whilst functional foods have been considered a promising area for human health (Starling, 2002), it has frequently been observed that consumers expect added-value products without substantial extra cost, suggesting that the development of low-cost approaches will be important (Dewhurst et al., 2006). Considering that soybean is an imported feedstuff in the majority of the countries and given the current prices in the market, the objective of this study was to evaluate the effect of a dietary supplement with different quantities of extruded whole soybean (0.5 and 1.0 kg d -1 ) on milk yield, CLA isomers content and metabolic profile in Jersey cows maintained in pasture-based systems. Animals and diets The experiment was carried out in a farm with a Jersey herd based on grazing plus the use of concentrate, located in the Entre Lagos sector (district of Osorno, Chile), approximately 72° 36' 24" W 40° 41' 26" S, 250 m above sea level. The farm lies in the pre-mountain range of the Chilean Andes, Region X, which is characterized by an average rainfall of 2250 mm per year, with an average daily temperature of 21.8°C in January and 3°C in August. The experiment was developed in compliance with the principles and specific guidelines on animal care and welfare as required by Chilean law (SAG, 2010). The duration of the experiment was 75 d, between 15th November 2005 and 25th January 2006 (spring period). The first 15 d were for adaptation to the experimental diets and the experimental period was from day 15 to day 75. Twenty-one healthy cows between 2 and 7 calvings, with a range from 60 to120 days in milk (92 ± 5 DIM) and a body condition score of 2.75 ± 0.7 were used in the study. An average of 18.4 ± 3.73 kg d -1 of milk production was recorded. Cows were selected for the study based on previous milk production in order to make three ho- Introduction Conjugated linoleic acid (CLA) represents between 20 and 28 isomers of linoleic acid C18:2 (Lock & Garnsworthy, 2003) that has been indicated as one of the most beneficial fatty acids for human health (Pariza & Park, 2001). Likewise, ruminant products as milk and meat constitute the principal source of CLA for humans. Of all possible isomers, only cis-9, trans-11 and trans-10, cis-12 have shown an interesting biological activity (Wahle et al., 2004). The cis-9, trans-11 isomer, also known as rumenic acid, has been documented to have an anticarcinogenic (Ha et al., 1987;Visonneau et al., 1997;Aro et al., 2000) and antioxidant effect (Devery et al., 2001), whereas the trans-10, cis-12 isomer is capable of decreasing body fat and increasing lean body mass. Diet has a major influence on milk fat CLA and it has been extensively investigated . Several nutritional studies have been addressed to increase CLA content in animal products and to improve their nutritional properties. For instance, it has been reported that fresh forage and oil-rich feeds increase CLA concentration (Khanal et al., 2005;Dewhurst et al., 2006). Soybean is widely used in total mixed ration (TMR) in different proportions, and it has been observed that seed treatment (roasting or extrusion) results in a higher increase on CLA content than that observed with intact seed (Chouinard et al., 2001). However, contradictory results has been found on the effects of soybean supplementation and CLA content in milk on feeding systems based on fresh forage. Some studies (Bartolozzo et al., 2003;Khanal et al., 2005) did not found effects on milk CLA concentration in dairy cows, while others (Lawless et al., 1998;Paradis et al., 2008) observed an increase in the CLA using dairy and beef cattle. On the other hand, the effects of other factors such as breed, lactation and parity on CLA content in milk fat have received less attention. Kelsey et al. (2003) reported that breed (Holstein vs. Brown Swiss), parity, and days in milk accounted for < 0.1, < 0.3, and < 2.0% of the total variation in CLA concentration in milk fat, respectively. The incorporation of Jersey cattle in dairy farms has been increased in the last decade in Chile due to their high level of total milk solids produced CLA content in milk of Jersey cows supplemented with extruded whole soybean on pasture mogenous groups and they were randomly assigned (n=7/per group) to receive a dietary concentrate or treatment with different quantities of extruded whole soybean: T1= control diet without supplementation (0-SB), T2 = 0.5 kg d -1 (0.5-SB) and T3 = 1 kg d -1 (1-SB). Each animal was fed with 5 kg d -1 of isoenergetic dietary concentrate (Table 1), distributed in two visits to the milking parlour, at 06:00 h in the morning and in the afternoon at 16:00 h. Visual observation of feed intake indicated that cows consumed all concentrate offered. The animals grazed in two paddocks of 18 hectares each, and managed with rotational stripgrazing and electric fencing. The pasture was an improved natural pasture with grasses being the predominant species (70% Lolium perenne, 25% Trifolium repens, 3% Bromus sp). The animals were transferred from one strip to the next every 24 h. The diets were formulated according to the animal requirements established by NRC (2001). Chemical composition and nutritional value of feedstuff Samples of the pasture and concentrates were taken every 10 days to determine their chemical and nutritional composition (Tables 1 and 2). Representative samples of pasture forage were collected from the paddock before grazing at a height of 8 cm above the ground, using a 1-m 2 quadrant. Dry matter contents of the pasture were determined by forced air oven at 60°C for 48 h. Samples of pasture and concentrates were ground to pass a 1-mm screen in a Willey mill before analysis. Dry matter (method 934.01), ash (method 1 Control without extruded whole soybean (0-SB), with extruded whole soybean at 0.5 kg d -1 (0.5-SB), and with extruded whole soybean at 1 kg d -1 (1-SB). 942.05), ether extract (method 920.39), N (method 984.13) and crude fiber (method 978.10) were determined according to AOAC (2005) methods. The N values determined by the Kjeldahl procedure, and converted to crude protein by multiplying by a factor of 6.25. The analyses of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were carried out according to Van Soest et al. (1991), and both NDF and ADF were expressed exclusive of residual ash. All fiber fractions were analyzed on a Fibertec 1030 Hot Extractor (Tecator, Sweden). The fat content was measured by extraction with petroleum ether (boiling point, 40 to 60°C) on a Soxtec System 1040 Extraction Unit (FOSS Tecator AB, Sweden). The metabolizable energy (ME) of the supplemented dietary concentrates was estimated according to NRC (2001). The in vitro dry matter digestibility (IVDMD) of the pasture was determined according to the procedure described by Tilley & Terry (1963) modified by Van Soest (1991) and the ME was estimated according to the equation (Garrido, 1981): ME = 0.279 + 0.0325 × IVDMD. Milk yield and quality Cow milk production was determined using a Waikato® measuring equipment, on days 1, 15, 30, 45, 60 and 75. At each control, a milk sample of 30 ml was taken (to which was added 0.03 g of potassium dichromate at 0.1% as a preservative), and the contents of fat, protein and urea were determined automatically using an infrared spectrophotometer (Foss 4200 Milko-scan; Foss Electric, Denmark). CLA content and composition At each milk control, milk samples of 100 mL were taken and sent to the laboratory in thermally insulated containers at 4°C for analysis of CLA isomers (cis-9, trans-11; trans-10, cis-12; cis-10. cis-12). Total lipids were extracted by the method of Folch et al. (1957), using a mixture of chloroform and methanol (2:1, v:v). The methylation of the fatty acids of the samples was done using the method described by Morrison & Smith (1964). Fatty acid methyl esters were analyzed by gas chromatography (HP 6890, Hewlett Packard, Surrey, UK), Flame Ionization Detector (FID), a capillary column SP-2560 (100 m, 0.25 mm i.d. with 0.20 μm thickness in the stationary phase; Supelco Inc., Bellefonte, Pennsylvania, USA) using He as the tracer gas. Gas chromatography conditions were as follow: the injection volume was 0.5 μL, a split injection was used (70:1, v:v); ultrapure hydrogen was the carrier gas; and the injector and detector temperatures were 250 and 300°C, respectively. The initial temperature was 70°C (held for 1 min), increased by 5°C per min to 100°C (held for 3 min), increased by 10°C per min to 175°C (held for 40 min), and then increased by 5°C per min to 220°C (held for 19 min) for a total run time of 86.5 min. Data were then quantified using the HPCHEM Stations software, and expressed as a percentage of area according to the total fatty acids identified. Metabolic profile At the beginning and at the end of the experiment blood samples were taken (5 mL animal -1 ) by coccygeal venipuncture flow and placed in tubes with sodium heparin. The samples were then centrifuged for 3 min at 3000 rpm and the plasma was aliquoted and frozen (-18°C) in microtubes of 1.5 mL. For each sample, the following plasma traits were determined: cholesterol (cholesterol-oxidase method, Cholesterol Liquicolor 10028 Human), albumin (Albumin Liquicolor Method BCG-Bromo Cresol), total protein (Total Protein Liquicolor-Biuret Method), calcium (Arsenazo III AA), Mg (Mg-color AA), phosphorus (Fosfataria UV AA), aspartate aminotransferase (IFCC Mod. LiquiUV test) and urea (ureasa/NADH method, UREA LiquiUV 10521 Human). All plasma traits were determined automatically by biochemical analyser (SelectraVitalab, Merk, Darmstdt, Germany). Statistical analysis Data of milk production, milk's constituents and metabolic profile were analysed as repeated measures, using the general linear model (GLM) of the SPSS for Windows 18.0 package (SPSS Inc., Chicago, IL, USA). The linear model used for each parameter was as follows: where Y ijk = observations for dependent variables; μ = overall mean; T i = fixed effect of treatment group or CLA content in milk of Jersey cows supplemented with extruded whole soybean on pasture dietary concentrate; A ij = random effect of animal j for the i treatment; W k = fixed effect of the k week of lactation; T × W = interactions among these factors for the i treatment and k week of lactation, and ε ijk = random effect of residual. Pairwise comparisons of means were carried out, where appropriate, using Tukey's honest significant difference tests. The level of significance for the analyses was 5%. The Pearson correlation coefficient between the milk fat concentration and the content of trans-10, cis-12 isomer was also determined. Milk yield and quality In the initial day no differences in milk production (p = 0.390) and quality were observed among the three experimental groups (data not shown). In the experimental period (from day 15 to day 75) milk production (p = 0.706) and fat corrected milk production (FCM, kg d -1 ) (p = 0.241) were similar among groups (Table 3). The amounts of milk fat (kg d -1 ) (p = 0.010), as well as protein (p = 0.045) and milk fat (p = 0.015) concentrations were lower in the 1-SB treatment, while the quantities of protein (p = 0.926) and urea (p = 145) were similar among all treatments. The patterns of milk production and basic composition throughout lactation were affected by the lactation day for all the components (Table 3). Milk yield significantly decreased as a function of the week and for the chemical composition, the highest values for these components were found in the last weeks (data not shown). CLA content and composition In the initial day no differences in total CLA (p = 0.791) and of each of its isomers were observed among the three experimental groups (data not shown). In the experimental period, there was no effect of the inclusion of extruded soybean on total CLA content (p = 0.290) or the content of cis-9, trans-11 (p = 0.582), trans-10, cis-12 (p = 0.136) and cis-10, cis-12 (p = 0.288) isomers (Table 3). Although the highest values were found for the cis-9, trans-11 isomer (53-59% of total CLA), the trans-10, cis-12 and cis-10, cis-12 isomers presented higher values (17-23% and 20-25% of total CLA, respectively) than normally reported in the literature. The pattern of fatty acid composition throughout lactation was affected by the lactation day for all components (Table 3; Fig. 1). For the content of total CLA and of each of its isomers, a similar trend is observed in all the treatments. The lowest CLA values were obtained in the lasted weeks, when the herbage presented the poorest nutritional quality (see Table 2). The cis-10, cis-12 isomer was the only one that diminished from day 1 to day 45, and increased after day 60. Metabolic profile Throughout the trial the cows were in good health and did not show any relevant pathology. All the me-tabolites evaluated, except blood urea at the end of the trial, were found to be within the normal range, with no significant differences observed between treatments (Table 4). Milk yield and quality Although crude protein of supplemented concentrate ranged between 17 and 21%, in all groups the pasture contributed an adequate level of protein and energy in accordance with NRC (2001) recommendations based on milk production and milk urea content (see Table 3). The reduction in the percentage and amount of fat (kg d -1 ) in the group fed with a higher quantity of soybean (1 kg d -1 ) may result from the extrusion process, which breaks up the micelles of fat in the seed, allowing a rapid release of the lipids in the rumen and reducing milk fat content (Mohamed et al., 1988;DePeters & Cant, 1992). Low milk fat syndrome has been recognized for many years, but the exact mechanism is still unclear. Data from several studies revived the theory of trans fatty acids, coming from ruminal biohydrogenations and from desaturation by the mammary gland, as the central mechanism of milk fat depression (Griinari & Bauman, 2003;Loor et al., 2005). In particular, the increase of C18:1 trans-10 and CLA trans-10, cis-12 isomers in the mammary gland has been associated with a reduction in the de novo synthesis of short and medium chain fatty acids (Banks et al., 1980;Grummer, 1991;Baumgard et al., 2000). The CLA trans-10, cis-12 isomer was found in the highest quantity (though such difference was not significant) in the 1-SB treatment. Also, in the present study there were an inverse linear relation (R 2 = 0.11, p = 0.04) between milk fat concentration and the content of this isomer. The milk protein content was lower in the 1-SB diet when compared with 0-SB and 0.5-SB diets. Although the dietary fat and protein were highest in the 1-SB group, Theurer et al. (1995) suggested that increasing the amount of dietary protein within a constant dietary energy level has little effect on milk protein synthesis and whenever dietary protein level increases milk protein yield, the effect seems to be associated with an increase in milk yield. However, in this study no differences between groups were found both in milk protein yield and in milk yield. The decrease in milk protein content might have been due to an increased availability of fat in the rumen in the 1-SB diet (Chouinard et al., 1997). The reduction in the percentage of protein observed in most of studies in animals fed with diets with a high fat content appears to be associated with negative effects on the growth of ruminal micro-organisms and the production of microbial protein (Solomon et al., 2000). In addition, when cows are fed fat, the energetic efficiency of milk synthesis is increased. Cows fed high fat diets required less liters of blood flowing to the mammary gland per kg of milk produced (Cant et al., 1993). Because mammary uptake of amino acids is dependent upon amino acid concentration in the blood and blood flow to the mammary gland, these data suggest that the decrease in blood flow per volume of milk produced would limit the uptake of amino acids for milk protein synthesis. However, there are studies that did not found any effect (Guillaume et al., 1991) or even others that found an increase of protein concentration in milk (Block et al., 1981). The differences in feeding (mainly due to ingestion and nutritional composition of the herbage) and lactational effects can explain the changes on milk production and components across the weeks of the study. However, since total forage ingestion was not monitored in this study this will have to be tested in future studies. CLA content and composition As in our study, Khanal et al. (2005) did not found effects of a mixed supplement containing 2.4 kg d -1 of extruded soybean on CLA concentration in the milk of Holstein cows on pasture (1.63 and 1.69% of total FA for groups fed on pasture alone and pasture + supplement mixed with extruded soybean, respectively; these values include the cis-9, trans-11 isomer). The values obtained by these authors are somewhat higher than those found herein for the three CLA isomers together. Bartolozzo et al. (2003), in Friesian cows fed on pasture and fed a mixed supplement containing 2.6 kg of raw soybean, also obtained a high quantity of CLA (0.96%) as compared to TMR diets with raw or extruded soybean (0.52%), which is in agreement with the results of White et al. (2001). All these results indicate a greater influence of the pasture than that of the source or level of soybean incorporated into the diet on the CLA milk content. The different CLA values found in the literature may be related to differences in the nutritional composition of the pasture derived from the different botanical and agronomic characteristics of the herbage used in the various studies (Dewhurst et al., 2006) and, to a lesser degree, to the influence of other factors such as the breed (White et al., 2001;Kelsey et al., 2003). In this respect, White et al. (2001) observed 18% less CLA in milk from Jersey cows compared with milk from Holsteins. On the contrary, other authors observed an increase in the CLA milk content as compared to the control groups, with values up to 2.2% for all the CLA isomers in dairy cows on pasture supplemented with 3.1 kg d -1 roasted soybean (Lawless et al., 1998), and 2.4% for only the cis-9, trans-11 isomer in beef cattle on pasture supplemented with 2 kg d -1 extruded soybean (Paradis et al., 2008). However, the exact influence of breed related to dietary supply, and possible interactions need to be determined in further studies. Rumenic acid is typically the most abundant CLA isomer, with values greater than 80% of total CLA (Palmquist et al., 2005). The cis-10, cis-12 isomer, on the other hand, was found in very low quantities and has no known physiological function (Khanal & Olson, 2004). In the present study, the trans-10, cis-12 and cis-10, cis-12 isomers presented higher values than normally reported in the literature. The regulation of isomer balance is largely unknown. Nevertheless the cis-9, trans-11 isomer is mainly generated from vaccenic acid in the mammary gland (Mosley et al., 2006), while the trans-10, cis-12 is a minor intermediate of rumen biohydrogenation (Walker et al., 2004) and is relatively unaffected by changes in the diet except at very high levels of concentrate feeding (Chilliard et al., 2007). Therefore future studies are necessary to determine its biological function and metabolic production routes. In the temporal pattern ( Fig. 1) for the content of total CLA and of each of its isomers, a similar trend is observed in all the treatments, which would indicate that the influence of the herbage on the CLA content of the milk is greater than that of the different dietary concentrates supplemented. This may be related to differences in the nutritional composition of the herbage, which has also been shown to affect the fatty acid composition of milk (Dewhurst et al., 2006). In this respect, lower CLA contents in milk have been observed with more mature pasture, and this effect has been attributed to the declining quality and quantity of the herbage (Lock & Garnsworthy, 2003;Ward et al., 2003). This is in agreement with the present work, in which the lowest CLA values were obtained in late December and January, when the herbage presented the poorest nutritional quality (see Table 2). The cis-10, cis-12 isomer was the only one that diminished from day 1 to day 45, and increased after day 60. At present, it is difficult to explain both the higher quantity and the evolution of this isomer, as observed in the present study. Metabolic profile All the metabolites evaluated, except blood urea at the end of the trial, were found to be within the normal range, in agreement with the values for healthy lactating dairy cows (Bertoni & Piccioli, 1999). Previous studies (Pulido, 2009) have shown an increase in blood urea when diets present high levels of degradable protein, which is the case with animals fed to pasture on grass (L. perenne). Under this conditions, highly soluble protein is associated with low levels of NDF and high leaf/stalk proportions at the beginning of spring (Van Vuuren et al., 1991), resulting in incomplete use of the nitrogen in the rumen and high levels of blood urea during the spring and early summer (Wittwer et al., 1993). These levels may exceed the normal range, especially at the beginning of spring, and this is considered normal in Chile (Wittwer et al., 1993). Conclusions The dietary concentrate with different quantities of extruded soybean (0.5 and 1.0 kg d -1 ) fed to Jersey cows, on pasture-based systems, did not influence milk production or total CLA content or its cis-9, trans-11, trans-10, cis-12 and cis-10, cis-12 isomers. However, CLA contents were affected by the nutritional quality of the pasture, with lower values observed at greater maturity stages of pasture. In the present study, high quantities of the trans-10, cis-12 and cis-10, cis-12 isomers were obtained in comparison to those normally found. The cis-10, cis-12 isomer does not appear in the scientific literature, therefore future studies are necessary to determine its biological function and metabolic production routes. The failure of the dietary concentrate with different quantities of extruded whole soybean supplemented in Jersey cows on pasture to increase the concentration of CLA cis-9, trans-11 in milk fat requires further investigation.	v3-fos-license
2020-08-27T09:03:46.096Z	2020-08-21T00:00:00.000	225295874	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2020/ra/d0ra03910a", "pdf_hash": "5408015ca88de4a1ff079f4ae05cbd4031abc1d7", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1416", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "e6a2e436b28c99df3c377534885ed424bf22b215", "year": 2020 }	pes2o/s2orc	Synthesis of highly functionalized thiazolo[3,2-a]pyridine derivatives via a five-component cascade reaction based on nitroketene N,S-acetal A highly efficient and straightforward synthesis of N-fused heterocyclic compounds including 5-amino-7-(aryl)-8-nitro-N'-(1-(aryl)ethylidene)-3,7-dihydro-2H-thiazolo[3,2-a]pyridine-6-carbohydrazide derivatives is successfully achieved via a five-component cascade reaction utilizing cyanoacetohydrazide, various acetophenones, aromatic aldehydes, 1,1-bis(methylthio)-2-nitroethylene and cysteamine hydrochloride in ethanol at reflux conditions. The new approach involves domino N,S-acetal formation, Knoevenagel condensation, Michael reaction, imine–enamine tautomerization and N-cyclization sequences. The prominent advantages of this protocol include: facility of operation, available and economical starting materials, no need for toxic solvents, high yields and tolerance of a wide variety of functional groups. Introduction The thiazolopyridine moiety is found in a wide spectrum of biologically active compounds. Thiazolo [3,2-a]pyridines are an important category with notable antibacterial and antifungal activity 1 and other considerable bioactivities including as a beta-amyloid production inhibitor, 2 potent CDK2-cyclin A inhibitor, 3 potential uterus stimulant, 4 coronary dilator, antihypertensives, and muscle relaxant. 5 Also they are useful for chemotherapy of various cancers, such as leukemia, lung cancer, and melanoma. [6][7][8] Some biologically active compounds with this nucleus are presented in Fig. 1. [9][10][11] Obviously, the synthesis of new classes of thiazolo[3,2-a] pyridines may give a library of compounds as possible candidates for various biological activities. Cyclic ketene N,S-acetal structures are as such used as drugs for the treatment of hypertension diseases and usually employed as probes for nucleic acids to study the interaction between G4 (G-quadruplex) and its ligands (Fig. 1, I-III). 12 It's interesting that the cyclic nitroketene N,S-acetal nithiazine IV was the rst reported compound of neonicotinoid insecticides 13 and is widely used as a common insecticide around the world (Fig. 1). Synthetically, the cyclic nitroketene N,S-acetals have a rigid structure and act as Michael donor 1,3-N,C dinucleophiles for the generation of nitrogen-containing heterocyclic compounds. The ethylene motif has a polarized pushpull type of alkene, therefore the one end expands an electrophilic character, whereas the other end develops a nucleophilic character. This feature of nitroketene N,S-acetals make them highly useful to apply in the Michael addition, annulation and multicomponent reactions. 14 Today, multicomponent reactions (MCRs) have become a prominent strategy and are selected over stepwise synthesis due to the following reasons: reduced synthetic time, labor and cost, minimal utilization of toxic and harmful chemicals, simple workup of products, high yields, straight forward and simplicity of experimental procedures and economic viability; therefore, MCRs are a powerful approach to promotion of green chemistry by reducing the formation of large quantities of waste. [15][16][17][18][19] The ve-membered cyclic nitroketene N,S-acetal and commercially available six-membered nithiazine have been remarkably explored in the literature and their reactions with different Michael acceptors are most expected. Here we report the some synthesis of thiazolo[3,2-a]pyridine compounds performed with cyclic ketene N,S-acetals (Scheme 1). In 2005, Chakrabarti et al. described the reactions between diverse cyclic N,S-and N,N-ketene acetals and itaconic anhydride (A). 20 In 2010, Yan et al. reported one-pot synthesis of functionalized bicyclic pyridines under solvent-and catalyst-free conditions with triethoxymethane, ethyl 4,4,4-triuoro-3-oxobutanoate and various ketene aminals (B). 21 In 2011, Altug et al. developed the synthesis of thiazolo[3,2-a]pyridines via a one-pot reaction between 2-(nitromethylene)thiazolidine, aromatic aldehydes and ethyl 2-cyanoacetate, malononitrile or 2-(phenylsulfonyl) acetonitrile (C). 22 In 2018, our research group synthesized fused thiazolo[3,2-a] pyridines utilizing the ve-membered cyclic nitroketene N,Sacetal, dimedone and different aromatic aldehydes (D). 23 Also we reported the synthesis of indenone-fused thiazolo [3,2-a] pyridines via a one-pot reaction between 2-(nitromethylene) thiazolidine, aromatic aldehydes and 1,3-indandione (E). 24 In addition, we were able to produce the desired products using cyanoacetamide, aromatic aldehydes and 2-(nitromethylene) thiazolidine (F). 25 Moreover, in 2018, the reaction of cyanoacetohydrazide with aromatic aldehydes and 2-(nitromethylene) thiazolidine/oxazolidine resulted in functionalized thiazolo/ oxazolo pyridine derivatives (G). 26 Following our efforts to synthesize the new heterocyclic compounds using cyanoacetohydrazide and based on previous works, we designed new reactions utilizing 2-(nitromethylene) thiazolidine as heterocyclic ketene aminal. In this article we report an efficient synthesis of highly functionalized 2H-thiazolo[3,2-a]pyridine-6-carbohydrazide compounds via a one-pot ve-component domino reaction. To the best of our knowledge, there is no report on the synthesis of these structures. In general, due to the variable reactivity of cyanoacetohydrazide (based on its specic structure) and on the other hand due to the ve-component nature of the dened reactions, great efforts were made to obtain the desired products with high purity. At rst ethanol was examined and the experimental results showed when ethanol was used as solvent with triethylamine at reux conditions, the yield of desired product 6a was 93% (Table 1, entry 1). It should be noted that the catalyst used (NEt 3 ) is not working on the rate-limiting step. To prepare 2-(nitromethylene)thiazolidine solution (from 1,1bis(methylthio)-2-nitroethene and cysteamine hydrochloride, which is mentioned in the Experimental section), it is necessary to use triethylamine to separate cysteamine from its salt. 23,27 No reaction will occur without the use of triethylamine (entry 4). The use of other catalysts is related to the whole reaction. In order to increase the reaction rate, two types of catalysts were used. With piperidine, the reaction efficiency decreased slightly (entry 2) and with acetic acid, the product did not form (entry 3). According to the investigations, it was determined that in basic and acidic medium, other products are formed (two-, three-and four-component products). The percentage of each was different for various derivatives. In general, it was found that the slightest change in the reaction conditions (even in ethanol amount) leads to a decrease in the efficiency of the desired product or oen its non-formation. In addition, we observed the formation of a four-component by-product in two cases, which are described in the general procedure section. However, we also studied the effect of other solvents. The use of water or acetonitrile did not result in the desired product (entry 5 and 7), and when the mixture of water and ethanol was used (overall 1 : 1, v/v), the efficiency decreased (entry 6). With chloroform, methanol and DMF, in reux conditions the desired products were not formed (entry 8, 9 and 10). With information obtained from optimization conditions table, we could synthesize target compounds (E)-5-amino-7-(aryl)-8-nitro-N'-(1-(aryl)ethylidene)-3,7-dihydro-2H-thiazolo[3,2a]pyridine-6-carbohydrazide 6a-p in good to high yields (70-95%) using cyanoacetohydrazide 1, acetophenone derivatives 2, various aromatic aldehydes 3, 1,1-bis(methylthio)-2-nitroethene 4 and cysteamine hydrochloride 5 as starting materials (Scheme 2). The reactions were completed aer 24 h to afford the corresponding heterocyclic structures. The results are summarized in Table 2. Scope and limitations This reaction was performed with ortho derivatives of benzaldehyde (2-chloro, 2-hydroxy and 2-nitro) under the same conditions, which did not result in the product probably due to steric effects. Also the use of acetophenone and 4-methoxyacetophenone did not lead to the favorable products. The reaction was also used with aliphatic ketones instead of acetophenone derivatives and aliphatic aldehydes instead of aromatic aldehydes which resulted in no product formation. It was found that the major by-product of this reaction is a fourcomponent structure that was previously synthesized using two equivalents of aldehyde 26 which will prevent its formation by performing the correct reaction steps (see Experimental section). Structure determination The structures of all new compounds 6a-p were supported by means of IR, 1 H NMR, 13 C NMR spectroscopic and mass spectrometric data (see the ESI †). The 1 H NMR spectrum of 6a showed NH group at d 9.35 ppm. The NH 2 group appeared at d 8.15 ppm. The proton of CH at pyridine ring was seen at d 5.66 ppm. Four protons of two methylene groups appeared at d 4.22 to 4.38 ppm as two multiplets. The signal at d 2.11 ppm was related to methyl group. The 1 H-decoupled 13 C NMR spectrum of 6a indicated 18 distinct resonances in accordance to desired structure. The characteristic signals of four aliphatic carbons (CH 3 , CH and two CH 2 groups) were seen at d 13.8, 37.7, 27.6 and 50.8 ppm respectively. Characteristic signal at d 81.8 ppm was related to C]C-CO. The carbonyl group appeared at d 165.7 ppm (Fig. 2). The IR spectrum of 6a showed absorption bands at 3141 and 3284 cm À1 due to NH and NH 2 groups, strong absorption of carbonyl group at 1626 and C-N band at 1237 cm À1 . Two absorption bands due to nitro group appeared at 1514 and 1302 cm À1 . Mechanism A general plausible mechanism for the formation of thiazolo [3,2-a]pyridine carbohydrazides is shown in Scheme 3. The condensation of cyanoacetohydrazide 1 with acetophenone 2 leads to the hydrazide-hydrazone structures 7. On the basis of well-established chemistry of 1,1-bis(methylthio)-2-nitroethene, on the other hand, addition of cysteamine hydrochloride 5 to 1,1-bis(methylthio)-2-nitroethene 4 leads to the formation of ketene N,S-acetal 9. 23,27 The formation of b-nitrothiazolidine 9 occurs in the presence of an equivalent amount of triethylamine base for releasing cysteamine salt. Further, with adding aldehyde 3, the Knoevenagel condensation affords intermediate 8. 6-carbohydrazide derivatives A mixture of cysteamine hydrochloride (0.113 g, 1 mmol), 1,1bis(methylthio)-2-nitroethylene (0.165 g, 1 mmol), Et 3 N (140 mL, 1 mmol) and 10 mL EtOH in a 50 mL ask was reuxed for 5 hours. In another 50 mL ask the stoichiometric mixture of cyanoacetohydrazide (1 mmol, 0.099 g) and acetophenone derivative (1 mmol) in EtOH (10 mL) was reuxed for 3-5 hours depending on the type of acetophenone. Aer these times, TLC shows the consumption of the starting components. Then, aromatic aldehyde (1 mmol) and the rst solution (HKA), were added to the second mixture simultaneously. The progress of the reaction was monitored by TLC using ethyl acetate/n-hexane (1 : 1). Aer completion of the reaction (24 hours), without the need for chromatography or recrystallization, the precipitated product was collected by ltration and washed with warm ethanol to give the pure products 6a-p in excellent yield. To achieve the pure products, it was necessary to complete the reaction of cyanoacetohydrazide and acetophenone derivatives in ethanol at reux conditions in sufficient time (3 hours for 4-nitroacetophenone and 5 hours for 4-chloro and 4-bromoacetophenone), then with no need for product separation, nitroenamine solution and aromatic aldehyde were added to two-component hydrazone mixture at the same time. We found two distinct cases (6g and 6h) that led to a mixture of two products: the desired product and the product without participation of acetophenone derivative. 26 (E)-5-Amino-7-(4-chlorophenyl)-N'-(1-(4-chlorophenyl)ethylidene)-8-nitro-3,7-dihydro-2H-thiazolo[3,2-a]pyridine-6carbohydrazide (6a). Yellow solid; yield: 0.468 g (93%); mp: 252- Scheme 3 Proposed mechanism for the formation of products 6.	v3-fos-license
2019-04-26T13:36:11.524Z	2019-03-31T00:00:00.000	132400820	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://jidc.org/index.php/journal/article/download/32040454/2028", "pdf_hash": "9d57c843e010f49548b15c025a20b50e566e188c", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1424", "s2fieldsofstudy": [ "Biology" ], "sha1": "9d57c843e010f49548b15c025a20b50e566e188c", "year": 2019 }	pes2o/s2orc	In vitro anti-Trichomonas vaginalis activity of Haplophyllum myrtifolium Introduction: In the classic treatment of Trichomonas vaginalis infection, although metronidazole has been used since the 1960s, there has been an increase in MTZ-resistant T. vaginalis strains and failure in the treatment of trichomoniasis causes serious concerns. Therefore, the present study aimed to investigate the in vitro antitrichomonal activities of extracts (ethanol and total alkaloid) and pure compounds (chrysosplenetin, dictamnine, gamma-Fagarine, skimmianine) of H. myrtifolium against T. vaginalis. Methodology: H. myrtifolium was collected from the town of Honaz in Denizli, located in the Aegean region of Turkey, and preparation of extracts and isolation and structure elucidation of pure compounds were performed. Later, different concentrations of extracts and pure compounds were incubated with T. vaginalis trophozoites isolated from Turkey, which are known to be sensitive to metronidazole. Results: It was found that ethanol extract caused a more effective lysis on T. vaginalis trophozoites compared with total alkaloid extract (P < 0.05). No compounds except for furoquinoline alkaloid skimmianine prepared above 37.5 μg/mL were found to have any inhibitory effect on T. vaginalis trophozoites. Conclusion: The ethanol extract of H. myrtifolium and skimmianine can be considered as potential candidates for antitrichomonal drug development. Introduction Trichomoniasis, caused by the pathogenic trichomonad Trichomonas vaginalis, is the most common, non-viral sexually transmitted disease (STD) worldwide, with 276 million new cases each year [1]. The natural host of this flagellated protozoan is humans, and it can cause urogenital tract infections both in females and males [2].In females, T. vaginalis leads to vaginitis, showing symptoms of vaginal itching, odor, and discharge, whereas in males, symptoms are not generally noticeable, although urethritis and swelling of the prostate gland can be observed [3].In addition, trichomoniasis can give rise to serious health consequences in females such as adverse pregnancy outcomes resulting in low-birth-weight infants and premature rupture of placental membranes, pelvic inflammatory disease, cervical cancer, and infertility.It has also been reported in previous studies that trichomoniasis can increased risk of transmission of HIV [2,[4][5][6][7][8]. The incidence rates for STDs vary according to age, sexual activity, number of sexual partners, menstrual cycle, methods of diagnosis, and socioeconomic status [9].A limited number studies have investigated the prevalence of trichomoniasis among female and male patients living in Turkey [10].According to results obtained from a few studies, the prevalence of T. vaginalis among female patients with vaginal discharge is 7-8.69%,whereas the prevalence in male patients with suspected urinary system infection is 6.5% [10][11][12]. In the classic treatment of T. vaginalis infection, metronidazole (MTZ), the only effective approved drug, has been used since the 1960s.On the other hand, there has been an increase in the number of MTZresistant T. vaginalis strains, and failure in the treatment of trichomoniasis causes serious concerns [2].Erroneous cases are generally treated using increased doses of MTZ, which may cause an increase in the rate of adverse effects [2].In addition to this, poor absorption and ineffective delivery of drug on the infection site are accepted as additional negative factors in the failure of trichomoniasis treatment [13].These reasons are sufficient to investigate new natural antitrichomonal therapeutic agents that have fewer adverse effects and are relatively inexpensive. A study conducted in 2018 listed natural plants that showed anti-Trichomonas vaginalis activity [2].According to the list, dried bean seed coats, lectins of Phaseolus vulgaris, Perla black bean, leaves of Voacanga globose, leaves of Cussonia species L., leaves of Eucalyptus camaldulensis, seeds and oil of Nigella sativa, roots of Polygala decumbens, leaves of Campomanesia xanthocarpa, and seeds of Persa americana and Pistacia lentiscus were shown to be effective against T. vaginalis.In addition, natural secondary metabolites such as flavonoids, alkaloids, cumarins, saponins, and glycosides were also reported to have anti-Trichomonas vaginalis activity [13]. To the best of our knowledge, there has been no previous work on the anti-T.vaginalis activity of H. myrtifolium, a medicinal plant endemic in Turkey.Therefore, the present study aimed to investigate the in vitro anti-Trichomonas vaginalis activities of extracts and pure compounds (chrysosplenetin, dictamnine, gamma-Fagarine, skimmianine) of H. myrtifolium against T. vaginalis in comparison with MTZ, which is the reference drug for the treatment of trichomoniasis. Plant material and preparation of extracts H. myrtifolium was collected from the town of Honaz, Denizli, located in the Aegean region of Turkey.The plant was taxonomically identified and deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Izmir, Turkey. The preparation of extracts and isolation and structure elucidation of pure compounds were performed as previously described [14][15].Briefly, the aerial and underground parts of the plant were cut into small pieces, dried and powdered in a grinder.For ethanol extraction, the powdered material was first dissolved in ethanol at room temperature.Later, extraction solvent was filtered and evaporated under reduced pressure in a rotary evaporator (Buchi, Essen, Germany).Finally, a part of extract was lyophilized and used as ethanol extract.In the next step, for total alkaloid extraction, the remaining extract, a dark syrupy material, was acidified using 2% hydrochloric acid and filtered.Later, 25% aqueous ammonium hydroxide was added to the filtrate to adjust to pH 9-10 and the filtrate was extracted using chloroform.Finally, the chloroform was evaporated to yield the total alkaloid fraction.The extracts and pure compounds were kept below -80ºC until required for analysis. Parasite cultivation T. vaginalis trophozoites isolated from Turkey that are known to be sensitive to metronidazole were used in this study.T. vaginalis trophozoites were axenically grown at 37°C using Trypticase yeast extract-maltose medium (TYM, pH: 6) supplemented with 15% (v/v) fetal calf serum (FCS, Sigma-Aldrich, Taufkirchen, Germany) and penicillin/streptomycin at a dilution of 1:100, as previously described [3]. T. vaginalis trophozoites in the logarithmic phase of growth were transferred to a 24-well plate (10 5 parasites/well) containing TYM medium supplemented with 15% (v/v) FCS and penicillin/streptomycin (Sigma-Aldrich, Buchs, Switzerland) at a dilution of 1:100.Thereafter, different concentrations of extracts and compounds were added to each well and incubated at 37°C.As a reference drug, metronidazole prepared in DMSO (25 µg/well) was used.For untreated groups, two wells were used; one well contained DMSO (25 µg/well) in TYM medium with 15% FCS and T. vaginalis trophozoites (10 5 parasites/well), and the other contained only TYM medium with 15% FCS and T. vaginalis trophozoites (10 5 parasites/well). The number of parasites was counted using a hemocytometer under a light microscope (Olympus, CK 40, UK) at the 12, 24, and 72 hours.All in vitro experiments were run in triplicate and the results are expressed as mean percentages of the number of parasites. Results No immotile T. vaginalis trophozoites or active lysis was observed when ethanol and alkaloid extracts were applied at a concentration of 50 µg/mL and 100 µg/mL at 24, 48, and 72 hours of incubation.T. vaginalis trophozoite motility disappeared at 48 hours after exposure with 200 μg/mL of ethanol extract and 400 μg/mL of alkaloid extract.Trophozoite lysis was observed for the first time at 48 and 72 hours when 400 µg/mL of ethanol extract and the highest dose (800 µg/mL) of alkaloid extract were used, respectively (Table 1). When the percent lysis values obtained from extracts or pure compounds were compared with the control groups at the first 24 hours, no statistically significant difference was found (P > 0.05).On the other hand, use of ethanol extract at a concentration of 400 µg/mL resulted in the lysis of 50% and 100% of T. vaginalis trophozoites at 48 and 72 hours of incubation, respectively.Also, it was found that the highest doses (800 µg/mL) of ethanol and alkaloid extracts led to the lysis of 100% of T. vaginalis trophozoites at 48 hours. The minimum inhibitory concentration (MIC) for the ethanol extract and alkaloid extract at 48 hours was 200 μg/mL and 400 μg/mL, respectively.The minimal lethal concentration (MLC) was 400 μg/mL at 72 hours for the ethanol extract and 800 μg/mL for alkaloid extracts at 48 hours. Among the pure alkaloid compounds, dictamnine and gamma-Fagarine showed no activity against T. vaginalis trophozoites.Similarly, skimmianine and the flavonoid compound chrysosplenetin had no anti-T.vaginalis activity at concentrations of 12.5 µg/mL-37.5µg/mL.Interestingly, although no motile T. vaginalis trophozoites were observed at 48 and 72 hours when chrysosplenetin was applied over 37.5 µg/mL, lysis was not detected.Otherwise, skimmianine from 50 µg/mL to 150 µg/mL showed anti-T.vaginalis activity at 48 and 72 hours (Table 2). At 48 hours of incubation, it was found that skimmianine at a concentration of 100 µg/mL caused the lysis of 50% of T. vaginalis trophozoites.Also, 100% of T. vaginalis trophozoites were observed to be lysed when skimmianine concentration was increased to 150 µg/mL. The MIC values at 48 hours for skimmianine and chrysosplenetin were 50 μg/mL.The MLC value at 48 hours for skimmianine was 150 μg/mL.The MLC value was not estimated for chrysosplenetin because it had no lethal effect. MTZ at concentration of 50 µg/mL caused the lysis of 100% of T. vaginalis trophozoite at 72 hours.DMSO prepared in a final concentration of 0.5% (v/v) showed no statistically important anti-T.vaginalis activity when compared with the negative control groups (P > 0.05). Discussion MTZ has long been used in the classic treatment of T. vaginalis infections.However, new alternative drugs obtained from natural sources are being investigated for the treatment of trichomoniasis due to occurrence of MTZ-resistant T. vaginalis strains in approximately 9.6% of cases, in addition to the adverse effects of MTZ [16]. Plant materials are being used as an alternative approach for the treatment of various diseases, including parasitic diseases, because they have useful therapeutic activities.Accordingly, several studies have been conducted to develop alternative drugs for the treatment of trichomoniasis and it was found that flavonoids, alkaloids, cumarins, saponins, and glycosides obtained from plant materials showed anti-T.vaginalis activity [2,13]. H. myrtifolium, which was used in this study, is a member of the Rutaceae family.This family is represented by 161 genera and about 1900 species around the world.Its most characteristic features are secretory cavities that contain volatile oils, alkaloids, resin, hesperidin, and some other chemical compounds [17]. A study reported that ethanol and alkaloid extracts of H. myrtifolium showed anti-leishmanial activity against Leishmania tropica, which is a causative agent of cutaneous leishmaniasis [15].To the best of our knowledge, the antitrichomonal effect of H. myrtifolium, which contains biologically active compounds such as alkaloids, lignans, and glycosides, has not been studied before.In the present study, the antitrichomonal effect of H. myrtifolium was investigated for the first time and ethanol extract was found to be more effective for the lysis of T. vaginalis trophozoite than the alkaloid extract.This result is not surprising because the ethanol extract contains therapeutics such lignans, coumarins and flavonoids, in addition to alkaloids. In addition, the antitrichomonal impact of furoquinoline alkaloids such as dictamnine, gamma-Fagarine, and skimmianine was investigated in this study, and skimmianine over 37.5 µg/mL was found to have an inhibitory effect on T. vaginalis trophozoites.Also, it was observed that chrysosplenetin only prevented the movement of T. vaginalis trophozoites when used at its highest dose (150 µg/mL). Similar to our study, the results of different studies indicated that alkaloids showed an anti-T.vaginalis effect.In a previous study, alkaloids obtained from seeds of Crolatalaria pallida were indicated to have an inhibitory effect on T. vaginalis trophozoites [18].In another study, it was reported that alkaloids from Hippeastrum breviflorum showed an anti-T.vaginalis effect [19].It was shown that furoquinoline alkaloids extracted from Teclea afzelii (Rutaceae) plants had anti-plasmodial activity [20].Another study reported that some alkaloids (furoquinoline and acridone) obtained from plants form the Rutaceae family had antiplasmodial activity against Plasmodium falciparum [21]. Conclusion The results of this study indicated that the ethanol extract of H. myrtifolium and isolated compound skimmianine could serve as an efficacious alternative therapeutic agent for the treatment of T. vaginalis infection. Table 1 . Effect of ethanol and alkaloid extracts of H. myrtifolium on T. vaginalis trophozoites. Table 2 . Effect of skimmianine and chrysosplenetin pure compounds on T. vaginalis trophozoites.	v3-fos-license
2016-05-04T20:20:58.661Z	2015-01-22T00:00:00.000	10721692	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://downloads.hindawi.com/journals/bmri/2015/870389.pdf", "pdf_hash": "19caba31d2e031ac286065fca4c4994257e88786", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1428", "s2fieldsofstudy": [ "Medicine", "Chemistry", "Computer Science" ], "sha1": "74d9d4cfbd0ee2e054dc68b01d1524a07d6100f6", "year": 2015 }	pes2o/s2orc	Virtual Screening of Acetylcholinesterase Inhibitors Using the Lipinski's Rule of Five and ZINC Databank Alzheimer's disease (AD) is a progressive and neurodegenerative pathology that can affect people over 65 years of age. It causes several complications, such as behavioral changes, language deficits, depression, and memory impairments. One of the methods used to treat AD is the increase of acetylcholine (ACh) in the brain by using acetylcholinesterase inhibitors (AChEIs). In this study, we used the ZINC databank and the Lipinski's rule of five to perform a virtual screening and a molecular docking (using Auto Dock Vina 1.1.1) aiming to select possible compounds that have quaternary ammonium atom able to inhibit acetylcholinesterase (AChE) activity. The molecules were obtained by screening and further in vitro assays were performed to analyze the most potent inhibitors through the IC50 value and also to describe the interaction models between inhibitors and enzyme by molecular docking. The results showed that compound D inhibited AChE activity from different vertebrate sources and butyrylcholinesterase (BChE) from Equus ferus (EfBChE), with IC50 ranging from 1.69 ± 0.46 to 5.64 ± 2.47 µM. Compound D interacted with the peripheral anionic subsite in both enzymes, blocking substrate entrance to the active site. In contrast, compound C had higher specificity as inhibitor of EfBChE. In conclusion, the screening was effective in finding inhibitors of AChE and BuChE from different organisms. Introduction Alzheimer's disease (AD) was first reported by the pathologist Alois Alzheimer in 1907. It is a neurological disorder characterized by a significant decrease in hippocampal and cortical levels of the neurotransmitter acetylcholine (ACh) [1] with formation of extracellular amyloid plaques and intracellular neurofibrillary tangles that lead to neurotoxicity [2]. The AD affects up to 5% of people over 65 years, rising to 20% of those over 80 years [3]. One of the major therapeutic strategies adopted for AD treatment is based on the cholinergic hypothesis. Clinically, AD is associated with cognitive, functional, and behavioral symptoms, which can be explained by the cholinergic neurotransmission deficit with the loss of cholinergic neurons [4]. The neurotransmitter ACh plays a key role in learning and memory processes by activating nicotinic and muscarinic receptors of the central nervous system (CNS) [5]. The acetylcholinesterase (AChE) is an enzyme that hydrolyzes ACh to acetate and choline in the synaptic cleft, terminating the ACh neurotransmission [6]. The inhibitory effect on AChE activity increases ACh in synaptic cleft with overactivation of the cholinergic transmission [7]. Since disruption of cholinergic neurotransmission is involved in different brain functions, the search for new acetylcholinesterase inhibitors (AChEIs) is relevant as an early step to select molecules that can be used in preclinical trials as potential pharmacological agents or even to synthesize other kinds of compounds, such as insecticides. Virtual screening is established as an effective method for filtering compounds in the course of new drug discovery The nonpolar hydrogen atoms were omitted. [8]. During this long process, it is possible to search for compounds with specific features that can, potentially, lead to the development of effective therapeutic agents. For instance, the consideration of the Lipinski's rule of five [9], that is, molecular weight lower than 500 Da, number of donor hydrogen bonds less than 5, number of acceptor hydrogen bonds less than 10, and the log lower than 5, is of importance for the screening of drugs with pharmacological activity. The molecular docking is a method that can predict the most favorable orientation of a molecule (ligand) when interacting with a macromolecular target, such as an enzyme or a receptor, to form a stable complex. The crucial thermodynamic parameter involved in this method is the binding free energy (Δ binding ), which checks the theoretical stability of the ligand-protein complex [10]. Based on this principle, the main objective of the present study was to propose a strategy of virtual screening to unravel potential new AChEIs by using the virtual molecules ZINC bank and selecting compounds that obey the Lipinski's rule of five which have quaternary ammonium atom (because of the similarity with ACh). We also assessed the inhibitory activity of selected compounds in vitro, in order to check which compounds have the highest inhibitory activity using different AChE sources, purified AChE from Electrophorus electricus (EeAChE), AChE from Danio rerio (DrAChE), and human AChE (HsAChE). Furthermore in order to verify whether the selected compounds could also inhibit butyrylcholinesterase (BChE) activity, we investigated the effects of these compounds on purified BChE from Equus ferus (Ef BChE). In Silico Analysis. To search for new drugs with binding affinity to AChE, we used the virtual molecules ZINC bank (http://zinc.docking.org/), where approximately 5.5 million of different molecular structures are deposited [11]. First, we selected only tridimensional structures of compounds with quaternary ammonium atom that were in accordance with the Lipinski's rule of five. In addition, another rule was also included: the number of rotatable bonds had to be less than 10 [12]. The molecules obtained were downloaded and their geometry optimized using the software Avogadro 0.9.4 following the MMFF94 method. The molecular docking simulation was used as a second screening, aiming to search for compounds with higher inhibitory capacity and to propose an interaction model. We used different crystallographic structures of AChE from Protein Data Bank (PDB) (http://www.pdb.org/). The CHIMERA 1.5.3 software was used to remove molecules, ions, and water and to minimize the structure of proteins, using the Gasteiger charges with 500 steps of minimization. After obtaining the ligands and enzymes, their structures were converted to pdbqt format, using the Auto Dock Tools 1.5.4 program, in which all the rotatable bonds of ligands were allowed to rotate freely, and the receptors were considered rigid. For docking studies, we used the Auto Dock Vina 1.1.1 [13], with 1Å of spacing between the grid points. The grid box was centered on the active site of the enzymes with high resolution, allowing the program to search for additional places of probable interactions between the ligands and the receptor. Other configurations were considered default. The type of enzyme, species, PDB code, RMSD value, coordinates, and size of the grid box are shown in Table 1. Importantly, some enzymes do not present the RMSD value because they do not have inhibitor on their structures. The figures of structures with RMSD are represented in Figure 1. The RMSD value (less than 2Å) is a criterion often used for correcting bound structure prediction [14]. The redockings were performed with the same configurations of the previous performed dockings. For in vitro assays, we selected the compounds that presented lower binding energy (Δ binding ) in all enzymes used for the screening. The interactions between ligand-protein were visualized by Accelrys Discovery Studio Visualizer 2.5. In Vitro Analysis. The compounds selected as inhibitors of AChE activity were obtained commercially from MolPort (http://www.molport.com/buy-chemicals/index). They were dissolved in dimethyl sulfoxide (DMSO), at a final concentration of 0.1%. The cholinesterase activities were measured based on Ellman et al. 's method [15]. The increase of absorbance was monitored at 412 nm in a reaction mixture containing Haemoglobin-free erythrocyte ghosts were prepared according to the method previously described [16]. Blood of nonfasted healthy voluntary donors was collected. Heparinized human blood was centrifuged at 3000 g for 10 min. The packed erythrocytes were diluted in 20 volumes (w/v) of hypotonic sodium/potassium phosphate buffer (6.7 mM, pH 7.4) to facilitate the hemolysis, followed by centrifugation at 30.000 g for 30 min at 4 ∘ C. The supernatant was removed and the pellet resuspended in hypotonic phosphate buffer. After two additional washing cycles, the pellet was resuspended in sodium/potassium phosphate buffer (0.1 M, pH 7.4) and then centrifuged again at 30.000 g for 30 min at 4 ∘ C. The supernatant was gently removed and the pellet was stored. Aliquots of the erythrocyte ghosts were stored at −20 ∘ C until usage within one week. The sample was diluted 10 times for AChE activity measurement. Fifty L of the stored ghost preparation, in a final volume of 200 L, was used for the assay. Hemoglobin content from ghost membranes was measured at 540 nm as the cyano-met-Hb form, but no hemoglobin was detected. The DrAChE assay was performed as previously described by Rosemberg et al. (2010) [17]. Briefly, zebrafish brains were homogenized on ice in 60 volumes (v/w) of Triscitrate buffer (50 mM Tris, 2 mM EDTA, 2 mM EGTA, and pH 7.4, with citric acid) using a Potter-Elvehjem-type glass homogenizer. Samples (0.5 g protein) were preincubated for 10 min at 25 ∘ C and the enzyme activity was further assessed in the absence and the presence of the selected compounds. Statistical Analysis. The IC 50 values were determined by nonlinear regression (log concentration-inhibition curves). Data were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test. Statistical significance was set at < 0.05. The statistics have been performed using GraphPad Prism 5 (version 5.01, GraphPad Software, Inc., USA). Results and Discussion The first virtual screening retrieved 382 compounds that obey the Lipinski's rule of five and have the ammonium quaternary atom. The retrieved compounds were docked with the enzymes listed in Table 1 (second screening). We obtained the mean value of the lower binding free energy for each molecule resulting in 7 compounds (Table 2). These compounds were further obtained commercially for in vitro assay. The in vitro assay was carried out as a third screening step, in which we identified that the compound "D" presented the higher anti-AChE activity (for EeAChE, DrAChE, and HsAChE), being also able to inhibit the Ef BChE. These results suggest that the respective compound is not specific to acetylcholinesterases, because its IC 50 was very similar to all the enzymes tested in this study, with values ranging from 1.69 ± 0.46 to 5.64 ± 2.47. On the other hand, the compound "C" presented a higher inhibitory potency against Ef BChE, with an IC 50 value of 0.75 ± 0.18, than with AChEs IC 50 values ranging from 92.08 ± 39.73 to 761.17 ± 127.6. These data demonstrate that even if a strategy is adopted to select specific AChEIs using in silico analysis, it is relevant to assess whether the potential inhibitors may also alter BChE activity in vitro. The IC 50 values for both enzymes tested are shown in Table 3 and the graphics for purified EeAChE and Ef BChE are depicted in Figure 2. We further proposed an interaction model for the compounds with AChEs enzymes (TcAChE: PDB: 1EA5 and HsAChE: PDB: 1B41) and HsBChE (PDB: 2BDS), to compare the interactions of the compounds in each enzyme and to investigate the putative mechanisms of inhibition ( Figure 3). The TcAChE and compound "C" (Figure 3(a)) have only two cation-interactions (in orange) with Trp84, indicating a lower affinity with the enzyme, and this fact was confirmed by its IC 50 value for EeAChE (761.17 ± 127.6 M). However, for human AChE (Figure 3(b)), we found a larger number of -interactions (in green) of Tyr341 and Trp286 (peripheral anionic subsite) with the molecule "C, " indicating a slight increase in affinity and a decrease in the IC 50 value (92.08 ± 39.73 M). For HsBChE (Figure 3(c)) it was possible to detect one hydrogen bond (H-bond) between the compound "C" and Pro285. This H-bond is stronger when compared with the -or cation-interactions. Furthermore, the results showed that -and cation-interactions occur stacking between the compound "C" and the enzyme in the anionic subsite (Trp82). All these interactions could explain, at least partially, the high inhibitory potency of the molecule "C" for HsBChE (IC 50 = 0.75 ± 0.18 M). These results indicated that the compound "C" is more specific to HsBChE. On the other hand, the molecule "D" was able to inhibit the EeAChE, DrAChE, HsAChE, and HsBChE with a similar potency (see Table 3). The docking results suggest that for TcAChE occur a larger number of -and cation-interactions, mainly with the anionic subsite (Trp84), catalytic triad (His440), and peripheral anionic subsite (Tyr334)- Figure 3(d). In the presence of the compound "D, " a very similar conformation was detected for HsBChE (Figure 3 H-bond occurs between oxadioazole group and Tyr337 (peripheral anionic subsite) (Figure 3(e)). In addition, astacking was observed between Trp286 (peripheral anionic subsite) and the compound "D. " The different model of which "D" interacts with the HsAChE (Figure 3(e)) could be responsible for its more potent inhibitory effect on the enzyme. In all these models proposed above, the interactions of inhibitors with ChEs are expected to prevent the entrance of ACh in the activity site from AChEs and HsBChE, consequently, causing their inhibition. It is possible to observe that both compounds (C and D) interact rather with peripheral anionic and anionic subsites, probably due to the presence of aromatic residues in both peripheral anionic and anionic subsites. Importantly, similar observations were made by other studies involving AChEIs and molecular modeling [18,19]. The IC 50 values found for the compounds "C" and "D" are in the range of M; that is, they are 1-3 orders of magnitude higher than those of tacrine and donepezil (IC 50 = 205 ± 18 nM and 11.6 ± 1.6 nM, resp. [19]), which are two commercial drugs for treating AD. Moreover, according the thermodynamic data (Δ binding ), we observed that no type of correlation occurred with the IC 50 values. An overall scheme of the strategy used for this study is depicted in Figure 4. Conclusion In this study, we carried out a total of tree hierarchical screening steps (two in silico and one in vitro) in order to search for potential molecules able to act as AChEIs. We found one compound "D" with relevant anti-AChE and anti-BChE activity. To our surprise, we found one molecule "C" which inhibited Ef BChE more significantly than it did with AChEs. These results suggest that selecting compounds with pharmacophoric properties (Lipinski's rule of five) and performing the molecular docking screening to search potential inhibitors are interesting strategies that could be used for high throughput screenings aiming to detect new compounds with desirable biological activity. We also reported the importance of aromatics rings in the inhibitors. These aromatic moieties in the ligands perform -and cation-interactions with several aromatic residues located in the gorge of AChE (for instance, Trp84, Trp279, Phe330, Phe331, and Tyr334). These molecules interact at the peripheral anionic subsite and anionic subsite of AChE, preventing the hydrolysis of ACh. Despite the fact that some compounds act as inhibitors of AChE and BChE, we emphasize that other approaches, such as in vivo studies, are necessary to validate the pharmacological and toxicological properties of these compounds.	v3-fos-license
2022-09-03T00:01:18.833Z	2020-01-01T00:00:00.000	230524286	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://www.researchsquare.com/article/rs-123795/v1.pdf?c=1631880839000", "pdf_hash": "34b6b83f66577d53555433aa3a1517bc017112fb", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1431", "s2fieldsofstudy": [ "Materials Science" ], "sha1": "34b6b83f66577d53555433aa3a1517bc017112fb", "year": 2020 }	pes2o/s2orc	Antifungal effects of ZnO, TiO2 and ZnO-TiO2 nanocomposite on Aspergillus avus This study aimed to synthesis ZnO, TiO 2 and ZnO–TiO 2 (ratio weight of 1/1 for Zn/Ti) nanoparticles using zinc acetate and titanium isopropoxide through the sol-gel method. Physicochemical and morphological characterization and antifungal properties evaluation like minimum inhibition concentration (MIC) and minimum fungicide concentration (MFC) of nanopowders were investigated against Aspergillus avus at in vitro. All synthesized nanoparticles (50 µg/ml) showed fungal growth inhibition while ZnO-TiO 2 showed higher antifungal activity against A. avus than pure TiO 2 and ZnO. TiO 2 and ZnO-TiO 2 (300 µg/ml) inhibited 100% of spur production. Pure ZnO and TiO 2 showed pyramidal and spherical shapes, respectively whereas ZnO-TiO 2 nanopowders illustrated both spherical and pyramidal shapes with grown particles on the surface. Based on our ndings, low concentration (150 µg/ml) of ZnO-TiO 2 showed higher ROS production and stress oxidative induction thus fungicide effect as compared to alone TiO 2 and ZnO. In conclusion, ZnO-TiO 2 nanostructure can be utilized as an effective antifungal compound but more studies need to be performed to understand the antifungal mechanism of the nanoparticles rather than ROS inducing apoptosis. Introduction ZnO and TiO 2 have been used in various biomedical applications due to photocatalytic, antimicrobial and antifungal properties [1,2]. Doping and nanocomposite manufacturing have been previously utilized as the processes for enhancing the antifungal activity of this kind of nanoparticles. Among the various semiconductor nanomaterials, titanium dioxide (TiO 2 ) and zinc oxide (ZnO) have achieved more attention due to their high chemical stability, nontoxicity, relatively low cost and high antimicrobial activity [3,4]. The metal oxide NPs antibacterial and antifungal properties have been previously studied [5][6][7] and the ndings showed the ZnO antibacterial activity as well its capability to increase of induction of reactive oxygen species (ROS) production by decreasing its particle size. The zinc oxide antifungal activity is related to the formation of free radicals on the surface of nanoparticles that damage the fungal cell membrane lipids, which lead to protein leakage through the membrane disruption [8][9][10][11][12]. TiO 2 NPs possess the antimicrobial properties even at low concentrations through the photocatalytic process that causes fatal damage in treated microorganisms [13][14][15]. Based on TiO 2 nanoparticle antimicrobial properties, these nanoparticles in the anatase and rutile phase show the excellent antifungal properties [16]. The titania owned enormous applications because of its high thermal/chemical stability, and high photocatalytic activity. The toxicity of titania nanoparticles originates from its physical properties, not its chemical structure. These nanoparticles can permeate from biological barriers that can damage the cells or even organs. Some methodologies have been previously applied for improving the titania NPs' antimicrobial activities on simple microorganisms such as bacteria and viruses [17][18][19][20][21]. By considering the Aspergillus species as the deadliest opportunistic fungal infections, these fungi are the main threat to human health. Among the 600 species of Aspergillus, the avus, fumigatus, and niger species possess the pathogenicity for humans and growing on crops can cause the occurrence of some disease [22,23]. Upon the previous quantitative reports on ZnO and TiO2 fungal growth inhibition, these nanoparticles possess fungicidal effects on Candida albicans, Aspergillus niger, and Penicillium sp. fungus. We showed previously the increase of ZnO and TiO 2 antibacterial activity by increasing the concentration of dopant in doped ZnO and TiO 2 [24,25]. This study aimed to synthesize the ZnO, TiO 2 , and ZnO-TiO 2 nanostructures using the sol-gel methodology, physicochemical characterization of nanopowders, and antifungal assays against Aspergillus avus to nd the highly effective antifungal concentration at dark condition. Nanostructures synthesis ZnO and TiO 2 nanostructures were synthesized using the sol-gel method as described by Najibi [25]. For the preparation of ZnO-TiO 2 nanostructures, separately prepared ZnO and TiO 2 sols were mixed at the same molar ratio of Zn:Ti then the mixture was stirred at ambient temperature for 2 h and the stirred solution was remained for 24 h to obtain a gel. Prepared gel was dried at 100 °C and was calcined at 500 °C for 2.5 hours. Material characterization method The XRD pattern and phase identi cation of nanopowders were determined by X-RAY diffraction analysis (Philips-MPD XPERT, λ: CuKα=0.154 nm) and 20-70˚ range of scanned samples were considered as 2Ө. The scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analyzer (N5, Backman, USA), and zeta potential analyzer (Malvern Zeta-sizer 3000, Malvern Instrument Inc., London, UK) were utilized for morphological, size, and zeta potential characterization of all samples, respectively. Fourier Transform Infrared (FTIR) Spectroscopy was used to identify organic, polymeric, and in some cases, inorganic materials. Fourier transforms infrared (FTIR) spectra were obtained using a Bruker IFS 48 instrument (Bruker Optik GmbH, Germany). All spectra were taken under air as a function of time with 16 scans at a resolution of 4 cm − 1 and a spectral range of 4000-5000 cm − 1 . Antifungal assay A. avus, purchased from the Iranian biological resource center (IBRC), were cultured on Sabouraud dextrose agar (SDA; Merck, Darmstadt, Germany) at 25 °C and the dark condition. The autoclaved SDA media containing ZnO, TiO 2 and ZnO-TiO 2 NPs at concentrations of 0, 37, 75,150 and 300 µg ml − 1 and an NP-free solution were poured onto the 6 cm diameter Petri dishes. To determination of minimum inhibition concentration (MIC) of nanoparticles for each treatment group, the CLSI-M38 standard method was used for the time intervals of 7 days by measuring the diameter of fungal colonies opacity. To determine the minimum fungicide concentration (MFC), the higher concentrations than MIC for each nanostructure were used on SDA medium similar to the MIC determination experiment and the minimum concentration that killed A. avus considered as MFC. To detect the production of ROS after each time point of treatment, 2′-7′-Dichlorodihydro uorescein diacetate (DCFH-DA) solubilized in ethanol (5 µM nal concentration) was added to the cultures and incubated on a shaker at room temperature at the dark condition for 1 h. DCFH-DA, a nonpolar dye, is converted to the non uorescent polar derivative DCFH by cellular esterases. DCFH can switch to highly uorescent DCF through oxidization by intracellular ROS and possessing an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. After incubation time, samples were subjected to uorescence microscopy (Biozero BZ-8000; Keyence, Osaka, Japan) equipped with the following lter set EX 495 nm EM 510 nm, and uorescence spectrophotometric (RF-5000, Shimadzu, Kyoto, Japan) analysis at room temperature. The XRD patterns of ZnO and TiO 2 showed a single high-intensity peak that implies a highly oriented and single-crystalline nature of the samples. As shown in Fig. 1, the intensity of TiO 2 peaks considerably decreased after the addition of TiO 2 into the structure of ZnO in the ZnO-TiO 2 composite that indicates the greater crystallinity of pure TiO 2 NPs compared to ZnO-TiO 2 NPs [27]. Pro le broadening also indicated the small crystalline domain sizes of wurtzite and anatase indicating that the ZnO-TiO 2 composite hinders the growth of particles during calcination. The main peaks of each sample in the range of 2θ = 20-50° speci ed some peaks belonging to anatase (Fig. 1). Table 1 PSA and Zeta potential analysis The zeta potential is an important indicator of the stability of dispersed particles in the suspension solution. The zeta potential determines the repulsion of dispersed particles in the solution. Small particles require the high zeta potential for superior stability, and low zeta potential causes to particle accumulation. The zeta potential of a particle alters by the particle surface chemical composition, the pH and ionic strength of the solution. Zeta potential of ZnO, TiO 2 , and ZnO-TiO 2 were − 11.6, -36.4, and − 12 mV, respectively ( Fig. 2 and Table 1). Based on our ndings, TiO 2 and ZnO-TiO 2 showed the highest and lowest stability in aqueous suspension, respectively. Larger particle sizes for ZnO (608 nm), TiO2 (299 nm), and ZnO-TiO2 (983 nm) were determined by PSA analysis showing the agglomeration of nanoparticles. SEM and TEM analysis As shown in Fig. 3, the ZnO and TiO 2 nanoparticles illustrated hexagonal-pyramidal and spherical shape with grown articles on surfaces, respectively. The wurtzite-structured ZnOcrystal is described as several alternating planes composed of four-fold tetrahedrally-coordinated O 2− and Zn 2+ ions stacked alternatively along the c-axis [28]. The oppositely-charged ions produce positively-charged Zn (0001) and negatively-charged O(0001 ) surfaces, resulting in a normal dipole moment and spontaneous polarization along the c-axis, as well as a divergence. In the ZnO-TiO 2 nanostructures, the morphology was a mixture of pyramidal and spherical with more agglomeration while the particle sizes were smaller than alone titanium and zinc oxide particles. Upon the EDX analysis, the strong signals of Zn, Ti and Zn-Ti were observed in ZnO, TiO 2 , and ZnO-TiO 2 nanostructures, respectively (Fig. 3). The TEM images of nanostructures clari ed the regular growth of all nanostructures and illuminated the TiO 2 (5 nm) particle size smaller than ZnO (10 nm) and ZnO-TiO 2 (35 nm) nanoparticles with lower agglomeration rate (Fig. 3). related to the hydroxyl groups. Also, water molecules in the bending band at 1630 cm − 1 are visible [31]. The presence of some bands can be associated with the organic phase of solid, despite the use of organic compounds in the synthesis of nanoparticles (Fig. 4). Antifungal properties of nanostructures As shown in Table 1, ZnO-TiO 2 nanostructure exhibits better antifungal effects against A. avus than other nanoparticles due to its high speci c surface area. By increasing the speci c surface area, the possibility of chemical reactions and the production of reactive oxygen species on the surface were increased [32]. The MIC for ZnO-TiO 2 , ZnO, and TiO 2 against A. avus was determined 39, 156, and 78 µg/ml, respectively. Because of the small particle size, the best cell internalization, and the ability to produce more reactive oxygen species, TiO 2 showed a higher fungicide than ZnO. The MFC for ZnO, TiO 2 , and ZnO-TiO 2 was 312, 156 and 78 µg/ml, respectively. The particle size of the ZnO-TiO 2 nanostructure possessed a sharp structure with smaller particles than the cell membrane that can inhibit the growth of the fungus by entering the cell membrane and injuring the cell wall thus resulting in the high toxicity. Figure 5 illustrated the inhibition zone of ZnO, TiO 2 , and ZnO-TiO 2 at 37.5, 75, 150, and 300 µg/ml concentrations. By increasing the concentration of nanoparticles, inhibition zone diameter of growth increased and 100% of inhibition was achieved at 300 µg/ml for TiO 2 and ZnO-TiO 2 treated groups. The minimum fungal growth (72%) was obtained at 37.5 µg/ml for ZnO-TiO 2 while for ZnO was 50% at the same concentration showing that the TiO 2 synergistic effect into the mixture [33]. Among all nanoparticles, ZnO nanoparticles showed the lowest fungicide activity compared to others whereas it signi cantly increased the antifungal activity in ZnO-TiO 2 nanocomposite. The destructive changes were observed on the shape and growth of the treated A. avus (at a concentration of 37.5 µg/ml for all samples) compared to the untreated control group. As shown in Fig. 6, the untreated control fungus produced the highest count of fungal spores while treated groups showed a lower count of spores and damaged tubular laments, in instance deformation, smoothness, and noticeably thinner in hyphae compared to the untreated group. Upon the previous reports, increasing the hyphae causes to form whiter medium [34] and our ndings agreed to color changes based on the used nanoparticles (Fig. 6). Among the reactive oxygen species, hydrogen peroxide and hydroxyl radicals as the strong and nonselective ROSs can damage all types of biomolecules including carbohydrates, acids, lipids, proteins, DNA, RNA, and amino acids through inducing the oxidative stress [35]. The production rate of the three [36]. There is a direct dependency between increasing the formation of ROS and the fungicide of nanoparticles. As shown in Fig. 7, all nanoparticles raised the ROS production in treated A. avus compared to untreated control with order ZnO-TiO 2 > TiO 2 > ZnO > untreated control. The production of intracellular ROS was in uenced by the type and speci c surface of nanoparticles. Titania can produce ROS higher than zinc oxide [37], our ndings also con rmed the highest ROS production through stronger uorescence intensity in ZnO-TiO 2 treated group. In ZnO-TiO 2 nanostructures, the speci c surface area is higher than other nanoparticles (TiO 2 and ZnO) and accordingly high ROS generation. Oxidative stress induced by reactive oxygen species generation in ZnO-TiO 2 nanostructures is thought to be the main mechanism of antifungal activity. The suggested mechanism for the antifungal activity of these compounds can be based on the formation of high levels of reactive oxygen species (ROS) that disrupt the integrity of the fungal cell membrane, which assists in the damage of microbial enzyme bodies thus killing the fungi [38]. Conclusion This study aimed to compare the antifungal properties of TiO 2 and ZnO versus ZnO-TiO 2 nanocomposites to select the compound with the highest antifungal activity. Based on our ndings, low concentration (150 µg/ml) of ZnO-TiO 2 showed higher fungicide and stress oxidative induction through ROS production as compared to TiO 2 and ZnO. In conclusion, ZnO-TiO 2 nanostructure composition can be used as an effective antifungal compound but more studies need to be performed to deeply understand the antifungal mechanism of the nanoparticles rather than stress oxidative induction. Declarations Contributions NNI carried out most of the experiments and wrote this paper. MM participated in this project and proposed the idea. AYKH designed and supported the project edited and revised the paper. All authors read and approved the nal manuscript. Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable.	v3-fos-license
2020-08-01T13:13:15.933Z	2020-07-28T00:00:00.000	220885964	{ "extfieldsofstudy": [ "Chemistry", "Medicine", "Biology" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.jbc.org/article/S0021925821001551/pdf", "pdf_hash": "06647738132178699e4e152ec58eb884c6b2f8dc", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1474", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "ccb271ff40ee8aa5e9d7ebffd09753cd6dc79f65", "year": 2021 }	pes2o/s2orc	Insights into the catalytic properties of the mitochondrial rhomboid protease PARL The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min−1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated β-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design. The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min −1 . In contrast, PGAM5 is cleaved with the highest efficiency (k cat /K M ) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated β-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design. Mitochondria play an essential role in cellular respiration but also play an equally important role in modulating cell death (1). These functions rely on the selective quality control of mitochondrial protein homeostasis (2) that includes the controlled turnover of regulators in the mitochondria by the mitochondrial intramembrane protease PARL. This enzyme was originally named Presenilin-Associated Rhomboid-Like protease after discovery in a yeast-two hybrid screen (3,4). PARL cleaves various safeguards of mitochondrial health, including the kinase PINK1 (phosphatase and tensin (PTEN)induced putative kinase 1) (5-7) and the phosphatase, PGAM5 (phosphoglycerate mutase family member 5) (8), both of which are known to play roles in mitophagy, the selective removal of damaged mitochondria (9). Hence, PARL has been renamed, and the acronym now corresponds to PINK1/PGAM5 Associated Rhomboid-Like protease (10). Additional substrates of PARL have been identified in a recent proteomic analysis including the proapoptotic factor Smac/Diablo (11). The PARL knockout (KO) mouse exhibits a severe respiratory defect, similar to Leigh's syndrome, which is a consequence of misprocessing of the nuclear encoded substrate TTC19, a subunit of complex III (12). These PARL KO mice present with a severe motor defect, the loss of gray matter (cell bodies of neurons) in the cortex, and early lethality. The mitochondria of the PARL KO mice have a distinct morphology lacking cristae that precedes neurodegeneration in gray matter (13,14). When the PARL orthologue Pcp1/Rbd1 is knocked out in yeast, similar disturbances are also observed where the cristae and protein-mtDNA assemblies in the matrix dissipate (15,16). These studies emphasize the essential nature of the PARL-type proteases for cell viability across evolution. The PARL protease is a member of the rhomboid intramembrane protease family (17,18), which are membraneembedded serine peptidases. Their functions range from cleavage and release of membrane-tethered signaling molecules to membrane protein degradation (17,19). Regulation of PARL activity at the molecular level is thought to occur via posttranslational modifications. Different forms of PARL have been identified in various tissues as a result of processing events; these include a protein with a mitochondrial matrix targeting sequence (MTS), a full-length mature form after removal of the MTS (PARLΔ55), and a truncated form derived from cleavage at residue S77 (PARLΔ77), referred to as βcleavage (20). Ectopic expression of this truncated form of PARL in tissue culture was shown to alter mitochondrial morphology leading to fusion defects and hence has been suggested to be more active (20). In contrast, it was shown by others that truncation of PARL leads to decreased processing of PINK1 (7). The truncation site at S77 was shown to be phosphorylated in response to stress, an event that influences β-cleavage of PARL and its activity in tissue culture cells (21). The mechanism of this putative regulatory switch remains to be determined, and it has not yet been established if PARLΔ77 is generated by PARL itself or other mitochondrial proteases. Despite this, PARL has not been characterized at the molecular level and thus the importance of β-cleavage in its regulation remains unclear. Our analysis with human recombinant PARL, comparing full-length and the truncated β-cleavage form, allows us to determine kinetics of substrate cleavage and examine the parameters influencing PARL activity. We evaluated PARL activity using SDS-PAGE and FRET-based fluorescent assays with peptide substrates. We observe that β-cleavage increases the catalytic activity of PARL. When PARL is reconstituted in a lipid environment similar to that found in the inner mitochondrial membrane (IMM), we reveal similar substrate specificities yet at an enhanced catalytic rate of cleavage of all substrate peptides when compared with those measured with the enzyme in detergent micelles. In addition, we observe that PARL activity was increased by cardiolipin (CL). Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from the small side chain residues recognized by most bacterial rhomboids. Together, this work provides characterization of the PARL protease and further extends our mechanistic understanding of this important safeguard of mitochondrial homeostasis. Recombinant human PARL expressed in yeast is active To examine the molecular features that determine PARL activity, we took an approach to express and purify Figure 1. Recombinant human PARL protease is active. A, cartoon representation of the PARL protease topology and truncations. Recombinant human PARL was expressed in P. pastoris to generate full-length (FL) starting at residue 55 or the β-cleavage form truncated at residue 77. An inactive PARLΔ77-S277A mutation was also generated. B, incubation of recombinant PARLΔ77 with MBP-PGAM5 reveals an expected shift on SDS-PAGE resulting in N-and Cterminal fragments. Asterisk in B indicates minor contaminant from PARLΔ77 preparation. C, a cartoon representation of the substrate construct with residues 70-134 of PINK1 flanked by the CyPet/YPet fluorescence reporter pair. D, cleavage of FRET-PINK1(70-134) by detergent-solubilized PARLΔ77 in the presence of increasing lipids. N = 4 * p < 0.05; p < 0.005, * p < 0.0005. E, representative Michaelis-Menten curves for FRET-PINK170-134 cleavage by PARLΔ55, PARLΔ55-S77N, and PARLΔ77 N = 3. F, representative Michaelis-Menten kinetic curves for IQ-PGAM5 substrate cleavage by PARLΔ77 and PARLΔ55. N = 3. Values are represented as mean ± SEM. n.s., no significance. recombinant PARL to study it in vitro. Human PARL was cloned into a His-tagged pPICZ expression vector where we added a C-terminal Green Fluorescent Protein (GFP)-tag, which allowed us to monitor protein expression in Pichia pastoris (22). The PARL precursor, with the MTS intact, resulted in poor yield (results not shown). The full-length mature form (PARLΔ55), a truncated version representing βcleavage (PARLΔ77), and a mutant with impaired-β-cleavage PARLΔ55-S77N were successfully expressed (Figs. 1A and S1). In addition, the active site mutant PARLΔ77-S277A was also generated. Recombinant PARL proteins were purified using affinity chromatography from dodecylmaltoside (DDM)-solubilized membrane fractions, followed by removal of the GFP-His-tag. Milligram quantities of all PARL variants were obtained using this expression system. The oligomeric states of expressed PARL proteases were examined using SEC, which revealed that PARL existed in a monomeric form in detergent solution (Fig. S2). To assess if the recombinant PARL was active, we first examined the cleavage of the transmembrane (TM) domain of PGAM5 fused to an N-terminal maltose binding protein (MBP) and a C-terminal Thioredoxin 1 domain (Fig. 1B). The approach of using a substrate TM segment fused to MBP was undertaken before for both eukaryotic and prokaryotic rhomboids and the presence of the tag did not affect their ability to cleave the substrates (23)(24)(25). Upon incubation of the MBP-PGAM5 fusion protein with PARLΔ77 in the presence of CL to increase rhomboid activity (see below), new bands, representing the N-and C-terminal cleavage products, were observed on SDS-PAGE. These bands are not present following incubation of MBP-PGAM5 with PARLΔ77-S277A (Figs. 1B and S3). This assay confirmed the functionality of human mitochondrial PARL generated in the P. pastoris system. Lipids enhance PARL activity To assess the catalytic properties of PARL protease and factors influencing its activity, we designed a FRET-PINK1 70-134 substrate with fluorophores that were previously used to measure bacterial rhomboid protease activity in vitro (26,27). Residues 70-134 of PINK1, encompassing the predicted TM segment (residues 89-111) and adjacent residues, were cloned between two fluorescent protein reporters, YPet and CyPet, to allow for FRET activity measurement upon cleavage (Fig. 1C) (28). Given the fact that PARL protease has never been expressed and studied in vitro, all parameters of the activity assay were optimized. First, we examined the pH dependence of PARL activity using FRET-PINK1 70-134 substrate and determined that the optimum was pH 7.0 (Fig. S4). Lipids are known to influence membrane protein function (29). In mitochondria, for example, increased CL amounts are observed during mitochondrial stress, which influences protein function at the molecular level (30). Therefore, we assessed the effect of the three primary lipids present in the IMM, namely CL, phosphatidylcholine (POPC), and phosphatidylethanolamine (POPE) on PARLΔ77 activity with the FRET-PINK 70-134 substrate (Fig. 1D) (31). The lipid-free conditions were used as a baseline and consisted of PARLΔ77 reconstituted in detergent (DDM) micelles, while all lipid conditions consisted of a mixed detergent-lipid micelle system. First we assess the effect of different classes of lipids. Compared with conditions with no lipid added, POPE at a molar ratio of 50:1 and 100:1 did not significantly increase PARL activity while POPC at a molar ratio of 50:1 increased activity by fourfold. CL at a molar ratio of 50:1 resulted in a significant increase in PARL activity and to a lesser extent at a molar ratio of 100:1 (Fig. 1D). In order to precisely determine the optimal concentration of CL for PARL activity, the initial velocities of PARL-mediated cleavage of FRET-PINK1 70-134 were measured in the presence of increasing concentrations of CL. The influence of CL on PARL activity resulted in a bellshaped curve with the 25:1 CL: PARL molar ratio having the most significant effect (Fig. S5). This data indicates that CL modulates the activity of the mitochondrial rhomboid protease PARL by enhancing its overall structural stability through protein-lipid interactions, similar to other mitochondrial proteins (32,33). Therefore, CL was included in all subsequent protein preparations of detergent-solubilized PARL at the last purification step. Next, we used this optimized preparation and assay to determine the catalytic parameters of PARL. The cleavage of FRET-PINK1 70-134 by DDM-solubilized PARLΔ55 and PARLΔ77 in the presence of CL obeyed Michaelis-Menten kinetics ( Fig. 1E and Table S1) and revealed slow rates of cleavage, 0.43 ± 0.09 h −1 and 0.73 ± 0.06 h −1 , respectively. This, however, reflects the tendency of intramembrane proteases to have slow turnover rates (26,34,35). The truncation of PARLΔ55 to PARLΔ77 was proposed to be autocatalytic (22); however, this has not been confirmed in vitro. To ensure the integrity of PARLΔ55 in our kinetic assays, we tested if self-truncation occurred in vitro under the conditions of activity measurements. Purified PARLΔ55 was incubated at 37 C at the concentration of 0.8 mg/ml for 4 h (the longest time used for kinetic assay), and protein samples taken after 2 and 4 h were run on SDS-PAGE (Fig. S6). No additional band corresponding to molecular weight of PARLΔ77 was observed after the time of incubation, suggesting that no autocleavage occurs under these conditions. Furthermore, the catalytic parameters of PARLΔ55S77N, the mutant that prevents β-cleavage, were similar to PARLΔ55, which again suggests that PARLΔ55 does not undergo auto processing since one would expect different catalytic parameters for PARLΔ77 (Fig. 1E). To further evaluate the cleavage of other known substrates of PARL, we adopted a more facile system to examine cleavage of multiple substrates and generated internally quenched (IQ) peptide substrates (25,(36)(37)(38) based on the amino acids flanking the PARL cleavage sites of PINK1 (39), PGAM5 (8), and Smac/Diablo (11). Kinetic analysis using both full-length and β-truncated PARL with IQ-PINK1 99-108 , IQ-PGAM5 [20][21][22][23][24][25][26][27][28][29] , and IQ-Smac/Diablo 51-60 peptide substrates was first performed in detergent, which revealed similar Michaelis-Menten kinetics for all peptides (Fig. 1F). The assay allowed us Catalytic properties of rhomboid protease PARL to determine the catalytic parameters for the three primary PARL substrates and examine the substrate specificity (Fig. 2, Tables S2-S4). Similar to previously shown kinetic parameters for FRET-PINK1 70-134 cleavage, a slow turnover rate for all substrates was observed, with the k cat values ranging from 0.5 to 1.3 h −1 with the Smac/Diablo peptide being the fastest cleaved and PGAM5 being the most efficiently cleaved based on k cat /K M value. These data also revealed that the turnover rates of PARL proteases, with the short IQ-PINK1 99-108 peptide and longer FRET-PINK1 70-134 substrate, were comparable, being, for example, 0.42 ± 0.03 h −1 with PINK1 peptide and 0.46 ± 0.09 h −1 with FRET-PINK1 70-134 for full-length PARLΔ55 (Fig. 1E, Tables S1 and S2). We conclude that the regions adjacent to the PINK1 cleavage site do not influence the cleavage process, and thus the IQ-peptide substrates are suitable for performing further kinetic studies. PARL shows enhanced catalytic rate in liposomes To assess the activity of recombinant PARL toward known substrates in a lipid bilayer, full-length and β-truncated PARL were reconstituted in proteoliposomes (PLs) using Escherichia coli lipids that closely resemble the composition of the IMM (31). We determined that the EDANS/Dabcyl -FRET tags prevented incorporation of the substrates into PL; however, the substrate could still be cleaved since the active site is proposed to be near the lipid/water interface (40,41) analogous to bacterial rhomboid proteases (Fig. 1). To calculate the specific activity of the protease, we quantified the fraction of PARL in the PLs with an outward facing active site using a membrane semipermeable activity-based TAMRA-labeled fluorophosphonate probe (42). In PLs 70% of PARL could be labeled with the probe revealing the proportion with the active site in an outward-facing manner (Fig. S7). These values were used to calculate specific activity of the PARL protease. Next, we compared cleavage of three IQ-peptide substrates by reconstituted full-length mature PARLΔ55 and the β-truncated form, PARLΔ77 (Fig. 3, Tables S5 and S6). Overall, we observed that the lipid environment increased the activity of both forms of PARL toward all substrates and the reaction still displayed Michaelis-Menten kinetics (Fig. 3, A and C and Table S5 and S6); only negligible background activity was detected for inactive PARLΔ77-S277A (Fig. S8). Of all peptides assessed, cleavage of Smac/Diablo by β-truncated PARLΔ77 was the fastest with a turnover rate of 58 ± 7 h −1 , or 1.0 ± 0.1 min −1 , and PGAM5 was the preferred substrate with the lowest K M and the highest catalytic efficiency (k cat /K M ) of 26 ± 8 μM −1 h −1 (Table S6), which agrees with the data obtained in a detergent environment. To examine if CL influences PARL activity in a membrane environment, we prepared PL samples containing β-truncated PARL and conducted detailed kinetic analysis with the three IQ-peptide substrates (Fig. 3B). Two independent PL samples were generated: the first consisted only of POPC and POPE, while the second also contained CL. The turnover rates for the PINK1, PGAM5, and Smac/Diablo peptide substrates with the two PL samples demonstrated a trend similar to that observed in the detergent environment (Fig. 3C). When CL was omitted from the PL, we observed two to tenfold slower turnover rates of cleavage for IQ-peptide substrates by β-truncated PARL ( Fig. 3B and Table S7). This result again demonstrates that CL, which is specific to the inner mitochondrial membrane where PARL resides, influences the proteolytic activity of PARL. β-Cleavage influences the activity of PARL The influence of PARL truncations on activity has been controversial. PARL processing has been proposed to be a regulator of its enzymatic activity (43). Cellular studies with PARLΔ55-S77N, a form of PARL unable to undergo Catalytic properties of rhomboid protease PARL β-cleavage, have shown impaired cleavage of PINK1, with additional data suggesting that the longer form is less active toward PINK1 (7,21). Another study showed that the β-truncated PARL (PARLΔ77) induces mitochondria fragmentation in cells (20). We used our kinetic analysis to examine whether β-cleavage influences the activity of PARL in vitro. Kinetic parameters of full-length and β-truncated PARL in both detergent (Fig. 2, Tables S3 and S4) and PL (Fig. 3, Tables S5 and S6) were determined using three IQ-peptide substrates: PINK1, PGAM5, and Smac/Diablo. In detergent, turnover rates increased (PINK1 and PGAM5) or remained unchanged (Smac/Diablo) with βtruncated PARL when compared with full-length-PARL (Fig. 2). In PL, mimicking a bilayer environment, a similar trend was observed with the turnover rate being significantly enhanced for Smac/Diablo with β-truncated PARL ( Fig. 3 and Table S8). The direct influence of PARL truncations on substrate cleavage has not been examined in vitro before and with this data we confirm that PARL is catalytically active in either form, thus indicating that processing to the β-cleavage form is not required for proteolytic activity or PARL functionality as was once speculated (7). PARL is weakly inhibited by commercial inhibitors Rhomboids were initially discovered to be serine proteases because the first identified rhomboid protease from Drosophila, Rhomboid-1, was sensitive to serine protease inhibitors dichloroisocoumarin (DCI) and tosyl phenylalanyl chloromethylketone (TPCK) (44). Further, the crystal structures of bacterial rhomboids with serine protease inhibitors-diisopropylfluorophosphate and isocoumarins aided in revealing structural insight about their molecular mechanism of catalytic reaction (17). However, the broad-spectrum serine protease inhibitor PMSF does not act on bacterial rhomboid proteases (44)(45)(46). Inhibition of PARL has not been previously explored. . Enhanced catalytic rate is observed with PARL in proteoliposomes with cardiolipin. A, representative Michaelis-Menten curve for PARLΔ77 cleavage of IQ-PINK199-108. B, the effect of CL on PARLΔ77 activity in proteoliposomes with IQ-peptide substrates. C, bar graphs for turnover rates and catalytic efficiencies of proteoliposome-reconstituted PARLΔ55 and PARLΔ77 with IQ-peptide substrates. Experiments were conducted in duplicate with an N = 4. Individual data points (black dots) are indicated on the bar graphs, representing the mean ± SEM (* p < 0.05; ** p < 0.005). n.s., no significance. Catalytic properties of rhomboid protease PARL We examined whether inhibitors belonging to three standard serine protease inhibitors families-sulfonyl fluoride (PMSF), chloromethyl ketone (TPCK), and coumarin (DCI)were able to inhibit PARL when tested with IQ-PINK1 and IQ-PGAM5 substrates. In detergent and PL, for both PINK1 and PGAM5 peptide substrates, we show that PARL activity is not inhibited by 100 μM PMSF, whereas 100 μM TPCK partially inhibits and 100 μM DCI has the largest inhibitory effect (Fig. S9), which is in agreement with previously shown effect for bacterial rhomboid proteases (47). Overall this supports the view that for PARL, specific inhibitors will need to be developed similar to bacterial rhomboid proteases (48,49). PINK1, PGAM5, and Smac/Diablo are competing for the same binding site With multiple substrates discovered, it is obvious that PARL has pleotropic roles in mitochondria and that substrate cleavage must be precisely regulated (50), whether through compartmentalization (51) or differential substrate binding. It was shown that PARL-mediated differential cleavage of PINK1 and PGAM5 depends on the health status of mitochondria (51). Further studies suggested that the rate of PINK1 cleavage in cells is influenced by PGAM5, indicating that PINK1 and PGAM5 may compete for cleavage by PARL (52). However, it has been speculated that PINK1 and PGAM5 are not competitive substrates in vivo since reducing the expression of PINK1 by siRNA did not increase cleavage of PGAM5 by PARL (8), highlighting that function of PARL changes in response to ΔΨ m loss. This raises questions whether PARL substrates bind to the same residues in the active site or alternative binding sites might exist on enzyme's surface. To determine if substrates bind to the same site, we performed competition binding assays using fluorescent IQ-PGAM5 20-29 and IQ-Smac/Diablo 51-60 as the main substrates and nonfluorescent PINK1 89-111 ( 89 AWGCAGPCGRAV-FLAFGLGLGLI 111 ) as a competing substrate (Fig. 4). The longer version of substrate was used in this assay in order to reveal possible exosite interactions of substrate with PARL, assuming that regions surrounding the cleavage site might be involved in a primary substrate binding with a putative exosite. The Michaelis-Menten curves of PARL-mediated cleavage of IQ-PGAM5 20-29 and IQ-Smac/Diablo 51-60 were obtained in the presence of different concentrations of PINK1 89-111 . The data sets were fitted globally to competitive, noncompetitive, and mixed inhibition with strong preference for competitive inhibition for both substrates and global R 2 of 0.93 and 0.90 for IQ-PGAM5 20-29 and IQ-Smac/Diablo 51-60, respectively. The determined K d for IQ-PINK1 89-111 (represented by K i of the PARL-PINK 89-111 complex) was 2.4 ± 0.6 μM when IQ-PGAM5 20-29 was used as the main substrate and 2.3 ± 0.7 μM when IQ-Smac/Diablo 51-60 was used (53). These competitive inhibition parameters between the two substrates revealed that the assessed PARL substrates bind to the same binding site with similar affinities and are exclusive to each other. These results suggest that the inverse regulation of PINK1, PGAM5, and Smac/Diablo cleavage observed in cells is controlled by other mechanisms such as compartmentalization, involvement of protein partners for substrate presentation or different accessibility of the scissile bond in response to different membrane conditions. PARL has bulky substrate specificity preferences distinct from bacterial rhomboid proteases Bacterial rhomboid proteases are known to cleave substrates with a certain specificity for small side chain residues in the P1 position and bulky hydrophobic residues in the P4 position (23). Analysis of the C-terminal cleavage sites of PARL-generated cleavage products isolated from cell extracts by Edman degradation (8,39) or mass spectrometry (11) so far revealed no consensus. Using recombinant PARL, we now assessed the cleavage site specificity using a library of 228 synthetic peptides that are each 14 amino acids in length. This library was developed to have all pairwise combinations of neighbor and near-neighbor amino acids. We have previously confirmed that many peptide substrates of this library are cleaved by the bacterial rhomboids from Providencia stuartii and Haemophilus influenza (36). PARL in buffer containing DDM or reconstituted in proteoliposomes was incubated with an equimolar mixture of peptides and 70 common cleavage products by the enzyme under these two different assay conditions were identified (Fig. 5). We analyzed the location of cleavage within the peptide substrates and discovered that when PARL was assayed in proteoliposomes, it cleaved many peptides near the amino terminus, while PARL in DDM did not (Fig. 5B). These data indicated that the lipid environment for PARL may alter its cleavage preference of the enzyme or prevent access of certain substrates to the active site. Importantly, small amino acids were not found in the P1 position when PARL was assayed in either DDM or PL. In DDM, PARL preferentially cleaved peptides with norleucine (Nle), Tyr, Phe, Arg, and Lys in the P1 position while Phe and Ile are most frequently found in the P1ʹ position (Fig. 5C). In addition, hydrophobic amino acids are found at the P4 and P2 position. When PARL was assayed in PL, Phe and Nle were found most frequently in the P1 and P1ʹ positions (Fig. 5D). Lys was also significantly enriched in the P1 position, while hydrophobic amino acids were found in the P4, P2, and P4ʹ positions. When these profiles were compared with the sequences surrounding the putative PINK1 cleavage site, VFLA-FGLG, only P1ʹ-Phe and P4-Val are well tolerated from this sequence. In fact, Ala at P1 and Gly at P2ʹ are disfavored Catalytic properties of rhomboid protease PARL by PARL when assayed in DDM and PL, respectively. However, a GlpG-based homology model of human PARL reveals a putative pocket in the P1 position that could accommodate such a bulky side chain (Fig. 5E). The P4 position has a consistent bulky residue similar to that for bacterial rhomboid proteases. When PARL was incubated with the full TM region of MBP-PGAM5, N-terminal sequencing revealed that cleavage occurred between Phe-23 and Ser-24 (Fig. 5F). Interestingly, this cleavage site determined in our in vitro assay is different from the site previously determined in tissue culture cells, which for PGAM5 was between Ser-24 and Ala-25 (8). Readout of an in vitro assay is more direct than determination of N terminus of cleavage fragments isolated from tissue culture cells, so we suggest that the in vivo cleavage fragments may become subject to further trimming by additional proteases. In addition, the newly revealed PGAM5 cleavage site, originally thought to be in the center of the TM domain, topologically, is now placed closer to the matrix-exposed rhomboid active site. Taken together with the results from the peptide library (Fig. 5), the in vitro determined PGAM5 cleavage sites support the preference for PARL to cleave proteins and peptides with a bulky amino acid such as Phe in the P1 position. This is the first substrate specificity study of PARL, which shows an interesting preference for Phe in the P1 position, which is distinct from most bacterial rhomboid proteases that prefer small side chain residues in the P1 position (23). Discussion In this study, we provide multiple lines of evidence that recombinantly produced human PARL protease is active in both detergent and lipid environments, allowing for a significant advance in our understanding of the regulation of PARLmediated catalysis. We established a FRET-based assay to monitor proteolytic activity of PARLΔ55 and PARLΔ77 in a continuous manner, which allowed us to gather catalytic parameters of cleavage of three unique substrates, PGAM5, PINK1, and Smac/Diablo, by each PARL construct. Taken together, this is the formal proof that PARL is indeed able to cleave substrates that have been previously identified in cellular assays. When comparing the catalytic parameters, we revealed that the cleavage rates for three known PARL substrates are different with PGAM5 being preferred. We consistently observe that the K M value is the lowest for PGAM5, indicating a higher affinity with the greatest catalytic efficiency. This suggests that substrate specificity or substrate preference might have a role in regulating PARL-catalyzed cleavage. Overall PARL exhibited a very slow catalytic rate for proteolytic reactions. The bacterial rhomboid proteases from Haemophilus influenzae (HiGlpG) and P. stuartii (AarA) cleave at a rate of roughly two per minute for their preferred substrates in DDM, while the E. coli rhomboid protease GlpG cleaves slowly in DDM, much like PARL, at a rate of approximately six per hour and with a rate of 2.5 min −1 in PLs (26,34,35). Studies on other intramembrane proteases also suggest that these slow turnover rates are common for intramembrane proteolytic assays performed in vitro; intramembrane aspartyl proteases have been found to cleave a physiological FRET peptide substrate at a rate of approximately two per hour, which is not considerably different than what we see for PARL (26,34,35,38). Regardless of the slow rate of substrate turnover, the catalytic parameters are still able to provide valuable information regarding the unique enzymesubstrate interactions for each substrate assessed. With the recombinant protein and established activity assays in hand, we were able to conduct the competition binding studies to determine if the active site of PARL is the only molecular determinant regulating the substrate selection or if allosteric interactions are involved in the mechanism of cleavage of multiple substrates. The fact that we see competitive inhibition between all three substrates demonstrates that they are binding to the same binding sites on PARL molecule and the existence of allosteric sites is not supported by our data. Processing of PARL to either its mature Δ53 form or the further truncated Δ77 form has been proposed to be a modulator of protease enzymatic activity. Cellular studies have provided conflicting evidence: an impaired PARL activity is observed when mutation at Ser77 prevents β-cleavage to the PARLΔ77 form, though the PARLΔ53 form appears to be more active toward the PINK1 substrate (7,21). Using recombinant PARLΔ55 and PARLΔ77, we assessed the cleavage of three unique peptide substrates, IQ-PGAM5, IQ-PINK1, and IQ-Smac. We validated that PARL is catalytically active in either form, thus indicating that processing to the Δ77 form is not required for its proteolytic activity or functionality as was once speculated (7). While these truncations do not serve as an activation switch for the protease, we found that there are measurable differences in the catalytic parameters of cleavage between the two forms. This suggests that PARL truncations identified in vivo might regulate aspects of their activity. PARLΔ55 demonstrated significantly lower substrate turnover for IQ-PGAM5 and IQ-PINK1. PGAM5 is known to be preferentially cleaved by PARL upon mitochondrial depolarization, when enhanced β-cleavage and PARLΔ77 formation is also observed (8,21). Previous studies suggest that PARLΔ77 is catalytically less active toward PINK1 than the longer form of the protease (21), though our data suggest otherwise. This contradictory speculation could be explained by the fact that these cellular studies were performed during times of mitochondrial stress, in which PARLΔ77 formation is enhanced, but PINK1 import to the IMM is impaired, therefore even if PARLΔ77 is more active toward PINK1, it does not have access to the substrate. Interestingly, with the IQ-Smac/Diablo peptide, no significant difference was noticed in any of the catalytic parameters when cleaved by PARLΔ55 or PARLΔ77 in detergent, but we see an increase in proteolysis with PARLΔ77 form in PLs. While we were able to compare the cleavage of substrates mediated by either PARLΔ55 or PARLΔ77, many questions remain unanswered in regard to these forms of PARL. It is important to note that varying amounts of PARL truncations are detected in different tissues, suggesting that β-processing and even function of specific PARL forms could be tissuespecific (20). This raises questions on the role of the roughly 20 amino acid N-terminal region of protease molecule that is removed upon β-cleavage. We can make several speculations on its potential function, which may include protein stabilization or aiding in substrate recognition. Based on its localization to the matrix side of the IMM, it may be involved in mediating interactions with proteins that reside in the mitochondrial matrix. PARL is a member of a larger proteolytic hub in the IMM consisting of PARL protease, the i-AAA protease YME1L, and the scaffold stromatin-like protein 2 (51); however, it is still unknown what form of PARL associates with the complex. Plausibly, N-terminal region is required for protein-protein interactions between PARL and the SLP2 scaffold protein or truncation of PARL may alter associations with YME1L. We also demonstrated that cardiolipin has a significant effect on the activity of PARL, which presents the first evidence that lipids may modulate the activity of the mitochondrial rhomboid protease. Cardiolipin is the lipid exclusive to the IMM of eukaryotic cells, the membrane in which PARL is localized. In the IMM, CL represents approximately 10% of the total lipid, which is known to be essential to the activity of numerous IMM proteins (54). The finding that CL can influence the proteolytic activity of PARL is not overly surprising as there is considerable evidence that the activity of rhomboids can be modulated by lipids and that proteins of the IMM are influenced by the presence of CL. We determined that a 25:1 M ratio of CL to PARL results in the greatest increase in proteolytic activity compared with the no-lipid condition. Such increase in activity may be explained either by enhancement of protease stability or by CL binding to a specific site on PARL molecule, thereby inducing subtle conformational changes that facilitate substrate binding or substrate entrance to the active site. There are currently over 60 different proteins, many from the mitochondria, reported to interact with CL, and for over 20 of these high-resolution structures have been determined with at least one CL molecule present (55). It is worth noting that CL is often seen as an interactor within protein complexes in the IMM, exemplified by its critical role in both stability and function of the respiratory supercomplexes; there are predicted to be 200-400 cardiolipin molecules associated with the respiratory supercomplexes from bovine heart (55). Given the fact that PARL is thought to interact with YME1L-SLP2 complex within the IMM, CL might facilitate the formation, stability, and organization of such a complex. Characterization of PARL protease revealed a unique substrate specificity different from most bacterial rhomboid proteases. The preference for a large hydrophobic residue, particularly Phe, in the P1 position is in stark contrast to the substrate specificity of most bacterial rhomboid proteases, which allow only the small nonpolar residue Ala in the P1 position (23,56). In fact, no cleavage of the TatA substrate occurs by P. stuartii rhomboid protease AarA when Phe is mutated into the P1 position of the substrate cleavage site (23). Analysis of the E. coli rhomboid protease GlpG with a peptide substrate transition analog revealed a similar preference (56). Thus far, YqgP from B. subtilis is the only bacterial rhomboid protease known to cleave with Phe at the P1 position (23). YqgP is evolutionarily distinct from the E. coli GlpG (18), which suggests evolutionary pressure on substrate specificity. However, we still see that a hydrophobic Phe residue is conserved in the P4 position between bacterial rhomboids and PARLΔ77. Previous proteomics study identified six PARL substrates with three having an Ala, while the others either a Ser or Cys residue in the P1 position (11). The contradiction with our data could be explained by the fact that in the previous report lysates of HEK293 cells were used as opposed to purified protease for substrate identification. Structural modeling of PARL also supports its preference for a bulky amino acid at P1 position. When looking at the surface representation of a homology model of PARL based on the bacterial rhomboid protease HiGlpG structure, a large substrate binding pocket that can easily facilitate the entrance of a bulky residue, such as a Phe, is observed within the catalytic core of the enzyme (Fig. 5E). We see that negatively charged amino acids are highly unfavorable within the P4 to P4' positions; this is likely due to disruption of the oxyanion hole that would result from a negative charge entering into the catalytic core of the enzyme (57). The substrate specificity profile for PARL also suggests that the enzyme has overall broad substrate specificity toward TM substrates based on the preference for residues such as Phe, Ala, Val, Ile, and Pro, which are commonly associated with TM regions of a protein. Furthermore, in the region directly Cterminal to the cleavage site, there is a preference for the helixdestabilizing or helix-breaking residues Pro and Gly (6), which supports the evidence gathered for bacterial rhomboids suggesting that helix-destabilizing residues are required to facilitate unwinding of the helical TM substrate segment for better access to the cleavage site (58). It also indicates that there are likely other factors that regulate intramembrane proteolysis, rather than a highly specific substrate recognition motif. The broad substrate specificity obtained for PARL supports previous work on the yeast mitochondrial rhomboid that demonstrated large sequence variability in cleavable substrates (59). Other intramembrane proteases, such as γ-secretase, have also been established to have broad specificity, with γ-secretase sometimes being referred to as the "proteasome of the membrane" with over 100 identified substrates (60,61). Most likely for mitochondrial rhomboids, there are also numerous substrates that have yet to be identified. Our study characterized several aspects of PARL-mediated cleavage that were addressed by using in vitro proteolytic assays with a recombinant enzyme. We established activity assays with specific FRET-peptides, which could be used for downstream applications such as inhibitor screening. These assays confirmed that our recombinant protease retained activity after purification and determined the catalytic parameters of cleavage of three main substrates. Our in vitro studies present a significant advancement in the field as the majority of previous kinetic studies on rhomboid proteases have been Catalytic properties of rhomboid protease PARL limited to the bacterial rhomboid proteases and provide new methods for characterizing regulatory and mechanistic aspects of PARL's proteolytic functions. Experimental procedures Expression and purification of recombinant PARL PARL gene (PARLΔ77, PARLΔ77-S277A, or PARLΔ55) was cloned into pPICZA vector, followed by a TEV cleavage site, with a C-terminal GFP and hexahistidine-tag (22). The S277A mutant was created using site-directed mutagenesis technique (Q5 Site-Directed Mutagenesis Kit, NEB) with GTCAT-GATGGCACCAGCT GCACCAAGTGATGGT as forward primer and ACCATCACTTGGTGCAGCTGGTGCCAT-CATGAC as a reverse primer. An identified high-expressing clone was grown overnight at 28 C in 100 ml of BMGY media to an OD 600 of 4. A total of 6 L of BMGY media was subinoculated to a starting OD 600 of 0.03 and grown for 20 h at 28 C. Cells were harvested by centrifugation and cell pellets were resuspended in an equal volume of BMMY induction media. Cultures were induced for 48 h at 24 C, with fresh methanol being added after 24 h to a final concentration of 1% (v/v). Cells were harvested and resuspended in TBS buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl). Cells were resuspended in TBS with PMSF and lysed by passage through a Constant Systems cell disruptor at 38.2 kPSI, and membranes were isolated by ultracentrifugation. Membranes were homogenized in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% glycerol, 20 mM imidazole, 1 mM PMSF, and solubilized using 1.2% Triton X-100. Insoluble material was pelleted by ultracentrifugation and the supernatant bound to HisPur cobalt resin (Thermo Fisher) by gravity flow-through column. The protein-bound resin was washed with 10 mM imidazole and eluted with imidazole (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol, 0.1% DDM [Anatrace], 1 M imidazole). The purified PARL-GFP fusion protein was digested by incubation with TEV protease and 1 mM TCEP overnight at 4 C. Dialysis was performed for 2 h to remove imidazole and TCEP (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol). PARL was purified from GFP and TEV using HisPur Ni-NTA agarose resin (Thermo Fisher). Flow-through was collected and concentrated using a 10000 MWCO concentrator (Millipore). Protein concentration was determined by BCA assay (Pierce BCA Protein Assay Kit, ThermoFisher). Purified protein, at a concentration of 1 mg/ml, was incubated on ice with dried cardiolipin (Sigma-Aldrich) with protein: cardiolipin molar ratio of 25:1 Protein-lipid sample was aliquoted, flash-frozen, and stored at -80 C. After each purification, the quality of purified PARL was controlled by measuring its enzymatic activity. FRET-PINK1 purification Residues 70-134 of Human PINK1-WT were cloned into the pBad/HisB vector that already encoded for the engineered FRET pair, CyPet and YPet (derived from cyan fluorescence protein and yellow florescence proteins); this new pair exhibited 20-fold energy transfer efficiency when compared with the parental pair (62). The vector was transformed into TOP10 chemically competent E. coli cells (Thermo Fisher). Transformed cells were grown overnight at 37 C on LB agar plates containing 100 μg/ml ampicillin. One transformant colony was selected and grown overnight at 37 C in 120 ml of LB medium containing 100 μg/ml ampicillin. A total of 6 L LB media was subinoculated with 20 ml of overnight culture and grown to an OD 600 of 0.7 at 37 C. Cultures were induced by addition of 0.02% (v/v) L-arabinose for 8 h at 24 C. After induction, cells were harvested by centrifugation in a Beckman JLA8.1000 rotor (6900g, 20 min, 4 C), flash-frozen in liquid nitrogen, and stored at -80 C. Harvested cells were thawed on ice and resuspended in a 4:1 buffer volume to cell pellet weight ratio in resuspension buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20% glycerol, 10 μg/ml DNase, 1 mM PMSF, two EDTA-free protease inhibitor cocktail tablets). Resuspended cells were lysed using an Emulsiflex with a maximum pressure of 40 kPSI. Following cell lysis, the lysate was subjected to centrifugation using a Beckman TI45 rotor (31,300g, 20 min, 4 C) to pellet cell debris and unlysed cells. The supernatant was incubated with 1% (v/v) Triton X-100 at 4 C for 30 min with stirring. Supernatant was then passed through 1 ml settled HisPur cobalt resin (ThermoFisher) by gravity flow to allow binding of FRET-PINK1-His to the resin. Protein was eluted (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20% glycerol, 250 mM imidazole), pooled, and concentrated for loading onto the Superdex 200 column for size-exclusion chromatography. Size-exclusion chromatography fractions were analyzed by SDS-PAGE and fractions containing FRET-PINK1 protein were pooled and concentrated. Concentrated sample was aliquoted, flash-frozen with liquid nitrogen, and purified HsFRET-PINK1(70-134) was stored at -80 C for subsequent use. PINK1 TM expression and purification The sequence of PINK1 TM domain (amino acids 89-111) was codon optimized for E. coli expression and cloned into pMAL-c2 vector (New England Biolab) with N-terminal Maltose Binding Protein (MBP) followed by a tobacco etch virus (TEV) cleavage site. The vector was transformed into DH5α cells and the protein was induced with 0.5 mM IPTG and expressed for 3 days at 24 C. Cells were harvested, resuspended in 20 mM KPO 4 (pH 8), 120 mM NaCl, 50 mM glycerol, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, and lysed using an Emulsiflex with a maximum pressure of 40 kPSI. 0.5% Triton X-100 was added postlysis and cell debris was removed by centrifugation at 40,000g for 30 min at 4 C. The supernatant was loaded onto amylose resin (Amylose Resin High Flow, NEB), equilibrated with 20 mM KPO 4 , pH 8.0, 120 mM NaCl, 1 mM EDTA buffer, and the protein was eluted with 40 mM maltose in equilibration buffer. MBP tag was cleaved off by MBP-PINK1 incubation with recombinant TEV protease (1.5 mg of TEV per 30 mg of fusion protein) at 16 C for 4 to 8 days. To extract PINK1 TM segment 1/6 of the sample volume of 60% w/v trichloroacetic acid (TCA) was added to protein mixture and incubated for 30 min on ice. The precipitate was pelleted for 10 min at 10,000g, rinsed three times with ddH 2 O, resuspended in 50:50 isopropanol: chloroform, and mixed with a homogenizer. To this mixture, 1-2 ml of ddH 2 O was added into each tube and incubated overnight allowing for separation of the organic and aqueous layers. The organic layer was transferred into a clean tube and fresh 1-2 ml of was aliquoted into a sample and left overnight at room temperature. This separation was repeated until all white precipitate was removed and organic phase was considered clean. Organic layers were combined and dried down under nitrogen or argon gas. The PINK1 peptide was resuspended in 6-8 ml of 7 M guanidine-HCl, 50 mM KPO 4 buffer (pH 8) and injected onto an Agilant Zorbax SB-300 C8 silica-based, stainless steel 25 cm × 1 cm column, which was preheated to 60 C. The column ran at 60 C with a flow rate of 1 ml/min. An isopropanol gradient (20%-80%) against 0.05% TFA/water was used to elute the protein. PINK1 TM typically eluted at 50% isopropanol. Determination of fractions containing the peptide was established by running 6% urea gels, which were visualized through silver staining. MBP-PGAM5 expression and purification The sequence of the PGAM5 TM region (amino acids 1-46) was cloned into E. coli expression vector pET-25b(+) (Novagen) with N-terminal MBP and C-terminal Thioredoxin 1 followed by a triple FLAG-tag and a C-terminal hexahistidine-tag. The vector was transformed into chemical competent Rosetta 2 (DE3) cells (Novagen), grown in LB medium. Expression of the protein was induced with 0.3 mM IPTG and expressed for 2 h at 37 C. Cells were harvested by centrifugation at 3500 rpm for 15 min at 4 C and resuspended in 20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 1 mM PMSF, 5 mM β-mercaptoethanol. Prior to lysis, 200 μg/ml lysozyme, 1 mM PMSF, and benzonase (2.5 ku, Merck Millipore) were added and cells were lysed using Emulsiflex (Avestin) with a maximum pressure of 15 kPSI (100 MPa). Crude membranes were obtained by ultracentrifugation at 29,000 rpm for 45 min at 4 C. The membrane pellet was resuspended in 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 1 mM PMSF, 5 mM β-mercaptoethanol. MBP-PGAM5 was solubilized from the crude membranes with 1.5% DDM for 1 h on a rotating wheel at room temperature. Extraction of MBP-PGAM5 from membrane debris was done by ultracentrifugation at 29,000 rpm for 1 h at 4 C. Cleared extract was batch incubated with Ni-NTA beads (Macherey-Nagel) for 1 h on a rotating wheel at room temperature for His-tag affinity purification. Bound MBP-PGAM5 was washed with 50 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 50 mM imidazole, 0.05% DDM and eluted with 50 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 400 mM imidazole, 0.05% DDM. Determination of fractions containing the peptide was established by SDS-PAGE running 12% acrylamide gels, which were visualized through Coomassie staining. N-terminal sequencing by Edman degradation In total, 8-16 μg of E. coli purified MBP-PGAM5 was incubated with 0.4 μg of P. pastoris purified PARL for 2 h at 37 C in cleavage buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 0.3% DDM. Protein fragments were separated by SDS-PAGE running 12% acrylamide gels and transferred to a PVDF membrane by wet blot (glycine buffer) for 1 h at 100 V. Protein fragments were stained with Coomassie overnight and the C-terminal fragment (CTF) was then analyzed in four cycles by Edman degradation (TOPLAB). FRET-based protease kinetic assay Assays with FRET-PINK1 70-134 were conducted as previously described (26). For EDANS/Dabcyl 10-mer IQ peptides (PINK1, PGAM5, Smac/Diablo), lyophilized peptides were initially dissolved in DMSO to obtain a stock solution. The IQ peptide substrates in a concentration range of 0.1-70 μM were incubated with activity assay buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 10% glycerol, 0.1% DDM) in a 384-well blackbottomed plate at 37 C for 30 min in a multiwell plate reader (SynergyMx, BioTek). For all concentrations of IQ peptide, the DMSO was kept constant at 5%. Following preincubation, PARL was added to a final concentration of 0.8 μM to initiate the cleavage reaction. Fluorescence readings were taken every 3 min over a 3 h time course at ƛ ex = 336 nm and ƛ em = 490 nm. The initial velocity was determined from the fluorescence readings over the time course. For each substrate concentration, a no-enzyme control was subtracted to eliminate background fluorescence changes not related to substrate cleavage. Relative fluorescence units were converted to concentration (μM) by determining the maximum change in fluorescence observed for each substrate concentration when fully digested. GraphPad Prism software was used for Michaelis-Menten analysis of kinetic curves. All kinetic data were obtained using at least three biological replicates (different enzyme preparations) with technical duplicates for each experiment. For kinetic measurements in detergent with all three substrates, the activity assays were repeated at least five times. In addition, after each protease purification, the quality control of purified PARL was controlled by measuring its enzymatic activity. Minimum of three experimental replicates with two technical replicates were used for data analysis. Reconstitution in proteoliposomes E. coli polar lipids (Avanti), 400 μg in chloroform, were dried under nitrogen stream in a glass tube to yield a thin film Catalytic properties of rhomboid protease PARL of lipid. The tube was incubated overnight in a desiccator to completely remove all traces of solvent. In total, 50 μl of water and DDM detergent was added to the lipid film for resuspension at room temperature for 10 min, followed by the addition of purified PARL (400 μg) in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 20% glycerol, 0.1% DDM, to yield the final weight ratio of 1 PARL:1 lipid:2 detergent. The detergent was slowly removed by the addition of SM2 Biobeads (Bio-Rad) while stirring on ice for 6 h to allow for the generation of proteoliposomes (PL); the process was controlled by the regimen of Biobead addition. To purify the PL, 50%:20% sucrose density gradient ultracentrifugation was used. A TAMRA probe (ThermoFisher) was used to determine the orientation of reconstituted PARL, which specifically and covalently labels serine residues of an enzymatically active serine protease. In total, 2 μM of TAMRA probe was added to PL samples and incubated for 1 h, allowing the TAMRA probe to label only the outward-facing accessible active sites. The same amount of PL, but with 1% DDM added to dissolve lipid vesicles incubated with 2 μM of TAMRA probe at the same conditions, was used as a benchmark for 100% accessible protein amount. The reaction was quenched with addition of SDS-containing sample buffer and the protein samples were visualized with SDS-PAGE followed by fluorescent gel scanning. The bands were quantified by densitometry analysis. The difference in labeling between the first and the second samples gave us the proportion of accessible protein in PLs versus the whole amount of protein. Coomassie staining was used to normalize the amount of protein loaded. Activity assay in proteoliposomes The activity assay with PARL reconstituted in PL was performed the same way as for PARL in DDM with the only difference being the activity buffer, where DDM was omitted (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 10% glycerol). For inhibitory studies, PARL in PL (0.8 μM) was incubated with inhibitors (20 μM) in activity buffer for 30 min, and then the proteolytic reaction was started with the addition of substrate (5 μM). Molecular modeling Human PARL, isoform 1, was modeled using iTASSER with HiGlpG as the template (63), without any additional restraints (63). Residues 1-167 were removed from the modeling due to low homology with bacterial rhomboid protease crystal structures. Multiplex substrate profiling by mass spectrometry Multiplex substrate profiling by mass spectrometry (MSP-MS) assays was performed in quadruplicate. In total, 1 μM of PARLΔ77 was incubated with an equimolar mixture of 228 synthetic tetradecapeptides at a final concentration of 0.5 μM for each peptide in 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 10% glycerol, with or without 0.1% DDM. The sequence of each peptide is listed in Supplementary Data File. These peptides have been validated as substrates for a wide variety of proteolytic enzymes (PMID 23023596, 24073241, 25944934) including bacterial rhomboid proteases (30705125). For each assay, 20 μl of the reaction mixture was removed after 0, 60, and 240 min of incubation. Enzyme activity was quenched by adding GuHCl (MP Biomedicals) to a final concentration of 6.4 M, and samples were immediately stored at -80 C. All samples were desalted using C18 spin columns and dried by vacuum centrifugation. Approximately 2 μg of peptides was injected into a Q-Exactive Mass Spectrometer (Thermo) equipped with an Ultimate 3000 HPLC. Peptides were separated by reverse-phase chromatography on a C18 column (1.7 μm bead size, 75 μm × 25 cm, 65 C) at a flow rate of 300 nl/min using a 60min linear gradient from 5% to 30% B, with solvent A: 0.1% formic acid in water and solvent B: 0.1% formic acid in acetonitrile. Survey scans were recorded over a 150-2000 m/z range (70,000 resolutions at 200 m/z, AGC target 3 × 10 6 , 100 ms maximum). MS/MS was performed in data-dependent acquisition mode with HCD fragmentation (28 normalized collision energy) on the 12 most intense precursor ions (17,500 resolutions at 200 m/z, AGC target 1 × 10 5 , 50 ms maximum, dynamic exclusion 20 s). Data was processed using PEAKS 8.5 (Bioinformatics Solutions Inc). MS 2 data were searched against the 228 tetradecapeptide library sequences with decoy sequences in reverse order. A precursor tolerance of 20 ppm and 0.01 Da for MS 2 fragments was defined. No protease digestion and modification were specified. Data were filtered to 1% peptide-level false discovery rates with the target-decoy strategy. Peptides were quantified with label-free quantification and data are normalized by medians and filtered by 0.3 peptide quality. Missing and zero values are imputed with random normally distributed numbers in the range of the average of smallest 5% of the data ± SD. Cleaved sequences were defined as peptide products that increase by a fold change of >8 and q value <0.05 (by Student t test) between 0 min and 240 min. IceLogo software was used for visualization of amino-acid frequency surrounding the cleavage sites. Amino acids that were most frequently observed (above axis) and least frequently observed (below axis) from P4 to P4ʹ positions were illustrated. Norleucine (Nle) was represented as "n" in the reported profiles. Mass spectrometry data and searching results have been deposited in MassIVE with accession number, MSV000085295. Data availability All data is located in the article. Raw data associated with the mass spectrometry can be found in the supplemental information. Supporting information-This article contains supporting information. protein. E. A. conducted proteoliposome assays, V. S. conducted MBP-PGAM5 cleavage assay and Edman degradation assay, Z. J. conducted substrate specificity profiling. H. S. Y. conducted EM analysis of liposomes. The article was written by M. J. L. and edited by all the authors.	v3-fos-license
2020-07-30T02:04:54.552Z	2020-07-24T00:00:00.000	220857982	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1420-3049/25/15/3357/pdf", "pdf_hash": "65d6ad3e2a7310772178b13acca17e47021419f2", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1485", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "774e361f3c4e36df34a288c5bc5436f93359dbf8", "year": 2020 }	pes2o/s2orc	Synthesis of Highly Oxygenated Bicyclic Carbasugars. Remarkable Difference in the Reactivity of the d-gluco and d-xylo- Derived Trienes 2,3,4-Tri-O-benzyl-D-xylopyranose was used as a starting material in the preparation of the corresponding triene, which underwent smooth cyclization to a polyhydroxylated hydrindane, as a single diastereoisomer. The analogous triene prepared from D-glucose did not undergo any cyclization even under high pressure. Introduction Carbasugars are glycomimetics obtained through a substitution of the endocyclic oxygen atom with a methylene group [1,2]. Due to their similarity to carbohydrates, these compounds often possess interesting biological properties, e.g., they may act as glycosidase inhibitors [3][4][5]. Some of these compounds found an application in medicine; for example, acarbose is commercialized to treat obesity and type 2 diabetes mellitus [6] and validamycin is an antibiotic [7]. Elaboration of the convenient synthetic methods for the preparation of carbobicyclic sugar mimetics is one of the leading trends in our laboratory [8]. We have proposed a useful route to polyhydroxylated decalins (such as 5) from sugar allyltins as shown in Scheme 1 [9]. The model synthesis was initiated from D-gluco-derivative 1, which-upon treatment with mild Lewis acid (preferably ZnCl 2 )-afforded dienoaldehyde 2 with the E-configuration across the internal double bond. This compound was converted into phosphonate 3 and-by the Horner-Wadsworth-Emmons (HWE) reaction with the corresponding aldehyde-into triene 4, subsequently cyclized to 5. The methodology proposed in Scheme 1 allowed the preparation of a number of diastereoisomeric polyhydroxylated bicyclic derivatives in optically pure form starting from different sugars: d-glucose, d-mannose, and d-galactose [9][10][11][12]. It is known that polyhydroxylated decalins might have significant biological activity [13][14][15], thus there is an interest in the preparation of such targets. Our methodology, however, starting from organostannanes-being assumed to be highly toxic-cannot be used for the preparation of potential therapeutics. Other, environmentally friendly method(s), for the synthesis of such important derivatives is needed. We have proposed an efficient method for the stereoselective preparation of sugar dienes with the E or Z geometry across the internal double bond (8-E or 8-Z; Scheme 2) from D-glucosep-derivative 6 (via intermediate 7) [16]. These compounds could be eventually used as convenient precursors for the Alcohol 21 was oxidized to the corresponding aldehyde 22 which reacted with phosphonate 23 [24], to afford triene 24 with the E-configuration across the newly created double bond (Scheme 6). This triene was subjected to the cyclization induced by Me 2 AlCl; no reaction was, however, noticed. Application of other Lewis acids, as well as, high pressure (10 kbar) also did not give any positive result. This lack of cyclization was very surprising, especially in the context of the results obtained by Evans. We prepared also two other trienes: 25 and 26 by reaction of aldehyde 22 with appropriate phosphoranes: Ph 3 P=CHCO 2 Me and Ph 3 P=CHCO 2 Me. None of the resulting trienes, however, underwent cyclization (Scheme 6). Why could the cyclization of compounds-similar to those successfully used in Evans' synthesis-not be realized? Is it a result of only the different configuration across the double bond in a diene system (Z in 24 in our case vs. E in 14 in Evans case)? Or maybe other reasons are responsible for that strange result. This phenomenon is not clear. We turned, therefore, our attention to shorter analogs being precursors of perhydroindane derivatives. Synthesis of Derivatives of D-Xylose with the Diene System at the C1 The synthesis was initiated from known lactone 27 [25], obtained by an oxidation of 2,3,4-tri-O-benzyl-D-xylose. It was reacted with the in situ generated complex of Me 3 Al and N,O-dimethylhydroxylamine which provided the Weinreb amide 28. Since this compound was relatively unstable (easily underwent decomposition to 27), it was converted immediately into silylated derivative 29 and then into aldehyde 30. Reactivity of 30 was similar to the previously obtained derivatives. Its conversion into 31 followed the same route as for D-glucose derivatives (depicted in Scheme 1); this silanol (31) was also unreactive under the acidic conditions and underwent conversion into Z-diene 32 with the simultaneous removal of the silyl block upon treatment with fluoride anion (Scheme 7). The configuration of the internal double bond in the diene part was proven by 1 H NMR data in which the coupling constant J = 11.2 Hz was observed (see Scheme 7). The synthesis was initiated from known lactone 27 [25], obtained by an oxidation of 2,3,4-tri-Obenzyl-D-xylose. It was reacted with the in situ generated complex of Me3Al and N,Odimethylhydroxylamine which provided the Weinreb amide 28. Since this compound was relatively unstable (easily underwent decomposition to 27), it was converted immediately into silylated derivative 29 and then into aldehyde 30. Reactivity of 30 was similar to the previously obtained derivatives. Its conversion into 31 followed the same route as for D-glucose derivatives (depicted in Scheme 1); this silanol (31) was also unreactive under the acidic conditions and underwent conversion into Z-diene 32 with the simultaneous removal of the silyl block upon treatment with fluoride anion (Scheme 7). The configuration of the internal double bond in the diene part was proven by 1 H NMR data in which the coupling constant J = 11.2 Hz was observed (see Scheme 7). Scheme 7. Stereoselective synthesis of bicyclic derivative 35 from D-xylose. Compound 32 was converted into aldehyde 33 and further into triene 34, with the E-configuration across the newly created double bond (J = 15.9 Hz). Cyclization of this triene induced with Me2AlCl provided bicyclic compound 35 as a single stereoisomer (Scheme 3); only two olefinic signals at δ = 128.0 and 124.3 ppm were seen in the 13 C NMR spectrum. The configuration of the product was assigned by the Nuclear Overhauser Effect (NOE) measurement ( Figure 1). Small NOE values between the H1-H7a and H3-H3a confirmed structure 35 and-at the same time-excluded the alternative one: 35a in which the strong H1-H7a and H3-H3a interaction should have been observed. Compound 32 was converted into aldehyde 33 and further into triene 34, with the E-configuration across the newly created double bond (J = 15.9 Hz). Cyclization of this triene induced with Me 2 AlCl provided bicyclic compound 35 as a single stereoisomer (Scheme 3); only two olefinic signals at δ = 128.0 and 124.3 ppm were seen in the 13 C NMR spectrum. The configuration of the product was assigned by the Nuclear Overhauser Effect (NOE) measurement ( Figure 1). Small NOE values between the H1-H7a and H3-H3a confirmed structure 35 and-at the same time-excluded the alternative one: 35a in which the strong H1-H7a and H3-H3a interaction should have been observed. General NMR spectra were recorded in CDCl3 (internal Me4Si) with a Varian AM-600 (600 MHz 1 H, 150 MHz 13 C) spectrometer (Sugar Land, TX, USA) at rt. Chemical shifts (δ) are reported in ppm relative to Me4Si (δ 0.00) for 1 H and residual chloroform (δ 77.00) for 13 C. All significant resonances (carbon skeleton) were assigned by COSY ( 1 H-1 H), HSQC ( 1 H-13 C), and HMBC ( 1 H-13 C) correlations. The relative configuration of the stereogenic centers was assigned on the basis of 1D-NOESY spectra. Mass spectra (ESI) were recorded with an Applied Biosystems 4000 Q-TRAP (low resolution) (Toronto, ON, Canada) and Waters AutoSpec Premier (Waters, Milford, MA, USA) or Waters MALDISynapt G2-S HDMS (high resolution) spectrometers (Manchester, UK). HPLC analyses were conducted on Merck-Hitachi apparatus (Darmstadt, Germany) equipped with Merck LiChrospher Small NOE values between the H1-H7a and H3-H3a confirmed structure 35 and-at the same time-excluded the alternative one: 35a in which the strong H1-H7a and H3-H3a interaction should have been observed. The scans of the NMR data for all compounds are provided as Supplementary Materials. The scans of the NMR data for all compounds are provided as Supplementary Materials. Olefin 19 Generation of the titanium reagent 18: To a cooled to −78 • C solution of allyltrimethylsilane (3.7 g, 32.4 mmol, 10.0 eq) in dry THF (32 mL), a solution of 2.5 M BuLi in hexane (12 mL, 29.1 mmol, 9.0 eq.) was added within 1h with a syringe pump; after another 30 min. the mixture became yellowish. Then, a solution of 1.0 M ClTi(O i Pr) 3 in methylene chloride (29 mL) was added within 30 min (syringe pump) and the red mixture was stirred for another 30 min. To such prepared reagent 18, a solution of 17 (2.10 g, 3.19 mmol) in dry THF (6.4 mL) was added within 1h, the mixture was stirred overnight at −78 • C (using immersion cooler with temperature control Huber TC100E, temperature range −100 to +40 • C) and partitioned between water (70 mL) and ether (70 mL). The white precipitate was filtered off and discarded; the organic layer was separated, washed with brine, and concentrated to afford a crude mixture of (anticipated) anti-isomers, pure enough to be used in the next steps ( Figure 3). Generation of the titanium reagent 18: To a cooled to −78 °C solution of allyltrimethylsilane (3.7 g, 32.4 mmol, 10.0 eq) in dry THF (32 mL), a solution of 2.5 M BuLi in hexane (12 mL, 29.1 mmol, 9.0 eq.) was added within 1h with a syringe pump; after another 30 min. the mixture became yellowish. Then, a solution of 1.0 M ClTi(O i Pr)3 in methylene chloride (29 mL) was added within 30 min (syringe pump) and the red mixture was stirred for another 30 min. To such prepared reagent 18, a solution of 17 (2.10 g, 3.19 mmol) in dry THF (6.4 mL) was added within 1h, the mixture was stirred overnight at −78 °C (using immersion cooler with temperature control Huber TC100E, temperature range −100 to +40 °C) and partitioned between water (70 mL) and ether (70 mL). The white precipitate was filtered off and discarded; the organic layer was separated, washed with brine, and concentrated to afford a crude mixture of (anticipated) antiisomers, pure enough to be used in the next steps ( Figure 3). Dienoalcohol 21 To a solution of crude 19 obtained above in THF (16 mL) a solution of 1.0 M TBAF in THF (16 mL, 16.0 mmol, 5.0 eq.) was added and the mixture was stirred at room temperature overnight. Then it was concentrated and the residue was subjected to column chromatography (hexane-ethyl acetate, 92:8→36:64) to afford the title product 21 (1.11 g, 73% over two steps) as an oil ( Figure 4). Dienoalcohol 21 To a solution of crude 19 obtained above in THF (16 mL) a solution of 1.0 M TBAF in THF (16 mL, 16.0 mmol, 5.0 eq.) was added and the mixture was stirred at room temperature overnight. Then it was concentrated and the residue was subjected to column chromatography (hexane-ethyl acetate, 92:8→36:64) to afford the title product 21 (1.11 g, 73% over two steps) as an oil (Figure 4). Generation of the titanium reagent 18: To a cooled to −78 °C solution of allyltrimethylsilane (3.7 g, 32.4 mmol, 10.0 eq) in dry THF (32 mL), a solution of 2.5 M BuLi in hexane (12 mL, 29.1 mmol, 9.0 eq.) was added within 1h with a syringe pump; after another 30 min. the mixture became yellowish. Then, a solution of 1.0 M ClTi(O i Pr)3 in methylene chloride (29 mL) was added within 30 min (syringe pump) and the red mixture was stirred for another 30 min. To such prepared reagent 18, a solution of 17 (2.10 g, 3.19 mmol) in dry THF (6.4 mL) was added within 1h, the mixture was stirred overnight at −78 °C (using immersion cooler with temperature control Huber TC100E, temperature range −100 to +40 °C) and partitioned between water (70 mL) and ether (70 mL). The white precipitate was filtered off and discarded; the organic layer was separated, washed with brine, and concentrated to afford a crude mixture of (anticipated) antiisomers, pure enough to be used in the next steps ( Figure 3). Dienoalcohol 21 To a solution of crude 19 obtained above in THF (16 mL) a solution of 1.0 M TBAF in THF (16 mL, 16.0 mmol, 5.0 eq.) was added and the mixture was stirred at room temperature overnight. Then it was concentrated and the residue was subjected to column chromatography (hexane-ethyl acetate, 92:8→36:64) to afford the title product 21 (1.11 g, 73% over two steps) as an oil (Figure 4). To a cooled to 0 • C solution of alcohol 21 (350 mg, 0.62 mmol) and TEMPO (1.0 mg; 6.0 µmol) in dry CH 2 Cl 2 (8 mL), trichloroisocyanuric acid (155 mg; 0.66 mmol; 1.1 eq) was added and the mixture was stirred for 15 min. Then it was filtered through short pad of Celite, the filtrate was diluted with Et 2 O (6.0 mL) and washed with 5% Na 2 S 2 O 3 (2.0 mL), 1M NaOH (5 mL), 1M H 2 SO 4 (5 mL), and water (3.0 mL). The organic phase was dried and concentrated, and the crude aldehyde was used immediately in the next step ( Figure 5). To a cooled to 0 °C solution of alcohol 21 (350 mg, 0.62 mmol) and TEMPO (1.0 mg; 6.0 μmol) in dry CH2Cl2 (8 mL), trichloroisocyanuric acid (155 mg; 0.66 mmol; 1.1 eq) was added and the mixture was stirred for 15 min. Then it was filtered through short pad of Celite, the filtrate was diluted with Et2O (6.0 mL) and washed with 5% Na2S2O3 (2.0 mL), 1M NaOH (5 mL), 1M H2SO4 (5 mL), and water (3.0 mL). The organic phase was dried and concentrated, and the crude aldehyde was used immediately in the next step ( Figure 5). Weinreb Amide 29 To a cooled to 0 °C suspension of MeNHOMe x HCl (5.85 g, 60.0 mmol, 3.0 eq) in dry CH2Cl2 (175 mL), a 2M solution of Me3Al in toluene (30 mL, 60 mmol, 2 eq.) was added dropwise during 30 min. by a syringe pump. The mixture was stirred for an additional 30 min., then lactone 27 (8.37 g, 20 mmol) in dry CH2Cl2 (25 mL) was added within 30 min. by a syringe pump, and the mixture was Weinreb Amide 29 To a cooled to 0 °C suspension of MeNHOMe x HCl (5.85 g, 60.0 mmol, 3.0 eq) in dry CH2Cl2 (175 mL), a 2M solution of Me3Al in toluene (30 mL, 60 mmol, 2 eq.) was added dropwise during 30 min. by a syringe pump. The mixture was stirred for an additional 30 min., then lactone 27 (8.37 g, 20 mmol) in dry CH2Cl2 (25 mL) was added within 30 min. by a syringe pump, and the mixture was Weinreb Amide 29 To a cooled to 0 • C suspension of MeNHOMe x HCl (5.85 g, 60.0 mmol, 3.0 eq) in dry CH 2 Cl 2 (175 mL), a 2M solution of Me 3 Al in toluene (30 mL, 60 mmol, 2 eq.) was added dropwise during 30 min. by a syringe pump. The mixture was stirred for an additional 30 min., then lactone 27 (8.37 g, 20 mmol) in dry CH 2 Cl 2 (25 mL) was added within 30 min. by a syringe pump, and the mixture was stirred at room temperature for 3 h. Aqueous H 2 SO 4 (1M solution, 100 mL) was carefully added and the organic phase was separated, washed with water (100 mL), brine (100 mL), and dried. Imidazole (4.08 g, 60 mmol, 3.0 eq) was added to this containing crude 28 and the resulting mixture was cooled to 0 • C. A solution of tert-butyldiphenylchlorosilane (7.8 mL, 30.0 mmol, 1.5 eq.) in CH 2 Cl 2 (20 mL) was added dropwise within 1 h by a syringe pump and the mixture was stirred for additional 16 h. Aqueous H 2 SO 4 (1M solution, 50 mL) was carefully added, the organic phase was separated, washed with water (100 mL), brine (100 mL), dried, and the crude product was purified by column chromatography (hexanes-ethyl acetate: 13:1→7:1) to give the title product 29 (12.35 g, 86% over two steps) as a colorless oil (Figure 9). Silanol 31 This compound was prepared analogously as 19, from 30 (2.79 g, 4.23 mmol) as a colorless oil as a mixture of (anticipated) two anti-isomers ( Figure 11). Conclusions We have proposed a convenient and simple route to optically pure bicyclic carbasugar derivatives from D-xylose derivative. The methodology is based on the introduction of the Z-diene system at the anomeric position of a sugar and an olefinic unit at the terminal position. The so obtained triene underwent smooth and highly stereoselective cyclization providing the bicyclic Figure 1 in the text).	v3-fos-license
2020-10-24T13:05:46.930Z	2020-10-01T00:00:00.000	225049830	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1420-3049/25/20/4816/pdf", "pdf_hash": "119623f147dbf07594db9e41bb56e0b16504dfab", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1567", "s2fieldsofstudy": [ "Chemistry", "Environmental Science", "Medicine" ], "sha1": "dbecd145365e8224373cb819ee155768e8976452", "year": 2020 }	pes2o/s2orc	Supercritical Extraction of Red Propolis: Operational Conditions and Chemical Characterization The objective of this study was to determine the best operational conditions for obtaining red propolis extract with high antioxidant potential through supercritical fluid extraction (SFE) technology, using carbon dioxide (CO2) as the supercritical fluid and ethanol as the cosolvent. The following parameters were studied: overall extraction curve, S/F (mass of CO2/mass of sample), cosolvent percentage (0, 1, 2 and 4%) and global yield isotherms as a function of different pressures (250, 350 and 450 bar) and temperatures (31.7, 40 and 50 °C). Within the investigated parameters, the best conditions found were an S/F of 131 and the use of ethanol at the highest concentration (4% w/w), which resulted in higher extract yields and higher content of antioxidant compounds. Formononetin, the main biomarker of red propolis, was the compound found at the highest amounts in the extracts. As expected, the temperature and pressure conditions also influenced the process yield, with 350 bar and 40 °C being the best conditions for obtaining bioactive compounds from a sample of red propolis. The novel results for red propolis found in this study show that it is possible to obtain extracts with high antioxidant potential using a clean technology under the defined conditions. Introduction Propolis is a natural compound defined as a complex resin matrix produced by bees from a mixture of exudates from different plants, wax and salivary secretions; it has major applications in the food, pharmaceutical and cosmetic industries [1,2]. Numerous studies have demonstrated its antioxidant [3,4], antimicrobial [5,6], anti-inflammatory [7,8] and antitumor [7,8] activities, among others [9][10][11][12]. These activities are attributed to the bioactive chemical compounds in propolis, such as phenolic acids, flavonoids, terpenes and sesquiterpenes [13][14][15]. Given this context, research has intensified on different extracts from natural matrices that have a wide variety of compounds with beneficial effects on health, such as propolis [16][17][18][19]. Among the various types of propolis, classified according to their physicochemical properties and geographical location, red propolis has gained prominence due to its composition and pharmacological properties [20][21][22]. Originating in Northeast Brazil, red propolis has biologically active flavonoids as its major compounds and may also contain cinnamic acid derivatives, esters and some terpenes [14,22,23]. Flavonoids are the most common and most varied group among Table 1. Determination of pilot extraction kinetics for red propolis using supercritical fluid extraction by CO 2 with the results for S/F; mass yield of extract ± standard deviation (SD); mass yield of accumulated extract ± SD; total phenolic compounds in mg/GAE/g ± SD; and antioxidant activity (%) ± SD (parameters: 7.5 g of sample; 40 • C; 100 bar; CO 2 flow rate 6.0 g/min). In studies on the extraction of compounds from the herbs Plantago major and Plantago lanceolata, Mazzuti et al. [44] obtained fully developed kinetic curves, reaching the diffusional period at 240 min, using 15 g of crude extract from the samples defined for a pressure condition of 240 bar and a temperature of 50 • C. The same kinetic curve profile was found by Teixeira et al. [46] when analyzing the impact of temperature and pressure on SFE tests using 15 g of pracaxi seed oil (Pentaclethra macroloba), reaching the diffusional period at 240 min under the conditions of 40 • C and 250 bar. Figure 1 shows the kinetic curve obtained for the mean accumulated extract mass versus the total extraction time of the propolis sample. According to different studies, extraction kinetics experimental data are fundamental for scaling up studies of the supercritical technology applied to obtain extracts of solid matrices [44,46,47]. Using the SFE technology in natural matrices, different studies have shown that the kinetic curve is not a linear function of time, and its shape is an indicator that different mechanisms control the mass transfer in the different extraction stages [44,48,49]. In the curve obtained for red propolis (Figure 1), a classical pattern that occurs in SFE was observed, that is, the presence of a period of constant extraction rate in the first hours. This is related to the extraction of substrates that are easily accessible to supercritical CO 2 (process solvent). Subsequently, there is a progressive reduction in the extraction rate over time, which represents the extraction of substrates that are difficult to access by the supercritical solvent [42,50]. Experiment Molecules 2020, 25, x FOR PEER REVIEW 4 of 22 Figure 1 shows the kinetic curve obtained for the mean accumulated extract mass versus the total extraction time of the propolis sample. According to different studies, extraction kinetics experimental data are fundamental for scaling up studies of the supercritical technology applied to obtain extracts of solid matrices [44,46,47]. Using the SFE technology in natural matrices, different studies have shown that the kinetic curve is not a linear function of time, and its shape is an indicator that different mechanisms control the mass transfer in the different extraction stages [44,48,49]. In the curve obtained for red propolis (Figure 1), a classical pattern that occurs in SFE was observed, that is, the presence of a period of constant extraction rate in the first hours. This is related to the extraction of substrates that are easily accessible to supercritical CO2 (process solvent). Subsequently, there is a progressive reduction in the extraction rate over time, which represents the extraction of substrates that are difficult to access by the supercritical solvent [42,50]. The extraction kinetics of red propolis using CO2 as a supercritical fluid are also represented by the overall extraction curves ( Figure 2). Figure 2a shows the yield of accumulated extract (accumulated mass) during the process as a function of S/F, while Figure 2b shows the global yield of phenolic compounds and antioxidant activity versus S/F considering the total extraction time. The extraction kinetics of red propolis using CO 2 as a supercritical fluid are also represented by the overall extraction curves ( Figure 2). Figure 2a shows the yield of accumulated extract (accumulated mass) during the process as a function of S/F, while Figure 2b shows the global yield of phenolic compounds and antioxidant activity versus S/F considering the total extraction time. Molecules 2020, 25, x FOR PEER REVIEW 4 of 22 Figure 1 shows the kinetic curve obtained for the mean accumulated extract mass versus the total extraction time of the propolis sample. According to different studies, extraction kinetics experimental data are fundamental for scaling up studies of the supercritical technology applied to obtain extracts of solid matrices [44,46,47]. Using the SFE technology in natural matrices, different studies have shown that the kinetic curve is not a linear function of time, and its shape is an indicator that different mechanisms control the mass transfer in the different extraction stages [44,48,49]. In the curve obtained for red propolis (Figure 1), a classical pattern that occurs in SFE was observed, that is, the presence of a period of constant extraction rate in the first hours. This is related to the extraction of substrates that are easily accessible to supercritical CO2 (process solvent). Subsequently, there is a progressive reduction in the extraction rate over time, which represents the extraction of substrates that are difficult to access by the supercritical solvent [42,50]. The extraction kinetics of red propolis using CO2 as a supercritical fluid are also represented by the overall extraction curves ( Figure 2). Figure 2a shows the yield of accumulated extract (accumulated mass) during the process as a function of S/F, while Figure 2b shows the global yield of phenolic compounds and antioxidant activity versus S/F considering the total extraction time. Based on the results presented for study step 1 and after analyzing the extraction results and behavior using CO 2 as a supercritical fluid under the extraction conditions defined, an S/F of 131 was determined because more than 80% of the phenolic compounds were extracted, representing 63% of the global yield in mass of the extraction. At this point of the relationship between the solvent mass and sample mass, an accumulated yield of 4.88% (63% of the total mass extracted) was obtained, where a total CO 2 volume of 0.540 m 3 with an approximate extraction time of 240 min was obtained. Albuquerque et al. [43] obtained extracts rich in tocotrienols and defatted bixin-rich seeds from annatto and determined an S/F ratio of 35. Similar behavior was observed by Machado et al. (2012) [33], who determined the extraction kinetics curve for green propolis with a total extraction time of 150 min and an S/F ratio of 110. In the study by Krakowska-Sieprawska et al. [26], the highest yields were obtained at 60 and 120 min of the process and reached 6.42% for extracts from yerba mate and 9.60% for extracts from yellow lupine, respectively. The low mass yield of propolis extract by SFE has been demonstrated in other studies, which may be related to the fact that propolis is not very soluble in supercritical CO 2 but can be much more soluble in a mixture containing CO 2 and ethanol [51][52][53]. SFE is considered a very efficient technique in terms of selectivity for the extraction of target compounds, as well as for the separation and fractionation of different classes of compounds; however, depending on the polarity of these compounds, it is necessary to add small amounts of a modifier or cosolvent to the system to improve process yield [54]. For this reason, the influence of ethanol as a cosolvent at different concentrations in obtaining red propolis extracts by SFE with high antioxidant capacity was also evaluated in this study. Determination of the Cosolvent Concentration Although CO 2 is the most commonly used solvent in SFE, one of the main limitations of its use is the reduced capacity to dissolve polar molecules, as is the case for the compounds present in red propolis, even at high densities [55]. Phenolic compounds and flavonoids are the bioactive components of propolis of major importance from a biological significance standpoint due to their pharmacological activities and their applicability in different areas [56]. It is well documented that the total content of phenolic compounds present in propolis varies according to several factors, including the extraction method and conditions used [57]. Regarding red propolis, previously studies have reported the presence of more than 300 components, which are representatives of terpenes, flavonoids, aromatic acids and fatty acids [22]. Flavonoids, are the most common and widely distributed group of phenolics in red propolis, being the most active compound in this natural matrix, with formononetin as the most relevant chemical marker of this type of propolis [58]. Due to the hydrophilic properties of phenolic compounds, to increase the extraction capacity of these compounds using SFE, soluble polar solvent (e.g., methanol, ethanol, and water) can be added to supercritical CO 2 to modify its properties during extraction. In the present study, the SFE conditions using ethanol as a cosolvent were also evaluated. Table 2 presents the results for the yields of phenolic compounds, flavonoids, antioxidant activity (IC 50 -DPPH•), and concentrations of the formononetin and kaempferol compounds using 1, 2 and 4% ethanol (cosolvent) in relation to the mass of CO 2 (w/w) and without the presence of a cosolvent. Based on these data, it was observed that the best extraction conditions to obtain a higher yield of the compounds of interest-formononetin and kaempferol-as well as the highest content of total phenolics and antioxidant activity (represented by the lowest IC 50 ) were achieved when 4% cosolvent was used (highest concentration investigated in this study). The extraction yields of total phenolics from red propolis can be increased by up to 57% with the presence of ethanol in the system as a cosolvent to supercritical CO 2 . Furthermore, the antioxidant capacity of the extracts was increased by 70%, thus demonstrating that it is possible to increase the solubility of antioxidant compounds in supercritical CO 2 by adding ethanol. This may be related to the increased polarity of the solvent together with the ability of ethanol to improve the extraction surface area in a natural solid matrix [59]. The expansion of the material, generated by the interactions between solids and solvents, and the affinity between the liquid solvent and the associative compounds in the raw material leads to greater miscibility of the gas in the sample, resulting in greater extraction efficiency, as reported in previous studies [59,60]. Table 2. Results of the yields of total phenolic compounds (mg GAE/g), flavonoids (mg QE/g), IC 50 (DPPH.) (µg/g), and the biomarkers formononetin (mg/g) and kaempferol (mg/g) using 1, 2 and 4% ethanol (cosolvent) in relation to the mass of CO 2 (w/w) and without the presence of cosolvent (mean ± SD) (50 • C; 250 bar; CO 2 flow rate of 6 g/min; S/F 131). Analyses Values followed by the same letter in the column are not significantly different (p < 0.05) according to Tukey's test at the 95% confidence level. Previous studies with various types of propolis have also shown that ethanol is an important system modifier [35,61]. In a comparative study of extraction methods to obtain extracts from green propolis by SFE, Monroy et al. [36] found that when using 32% of the solution containing 80% ethanol and water as a cosolvent, a 43% increase in yield was obtained compared to SFE without the cosolvent, in addition to 220 mg GAE/g of phenolic compounds (extraction conditions 50 • C and 250 bar). Paviani et al. [53], when adding 15% ethanol as a cosolvent to obtain extracts of green propolis (extraction conditions 50 • C and 250 bar), obtained an increase of 43.7% compared to the same process without the use of a cosolvent. The influence of the addition and concentration of cosolvent on supercritical CO 2 extraction was also shown for other natural matrices [62], demonstrating the importance of these modifiers to improve the extraction capacity of polar compounds. Cruz et al. [63] showed that the presence of hydrated ethanol (1/1, v/v) significantly increased the extraction yield of yacon leaves, obtaining extracts with higher antioxidant activity compared to extraction without the use of cosolvent, leading to higher yields. In a recent study, Guedes et al. [60] showed that as the concentration of ethanol added to the process was increased (0.5/1, 1.1/1 and 1.5/1, w/w), the yield and efficiency of SFE extraction in samples of Synadenium grantii increased. The compound mainly found in the red propolis extract analyzed in this study was formononetin in the concentration range of 1 ± 0.2 mg/g (control 0%) to 7 ± 2 mg/g (extract with 4% of cosolvent), followed by kaempferol (from 1 ± 0.1 to 2 ± 0.3) (Figure 3). A 518% increase in the extraction capacity of formononetin was observed in the presence of 4% cosolvent in the system. For kaempferol an increase of 122% was observed. Previous studies have also reported higher concentrations of phenolic compounds, such as formononetin, in extracts of red propolis, which is identified as its major component [4,15,22]. López et al. [64], when evaluating samples of red propolis from different regions by mass spectrometry, identified the presence of formononetin (m/z 267.06, retention time, rt 4.5 min) in all analyzed samples. Hanski et al. [65] demonstrated the antimicrobial effect of formononetin on Chlamydia pneumoniae, while Li et al. [66] demonstrated the protective effect of formononetin in vitro, decreasing the levels of TNF-α and IL-6. Thus, the pharmacological activities of red propolis may be closely related to their phenolic compounds due to their antioxidant, anti-inflammatory and microorganism proliferation inhibition capacity. Batista et al. [67] also associated the antioxidant and anti-inflammatory effects of red propolis with the involvement of polyphenols in the photoprotective activity observed in their study. These studies show the importance of obtaining extracts rich in formononetin for a greater biological capacity of the obtained product. Despite the knowledge that the chemical composition of propolis depends on the biodiversity, type and geographical location of the beehives [22] and that the extraction method influences the extract composition [20,33], the identification of ideal conditions, especially regarding the use of clean technologies with the presence of adequate concentrations of system modifiers, given the polar nature of the compounds present in red propolis, is of great importance to obtain extracts with high antioxidant capacity, especially with high levels of formononetin. Formononetin has been associated with the antioxidant [68], antitumor [66], antimicrobial [22] and anti-inflammatory [8] effects of red propolis extracts. Lower contents of formononetin (6.15 and 6.54 mg/g) and kaempferol (0.43 and 0.65 mg/g) were identified by Reis et al. [23] in ethanolic extracts (ultrasound-assisted or not) of red propolis from the same geographical origin (Barra de Sto. Antonio, Porto Calvo, Alagoas, Brazil). Bueno-Silva et al. [69] evaluated the effect of season on chemical composition and found lower formononetin contents (78.76 to 112.78 µg/g) than those found in this study for ethanolic extract of red propolis of the same origin. This may demonstrate that SFE (CO 2 as the solvent and ethanol as the cosolvent) may be an important and promising technology for obtaining red propolis extracts with potential for nutraceutical and cosmetic applications [70]. In the present study, it was possible to demonstrate that ethanol (as a cosolvent) played a role by causing an increase in the polarity and eluting power of supercritical CO 2 , maintaining the same process parameters without significantly changing selectivity. Previous studies have also reported higher concentrations of phenolic compounds, such as formononetin, in extracts of red propolis, which is identified as its major component [4,15,22]. López et al. [64], when evaluating samples of red propolis from different regions by mass spectrometry, identified the presence of formononetin (m/z 267.06, retention time, rt 4.5 min) in all analyzed samples. Hanski et al. [65] demonstrated the antimicrobial effect of formononetin on Chlamydia pneumoniae, while Li et al. [66] demonstrated the protective effect of formononetin in vitro, decreasing the levels of TNF-α and IL-6. Thus, the pharmacological activities of red propolis may be closely related to their phenolic compounds due to their antioxidant, anti-inflammatory and microorganism proliferation inhibition capacity. Batista et al. [67] also associated the antioxidant and antiinflammatory effects of red propolis with the involvement of polyphenols in the photoprotective activity observed in their study. These studies show the importance of obtaining extracts rich in formononetin for a greater biological capacity of the obtained product. Despite the knowledge that the chemical composition of propolis depends on the biodiversity, type and geographical location of the beehives [22] and that the extraction method influences the extract composition [20,33], the identification of ideal conditions, especially regarding the use of clean technologies with the presence of adequate concentrations of system modifiers, given the polar nature of the compounds present in red propolis, is of great importance to obtain extracts with high antioxidant capacity, especially with high levels of formononetin. Formononetin has been associated with the antioxidant [68], antitumor [66], antimicrobial [22] and anti-inflammatory [8] effects of red propolis extracts. Lower contents of formononetin (6.15 and 6.54 mg/g) and kaempferol (0.43 and 0.65 mg/g) were identified by Reis et al. [23] in ethanolic extracts (ultrasound-assisted or not) of red propolis from the same geographical origin (Barra de Sto. Antonio, Porto Calvo, Alagoas, Brazil). Bueno-Silva et al. [69] evaluated the effect of season on chemical composition and found lower Figure 4 shows the global yield isotherms for total yield, total phenolic compound content, flavonoid content and antioxidant activity (IC 50 ) determined for the red propolis extracts obtained under the different temperature (31.7, 40 and 50 • C) and pressure (250, 350 and 450 bar) conditions used (S/F 131-step 1; 4% ethanol-step 2). Global Yield Isotherms (GYI) Regarding yield, a higher extraction percentage (63.46%) was obtained at a pressure of 450 bar at 40 • C, while the highest content of phenolic compounds (380 mgEAG/g) and flavonoids (10 mg EQ/g) and higher antioxidant capacity (110 µg/mL) were obtained under 450 bar and 50 • C. Figure 4a shows that at constant pressures of 350 and 450 bar the increase in temperature significantly favors the extraction capacity, and consequently, the global yield of the process. In general, the solubility of solutes in supercritical fluids increases with temperature at constant pressure [71]. When keeping the temperature constant at 31.7 and 40 • C, increasing the pressure from 350 to 450 bar also favors the process yield. The lowest yields at constant pressure were observed at the highest temperature (50 • C). It is known that the effect of temperature on SFE is complex due to the increase in the solute vapor pressure and reduction in the density of the supercritical solvent [72]. Increased temperature increases the vapor pressure of the solute, promoting an increase in its solubility in supercritical CO 2 ; however, the temperature increase also promotes a reduction in the density of supercritical CO 2 , thus reducing the solubility of the solute in the solvent [73]. In this case, the effect of reduced supercritical CO 2 density was favored in relation to the increased solvent density (Figure 4a). Figure 4 shows the global yield isotherms for total yield, total phenolic compound content, flavonoid content and antioxidant activity (IC50) determined for the red propolis extracts obtained under the different temperature (31.7, 40 and 50 °C) and pressure (250, 350 and 450 bar) conditions used (S/F 131-step 1; 4% ethanol-step 2). Regarding yield, a higher extraction percentage (63.46%) was obtained at a pressure of 450 bar at 40 °C, while the highest content of phenolic compounds (380 mgEAG/g) and flavonoids (10 mg EQ/g) and higher antioxidant capacity (110 μg/mL) were obtained under 450 bar and 50 °C. Figure 4a shows that at constant pressures of 350 and 450 bar the increase in temperature significantly favors the extraction capacity, and consequently, the global yield of the process. Global Yield Isotherms (GYI) In general, the solubility of solutes in supercritical fluids increases with temperature at constant pressure [71]. When keeping the temperature constant at 31.7 and 40 °C, increasing the pressure from 350 to 450 bar also favors the process yield. The lowest yields at constant pressure were observed at the highest temperature (50 °C). It is known that the effect of temperature on SFE is complex due to the increase in the solute vapor pressure and reduction in the density of the supercritical solvent [72]. Increased temperature increases the vapor pressure of the solute, promoting an increase in its solubility in supercritical CO2; however, the temperature increase also promotes a reduction in the density of supercritical CO2, thus reducing the solubility of the solute in the solvent [73]. In this case, the effect of reduced supercritical CO2 density was favored in relation to the increased solvent density (Figure 4a). In general, at a constant pressure of 250 bar, temperature had no effect on the extraction of phenolic compounds and total flavonoids or on the antioxidant activity (without significant differences). At the intermediate pressure studied (350 bar), variations in temperature promoted an increase in the extraction of these compounds in a sample of red propolis. For example, for phenols (40 and 50 • C), total flavonoids (50 • C) and antioxidant activity (50 • C), higher temperatures contributed to a higher extraction yield of these compounds. The positive effect of the temperature increase at a constant pressure of 450 bar (highest studied pressure) was clearly evident. Thus, the effect of temperature increase had a positive influence on the vapor pressure of the solutes [74] and, therefore, the temperature of 50 • C was the most efficient for obtaining extracts with high antioxidant capacity. It is noteworthy that at constant pressure, the influence of temperature cannot be treated in such a simple way [75]. When evaluating the behavior of pressure in the 31.7 and 40 • C isotherms, it was observed that the increase in pressure from 250 to 350 bar contributes significantly to the increase in the extraction of phenolic compounds, flavonoids and antioxidant activity. However, a reduction in the levels of phenolic compounds and flavonoids, and consequently antioxidant capacity, was observed when the pressure increased from 350 to 450 bar in the 31.7 and 40 • C isotherms. Thus, at these temperatures, intermediate pressures (350 bar) may be more efficient for the extraction of phenolic compounds from red propolis. The best phenolic and flavonoid yields, as well as antioxidant activity, were observed in the 50 • C isotherm at a pressure of 450 bar. In the 50 • C isotherm, the increase in pressure significantly accelerated the mass transfer in the supercritical extractor bed and contributed to increasing the extraction yield of phenolic compounds from red propolis. As demonstrated by Barroso et al. [76] and Fujii et al. [77], the density of supercritical CO 2 is dependent on the pressure used in the extraction process; therefore, at higher pressures, supercritical CO 2 will have a higher density, and consequently, its solvation power will be higher. It is important to note that the isotherms crossed between 400 and 450 bar for total phenols (Figure 4b) and between 350 and 400 bar for antioxidant activity (Figure 4d). Below this pressure level, known as the crossover pressure, the solubility of the antioxidant compounds of red propolis decreases with increasing temperature. A crossover is observed near the critical region [78,79]; in these regions, the effect of temperature on the increase in vapor pressure compensates for the effect of temperature on decreasing solvent density [27]. Due to the existence of this point, the lowest and highest concentrations of phenolic compounds occurred in the same isotherm (50 • C). Azevedo et al. [80] evaluated the extraction of caffeine using SFE and found that near the critical point, below the crossover pressure, small increases in temperature result in a drastic decrease in the density of the solvent and, consequently, in its extraction capacity. For the extraction of artemisinin from Artemisia annua L., Rodrigues et al. [81] found a crossover pressure of 200 bar, whereas Cadena-Carrera et al. [82] evaluated the bioactive properties of the extracts from guayusa leaves (Ilex guayusa Loes.) and found a crossover pressure close to 200 bar. Figure 5 shows the global yield isotherms for the three compounds identified and quantified by HPLC-DAD ((a) formononetin; (b) naringenin; and (d) kaempferol)) in the red propolis extracts under the different temperature (31.7, 40 and 50 • C) and pressure (250, 350 and 450 bar) conditions employed (S/F 131-step 1; 4% ethanol-step 2) in this study. Figure 6 shows the chemical structure of each phenolic compound analyzed in this study. Formononetin (Figure 5a) was obtained at higher concentrations (13 mg/g) at a pressure of 350 bar and a temperature of 40 • C (intermediate conditions). It is possible to observe that at constant pressures of 250 and 350 bar, there is an increase in the concentration of formononetin extracted when the temperature increases from 31.7 • C (2 mg/g and 6 mg/g) to 40 • C (5 mg/g and 10 mg/g), but there was a reduction at 50 • C (3 mg/g and 9 mg/g). In this case, the increase in temperature increased the vapor pressure of the solute, positively favoring the extraction process. However, at high temperatures, the effect of solvent density reduction may favor both the degradation of formononetin and the decrease in extraction efficiency for this compound [83,84]. In addition to solubility, the effect of temperature on the conversion or degradation of formononetin should also be taken into consideration. At a constant pressure of 450 bar, the highest concentration was observed at the highest and lowest temperatures employed, with concentrations of 9 mg/g and 7 mg/g, respectively. Thus, the effect of the supercritical CO 2 solubility at 450 bar should be more strongly considered than the effect of conversion or degradation of formononetin in terms of extraction yield. In addition, a crossover pressure close to 400 bar was also observed. When evaluating the pressure behavior at a constant temperature of 40 • C, an extraction profile similar to the extraction of total phenolic compounds, flavonoids and antioxidant activity was observed ( Figure 5): when increasing the pressure from 250 to 350 bar, there is an increase in extraction, but when increasing the pressure from 350 to 450, extraction is reduced. Theoretically, the higher the pressure, the greater the density and solubility of the supercritical fluid, increasing the extraction efficiency [85]. However, the concentration of some of the target compounds may be reduced because at high pressures, other compounds can also be extracted, and thus, there is a reduction in specificity [86]. In general, high pressures may not be efficient for the extraction of formononetin because the effect of decreased diffusivity overlaps the effect of increased density, thus decreasing the extraction yield of this compound at 450 bar. A similar effect was found by Saito et al. [61] when obtaining phenolic compounds from green and red propolis, with higher yields (14%) observed at 60 • C and 200 bar and the lower yields Regarding the compound naringenin (Figure 5b), when analyzing the behavior of the isotherms at a constant pressure of 250 bar, it was observed that the temperature did not influence the extraction process. However, when the pressure increased to 350 bar, the effect of temperature is evident because by raising the temperature from 31.7 to 40 • C, an increase of 114% in extraction is obtained, and when raising the temperature from 40 to 50 • C, a reduction of 27.3% is observed. For this compound, crossing of the temperature curves was also observed at a pressure close to 400 bar, and a higher concentration (2 mg/g) at 40 • C and 350 bar. This behavior is in agreement with that observed for total phenolic compounds, flavonoids and antioxidant activity ( Figure 5). When analyzing the behavior of pressure at constant temperature, it is noted that for the temperatures of 31.7 and 50 • C, the pressure has a positive effect on the extraction process. Under these conditions, the increase in pressure increases the density of the supercritical fluid and the solvation power of the solute, consequently increasing the extraction yield. At a temperature of 40 • C, extraction was only significant at a pressure of 350 bar. Majdoub et al. [87] found similar effects when raising the pressure from 100 to 300 bar at a constant temperature of 50 • C for obtaining extracts of Daucus carota. Similar to what occurred for the compound naringenin, the temperature variation also did not influence the kaempferol extraction process (Figure 5c) at a constant pressure of 250 bar, and this compound may have its extraction reduced or become nonextractable under these conditions. However, at the pressure of 350 bar, there is an increase in extraction when the temperature increases from 31.7 to 40 and 50 • C (1 mg/g and 1.078 mg/g, respectively), but with no significant differences between the concentrations obtained at the higher temperatures. The isotherms cross at approximately 400 bar, and the influence of the crossover pressure on the extraction profile was evident because up to 400 bar pressure, the extraction is more effective at the lowest and highest temperatures studied, with no significant difference, and below this value, the extraction is more effective at 40 • C. Higher concentrations were obtained at a pressure of 450 bar and 50 • C (1 mg/g). Zordi et al. [51], in a study on SFE to obtain propolis extracts, also observed that the chemical composition of an extract is significantly influenced by pressure and temperature in both a linear and quadratic way, obtaining optimal operating conditions of highest yield (14.3%) in the isotherms of 317 bar and 45 • C. Therefore, the present study shows that the conditions of global yield isotherms in SFE affect the composition of the extract obtained and should be analyzed and defined according to the extraction objective. In general, the best yields of bioactive compounds isolated from red propolis were observed at the intermediate isotherms of 40 • C and pressure of 350 bar. Study Sample The sample of red propolis used in this study was kindly donated by the company Bee Product Natural (Barra de Sto. Antonio, Porto Calvo, Alagoas, Brazil) with "Mangroves of Alagoas" denomination of origin (IG201101) [88]. Approximately 1 kg of red propolis was ground (Cadence-Brazil) and sieved (270-325 µm diameter) to allow the homogenization of the sample in the extraction bed. Samples of 200 g of red propolis were kept at −30 • C in vacuum-sealed packaging away from light. Process Parameters for Red Propolis Extraction by SFE The process parameters used to obtain the extracts of red propolis was performed as previously described by Machado et al. [33] with modifications and using a SFT-110 Supercritical Fluid Extractor (Supercritical Fluid Technologies, Inc., Newark, NJ, USA) under the different conditions used in the study. The CO 2 flow rate in the system was 6.0 g/min in all experiments (Figure 7). USA) and trans-ferulic acid (CAS number 537-98-4) was purchased from Fluka (St Louis, MO, USA). Study Sample The sample of red propolis used in this study was kindly donated by the company Bee Product Natural (Barra de Sto. Antonio, Porto Calvo, Alagoas, Brazil) with "Mangroves of Alagoas" denomination of origin (IG201101) [88]. Approximately 1 kg of red propolis was ground (Cadence-Brazil) and sieved (270-325 μm diameter) to allow the homogenization of the sample in the extraction bed. Samples of 200 g of red propolis were kept at −30 °C in vacuum-sealed packaging away from light. Process Parameters for Red Propolis Extraction by SFE The process parameters used to obtain the extracts of red propolis was performed as previously described by Machado et al. [33] with modifications and using a SFT-110 Supercritical Fluid Extractor (Supercritical Fluid Technologies, Inc., Newark, NJ, USA) under the different conditions used in the study. The CO2 flow rate in the system was 6.0 g/min in all experiments (Figure 7). The extraction bed was packed to avoid the formation of preferential paths by the solvent (CO 2 ), and for this purpose, glass wool and beads were used to fully fill the bed (Figure 8). In this work, it 7.5 g of red propolis was used, as reported in previous optimization studies [33,53], mainly to avoid very long extraction times. The best extraction operational condition was determined in three steps. Figure 8 presents an illustrative summary of the process used with all parameters studied at each step, as well as the number of experiments and analyses performed. The results for the parameters evaluated and determined in this study were expressed as the mean ± standard deviation (n = 3), and all analyses were performed in triplicate. First Step: Overall Extraction Curve and S/F (Mass of CO 2 /Mass of Sample) In the present study, to obtain the overall extraction curve, a temperature and pressure of 100 bar and 40 • C were used, respectively, with the objective of guaranteeing the worst extraction scenario (under mild extraction conditions), a sample of 7.5 g propolis and 6 g/min CO 2 flow rate [33,49]. The experiment was performed as follows: the extracts were collected at predetermined periods in vials of previously known weight. The S/F value was calculated according to Equation (1). where: MCO 2 = total mass (g) of CO 2 used in the system at each extraction point (considering the volume and density of CO 2 in the system) M sample = total mass (g) of propolis used to feed the system The overall yield (X 0 ) was calculated in accordance with Equation (2). where: M extract is the mass (g) of extract obtained in each extraction M sample is the mass (g) of propolis used to compose the extraction bed For the extracts obtained at the predetermined times (in each collection vial), the yield, total phenolic content and antioxidant activity were determined. A total of 13 experiments were obtained for each assay. Molecules 2020, 25, x FOR PEER REVIEW 13 of 22 The extraction bed was packed to avoid the formation of preferential paths by the solvent (CO2), and for this purpose, glass wool and beads were used to fully fill the bed (Figure 8). In this work, it 7.5 g of red propolis was used, as reported in previous optimization studies [33,53], mainly to avoid very long extraction times. The best extraction operational condition was determined in three steps. Figure 8 presents an illustrative summary of the process used with all parameters studied at each step, as well as the number of experiments and analyses performed. The results for the parameters evaluated and determined in this study were expressed as the mean ± standard deviation (n = 3), and all analyses were performed in triplicate. In the present study, to obtain the overall extraction curve, a temperature and pressure of 100 bar and 40 °C were used, respectively, with the objective of guaranteeing the worst extraction scenario (under mild extraction conditions), a sample of 7.5 g propolis and 6 g/min CO2 flow rate [33,49]. The experiment was performed as follows: the extracts were collected at predetermined periods in vials of previously known weight. The S/F value was calculated according to Equation (1). = (1) where: MCO2 = total mass (g) of CO2 used in the system at each extraction point (considering the volume and density of CO2 in the system) Msample = total mass (g) of propolis used to feed the system The overall yield (X0) was calculated in accordance with Equation (2). where: Mextract is the mass (g) of extract obtained in each extraction Msample is the mass (g) of propolis used to compose the extraction bed Second Step: Influence of Cosolvent Percentage In this step, 80% ethanol was used as a cosolvent to obtain the extracts in relation to the yield (in mass), content of total phenolic compounds, antioxidant activity and concentration of the compounds of interest (formononetin, naringenin and kaempferol). The extracts were obtained under the following conditions: S/F of 131 (calculated in the previous step), temperature of 50 • C and pressure of 250 bar (CO 2 flow rate of 6 g/min) [33]. The extractions were performed using 0, 1, 2 and 4% of the cosolvent, calculated in relation to the mass of CO 2 used (S/F), totaling 4 experiments for this step. Ethanol (80%) was diffused in the system using a cosolvent pump with a flow rate of 0.05 (1%), 0.1 (2%) and 0.2 (4%) mL/min for a total time of approximately 140 min of extraction (considering S/F = 131). Third Step: Global Yield Isotherms The yield isotherms were studied using three temperatures (31.7, 40 and 50 • C) and three pressures (250, 350 and 450 bar). The S/F value used was obtained in the first step (131), and the percentage of cosolvent (80% ethanol) was determined in the previous step (4% w/w) (CO 2 flow rate of 6 g/min). The extracts obtained under the different conditions were evaluated for yield (mass), content of total phenolics, flavonoids, antioxidant activity (EC 50 ) and concentration of the compounds of interest (formononetin, naringenin and kaempferol). For this step, a total of 9 experiments were obtained for each test performed. Total Phenolic Compounds, Flavonoids Content and Antioxidant Activity by DPPH• (2,2-Diphenyl-1-picrylhydrazyl) The content of total phenolic compounds of the red propolis extracts was determined from the reaction with the Folin-Ciocalteu method [89,90]. The reaction was prepared as previously described by Devequi-Nunes et al. [20]. The results are expressed as milligram of gallic acid equivalent (GAE) per gram of sample (mg GAE/g). For this, a calibration curve (y = 0.0104x + 0.0688, R 2 = 0.9976) was determined using standard solutions of the gallic acid (concentrations 0 and from 10 to 200 µg/mL). The content of flavonoids of the extracts was determined using the method proposed by Meda et al. [91] with adaptations, as previously described by Machado et al. [21]. The same procedure was performed using standard solutions of quercetin (0 and from 1 to 75 µg/mL) to obtain a standard curve (y = 0.0311x + 0.0259, R 2 = 0.9987). The content of total flavonoids was expressed as milligram of quercetin equivalent (QE) per gram of sample (mg QE/g). To evaluate the antioxidant activity, the 2,2-diphenyl-1-picrylryrazine-reactive method (DPPH•) was applied [92,93], as previous described by Reis et al. [23]. The extracts were diluted to five concentrations (50,100,150,200,250 and 300 µg/mL) in triplicates. The free radical sequestration capacity was expressed as the percentage of radical oxidation inhibition (Equation (3)) (extracts obtained in the first and second study steps). where: AA % = antioxidant activity in percentage Ab sample = absorbance of the extract sample Ab blank = absorbance of the blank (without sample) The IC 50 value (effective concentration of the extract to sequester 50% of the DPPH• radical) was calculated based on the linear equation obtained from the extract concentrations and respective DPPH• radical sequestration percentages. For the extracts obtained in the third study step, the results are expressed as IC 50 . High-Performance Liquid Chromatography (HPLC): Identification and Quantification of Phenolic Compounds The red propolis extracts obtained by extraction with supercritical CO 2 in the second (cosolvent influence) and third (yield isotherms) study steps were evaluated by high-performance liquid chromatography (HPLC-Shimadzu, LC-20AT, Japan equipped with an automatic injector and diode array detector-DAD, Shimadzu, SPD-M20, Kyoto, Japan) as previously described by Reis et al. [23], Salgueiro and Castro [94] and Cabral et al. [95]. For this, an analytical standard curve formed by 13 phenolic standards was obtained, and the presence of these compounds in the extracts was investigated. A NUCLEODUR 100-5 C18 ec column (150 × 4 mm internal diameter; 5 µm particle size) was used in conjunction with a ZORBAX Eclipse Plus C18 precolumn (4.6 × 12.5 mm) (Agilent, Folsom, CA, USA). The chromatographic analysis conditions were tested with an elution gradient with a mobile phase of 5% acetic acid (Phase A) and methanol (Phase B) at different proportions and with a total analysis time of 42 min (from 0 to 35 min [0-92% B]; 35 to 40 min [92-0% B]; 40 to 42 min [0% B]). The injection volume was 20 µL, and the flow rate was 1 mL.min −1 . The device was operated at a temperature of 40 ± 2 • C. The DAD reading was adjusted in the range of 190 to 800 nm, and chromatographic acquisition was defined between 280 and 370 nm [23]. The compounds were identified by comparing the retention time (RT) and the ultraviolet spectrum between samples and standards. The working range for all investigated compounds was 0.5 to 15 mg/g. The wave length (λ) ranged from 280 to 370. The RT ranged from 2.31 to 19.27 min, the detection limit (DL) ranged from 0.19 to 0.47 mg/g and the quantification limit (QL) ranged from 0.64 to 1.58 mg/g, and all these parameters were dependent on each compound. Figure 9 shows the chromatogram obtained for the construction of the analytical curve with the studied standards. with a mobile phase of 5% acetic acid (Phase A) and methanol (Phase B) at different proportions and with a total analysis time of 42 min (from 0 to 35 min [0-92% B]; 35 to 40 min [92-0% B]; 40 to 42 min [0% B]). The injection volume was 20 μL, and the flow rate was 1 mL.min −1 . The device was operated at a temperature of 40 ± 2 °C. The DAD reading was adjusted in the range of 190 to 800 nm, and chromatographic acquisition was defined between 280 and 370 nm [23]. Statistical Analysis The results were statistically analyzed using StatSoft 6.0 (StatSoft Inc., Tulsa, OK, USA). Analysis of variance (ANOVA) and Tukey's test at the 95% confidence level were performed to identify significant differences between the results obtained for each test (p < 0.05). Conclusions This novel study showed the feasibility of applying SFE to obtain red propolis extracts with high antioxidant potential and that it is important to consider aspects related to process parameters, such as the volume of CO 2 applied, addition of cosolvents and total yield isotherms. In the present study, the addition of ethanol as a cosolvent improved the extraction of bioactive compounds present in red propolis, including formononetin, the major compound of interest from this type of propolis. The best yield was obtained under the conditions of 40 • C and 450 bar using 4% ethanol as the cosolvent, a CO 2 flow rate of 6 g/min and S/F of 131. The best conditions to obtain extracts rich in phenolic compounds, flavonoids and antioxidant activity (represented by a low IC 50 ) were at a temperature of 50 • C and pressure of 450 bar. The intermediate conditions (40 • C and 350 bar) showed the greatest potential for obtaining high concentrations of formononetin and naringenin, as well as extracts with high antioxidant capacity. Formononetin is considered to be the compound with the greatest pharmacological interest in red propolis. In general, the presence of these compounds (formononetin, naringenin and kaempferol) at high concentrations in the extracts obtained in this study demonstrates, despite the low total yield, that SFE using CO 2 is a promising alternative to obtain red propolis extracts with high added value. However, to obtain the specific bioactive compounds investigated in the present study, it is necessary to evaluate the individual properties of each one.	v3-fos-license
2020-10-29T09:07:53.558Z	2021-06-10T00:00:00.000	228834377	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.19045/bspab.2021.100048", "pdf_hash": "1fec936551afc399429e9ed459e8bcb02ae5fa62", "pdf_src": "MergedPDFExtraction", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1575", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "d75f3b93d0b996eb3cb16733d5358c14ffa7a33a", "year": 2021 }	pes2o/s2orc	Experimental studies on re-hydration of vacuum freeze-dried Asparagus (Asparagus officinalis L.) with a hydrophilic substance Vacuum freeze-drying is one of the best water removal methods, with final products of the highest quality. The solid-state of water during freeze-drying protects the primary structure and the product's shape with minimal volume reduction. As the leading quality problem of dehydrated green Asparagus, this experiment was to study the technique of improving the rehydration of dehydrated green Asparagus by adding a hydrophilic substance (Maltodextrin, sucrose, salt) and controlling two ways in the process of vacuum freeze-drying. The mixed solution was soaked at the rate of three different concentration ratios, i.e., 1 (10%), 2 (15%), and 3 (20%) for maltodextrin, sucrose, and salts, respectively, using the L9 orthogonal and two-factor comparison experiment. It was concluded that increasing the mass of the Asparagus samples decreased the convective heat transfer coefficient. The evolution of drying months in the range of 1.78 4.74 W/m ̊C was recorded for the mass of Asparagus samples. The results noted that to dry the Asparagus by vacuum freeze-dryer from 09:00 to 18:00 hour decreases the product's drying rate up to 0.011g.(H2O).g (d. m).cm.hr and moisture level up to 8%. The study results noted that the pre-freezing condition was 23°C with the frozen time of 4 hours, which could remarkably improve the vacuum freeze-dried green asparagus rehydration. Finally, from the results, it was recommended that, from the actual production, to save energy, reduce costs; 23C was better for the precooling temperature with the pre-freezing time was 4 hours for drying green Asparagus. Introduction Green Asparagus is also an extremely perishable vegetable. Freshly harvested Asparagus deteriorates quickly, which results in a short shelf life under normal postharvest handling at room temperature. Drying is one of the most methods used for preservation. The drying of agricultural products has always been of great importance for preserving food by human beings. It is a primary preservation method and applies to a wide range of industrial and agricultural products. Adedeji et al. [1] reported that Asparagus (Asparagus officinalis L.) is commonly grown in temperate climates worldwide perennial plant with 100-150 cm tall, stout stems and soft vegetation. Asparagus's essential ingredients are energy, proteins, vitamins, fats, carbohydrates, etc. necessary in food with high nutritional value in the kitchen [2]. It is not only used to add food palatability, but it is also widely used in medicines, bakery products, wine and meat products, a soap product, etc. [3]. Asparagus is the most important cash crop globally, cultivated in China, Pakistan, Indian, Afghanistan, Uzbekistan, Japan, and Indonesia [4,5]. An et al. [6] stated that the People Republic of Chain producer 17 million tons a year of Asparagus and 45% of the total world's Asparagus contributing. Nearly half of the total production of Asparagus consumed as white and red Asparagus. In contrast, the remaining 30% converted into dry Asparagus for medicinal purposes, and 20% used as seed material [7]. Agricultural product drying has a vital role in preserving and shelf-life improvement after harvesting [8]. In developing countries, sundrying is a popular, effective, and economical method for drying food and herbal products. Sun-drying is a common food preservation technique used to control agricultural products' moisture content [9]. Traditionally, herbs, like Asparagus dried in the open sun, depend on sunshine availability and require ample drying space and long drying time [10]. Green Asparagus is very resistant to the storage; after harvesting of 1-2days, it will lose water, rot, and lose nutritional values [11]. The traditional processing method is to process green Asparagus into canned or frozen products. The research on the drying of green Asparagus is less; the main reason is that the drying of green Asparagus is reduced, which is also the key to dehydrated green quality asparagus [12]. The moisture content of the solar-dried unpeeled Asparagus found to be 7.0 %, unlike that of sun-drying, which could attain only 17.0 % moisture content [13]. Other researchers [14,15] have reported the drying behavior of Asparagus at four different drying air temperatures, i.e., 25 o C, 35 o C, and 45 o C, with the fixed air velocity of 1.3 m/s. The study results concluded that moisture content reduced from 87% to 6%, with a temperature of 45 o C on a wet basis. Blanching is the pre-treatment method used to arrest a few physiological processes. It helps in the inactivation of the enzymes, acceleration of drying rate, and reduced quality loss. It expels intercellular air from the tissues and softens it (16). Generally, the blanching of fruits and vegetables is done by heating in steam or hot water. Drying, a routine food preservation technique, is a crucial aspect of food processing [16,17]. The dried product's shelf life has been demonstrated to extend by reducing the water concentration at which microbiological and physicochemical deterioration is limited [18]. The drying method and processing conditions significantly affect the color, texture, density, porosity, and sorption characteristics of plant materials [19]. Therefore, the same plant raw material may yield a completely different product, depending on the type of drying and extraction methods employed [20]. In the past, it was mainly through controlling the soaking temperature, soaking time, and water consumption to improve the rehydration of dehydrated vegetables. In recent years, researchers have started to develop the ratio of rehydration in the view of pre-treatment. The pore of dehydrated vegetable infiltrated into maltodextrin, sucrose, and salt molecule, improving the rehydration ratio of dehydrated through the immersion of hydrophilic material green asparagus, which was much better than using the physical method alone. Material and Methods Description of the experimental procedure We took fresh green asparagus samples from the local market accessible in Nanjing, China, and washed them with distilled water for experimental work. The samples were cut cylindrical tube with a length of 3.6 mm and a 1.4 mm in diameter and placed on the weighing balance. Ullah et al. [21] reported that they are usually tiny and intense, growing 10-20 mm long and 3-7 mm in diameter. Asparagus samples were cut into cylindrical shapes with a length of 4mm, and a diameter of 6mm was also reported [22]. The data was recorded from 9:00 to 18:00 in June, July, and August 2016. The asparagus specimens were put in trays and placed on the digital electronic balance in each drying hour to determine water content discharge. After each hour of drying, the experimental observation data were recorded, as well as the evaporation was scrapped with the attained constant weight of the samples. The literature observed that the Asparagus dried from its average initial moisture content of 89% to the final moisture content of 8% [23]. The data obtained from the measurements of Asparagus weight used for drying kinetics and analysis of Asparagus in terms of moisture removal rate, and the drying was discontinued. The samples' constant weight was achieved. The difference in weight directly gave the quantity of water content evaporated during any time interval. Wet and dried Asparagus samples are shown in (Fig. 1). The moisture removal rate was expressed on a dry basis. Equation 1 was used for the determination of the moisture removal rate of the product. The moisture ratio of Asparagus during the drying can be obtained from equation 2. While the dry matter is the dry weight of the Asparagus can be calculated using equation 3, evaluated [24]. For determining the area of fruits, inch tape was used for recording the diameter before and after each hour of drying with the use of equation 4, reported [25]. Therefore drying rate is the evaporation of water content from the products in unit area unit time. It can be calculated from the dry matter of the product how much moisture was lost during the drying. Equation 5 was used to calculate the product's drying rate each hour studied by [26]. Similarly, the symbols used in the equations, "Dm" is Dry matter (g), "Wt" wet weight (g), "Minitial" is Initial moisture removing rate (%, dry basis), "Ww" is the weight of wet Asparagus (g), "Wd" represents the weight of dry Asparagus (g), "MR" is the moisture ration (%), "Mo" is the initial moisture content (%, dry basis), "Me" is the equilibrium moisture content (%, dry basis), "Ap" is the cross-sectional area of the product (cm 3 ), " " constant term 3.144, "r" radius of the product (cm), "Dr" is the drying rate of the product [(g (H2O).g -1 (d.m).cm -2 .h -1 )] and "Dt" denoted the drying time (hr). In (Table 1) it shows the moisture removal rate data, indoor and outdoor vacuum freezedryer temperature, product temperature, the surrounding temperature of the product, and ambient temperature during the experiment. Experimental methods The mixed solution was soaked at the rate of three different concentration ratios, i.e., 1 (10%), 2 (15%), and 3 (20%) for maltodextrin, sucrose, and salts, respectively, using the L9 orthogonal and two-factor comparison experiment. Determination of rehydration of green Asparagus with vacuum freeze-drying process, the fresh green Asparagus was selected for blanching treatment with maltodextrin's robust solution; sucrose, and salt concentrations (sodium Chloride, NaCl) (29) and soaked for 30min. In this experimental work, we used the treatment solution separately for determining the rehydration ratio. Kingsly et al., [29] studied the rapid HPLC method for the separation of isomaltulose (also known as Palatinose) from other common edible carbohydrates such as sucrose, glucose, and maltodextrins, commonly present in food and dietary supplements. After the treatment of blanching, we infiltrate the maltodextrin, sucrose, and salt molecules with the help of the osmotic process to improve the drying of green asparagus water are the best and suitable methods to increase the shelf life of products [30]. In this experiment, the concentration of maltodextrin, the sucrose level, and salt's strength was to select orthogonal analysis quality. Determination of water ratio in drying Asparagus repeated the quality times, using the SAS software reported by Vesali et al. [31] the variance of quality "L9" orthogonal experiment and "LSD," according to the results of multiple comparisons [32]. The experiment chooses the pre-freezing temperature and the precooling time as a factor. The drying ratio product to the measuring index influences its state of dehydrated products and then influences its rehydration factors. Lin & Brewer [33] According to the resistance method, the temperature of the eutectic point of green Asparagus was measured. The temperature of the 5-10 o C was lower than that of the eutectic point, so the highest temperature of green Asparagus was determined to be 23 o C. The pre-freezing time's relevant research data is less, and it needs to choose the wide horizontal range, and according to the result of the preliminary experiment. They used SAS software to analyze the variance of the experimental results and 1/2 multiple comparisons, a better pre-freezing process parameter chosen according to various comparisons. The rehydration capacity was used as a quality characteristic of the dried product [34] expressed in the rehydration rate -RR. Approximately 2g (± 0.01g) of the dried sample was placed in a 250ml laboratory glass (two analyses for each sample), 150ml distilled water was added, and the glass was covered and heated to boil within 3 minutes. The laboratory glass content was then gently boiled for ten (10) min more and then cooled. The cooled content was filtered for 5min under vacuum and weighed. The drying ratio was calculated from equation 6. At the same time, "Wr" is the drained weight (g) of the rehydrated sample, and "Wd" represents the weight of the dry sample used for rehydration T(o,v) is the temperature at vacuum freeze-dryer ( o C), Tc is the product temperature ( o C), Te is the product surrounding temperature ( o C), Mevp is the moisture evaporation (g), and M.removing rat is the moisture removing rat in the products with the unit of (%db). Results and Discussion Under natural convection mode, the handpeeled cylindrical shaped (diameter 1.4 mm, length 3.6 mm) mass of Asparagus samples is dry. Rect angular trays were used to conduct drying assessments of Asparagus sp ecimens. (Fig. 2) represents the comparison between the means of solar radiation (MSR) and the mass of the products of asparagus samples for the three months. Jamil et al. [36] studied bean moisture diffusivity and drying kinetics. They reported that the conditions of pre-freezing temperature and time would affect the size and quantity of products. Solar irradiation and product mass data collected for June, July, and August 2016 at a drying time of 60-minute duration under natural heat transfer solar energy drying, as shown in (Fig. 2). It has been observed that green asparagus re-hydration increases from morning to noon and decreases from noon to evening due to swelling and diminishing trend of solar irradiation in one day. The present results are in substantial agreement with the study's previous results reported by Deshmukh et al. [37]. They said that different products' rehydration process, i.e., apples, banana, green chili, red chili, green Asparagus, etc., started increasing from morning to noon. The study results agreed with Ismail [38]. They studied that the rehydration process increased with increasing solar irradiance. The data given in (Table 2) show the moisture removing rate, indoor and outdoor collector temperature, product and product surrounding temperature, and ambient temperature during the experiment. Table 2 shows the moisture removal rate is dependent on the total moisture present in the product mass. Hence, it has been observed that the moisture removal rate increases with an increase in green Asparagus samples mass and decreases significantly with the progression of drying days [39]. However, the moisture removal rate is also dependent on the ease of heat transfer [40]. (Fig. 3) shows the moisture lost and drying rate in Asparagus during dry. The products (Green Asparagus) were dried in the vacuum freezedryer with the process of rehydration, moisture loss, and drying rate was determined. Moisture lost in each hour of drying by a vacuum freeze-dryer is correlated with drying time. The drying rate is correlated with the change in percent moisture content to find the vacuum freeze dryer's promising performance as a drier for Asparagus's rehydration. The results show that moisture was lost with the higher temperature of the dryer. Before drying, 89% moisture was noted, and dried the product up to 8% moisture level with a dryer. Ullah and Kang [24] reported that the moisture content of the product was decreased with the highest temperature of the dryer. Similarly, the drying rate of the apples was starting to decrease from 0.032 g(H2O).g -1 (d.m).cm -2 .h -1 to 0.011 g(H2O).g -1 (d.m).cm -2 .h -1 with the increasing temperature of the dryer after 10hr of the drying process. Kohli et al., [15] stated that the drying rate of the product was reduced with the highest temperature of the dryer. The results are similar to [41], who found that the drying is directly related to the product's moisture content. Similarly, when the 15% (2) concentration of maltodextrin was applied, the water recovery was not significant. The second level of concentration of 15% (2) could be used as a balanced choice for other factors. The results agree with the findings [43]. They reported that decreasing the maltodextrin's concentration ratio in the products' rehydration process, the effect on the water recovery rate was not significant. The results showed that the difference between the first 10% (1) and the third 20% (3) levels of concentration is substantial, and the effect on the rehydration is not apparent. Table 2 shows the multiple comparisons of maltodextrin, sucrose, and salt concentration. When the sucrose concentration was at 20% (3), which improved the asparagus' rehydration, so optimum absorption of sucrose was 20%. The results are similar to the results [44]. The static results show in (Table 3) that when the sucrose concentration is the 10% (1) level, the difference between the water and the 10% (1) level is not significant, theoretically chosen as the object. Still, the influence of the other two factors should be considered. Serratosa et al., [45] conducted experimental results that are nearly matched with the present study results. At the 20% (3) level, the ratio of rehydration was significantly different, and the effect on the water recovery was not noticeable. The multiple salt concentration comparisons from ( Table 2) show that salt's frequency is 15% (2). The ratio of green Asparagus is higher than the average value, so, to improve the rehydration of dehydrated products, a better salt concentration should selected as 15%. These results are in agreement with the findings [46]. They reported that the concentration ratio of salt for the rehydration process of different products was 17%. Therefore, when the salt concentration was 10% (1) level, the proportion of water to the (2) level was not significant and could be used in balance with the other two factors. Singh et al., [47] reported that study results contradict the present study results. When the salt concentration was at the (3) level 20%, the rehydration effect was not significant, as shown in (Table 3). Determination of the concentration of hydrophilic substances found in preliminary experiments, when the frequency of maltodextrin was low, mostly under 10% concentration, there was little effect on the drying green Asparagus [48]. The main reason was that the low concentration maltodextrin's osmotic pressure was not enough to spread the maltodextrin to the inside of the material, only on the paste's surface. As a result, when the green Asparagus is dried and reused, the dextrin's surface first absorbs water to expand, instead of preventing it from spreading to the inside [49]. When the concentration of maltodextrin is more significant, especially when it is greater than 45%, the material is easily absorbed and melted during the dry process, resulting in shrinkage and deformation. Also, the dry products in the maltodextrin are very easy to moisture absorption, so that the storage becomes difficult, and there is too sticky feeling [50]. So in the appropriate concentration range, you can choose a higher concentration of maltodextrin, according to the results of multiple comparisons, the final determination of maltodextrin concentration of 20% [51]. Sucrose and salt are added to the maltodextrin to increase the solution's osmotic pressure [17]. However, the addition of sucrose and salt will affect the taste of dehydrated green Asparagus, after the experiment found that the sweet and salty ratio of 4:3 is more appropriate, so sucrose concentration of 20%, the salt concentration of 15% [52]. It is more suitable to be considered the final process parameter, whether from improving the rehydration or from a sweet-salty angle. Determination of pre-freezing process conditions The independence of the Eutectic Point of the pre-freezing cooling curve of green Asparagus is shown in (Fig. 4). As the temperature drops, the figure shows that the resistance changes very little; when the temperature drops to -11 o C to -13 o C, the resistance value suddenly increased. Due to the early freeze, green Asparagus inside there is a lot of water present, more charged ions can be moved freely, and with the temperature [53]. Most of the green asparagus water is converted to ice crystals, and when the temperature drops to -11 o C to -13 o C, the green Asparagus is frozen, and the resistance value suddenly increases [54]. Therefore, from the experimental result, the eutectic point temperature range for the green Asparagus is noted up to -11 o C to -13 o C. The results and analysis of the two factors of the water recovery ratio shown in (Table 4). They are using SAS software to analyze the variance of water recovery effects, shown in (Table 4). From the analysis of variance results in (Table 4), it can be seen that the Pvalue of the precooling temperature is less than 0.05, the P-value of the pre-freezing time is less than 0.01, and these two factors have a significant or significant effect on the rehydration ratio of the dehydrated green Asparagus [23]. Among them, pre-time is an essential factor, and the precooling temperature is the secondary factor. They are using the method of LSD to compare the prefreezing temperature and the pre-freezing time, taking the rehydration ratio of the dehydrated green Asparagus as the object of study, the proper conditions of precooling determined according to the results [14]. The multiple comparisons of the pre-freezing temperature ( Table 5) show that when the pre-freezing temperature -20 ( o C) and -30 ( o C), the green asparagus ratio is higher than the average is no significant difference between the two. So the precooling temperature is 23 o C and -30 o C, which is beneficial to improving the drying ratio of the green Asparagus [44]. When the pre-freezing temperature is the second level, the water recovery ratio's mean value is lower. There is a significant difference with the other two levels, which has no significant effect on improving the rehydration of the dehydrated green Asparagus. The multiple comparisons of pre-freezing time are shown in (Table 5). When the prefreezing time is 6 hours and 4 hours, the green asparagus ratio is higher, and the difference between them is not significant. . 6 hour is the first choice of parameters, the remaining three levels and above two levels of water ratio difference is significant, to improve the drying of green Asparagus. For analyzing the above factors, 6 hours and 4 hours is the pre-freeze time [20]. Conclusion The research reported in this paper includes the evaluation of re-hydration of vacuum freeze-dried Asparagus (Asparagus officinalis l) with hydrophilic substance (Maltodextrin, sucrose, salt) and moisture removing rate for the mass of asparagus samples. The experiment finally determined that, in the pre-treatment process, the green Asparagus soaked with 20% maltodextrin, 20% sucrose, and 15% salts in a mixed solution with the Pre-freezing temperature of 23 o C and 4 hours was the pre-freezing time can improve the absorption of dehydrated green Asparagus. The following observations and conclusions noted form the results: From the experiment results, the parameters of the pre-freezing process improving the ratio of water recovery. The experimental results noted that the convective heat transfer coefficient varies from 1.78 to 4.74 W/m 2˚C for green asparagus samples. The precondition of selecting the prefreezing process of precooling temperature was noted 23 o C and -30 o C with the prefreezing time 6 hours and 4 hours. The experiment results recommended that, from the actual production, to save energy, reduce costs; 23 o C was better for the precooling temperature. The pre-freezing time was 4 hours for the drying of green Asparagus. Authors' contributions Conceived and designed the experiments: F Ullah, Performed the experiments: F Ullah, Analyzed the data: F Ullah, Contributed materials/ analysis/ tools: F Ullah, Wrote the paper: F Ullah.	v3-fos-license
2020-03-19T10:39:10.066Z	2020-03-13T00:00:00.000	214702650	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2073-4344/10/3/324/pdf", "pdf_hash": "dcebcaf2a4363579989c873af0934f874311d56b", "pdf_src": "MergedPDFExtraction", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1658", "s2fieldsofstudy": [ "Environmental Science", "Chemistry", "Materials Science" ], "sha1": "a37014732279584bb2f2754c1c41ba51e0913dcc", "year": 2020 }	pes2o/s2orc	Flavin-Conjugated Iron Oxide Nanoparticles as Enzyme-Inspired Photocatalysts for Azo Dye Degradation In this work, a new photocatalytic system consisting of iron oxide nanoparticles (IONPs), coated with a catechol-flavin conjugate (DAFL), is synthesized and explored for use in water remediation. In order to test the efficiency of the catalyst, the photodegradation of amaranth (AMT), an azo dye water pollutant, was performed under aerobic and anaerobic conditions, using either ethylenediaminetetraacetic acid (EDTA) or 2-(N-morpholino)ethanesulfonic acid (MES) as electron donors. Depending on the conditions, either dye photoreduction or photooxidation were observed, indicating that flavin-coated iron-oxide nanoparticles can be used as a versatile enzyme-inspired photocatalysts. Introduction The treatment of industrial wastewater contaminated with synthetic organic compounds from the textile, cosmetic, pharmaceutical and other industries, is one of the most important tools in our fight against water pollution [1][2][3][4]. The textile industry specifically is one of the largest contributors to aquatic pollution. In fact, 17%-20% of the industrial contamination of water can be accredited to dyes being used in the textile industry [5]. Moreover, 10%-15% of these dyes are released into the environment without any pre-treatment [6] and many of them have been shown to be toxic to aquatic life [5]. The majority of the synthetic aromatic dyes used in the textile industry are azo dyes. They make up around two-thirds of all synthetic dyes used [7], and the demand for azo dyes such as amaranth (AMT) continues to increase [8]. Azo dyes are complex aromatic molecules characterized by one or multiple azo bonds (-N=N-), which are the main contributors to the toxicity of these dyes, in particular their cancerogenic properties [9]. For instance, the azo dye 4-aminoazobenzene (aniline yellow) was shown to display liver toxicity in male mice, in doses as small as 0.027-0.15 µmol/g body weight [10]. This dye has also been shown to induce tumor formation in rats when given orally or applied onto the skin [7]. Furthermore, the complexity of many azo dyes' structure results in nonbiodegradability and non-biocompatibility. Due to their demonstrated long term toxicity, many strategies have been developed and explored to degrade azo dye pollutants into less toxic products, among them some conventional chemical and physical techniques, such as coagulation, filtration, flocculation, adsorption and ozonation [11,12]. However, these strategies, besides being expensive and slow, often do not lead to the complete removal or degradation of the dyes. Although enzymes are specific and environmentally friendly catalysts, their application in water remediation is hindered due to their sensitivity to temperature and instability in alkaline conditions [5]. One strategy to overcome these limitations is to use a catalytic component, an organic cofactor instead of the whole enzyme, and repurpose it to afford the dye degradation, but removing the need for tightly controlled conditions and the use of additional cofactors such as nicotinamide dinucleotide (NADH). We initially explored such an approach by taking an advantage of the photocatalytic activity of flavin [19], and combining it with a polydopamine carrier, which acts as an active cofactor support. Such stabilized flavin was successfully used to mimic enzyme-like reactions using light instead of NADH [20,21]. Herewith, we report on a design of a catalyst that combines flavin and semiconducting, magnetic nanoparticles to afford a multifunctional system capable of effective dye degradation, while being easy to prepare and separate from the reaction mixture. Semiconductor-based photocatalysts, such as titanium dioxide (TiO2), zinc oxide (ZnO) [22,23], tungstate (WO3) [24], vandate (VO4) [25], and others [26,27] have already been successfully used for the removal of organic pollutants from water. Although they have high efficiency, as well as photostability, and some, such as TiO2 nanoparticles, are cheap to produce [8,12,28], they are difficult to separate and reuse. For example, wastewater remediation in bulk, using photocatalytically-active TiO2 nanoparticles, still requires the filtration of the catalyst after the photodegradation is complete. One way around this problem is immobilizing the catalyst onto a solid support that can be removed from the solution [12]. However, this can also be expensive and inconvenient on large scales. A viable alternative would be using magnetic nanoparticles, such as iron oxides (IONPs) that can be retrieved from solution with the help of magnets. Besides being magnetic, IONPs have some other advantages over TiO2 and other conventionally used photocatalytic metal-oxide nanoparticles. For example, IONPs have a band gap of 2.2-2.3 eV, which is lower than most commonly used semiconducting particles [29]. As a result, the particles can absorb light from the visible spectrum range, and do not have to rely on higher energy UV light for photocatalysis. Sundaramurthy et al. has shown that IONPs can photocatalytically degrade Congo red (CR) dye through formation of reactive oxygen species (ROS), with the help of photoexcited electrons [30]. In addition, they are easy to prepare on a larger scale, and are shown to be biocompatible, and have extensively been used in medicine [31]. Although enzymes are specific and environmentally friendly catalysts, their application in water remediation is hindered due to their sensitivity to temperature and instability in alkaline conditions [5]. One strategy to overcome these limitations is to use a catalytic component, an organic cofactor instead of the whole enzyme, and repurpose it to afford the dye degradation, but removing the need for tightly controlled conditions and the use of additional cofactors such as nicotinamide dinucleotide (NADH). We initially explored such an approach by taking an advantage of the photocatalytic activity of flavin [19], and combining it with a polydopamine carrier, which acts as an active cofactor support. Such stabilized flavin was successfully used to mimic enzyme-like reactions using light instead of NADH [20,21]. Herewith, we report on a design of a catalyst that combines flavin and semiconducting, magnetic nanoparticles to afford a multifunctional system capable of effective dye degradation, while being easy to prepare and separate from the reaction mixture. Semiconductor-based photocatalysts, such as titanium dioxide (TiO 2 ), zinc oxide (ZnO) [22,23], tungstate (WO 3 ) [24], vandate (VO 4 ) [25], and others [26,27] have already been successfully used for the removal of organic pollutants from water. Although they have high efficiency, as well as photostability, and some, such as TiO 2 nanoparticles, are cheap to produce [8,12,28], they are difficult to separate and reuse. For example, wastewater remediation in bulk, using photocatalytically-active TiO 2 nanoparticles, still requires the filtration of the catalyst after the photodegradation is complete. One way around this problem is immobilizing the catalyst onto a solid support that can be removed from the solution [12]. However, this can also be expensive and inconvenient on large scales. A viable alternative would be using magnetic nanoparticles, such as iron oxides (IONPs) that can be retrieved from solution with the help of magnets. Besides being magnetic, IONPs have some other advantages over TiO 2 and other conventionally used photocatalytic metal-oxide nanoparticles. For example, IONPs have a band gap of 2.2-2.3 eV, which is lower than most commonly used semiconducting particles [29]. As a result, the particles can absorb light from the visible spectrum range, and do not have to rely on higher energy UV light for photocatalysis. Sundaramurthy et al. has shown that IONPs can photocatalytically degrade Congo red (CR) dye through formation of reactive oxygen species (ROS), with the help of photoexcited electrons [30]. In addition, they are easy to prepare on a larger scale, and are shown to be biocompatible, and have extensively been used in medicine [31]. However, the main disadvantage of IONPs photocatalysts is their high degree of photoexcited electron-hole recombination [32], but this could be overcome by anchoring an appropriate photosensitizer to the particle surface. Herein, we present a novel heterogeneous photocatalytic system comprised of γ-Fe 2 O 3 IONPs functionalized with a novel catechol-flavin compound (DAFL). Such a IONP-DAFL hybrid system can afford efficient azo bond degradation in both aerobic and anaerobic conditions (Scheme 2), but this is a feature not widely exemplified in the literature for other photocatalytic systems. The catechol moiety coupled onto flavin species enables both an efficient flavin anchoring to the IONP surface and improved electron transfer between the flavin and γ-Fe 2 O 3 . The hybrid catalytic system is efficient, easy to prepare, and separate from the solution, and can be successfully used in the removal of water-polluting dyes. Catalysts 2020, 10, x FOR PEER REVIEW 3 of 17 However, the main disadvantage of IONPs photocatalysts is their high degree of photoexcited electron-hole recombination [32], but this could be overcome by anchoring an appropriate photosensitizer to the particle surface. Herein, we present a novel heterogeneous photocatalytic system comprised of γ-Fe2O3 IONPs functionalized with a novel catechol-flavin compound (DAFL). Such a IONP-DAFL hybrid system can afford efficient azo bond degradation in both aerobic and anaerobic conditions (Scheme 2), but this is a feature not widely exemplified in the literature for other photocatalytic systems. The catechol moiety coupled onto flavin species enables both an efficient flavin anchoring to the IONP surface and improved electron transfer between the flavin and γ-Fe2O3. The hybrid catalytic system is efficient, easy to prepare, and separate from the solution, and can be successfully used in the removal of water-polluting dyes. Some examples of the flavin-conjugated metal-oxide surface such as TiO2 and BiOCl have been reported, but not applied to the degradation of complex molecules, such as AMT, and to the best of our knowledge, none demonstrated both the reduction and the oxidation of the substrates as presented within this paper [33][34][35]. Synthesis of γ-Fe2O3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe2O3 IONPs were prepared using an oil in water (o/w) reverse micro-emulsion protocol, as reported by Benyettou et al. [36] using ferrous dodecyl sulfate (Fe(DS)2). Prepared IONPs were first characterized using Attenuated total reflection Fourier transform IR (ATR-FTIR ) and TEM ( Figure 1). Figure 1A shows bands at 962 cm −1 and 1619 cm −1 in the FTIR fingerprint region of the Fe(DS)2 spectrum that correspond to the S=O stretching vibrations of the sulfonic acid [37]. The sharp peaks at ~2919 cm −1 relate to the C-H stretching mode. The band at 3415 is a characteristic of the O-H bond vibration from the residual water that remained in the dried sample. The FTIR spectrum of the IONP shows characteristic bands at 556 cm −1 and 3270 cm −1 that, respectively, correspond to the Fe-O stretching vibration and the O-H stretching bond vibrations from the residual water molecules coordinated to the particle surfaces [38]. The absence of the strong, sharp peaks in the fingerprint region of the IONP spectrum indicates that only trace amounts of surfactant molecules are present [39]. Some examples of the flavin-conjugated metal-oxide surface such as TiO 2 and BiOCl have been reported, but not applied to the degradation of complex molecules, such as AMT, and to the best of our knowledge, none demonstrated both the reduction and the oxidation of the substrates as presented within this paper [33][34][35]. Synthesis of γ-Fe 2 O 3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe 2 O 3 IONPs were prepared using an oil in water (o/w) reverse micro-emulsion protocol, as reported by Benyettou et al. [36] using ferrous dodecyl sulfate (Fe(DS) 2 ). Prepared IONPs were first characterized using Attenuated total reflection Fourier transform IR (ATR-FTIR ) and TEM ( Figure 1). Figure 1A shows bands at 962 cm −1 and 1619 cm −1 in the FTIR fingerprint region of the Fe(DS) 2 spectrum that correspond to the S=O stretching vibrations of the sulfonic acid [37]. The sharp peaks at~2919 cm −1 relate to the C-H stretching mode. The band at 3415 is a characteristic of the O-H bond vibration from the residual water that remained in the dried sample. The FTIR spectrum of the IONP shows characteristic bands at 556 cm −1 and 3270 cm −1 that, respectively, correspond to the Fe-O stretching vibration and the O-H stretching bond vibrations from the residual water molecules coordinated to the particle surfaces [38]. The absence of the strong, sharp peaks in the fingerprint region of the IONP spectrum indicates that only trace amounts of surfactant molecules are present [39]. The zeta potential and hydrodynamic size of the nanoparticles were investigated using a Zetasizer Nano Range instrument (see Table S1). At a pH ~7, the IONPs have a surface charge of −19.25 ± 4.51 mv. The negative charges, due to hydroxyl groups on the nanoparticle surfaces, stabilize the suspension and stop the particles from aggregating. The IONPs displayed an average hydrodynamic size of 152.3 ± 2.45 nm, with a polydispersity index (PDI) of 0.211 ± 0.014 from dynamic light scattering (DLS) measurements. Transmission electron microscopy (TEM) was also used to study the IONPs size, distribution and morphology ( Figure 1B,C). The data shows rough spherical particles with an average diameter of 12.38 ± 1.12 nm. Particle diameters range from 10 to 15 nm with 12 nm, particles being the most frequently occurring. This disparity in particle size from DLS and TEM techniques is likely due to hydrate layers present on the IONPs in an aqueous medium [40] and the nanoparticle agglomeration in water due to magnetic attraction [41]. Surface Modification of γ-Fe2O3 Nanoparticles Although the functionalization of IONPs is well documented in the literature [44,45], the use of a catechol linker such as dopamine for functionalization, while avoiding the generation of the polydopamine coating, which could interfere with the electron transfer, has been reported less frequently. Herewith, a modified protocol described by Geiseler et al. [46] was used for the functionalization of the IONP surfaces with dopamine (DA) or dopamine-flavin (DAFL, see electronic supplementary information (ESI) for details on the synthesis). The dopamine-based linker was added to an IONP suspension at 23 °C (0.1 mg of DAFL for every 1 mg of IONP). The zeta potential and hydrodynamic size of the nanoparticles were investigated using a Zetasizer Nano Range instrument (see Table S1). At a pH~7, the IONPs have a surface charge of −19.25 ± 4.51 mv. The negative charges, due to hydroxyl groups on the nanoparticle surfaces, stabilize the suspension and stop the particles from aggregating. The IONPs displayed an average hydrodynamic size of 152.3 ± 2.45 nm, with a polydispersity index (PDI) of 0.211 ± 0.014 from dynamic light scattering (DLS) measurements. Transmission electron microscopy (TEM) was also used to study the IONPs size, distribution and morphology ( Figure 1B,C). The data shows rough spherical particles with an average diameter of 12.38 ± 1.12 nm. Particle diameters range from 10 to 15 nm with 12 nm, particles being the most frequently occurring. This disparity in particle size from DLS and TEM techniques is likely due to hydrate layers present on the IONPs in an aqueous medium [40] and the nanoparticle agglomeration in water due to magnetic attraction [41]. Surface Modification of γ-Fe 2 O 3 Nanoparticles Although the functionalization of IONPs is well documented in the literature [44,45], the use of a catechol linker such as dopamine for functionalization, while avoiding the generation of the polydopamine coating, which could interfere with the electron transfer, has been reported less frequently. Herewith, a modified protocol described by Geiseler et al. [46] was used for the functionalization of the IONP surfaces with dopamine (DA) or dopamine-flavin (DAFL, see electronic supplementary information (ESI) for details on the synthesis). The dopamine-based linker was added to an IONP suspension at 23 • C (0.1 mg of DAFL for every 1 mg of IONP). Since DA polymerizes into polydopamine (PDA) in the presence of O 2 , light and at basic pH [47], the linker coordination process was conducted in an Ar atmosphere, in the dark, and at a pH of~7. The coated IONPs were kept in the dark at room temperature in air. ATR-FTIR and TEM were used to characterize IONP-DAFL ( Figure 2) and the IONP-DA control ( Figures S2 and S3). The ATR-FTIR spectrum of DAFL ( Figure 2A) shows a sharp peak at 1535 cm −1 that is characteristic of flavin C=N modes of the isoalloxazine ring [48]. Moreover, combined bond vibrations of C=N and C=C are present at 1258 cm −1 [20]. These bond vibrations are also present in the IONP-DAFL FTIR spectrum, indicating a successful coordination of the linker on the particle surfaces. Furthermore, the band at 1388 cm −1 corresponds to the stretching vibration of the C-N bond, connecting the catechol to the flavin moiety. The vibrations at 647 cm −1 and 928 cm −1 correspond to the aromatic C-H bond vibrations [49]. Lastly, the peaks at 3241 cm −1 and 559 cm −1 in the IONP-DAFL spectrum correspond to the O-H and Fe-O bond vibrations, respectively. Catalysts 2020, 10, x FOR PEER REVIEW 5 of 17 Since DA polymerizes into polydopamine (PDA) in the presence of O2, light and at basic pH [47], the linker coordination process was conducted in an Ar atmosphere, in the dark, and at a pH of ~7. The coated IONPs were kept in the dark at room temperature in air. ATR-FTIR and TEM were used to characterize IONP-DAFL ( Figure 2) and the IONP-DA control ( Figures S2 and S3). The ATR-FTIR spectrum of DAFL ( Figure 2A) shows a sharp peak at 1535 cm −1 that is characteristic of flavin C=N modes of the isoalloxazine ring [48]. Moreover, combined bond vibrations of C=N and C=C are present at 1258 cm −1 [20]. These bond vibrations are also present in the IONP-DAFL FTIR spectrum, indicating a successful coordination of the linker on the particle surfaces. Furthermore, the band at 1388 cm −1 corresponds to the stretching vibration of the C-N bond, connecting the catechol to the flavin moiety. The vibrations at 647 cm −1 and 928 cm −1 correspond to the aromatic C-H bond vibrations [49]. Lastly, the peaks at 3241 cm −1 and 559 cm −1 in the IONP-DAFL spectrum correspond to the O-H and Fe-O bond vibrations, respectively. After functionalization with DA and DAFL, the zeta potential of the IONPs increases from −19.25 ± 4.51 mv to 27.33 ± 0.22 mV and 17.93 ± 1.04 Mv, respectively (Table S1). The shift from a negative to positive charge for both modified IONPs indicates successful surface functionalization, due to the introduction of amino moieties within the linker molecules. The size of the IONPs also increases from 152.3 ± 2.5 nm (PDI = 0.211) to 196.4 ± 4.7 nm (PDI = 0.320) for IONP-DA and 209.5 ± 1.4 nm (PDI = 0.320) for IONP-DAFL. This increase of hydrodynamic radius provides further evidence of successful catechol coating. Sizes obtained from DLS are compared to TEM images of the IONP-DAFL ( Figure 2B) and IONP-DA ( Figure S3). IONP-DAFL particles are spherical in shape, and have an average diameter of 16.17 ± 0.86 nm. They range in size from 14 to 19 nm, with 16 nm particles being the most frequently occurring. IONP-DA nanoparticles, on the other hand, have an average size of 17.69 ± 0.94 nm, with particles ranging from 15 to 20 nm in diameter. Thus, the size of both surface-modified IONPs is roughly the same. In order to confirm the presence and determine the loading percentage of DAFL on the IONP surface, XPS analysis was utilized ( Figure S4). The XPS survey spectrum ( Figure S4A) shows the presence of Fe, O, N and C, as expected. The atomic % of N was used to calculate the % loading of DAFL, as there are 5 N atoms per DAFL molecule, we can therefore assume the loading to be 1%. The high-resolution Fe 2p spectrum ( Figure S4B) shows two distinct peaks with binding energies of After functionalization with DA and DAFL, the zeta potential of the IONPs increases from −19.25 ± 4.51 mv to 27.33 ± 0.22 mV and 17.93 ± 1.04 Mv, respectively (Table S1). The shift from a negative to positive charge for both modified IONPs indicates successful surface functionalization, due to the introduction of amino moieties within the linker molecules. The size of the IONPs also increases from 152.3 ± 2.5 nm (PDI = 0.211) to 196.4 ± 4.7 nm (PDI = 0.320) for IONP-DA and 209.5 ± 1.4 nm (PDI = 0.320) for IONP-DAFL. This increase of hydrodynamic radius provides further evidence of successful catechol coating. Sizes obtained from DLS are compared to TEM images of the IONP-DAFL ( Figure 2B) and IONP-DA ( Figure S3). IONP-DAFL particles are spherical in shape, and have an average diameter of 16.17 ± 0.86 nm. They range in size from 14 to 19 nm, with 16 nm particles being the most frequently occurring. IONP-DA nanoparticles, on the other hand, have an average size of 17.69 ± 0.94 nm, with particles ranging from 15 to 20 nm in diameter. Thus, the size of both surface-modified IONPs is roughly the same. In order to confirm the presence and determine the loading percentage of DAFL on the IONP surface, XPS analysis was utilized ( Figure S4). The XPS survey spectrum ( Figure S4A) shows the presence of Fe, O, N and C, as expected. The atomic % of N was used to calculate the % loading of DAFL, as there are 5 N atoms per DAFL molecule, we can therefore assume the loading to be 1%. The high-resolution Fe 2p spectrum ( Figure S4B) shows two distinct peaks with binding energies of 710.9 eV for Fe 2p 3/2 and 724.5 eV for Fe 2p 1/2 [50]. The satellite peaks present at 718.8 eV and 732.7 eV are characteristic for Fe 3+ ions in Fe 2 O 3 [51]. The fitting also gives more detail as to the IONP composition, which shows both Fe 3+ and Fe 2+ ions present with the larger amount of Fe 3+ . This has been observed for other Fe 2 O 3 nanostructures containing both αand γ-phase Fe 2 O 3 [52]. The O 1s spectrum clearly shows the characteristic signals one would expect for the IONP-DAFL hybrid, including lattice Fe 2 O 3 at 530.1 eV, carbonyl C=O at 531.1 eV, a lattice hydroxyl signal at 532.7 eV that is commonly observed in Fe 2 O 3 , and another peak at around 536.6 eV, which could be ascribed to the catechol Fe-O bond [53]. The nitrogen 1s spectrum clearly shows a main C-N/C=N bond signal at 400.0 eV. Finally, the C 1s spectrum displays all characteristic signals associated that correspond to DAFL, including C-C at 285.0 eV, C-O bonding at 286.3 eV, carbonyl C=O at 288.3 eV, and the π-π* satellite can be observed at 290.4 eV. Amaranth Photooxidation (Aerobic Activity) The photocatalytic activity of IONP-DAFL towards AMT degradation was first carried out in an aerobic environment. In this condition, dye degradation is dependent upon the formation of reactive oxygen species (ROS) during the irradiation process [8]. These ROS are produced by semiconductors, such as IONP, through electron-hole (e-h) pair generation upon irradiation with photons with energy equal to or greater than the band gap energy of the material [54]. If the charge separation is maintained and the electron and holes do not recombine, electrons migrate to the conduction band, leaving a vacancy (a hole) in the valence band. The electron and hole can then recombine with O 2 and H 2 O, resulting in the production of the superoxide and hydroxyl radicals, as shown in Scheme 3. This process is limited by the rate of electron-hole recombination, which has been shown to be high for IONPs [29], so that a suitable sacrificial electron donor (ED) needs to be employed to quench generated holes, and an additional photoactive 'sensitizer' is required to improve activity. We envisioned that the addition of flavin-catechol, DAFL, would act as an IONP stabilizer and improve the visible-light IONPs catalysis. This is due to the direct charge transfer of electrons from flavin to the particle, as well as the enhanced ROS production by intermolecular charge and energy transfer to molecular oxygen, which results in the production of superoxide and singlet oxygen (Scheme 3). The direct charge transfer from flavin to NP through the catechol anchoring group could be expected as we observed a large degree of fluorescence quenching in dopamine-bound flavin (DAFL) in comparison to the NBoc-protected flavin, 3 ( Figure S6). The same phenomena was observed for other flavin-catechol conjugates that we have prepared [21]. In order to explore the efficiency of our system we chose two different classes of sacrificial electron donors: 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6, a zwitterionic buffer recently shown to have favorable electron donor properties with flavins [55], and ethylenediaminetetraacetic acid (EDTA) at pH 6, which is one of the most widely used electron donors for flavin photoreduction [56]. The electron donor was used in excess (0.1 M) to AMT and IONP-DAFL, since the rate of the reaction has been shown to be proportional to the concentration of the donor present [57]. Scheme 3. Possible oxidation pathways of AMT degradation. Flavin photoexcitation results in charge transfer (CT) to the conduction band (CB) of the IONPs in the presence of an electron donor (ED), as well as energy transfer (ET) and charge transfer processes with molecular oxygen to produce reactive oxygen species (ROS), singlet oxygen and superoxide, respectively, which then go on to degrade AMT. Photoexcitation of IONP electron-hole (e-h) pairs can interact with O2 to produce further superoxide radicals. AMT can also undergo a redox processes with these electron hole pairs initiating degradation through interactions with ROS. As a control, dopamine (DA) was attached to the surface of IONP in order to observe any catechol-mediated activity [58]. After 3 h of irradiation with MES, the C/C0 of AMT decreased by only 30% in the presence of the IONP (0.333 mg/mL) and IONP-DA (0.333 mg/mL) controls (Figure 3 A), showing that the catechol coating does not significantly enhance IONP photocatalytic activity, nor quenches ROS liberated from the IONP upon photoexcitation. However, both controls show much less activity in the presence EDTA after 3 h of irradiation with 450 nm blue light (λmax = 520 nm), proving that in the case of these controls, MES acts as a better electron donor than EDTA. ( Figure 3B) IONP-DAFL (0.333 mg/mL) and DAFL (0.003 mg/mL), on the other hand, degrade AMT by 91% and 60%, respectively, after 1 h of irradiation in the presence of MES. This clearly demonstrates the enhanced activity of the heterogeneous flavin-conjugated IONPs over the homogenous flavin alone. After 1 h of irradiation using EDTA, AMT is degraded by 86% in the presence of IONP-DAFL, slightly lower than that compared to MES, and surprisingly, by 98% in the presence of DAFL (see Figure S7 and S8 for UV-vis absorption spectra of AMT at each time point). This observation can be explained by the chemical nature of the electron donor used. It has been reported that photooxidation of EDTA leads to radical oxidation products that can degrade a substrate and the catalyst that generated them in the first place [59]. On the other hand, morpholine-based buffers, such as MES, not only act as electron donors, but have also been shown to inhibit oxidative flavin degradation through the formation of spin-correlated ion pairs that impede deleterious ROS [55]. In our case, this is clearly demonstrated by the relative activity of DAFL in MES and EDTA, the latter being much more efficient, most likely due to the additive effect of EDTA oxidation products degrading AMT. However, the combined IONP-DAFL system shows slightly better activity in the presence of MES, which can be attributed to the photoprotective effect of the morpholine buffer, which inhibits flavin-induced ROS formation, thus enhancing charge transfer to the IONP. For the rate and the quantum efficiency of the reaction, refer to the 'Quantum Efficiency' section of the ESI. , as well as energy transfer (ET) and charge transfer processes with molecular oxygen to produce reactive oxygen species (ROS), singlet oxygen and superoxide, respectively, which then go on to degrade AMT. Photoexcitation of IONP electron-hole (e-h) pairs can interact with O 2 to produce further superoxide radicals. AMT can also undergo a redox processes with these electron hole pairs initiating degradation through interactions with ROS. As a control, dopamine (DA) was attached to the surface of IONP in order to observe any catechol-mediated activity [58]. After 3 h of irradiation with MES, the C/C 0 of AMT decreased by only 30% in the presence of the IONP (0.333 mg/mL) and IONP-DA (0.333 mg/mL) controls (Figure 3 A), showing that the catechol coating does not significantly enhance IONP photocatalytic activity, nor quenches ROS liberated from the IONP upon photoexcitation. However, both controls show much less activity in the presence EDTA after 3 h of irradiation with 450 nm blue light (λ max = 520 nm), proving that in the case of these controls, MES acts as a better electron donor than EDTA. (Figure 3B) IONP-DAFL (0.333 mg/mL) and DAFL (0.003 mg/mL), on the other hand, degrade AMT by 91% and 60%, respectively, after 1 h of irradiation in the presence of MES. This clearly demonstrates the enhanced activity of the heterogeneous flavin-conjugated IONPs over the homogenous flavin alone. After 1 h of irradiation using EDTA, AMT is degraded by 86% in the presence of IONP-DAFL, slightly lower than that compared to MES, and surprisingly, by 98% in the presence of DAFL (see Figures S7 and S8 for UV-vis absorption spectra of AMT at each time point). This observation can be explained by the chemical nature of the electron donor used. It has been reported that photooxidation of EDTA leads to radical oxidation products that can degrade a substrate and the catalyst that generated them in the first place [59]. On the other hand, morpholine-based buffers, such as MES, not only act as electron donors, but have also been shown to inhibit oxidative flavin degradation through the formation of spin-correlated ion pairs that impede deleterious ROS [55]. In our case, this is clearly demonstrated by the relative activity of DAFL in MES and EDTA, the latter being much more efficient, most likely due to the additive effect of EDTA oxidation products degrading AMT. However, the combined IONP-DAFL system shows slightly better activity in the presence of MES, which can be attributed to the photoprotective effect of the morpholine buffer, which inhibits flavin-induced ROS formation, thus enhancing charge transfer to the IONP. For the rate and the quantum efficiency of the reaction, refer to the 'Quantum Efficiency' section of the ESI. Finally, in order to study the nature of the ROS being produced, an antioxidant assay using ROS quenchers was designed. Mannitol, (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO), and 1,4-diazabicyclo [2.2.2]octane (DABCO) have been shown to scavenge hydroxyl radicals, superoxide and singlet oxygen species, respectively [54,60]. 1.5 molar equivalents of each scavenger are added independently to three different vials, each containing 0.5 mL of AMT (0.1 mg/mL), 1 mL of 0.333 mg/mL IONP or IONP-DAFL, and 1 mL of MES (0.1 M). Since MES is a more appropriate electron donor for IONP-DAFL in an aerobic environment, it was chosen for this experiment. The reaction mixture is flushed with O2 before being irradiated for 3 h with aliquots being taken hourly, and analyzed ( Figure 4). The UV-vis absorption spectra at each time point for IONP and IONP-DAFL can be found in Figure S9 and S10 of the ESI, respectively. The scavengers are expected to quench the ROS being produced in solution, thereby slowing the reaction down. Therefore, the ROS being produced can be identified by the effects observed when a scavenger is added to the reaction. The addition of ROS scavengers to the bare IONP samples does not affect the particles' ability to degrade the AMT. ~30% degradation of AMT was observed after 3 h of irradiation in the presence of the scavengers (Figure 4 A). This aligns with the values obtained from the photooxidation of AMT in the presence of MES without the addition of the ROS quenchers ( Figure 3A). Therefore, sufficient ROS production requires charge transfer from an excited flavin molecule. Finally, in order to study the nature of the ROS being produced, an antioxidant assay using ROS quenchers was designed. Mannitol, (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO), and 1,4-diazabicyclo [2.2.2]octane (DABCO) have been shown to scavenge hydroxyl radicals, superoxide and singlet oxygen species, respectively [54,60]. 1.5 molar equivalents of each scavenger are added independently to three different vials, each containing 0.5 mL of AMT (0.1 mg/mL), 1 mL of 0.333 mg/mL IONP or IONP-DAFL, and 1 mL of MES (0.1 M). Since MES is a more appropriate electron donor for IONP-DAFL in an aerobic environment, it was chosen for this experiment. The reaction mixture is flushed with O 2 before being irradiated for 3 h with aliquots being taken hourly, and analyzed ( Figure 4). The UV-vis absorption spectra at each time point for IONP and IONP-DAFL can be found in Figures S9 and S10 of the ESI, respectively. Finally, in order to study the nature of the ROS being produced, an antioxidant assay using ROS quenchers was designed. Mannitol, (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO), and 1,4-diazabicyclo [2.2.2]octane (DABCO) have been shown to scavenge hydroxyl radicals, superoxide and singlet oxygen species, respectively [54,60]. 1.5 molar equivalents of each scavenger are added independently to three different vials, each containing 0.5 mL of AMT (0.1 mg/mL), 1 mL of 0.333 mg/mL IONP or IONP-DAFL, and 1 mL of MES (0.1 M). Since MES is a more appropriate electron donor for IONP-DAFL in an aerobic environment, it was chosen for this experiment. The reaction mixture is flushed with O2 before being irradiated for 3 h with aliquots being taken hourly, and analyzed ( Figure 4). The UV-vis absorption spectra at each time point for IONP and IONP-DAFL can be found in Figure S9 and S10 of the ESI, respectively. The scavengers are expected to quench the ROS being produced in solution, thereby slowing the reaction down. Therefore, the ROS being produced can be identified by the effects observed when a scavenger is added to the reaction. The addition of ROS scavengers to the bare IONP samples does not affect the particles' ability to degrade the AMT. ~30% degradation of AMT was observed after 3 h of irradiation in the presence of the scavengers (Figure 4 A). This aligns with the values obtained from the photooxidation of AMT in the presence of MES without the addition of the ROS quenchers ( Figure 3A). Therefore, sufficient ROS production requires charge transfer from an excited flavin molecule. The scavengers are expected to quench the ROS being produced in solution, thereby slowing the reaction down. Therefore, the ROS being produced can be identified by the effects observed when a scavenger is added to the reaction. The addition of ROS scavengers to the bare IONP samples does not affect the particles' ability to degrade the AMT.~30% degradation of AMT was observed after 3 h of irradiation in the presence of the scavengers (Figure 4 A). This aligns with the values obtained from the photooxidation of AMT in the presence of MES without the addition of the ROS quenchers ( Figure 3A). Therefore, sufficient ROS production requires charge transfer from an excited flavin molecule. The addition of TEMPO, DABCO and mannitol leads to a 2.5%, 14.09% and 20.17% photodegradation of AMT after 1 h of irradiation in the presence of IONP-DAFL ( Figure 4B). This is significantly less than the 91% degradation after 1 h of irradiation without any scavenger ( Figure 3A). Therefore, it could be concluded that hydroxyl radicals, superoxide and singlet oxygen, are all formed during the aerobic photodegradation of AMT. However, the photoreactions with both mannitol and DABCO proceed to near completion, while the addition of TEMPO only leads to a 24.76% degradation of AMT in 3 h. Therefore, superoxide radicals are the predominant reactive oxygen species. Amaranth Photoreduction (Anaerobic Activity) The photocatalytic degradation of AMT was also investigated under anaerobic conditions in an Ar atmosphere. In this case, we hoped to take advantage of the flavin photoredox catalysis via formation of the hydroquinone capable of reducing the azo substrate by hydride transfer [56], as well as a flavin-sensitized charge transfer to IONP and surface interaction of absorbed AMT [61] (see Scheme 4). Similarly, the reaction was conducted in the presence of either MES or EDTA solution as the electron donor. The addition of TEMPO, DABCO and mannitol leads to a 2.5%, 14.09% and 20.17% photodegradation of AMT after 1 h of irradiation in the presence of IONP-DAFL ( Figure 4B). This is significantly less than the 91% degradation after 1 h of irradiation without any scavenger ( Figure 3A). Therefore, it could be concluded that hydroxyl radicals, superoxide and singlet oxygen, are all formed during the aerobic photodegradation of AMT. However, the photoreactions with both mannitol and DABCO proceed to near completion, while the addition of TEMPO only leads to a 24.76% degradation of AMT in 3 h. Therefore, superoxide radicals are the predominant reactive oxygen species. Amaranth Photoreduction (Anaerobic Activity) The photocatalytic degradation of AMT was also investigated under anaerobic conditions in an Ar atmosphere. In this case, we hoped to take advantage of the flavin photoredox catalysis via formation of the hydroquinone capable of reducing the azo substrate by hydride transfer [56], as well as a flavin-sensitized charge transfer to IONP and surface interaction of absorbed AMT [61] (see Scheme 4). Similarly, the reaction was conducted in the presence of either MES or EDTA solution as the electron donor. After 3 h of irradiation, both bare IONPs and IONP-DA (both 0.333 mg/mL) show negligible activity in MES and EDTA, as seen in previous aerobic experiments. In MES, after 1 h of irradiation, the C/C 0 of AMT decreases by 33.4% in the presence of IONP-DAFL (0.333 mg/mL) and by 5.3% in the presence of DAFL (0.003 mg/mL) on its own ( Figure 5A). After 3 h of irradiation, the total AMT degradation is 39.5% and 5.7% in the presence of IONP-DAFL and DAFL, respectively (for UV-vis absorption spectra at each time point refer to Figure S11). However, after 1 h of irradiation in the presence of EDTA ( Figure 5B) the C/C 0 of AMT decreases by 92.2% in the presence of IONP-DAFL, and by 77.2% in the presence of DAFL (for UV-vis absorption spectra at each time point refer to Figure S12). After 2 h of irradiation, the photodegradation process in the presence of both IONP-DAFL and DAFL is complete. EDTA is therefore a much better electron donor than MES in anaerobic conditions, which could be attributed to the radical EDTA oxidation products that facilitate further AMT degradation [59]. Moreover, it is apparent that, irrespective of the electron donor, IONP-DAFL is a far more efficient photocatalyst than homogenous flavin, clearly demonstrating the benefit of our synergistic photocatalytic system. Refer to the 'Quantum Efficiency' section of the ESI for the rate of the reaction and quantum efficiency calculation. Catalysts 2020, 10, x FOR PEER REVIEW 10 of 17 After 3 h of irradiation, the total AMT degradation is 39.5% and 5.7% in the presence of IONP-DAFL and DAFL, respectively (for UV-vis absorption spectra at each time point refer to Figure S11). However, after 1 h of irradiation in the presence of EDTA ( Figure 5B) the C/C0 of AMT decreases by 92.2% in the presence of IONP-DAFL, and by 77.2% in the presence of DAFL (for UV-vis absorption spectra at each time point refer to Figure S12). After 2 h of irradiation, the photodegradation process in the presence of both IONP-DAFL and DAFL is complete. EDTA is therefore a much better electron donor than MES in anaerobic conditions, which could be attributed to the radical EDTA oxidation products that facilitate further AMT degradation [59]. Moreover, it is apparent that, irrespective of the electron donor, IONP-DAFL is a far more efficient photocatalyst than homogenous flavin, clearly demonstrating the benefit of our synergistic photocatalytic system. Refer to the 'Quantum Efficiency' section of the ESI for the rate of the reaction and quantum efficiency calculation. Figure S14A). IONP-DAFL, on the other hand, showed 34.02% AMT degradation in O2 ( Figure S14A) and a 25.05% degradation in Ar ( Figure S14B) after 3 h, which is most likely the result of electron donation from AMT itself, which then leads to its degradation, rather than water acting as the donor. Reusability of the Catalyst The reusability of the IONP-DAFL in the anaerobic photodegradation of AMT using EDTA ( Figure 6A) and the aerobic degradation of AMT with MES ( Figure 6B) were investigated. AMT (0.05mg/mL) was irradiated in the presence of IONP-DAFL (0.333 mg/mL) and EDTA or MES (0.1 M) for 1 h under Ar or O2, respectively. The IONP-DAFL was then removed from the solution with a magnet, washed, and re-used in another run. Heterogenous flavin-based photocatalysts have often shown poor recyclability in the literature [62], which is also confirmed in this study. Although the IONP-DAFL system can be easily removed from the solution with the use of a magnet, IONP-DAFL is not recyclable in either aerobic or anaerobic conditions. For the UV-vis absorption spectra for each run in aerobic and anaerobic conditions, refer to Figure S15A and S15B, respectively Figure S14A) and a 25.05% degradation in Ar ( Figure S14B) after 3 h, which is most likely the result of electron donation from AMT itself, which then leads to its degradation, rather than water acting as the donor. Reusability of the Catalyst The reusability of the IONP-DAFL in the anaerobic photodegradation of AMT using EDTA ( Figure 6A) and the aerobic degradation of AMT with MES ( Figure 6B) were investigated. AMT (0.05mg/mL) was irradiated in the presence of IONP-DAFL (0.333 mg/mL) and EDTA or MES (0.1 M) for 1 h under Ar or O 2 , respectively. The IONP-DAFL was then removed from the solution with a magnet, washed, and re-used in another run. Heterogenous flavin-based photocatalysts have often shown poor recyclability in the literature [62], which is also confirmed in this study. Although the IONP-DAFL system can be easily removed from the solution with the use of a magnet, IONP-DAFL is not recyclable in either aerobic or anaerobic conditions. For the UV-vis absorption spectra for each run in aerobic and anaerobic conditions, refer to Figure S15A,B, respectively. A significant loss in the catalyst's activity is observed after one run. The low recyclability of the material is most likely due to particle and flavin instability. Flavin photodealkylation at the N10 position has been reported in the literature [33]. In the case of IONP-DAFL, this would release the flavin moiety into the supernatant. To test this hypothesis, two samples of 1.0 mg/mL IONP-DAFL in H2O were irradiated with a 450 nm blue light in O2 and Ar for 1 h. The particles were then removed, and the fluorescence intensity of the supernatants of the samples were measured ( Figure S16). A strong fluorescence signal was obtained from both supernatants after excitation at 450 nm, indicating that flavin species were present in solution and no longer conjugated to the IONP surface after irradiation. Furthermore, a stronger fluorescence signal was obtained from the supernatant of the sample irradiated in O2, thus indicating that IONP-DAFL is less stable when irradiated in an O2-rich environment. Lastly, Figure S6C indicates that the fluorescence signal of DAFL is weak in comparison to NBoc-protected flavin, 3 due to quenching by the catechol moiety. Therefore, the fluorescence signals in Figure S16 can be attributed to lumiflavin that is a product of flavin photodegradation [63]. The regeneration of the catalyst was therefore not possible. For this reason, our current work looks at addressing this issue through investigating different anchoring groups, conjugation strategies and immobilization for flavin IONP hybrids with a wider range of sacrificial electron donors [59] that, unlike MES and EDTA, are stable in nonacid environments and at different temperatures. General Commercially available reagents were purchased in the highest purity from Acros Organics A significant loss in the catalyst's activity is observed after one run. The low recyclability of the material is most likely due to particle and flavin instability. Flavin photodealkylation at the N10 position has been reported in the literature [33]. In the case of IONP-DAFL, this would release the flavin moiety into the supernatant. To test this hypothesis, two samples of 1.0 mg/mL IONP-DAFL in H 2 O were irradiated with a 450 nm blue light in O 2 and Ar for 1 h. The particles were then removed, and the fluorescence intensity of the supernatants of the samples were measured ( Figure S16). A strong fluorescence signal was obtained from both supernatants after excitation at 450 nm, indicating that flavin species were present in solution and no longer conjugated to the IONP surface after irradiation. Furthermore, a stronger fluorescence signal was obtained from the supernatant of the sample irradiated in O 2 , thus indicating that IONP-DAFL is less stable when irradiated in an O 2 -rich environment. Lastly, Figure S6C indicates that the fluorescence signal of DAFL is weak in comparison to NBoc-protected flavin, 3 due to quenching by the catechol moiety. Therefore, the fluorescence signals in Figure S16 can be attributed to lumiflavin that is a product of flavin photodegradation [63]. The regeneration of the catalyst was therefore not possible. For this reason, our current work looks at addressing this issue through investigating different anchoring groups, conjugation strategies and immobilization for flavin IONP hybrids with a wider range of sacrificial electron donors [59] that, unlike MES and EDTA, are stable in nonacid environments and at different temperatures. General Commercially available reagents were purchased in the highest purity from Acros Organics (Pittsburgh, PA, USA), Alfa Aeser (Haverhill, MA, USA), Sigma-Aldrich (St. Louis, MO, USA), and TCI Chemicals (Tokyo, Kanto region, JPN) [20]. A Bruker (Billerica, MA, USA) 500 MHz DCH Cryoprobe Spectrometer was used for the 13 C and 1 H Nuclear Magnetic Resonance (NMR) spectroscopy measurements. UV-vis absorption spectra were obtained using an Agilent (Santa Clara, CA, USA) Cary 300 Spectrophotometer. Attenuated Total Reflection Fourier-Transform Infra-Red (ATR-FTIR) spectroscopy data was acquired from powder samples using a Perkin Elmer (Waltham, MA, USA) Spectrum One FT-IR Spectrometer. Fluorescence intensity measurements were done using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer. Dynamic Light Scattering (DLS) and Zeta potential measurements were obtained using a Zetasizer Nano Range instrument from Malvern Panalytical (Malvern, Worcs, UK). Synthesis of Ferrous dodecyl Sulfate (Fe(DS) 2 ) Briefly, a 100 mL solution containing 1 M sodium dodecyl sulfate (SDS) and 1 M iron (II) chloride (FeCl 2 ) in Milli-Q ® water is prepared and stored at 2 • C for 1 h. The resulting Fe(DS) 2 precipitate is then washed with 2 • C Milli-Q ® several times, and allowed to re-crystallize overnight. Synthesis of γ-Fe 2 O 3 Maghemite Iron Oxide Nanoparticles (IONPs) The γ-Fe 2 O 3 maghemite IONPs were prepared using a modified "one emulsion plus reactant" [64] micro-emulsion synthesis protocol described by Benyettou et al. [36]. 3.05 g of Fe(DS) 2 is dissolved in a 500 mL round-bottom flask containing 345 mL of Milli-Q ® water at 32 • C. Once the iron salt is dissolved, the temperature is reduced to 28 • C. 50 mL (40 wt%) dimethylamine, heated to the same temperature, is then added dropwise. The solution is left shaking for 2 h at the same temperature without the use of a magnetic stirrer. The flask is then placed on ice. The nanoparticles are then cleaned 10 times with Milli-Q ® water using a magnet. 4 mL of 4 M HCl is added before each wash for the first fourwashes. The IONPs are then stored in water at a pH of 7. Synthesis of Dopamine-Flavin (DAFL) Details on the synthesis of DAFL can be found in the Supplementary Information section. Surface Modification of γ-Fe 2 O 3 Nanoparticles Briefly, 5 mg of IONPs are suspended in a 5 mL solution of dopamine (DA) or dopamine-flavin (DAFL) (0.5 mg/mL in H 2 O) in an argon atmosphere at 23 • C. The solution is sonicated for 5 min. A vortex mixer is then used to shake the suspension for 2 h, while being protected from direct sunlight. The suspension is then washed 3-4 times on a magnet using Milli-Q ® water. The coated IONPs are kept in the dark at room temperature in air. General Procedure for Photoreduction and Photooxidation of Amaranth AMT (0.05 mg/mL) and an IONP sample (0.333 mg/mL) or DAFL (0.003 mg/mL) were added to a solution of either MES or EDTA buffer (0.1 M, pH 6, 3 mL), and then saturated with either O 2 gas or Ar for 15 min before being irradiated with a 450 nm blue LED (18 W; 34 mW/cm 2 ) in a HepatoChem (Beverly, MA, USA) EvoluChem™ PhotoRedOx Box photoreactor, equipped with a cooling fan to keep the temperature at~23 • C. 100 µL aliquots were removed from the reaction at specific time points and diluted to 1 mL with 0.1 M MES/EDTA. Any nanoparticles were removed by centrifugation or using a magnet before monitoring the degradation of AMT by UV-vis absorption spectroscopy, using λ max = 520 nm of AMT [8] to observe the relative concentration of remaining AMT in solution (C/C 0 ), where C 0 represents the initial concentration of AMT before irradiation. General Procedure for ROS Scavenging Experiments Mannitol, TEMPO or DABCO (1.5 equiv) were added to a solution of AMT (0.1 mg/mL), IONP-DAFL or IONP (0.333 mg/mL) in MES buffer (0.1 M, pH 6, 3 mL). The mixture was then saturated with O 2 gas and irradiated with a 450 nm blue LED (18 W; 34 mW/cm 2 ) in an EvoluChem™ PhotoRedOx Box photoreactor equipped with a cooling fan to keep the temperature at~23 • C. General Procedure for Recyclability Measurements The recyclability of IONP-DAFL was investigated using the general procedure described in Section 3.6 using MES buffer (0.1 M, pH 6) in an aerobic environment and with EDTA buffer (0.1 M, pH 6) under anaerobic conditions. One time point is taken at 1 h for each run before the catalyst was removed from the reaction via magnetic separation and washed with Milli-Q ® H 2 O for reuse. Four cycles for both samples were completed. Conclusions We have developed a new hybrid metal oxide-organic heterogeneous photocatalytic system capable of degrading azo bonds found in industrial dyes such as AMT. The hybrid flavin-IONP system (IONP-DAFL) is shown to enable both the photooxidation and photoreduction in an O 2 and Ar medium, respectively. While controls were found to be not photocatalytically active, IONP-DAFL and flavin-dopamine (DAFL) alone were shown to reduce AMT, with best results obtained in the presence of EDTA and in inert atmosphere. This was attributed to the synergistic action of the flavin-mediated photocatalysis and IONP photoexcitation. Lack of activity in IONPs controls in Ar can be explained by fast electron-hole recombination that commonly occurs in photoexcited IONPs [29]. In an O 2 environment, IONP-DAFL and DAFL were able to degrade the AMT dye in both electron donor buffers (MES and EDTA) through the release of ROS species shown to predominantly be superoxide radicals. The activity of IONP-DAFL was higher in MES, most likely due to spin ion pair correlation which prevents the flavin from degradation [55]. Contrary to IONP-DAFL, the photocatalysis of DAFL alone was more efficient in EDTA, probably due to an additive effect of EDTA's reactive oxidation products. The control photocatalysts, IONP and IONP-DA, displayed little catalytic activity both in EDTA and MES. In summary, we designed a novel hybrid photocatalyst that combines iron oxide nanoparticles and flavin moieties anchored to the nanoparticle surface using catechol linker. The catalysts mimic the activity of naturally-occurring azoreductatese enzymes, and facilitates azo-bond reduction in inert atmosphere. Interestingly, this activity can be switched to oxidation by the introduction of oxygen, and in air the hybrid mimics the activity of oxidases, such as laccase, which can oxidize the azo bond [65]. Moreover, unlike its enzymatic counterparts, the hybrid system is robust, and can be stored at room temperature over longer time, and can be efficiently removed from solution using a magnet, although not reused. Limited recyclability is attributed to the low stability of the flavin-IONP linkage, and our current efforts are focused on the redesign of the hybrid system by use of improved anchoring linkers and immobilization of additional electron donors. However, despite limited recyclability, photoactivation with blue light enables temporal and spatial control over the reaction, and the hybrid system is nontoxic, cheap, easy to prepare, and easy to remove from the solution. Therefore, we believe that it has a significant potential to be used in water remediation, and our future work with a look into the degradation of other contaminants, such as rhodamine B (RhB) and bisphenol A (BPA) [66], and the design of a suitable flow cell/membrane system, suitable for practical applications. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/3/324/s1, Figure S1: X-ray diffraction spectroscopy pattern of the -Fe2O3 IONPs; Figure S2: The ATR-FTIR spectrum of -Fe2O3 IONPs functionalized with dopamine (IONP-DA) in comparison to IONP and dopamine (DA); Figure S3: TEM images of IONP-DA at a scale of (A) 100nm and (B) 20nm. (C) The particle size distribution determined from the average of~100 measurements of particles; Figure S4 Figure S16: Fluorescence intensity measurements of aliquots taken from the supernatant of IONP-DAFL after irradiation with blue light in O2 or Ar for 1 h (λex = 450 nm), Table S1: DLS and zeta potential measurements of IONP, IONP-DA, and IONP-DAFL samples. The errors are calculated from the standard deviation of 3 repeats. Sizes from TEM images are the average of the particle size distribution of 100 particles of each sample. The error is the standard deviation.	v3-fos-license
2018-02-19T18:36:13.712Z	2017-10-30T00:00:00.000	3358557	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10482-017-0963-y.pdf", "pdf_hash": "98e57f9ab0bb5c2d32a06772f1e577e1292831ad", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1712", "s2fieldsofstudy": [ "Biology" ], "sha1": "aa7d4f0a3d6202ceb403011b8424e16cf3cbeb26", "year": 2017 }	pes2o/s2orc	Genetic redundancy in the catabolism of methylated amines in the yeast Scheffersomyces stipitis The catabolism of choline as a source of nitrogen in budding yeasts is thought to proceed via the intermediates trimethylamine, dimethylamine and methylamine before the release of ammonia. The present study investigated the utilisation of choline and its downstream intermediates as nitrogen sources in the yeast Scheffersomyces stipitis using a reverse genetics approach. Six genes (AMO1, AMO2, SFA1, FGH1, PICST_49761, PICST_63000) that have previously been predicted to be directly or indirectly involved in the catabolism of methylated amines were individually deleted. The growth of each deletion mutant was assayed on minimal media with methylamine, dimethylamine, trimethylamine or choline as the sole nitrogen source. The two amine oxidase-encoding genes AMO1 and AMO2 appeared to be functionally redundant for growth on methylated amines as both deletion mutants displayed growth on all nitrogen sources tested. However, deletion of AMO1 resulted in a pronounced growth lag on all four methylated amines while deletion of AMO2 only caused a growth lag when methylamine was the sole nitrogen source. The glutathione-dependent formaldehyde dehydrogenase-encoding gene SFA1 was found to be absolutely essential for growth on all methylated amines tested while deletion of the S-formylglutathione hydrolase gene FGH1 caused a pronounced growth lag on dimethylamine, trimethylamine and choline. The putative cytochrome P450 monooxygenase-encoding genes PICST_49761 and PICST_63000 were considered likely candidates for demethylation of di- and trimethylamine but produced no discernable phenotype on any of the tested nitrogen sources when deleted. This study revealed notable instances of genetic redundancies in the choline catabolic pathway, which are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0963-y) contains supplementary material, which is available to authorized users. Introduction Amines are organic nitrogen compounds common in the environment and consequently most microorganisms have evolved pathways to assimilate amines as a source of metabolic nitrogen. The ability to assimilate amines as a nitrogen source is nearly universal among budding yeasts (phylum Ascomycota, sub-phylum Saccharomycotina) but does not occur in the common model system Saccharomyces cerevisiae (van der Walt 1962;van Dijken and Bos 1981;Linder 2014). Due to the limited research hitherto conducted on non-Sa. cerevisiae yeasts, our understanding of the genetics governing the assimilation of amines in budding yeasts remains rudimentary. Previous biochemical studies on budding yeasts capable of assimilating amines have shown that the deamination of primary amines (RCH 2 NH 2 ) is catalysed by copper-containing amine oxidases (EC 1.4.3.6) to release ammonia, hydrogen peroxide and the corresponding alkylaldehyde (RCHO). Most budding yeasts appear to possess two types of amine oxidases, which are commonly referred to as methylamine oxidase and benzylamine oxidase, respectively (Haywood and Large 1981;Green et al. 1982). Methylamine oxidase (encoded by the AMO1 gene) appears to have higher affinity towards short-chain aliphatic amines while benzylamine oxidase (encoded by the AMO2 gene) has higher affinity towards amines with longer and bulkier side-chains (Haywood and Large 1981). The Amo1 methylamine oxidase contains an N-terminal type 2 peroxisomal targeting signal (PTS2) composed of the nonapeptide motif RLXXXXX H / Q L and localises to the peroxisome (Zwart et al. 1980;Faber et al. 1994) while the Amo2 benzylamine oxidase is thought to be localised to the cytosol. Secondary [(RCH 2 ) 2 NH] and tertiary [(RCH 2 ) 3 N] amines can also be assimilated by some yeasts (van Dijken and Bos 1981;Linder 2014) but the identity of the enzymes involved in the de-alkylation of these substrates into primary amines have yet to be established. Choline is one of the few quaternary (tetraalkylated) amines that is known to be de-alkylated and subsequently assimilated by budding yeasts (van Dijken and Bos 1981;Linder 2014). The catabolism of choline is thought to involve four sequential dealkylation steps via the intermediates trimethylamine, dimethylamine and methylamine before ammonia is released by amine oxidase (Zwart et al. 1980(Zwart et al. , 1983Fig. 1). Understanding of the genetics surrounding this pathway remains incomplete. A putative choline monooxygenase encoded by the CMO1 gene is thought to be responsible for the first de-alkylation step to produce trimethylamine and glycolaldehyde (Mori et al. 1988;Linder 2014). The yeast Cmo1 protein has not yet been biochemically characterised but the homologous plant protein (which converts choline into betaine aldehyde in the chloroplast) requires oxygen and ferredoxin (Brouquisse et al. 1989). Biochemical studies of the demethylation of diand trimethylamine indicate that two distinct enzyme activities are involved, which have characteristics typical of cytochrome P450 monooxygenases Large 1983, 1984;Fattakhova et al. 1991 ? ? Fig. 1 The proposed pathway for choline assimilation in budding yeasts final demethylation step involves one or both amine oxidases (Haywood and Large 1981). The present study set out to study the genetics surrounding the catabolism of choline and focused on six genes thought to be directly involved in either the amine de-alkylation steps or linked reactions such as detoxification of the significant amounts of formaldehyde generated by each demethylation reaction. The yeast Scheffersomyces stipitis was selected as a model system for this study as it can assimilate all the predicted intermediates of the choline pathway (Linder 2014). Sc. stipitis integration constructs Targeting cassettes for AMO1 (PICST_55523), AMO2 (PICST_83878), SFA1 (PICST_29252), FGH1 (PICST_65460), PICST_49761 and PICST_63000 were synthesised de novo by GenScript (NJ, USA) and inserted into either EcoRI/HindIII-cut or EcoRI/ StuI-cut pUC57 (GenBank accession Y14837; Fig. S1a). Each targeting cassette consisted of 500 bp sequence identical to the intergenic region immediately downstream (3 0 IGR) of the gene to be deleted (''PICST_xxxxx'') followed by 500 bp sequence immediately upstream (5 0 IGR) of the sequence to be deleted (Fig. S1b). The 3 0 and 5 0 IGR sequences in each targeting cassette were separated by a SwaI recognition site. Each targeting cassette was followed by a short polylinker to enable insertion of a selection marker. The full-length HIS3 gene was amplified from Debaryomyces hansenii CBS 767 genomic DNA with primers DhHIS3 fwd (5 0 GCG CGC GGA TCC TTT CAC CAG ATG GGA TCT AAT 3 0 ) and DhHIS3 rev (5 0 GCG CGC CTG CAG GCG CGC CAG TCG TAA TGT TTA TAG AAG A 3 0 ), digested with BamHI and PstI and inserted into each targeting construct, which had been digested with the appropriate enzymes to produce ends compatible with BamHI (either BamHI or BglII) and PstI (PstI or SbfI). The restriction sites used to insert the DhHIS3 gene into each targeting plasmid are indicated in Fig. S1a. Prior to transformation, the integration plasmids ( Fig. S1c) were digested with SwaI to produce linearised integration constructs (Fig. S1d), which were then purified into sterile water using the QIAquick PCR purification kit (Qiagen). Yeast transformation The transgenic yeast strains used in this study are listed in Table 1. The transformation methodology has been described previously (Linder 2014). The Sc. stipitis SF1 strain, which lacks a functional nonhomologous end-joining DNA repair pathway (Maassen et al. 2008), was kindly provided by Prof Ulrich Klinner (Aachen University, Germany). Correct chromosomal integration and the deletion of each targeted gene (''PICST_xxxxx'') was confirmed by PCR analysis of purified genomic DNA from each deletion strain ( Fig. S2) using the primers listed in Table S1. Nitrogen utilisation assays Sodium L-glutamate and the hydrochloride salts of methylamine, dimethylamine, trimethylamine and choline were purchased from Sigma Aldrich. A reduced sulfur/nitrogen-limited glucose medium (RSNLD) was used for assaying growth on individual amines. RSNLD medium is composed of 1.7 g Difco yeast nitrogen base without amino acids or ammonium sulfate l -1 (Becton, Dickinson and Company) and 20 g glucose l -1 . Prior to the nitrogen utilisation assay, individual yeast strains were pre-cultured in 3 ml minimal glucose medium (MMD) consisting of 6.7 g Difco yeast nitrogen base without amino acids l -1 (Becton, Dickinson and Company) and 20 g glucose l -1 . Pre-cultures were washed twice in RSNLD before being re-suspended in 2.97 ml RSNLD to a final OD 600 of 0.005 in a 50-ml tube. Individual nitrogen sources were added as 30 ll of a 1 M stock solution, making a final concentration of 10 mM. A non-supplemented sample with 30 ll deionised water was used as a control. Chloramphenicol (final concentration 15 mg l -1 ) was included to prevent bacterial contamination. Samples were incubated at 30°C in a rotary shaker set to 200 rpm with OD 600 measurements after 6, 12 and 18 days. OD 600 measurements were carried out with a 1 cm path length using an Ultrospec 1100 pro spectrophotometer (GE Healthcare). Each growth assay was performed in triplicate with each biological replicate using a separate pre-culture. The yeast strain Ogataea parapolymorpha CBS 11895 was purchased from Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Sequence alignment and phylogenetic analysis All BLASTP and TBLASTN searches applied an expect value cut-off of 10 -5 with the low-complexity region filter enabled. Protein sequences were aligned in MAFFT (Katoh et al. 2005; http://mafft.cbrc.jp/ alignment/server/index.html). Selection of sequence positions suitable for phylogenetic analysis was carried out in GBlocks (Castresana 2000; http://molevol.ibmb. csic.es/Gblocks_server/). The resulting amino acid positions were then used to construct a neighbourjoining tree in MEGA v. 6 (Tamura et al. 2013) using a JTT amino acid substitution model. Branch support was tested using 10,000 bootstrap replicates. Any nodes with bootstrap values equal or less than 50 were collapsed. The consensus trees were visualised in FigTree v.1.0 (http://tree.bio.ed.ac.uk/software/figtree/). Results and discussion Previous studies on the catabolism of choline and its putative downstream intermediates in budding yeasts have mainly employed biochemical and cell imaging methods (for example Zwart et al. 1980;Haywood and Large 1981;Zwart et al. 1983). Parallel analysis of gene deletion phenotypes under specific growth conditions (phenomics) is a useful tool for the identification of functional redundancies and genetic associations. Measuring cell growth in batch culture is particularly informative as it is possible to resolve the individual dynamic components of the typical sigmoidal cellular growth curve such as growth lag, growth rate and growth efficiency (Warringer et al. 2003). Very little reverse genetics have been done in Sc. stipitis to date due to a scarcity of selection markers and the low targeting frequency of integration constructs due to the domination of the non-homologous end-joining (NHEJ) pathway over homologous recombination. However, the development of a Sc. stipitis strain auxotrophic for histidine that also lacks a functional NHEJ pathway through deletion of the YKU80 gene has now enabled reverse genetic investigations in this yeast (Maassen et al. 2008). Targeting cassettes for each of the six genes to be deleted in this study (AMO1, AMO2, SFA1, FGH1, PICST_49761, PICST_63000) were synthesised de novo to contain 500 bp of flanking sequences adjacent to the deleted region (Fig. S1a). The D. hansenii HIS3 gene was inserted into each integration construct to enable selection for positive transformants on minimal medium lacking histidine (Fig. S1b-c). A Sc. stipitis Dyku80 strain with a regenerated endogenous HIS3 locus (Linder 2014) was used as a wildtype control in all growth assays. The first two genes to be investigated were the two amine oxidase-encoding genes AMO1 and AMO2. Each deletion mutant was cultivated in nitrogenlimited medium supplemented with either sodium Lglutamate, methylamine, dimethylamine, trimethylamine or choline. Growth was monitored every 6 days up until 18 days after initiation of the growth assay (Fig. 2). Both mutants displayed strong growth on sodium L-glutamate equivalent to the wildtype control. Interestingly both mutants displayed an identical growth lag on methylamine with strong growth detectable only after the 12-day time-point. In addition, both mutant strains displayed notably higher cell densities at day 12 and 18 on methylamine compared to the wildtype control. The reason for this effect was not immediately obvious but one possibility could be that the initial lag phase indirectly enabled more efficient energy metabolism once growth was initiated. The Damo2 strain was indistinguishable from the wildtype control on dimethylamine, trimethylamine and choline, which indicated that AMO2 was entirely dispensable for normal growth on these substrates. The Damo1 strain displayed a characteristic growth lag on dimethylamine, trimethylamine and choline, which indicated that AMO1 was only partially dispensable for growth on these substrates. It was notable that the Damo2 strain had no obvious growth defect on dimethylamine, trimethylamine or choline yet displayed a pronounced lag in growth on methylamine, which is a down-stream intermediate of the three other amines. One possibility is that when methylamine is supplied as an external nitrogen source, the activities of AMO1 and AMO2 must cooperate to achieve maximum growth while AMO2 does not play a significant role in the deamination of methylamine generated intracellularly through demethylation of di-and trimethylamine. Each of the three demethylation steps in the choline assimilation pathway produces an equimolar amount of formaldehyde to the amount of methylated amine (Fig. 1). The assimilation of methylated amines therefore generates significant amounts of highly reactive formaldehyde that must be appropriately metabolised by the cell to ensure viability. The principal pathway for the detoxification of formaldehyde in yeast is the cyclic glutathione-dependent formaldehyde oxidation pathway (Fig. 3a). Formaldehyde enters the pathway through a non-enzymatic reaction with glutathione to form S-hydroxymethylglutathione. S-hydroxymethylglutathione is then converted into S-formylglutathione by glutathionedependent formaldehyde dehydrogenase, which is encoded by the SFA1 gene in yeast (Sasnauskas et al. 1992). Glutathione is then regenerated through the action of S-formylglutathione hydrolase (encoded by the FGH1 gene) to release formic acid (Degrassi et al. 1999;Yurimoto et al. 2003). Formic acid is subsequently oxidised into carbon dioxide by formate dehydrogenase, which is encoded by the FDH1 gene (Allen and Holbrook 1995). The deletion of either SFA1 or FGH1 was therefore expected to cause rapid glutathione depletion and ultimately cell death under conditions of high levels of formaldehyde production, such as cultivation on medium where methylated amines were the sole nitrogen source. No obvious growth defect with sodium L-glutamate as nitrogen source was observed upon deletion of either SFA1 or FGH1 in Sc. stipitis (Fig. 3b). As expected, the Dsfa1 strain did not show any detectable growth when any of the methylated amines were the sole nitrogen source. This is in contrast with Fig. 2 The requirement for amine oxidase genes AMO1 and AMO2 for the utilisation of methylated amines as sole nitrogen sources. Sc. stipitis strains TLSS001 (wildtype control), TLSS005 (Damo1) and TLSS006 (Damo2) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD 600 0.005). Samples were incubated in a shaker set at 30°C, 200 r.p.m., and OD 600 was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation Antonie van Leeuwenhoek (2018) 111:401-411 405 previous work in the methylotrophic yeast Ogataea boidinii where the Dsfa1 cells displayed weak but detectable growth on both methylamine and choline (Lee et al. 2002). One possibility is that O. boidinii possesses a second functionally redundant enzyme capable of converting S-hydroxymethylglutathione into S-formylglutathione. Notably the Dfgh1 strain showed significant growth on all methylated amines and in the case of methylamine the Dfgh1 strain displayed noticeably higher cell densities than the wildtype control. However, the Dfgh1 strain displayed a pronounced growth lag on dimethylamine, trimethylamine and choline reminiscent of the growth dynamics observed with the Damo1 strain (Fig. 2). Direct comparison of the growth curves for the Dfgh1 and Damo1 strains showed a striking overlap in growth dynamics (Fig. S3), which could indicate a genetic link between the two genes. One possibility is that Amo1-dependent demethylation of methylamine derived from dimethylamine, trimethylamine and choline in Sc. stipitis requires Fgh1 while Amo2-dependent demethylation does not. In O. boidinii the Fgh1 protein has been shown to localise both in peroxisomes and the cytosol (Yurimoto et al. 2003). Assuming that the Sc. stipitis Fgh1 protein displays the same localisation pattern as its O. boidinii ortholog, the Fgh1 protein would available to both amine oxidase isoenzymes. The fact that SFA1 is essential for growth on methylated amines in Sc. stipitis but FGH1 is not would suggest there are additional pathways in this yeast for the regeneration of glutathione from S-formylglutathione. Previous studies of the FGH1 gene in O. boidinii have shown that deletion of FGH1 in this yeast results in detectable but retarded growth on both methylamine and choline (Yurimoto et al. 2003), which would suggest the existence of a functionally redundant enzyme in this yeast as well. Fig. 3 The requirement for the glutathione-dependent formaldehyde dehydrogenase gene SFA1 and the S-formylglutathione hydrolase gene FGH1 for the utilisation of methylated amines as sole nitrogen sources. a A simplified overview of the glutathione-dependent formaldehyde detoxification pathway. Only the thiol group of glutathione (-SH) is shown. b Sc. stipitis strains TLSS001 (wildtype control), TLSS007 (Dsfa1) and TLSS008 (Dfgh1) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD 600 0.005). Samples were incubated in a shaker set at 30°C, 200 r.p.m., and OD 600 was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation The enzymes responsible for demethylation of diand trimethylamine in budding yeasts remain to be identified (Fig. 1). Previous biochemical studies have suggested that cytochrome P450 monooxygenases may be responsible for these reactions Large 1983, 1984;Fattakhova et al. 1991). A survey of Sc. stipitis genes encoding proteins belonging to the cytochrome P450 (CYP) monooxygenase family was therefore conducted. A total of ten genes in the Sc. stipitis genome have been assigned to the CYP superfamily (Chen et al. 2014; Fig. S4). Three of the Sc. stipitis CYP genes appeared to be the orthologs of the Sa. cerevisiae genes DIT2 (N-formyltyrosine oxidase, family CYP56), ERG5 (C-22 sterol desaturase, family CYP61) and ERG11 (lanosterol 14ademethylase, family CYP51), respectively. The genes ERG5 and ERG11 are involved in the biosynthesis of the essential membrane steroid ergosterol (Turi et al. 1991;Kelly et al. 1995) and both genes have so far been found in all sequenced yeast genomes. The DIT2 gene is involved in the biosynthesis of dityrosine, which is a component of the spore cell wall (Briza et al. 1994). However, the DIT2 gene does not appear to be universally conserved among budding yeasts (data not shown). The remaining seven CYP-family genes lacked Sa. cerevisiae counterparts but five of them shared sequence similarity with n-alkane monooxygenases and fatty acid w-hydroxylases (ALK, family CYP52), which have been previously described in other yeasts (Sanglard and Loper 1989;Ohkuma et al. 1995;Huang et al. 2014). The two remaining Sc. stipitis CYP genes PICST_49761 and PICST_63000 have previously been assigned to CYP families CYP501 and CYP5217, respectively (Chen et al. 2014). Neither of these two CYP families have been biochemically characterised at the time of writing. To avoid the necessity of deleting all seven CYP genes, a bioinformatic survey was made of genomes of other budding yeasts to identify species capable of assimilating multi-alkylated amines while simultaneously possessing a smaller repertoire of CYP-family genes than Sc. stipitis. The genome of the yeast Ogataea parapolymorpha was found to contain five CYP-family genes in total (Chen et al. 2014; Fig. S4) as well as a homolog of the CMO1 gene (systematic gene name HPODL_01912), which predicted that this species should be able to assimilate choline and therefore di-and trimethylamine as well. O. parapolymorpha was therefore tested for its ability to use trimethylamine as a nitrogen source and was found to display strong growth (Fig. S5a). Phylogenetic analysis of the peptide sequences from the full CYP gene family complement of O. parapolymorpha, Sa. cerevisiae and Sc. stipitis identified two distinct clades of unknown function, which contained CYPfamily proteins from both O. parapolymorpha and Sc. stipitis but none from Sa. cerevisiae (Fig. S5b). The first clade contained the gene products of the O. parapolymorpha and Sc. stipitis genes HPODL_02874 and PICST_63000, respectively. The bootstrap support for this clade was moderate (68%) with the two proteins being 30% identical and 45% similar. A recent classification effort of fungal CYP genes placed the HPODL_02874 and PICST_63000 genes in separate CYP families CYP5223 and CYP5217, respectively (Chen et al. 2014). The second clade had strong bootstrap support (97%) and consisted of the gene products of O. parapolymorpha genes HPODL_02307 and HPODL_00882 as well as the gene product of the Sc. stipitis gene PICST_49761. HPODL_02307 formed an internal node with PICST_49761 with 83% bootstrap support and the protein sequences of the gene products were 37% identical and 54% similar. The HPODL_02307 and PICST_49761 genes are currently assigned to the CYP501 family while the HPODL_00882 gene has been placed in the CYP504 family (Chen et al. 2014). The assumption was made that if CYP-family enzymes were involved in the demethylation of di-and trimethylamine, these enzymes were expected to be shared between Sc. stipitis and O. parapolymorpha but lacking in Sa. cerevisiae. The only two Sc. stipitis CYP-encoding genes that satisfied this criterion were the two uncharacterised genes PICST_49761 and PICST_63000. The two Sc. stipitis genes PICST_49761 and PICST_63000 were therefore deleted and tested for growth on different nitrogen sources. However, neither deletion mutant displayed any obvious growth defect on methylated amines compared to the wildtype control (Fig. 4). This suggests that PICST_49761 and PICST_63000 do not encode either of the CYP-family enzymes or CYPlike enzymatic activities thought to catalyse the demethylation of di-and trimethylamine. These results suggest that the enzymes responsible for the demethylation of di-and trimethylamines may in fact belong to another enzyme family with biochemical properties similar to that of CYP monooxygenases that had been reported previously Large 1983, 1984;Fattakhova et al. 1991). Another possibility is that a diversity of pathways exists in different yeasts for the demethylation of di-and trimethylamine, where one or more alternative enzyme families are responsible for these enzymatic steps in Sc. stipitis while CYP-family enzymes catalyse these reactions in the yeast species studied previously. A third possibility is that there is functional redundancy within the CYP superfamily so that the Sc. stipitis CYP52 family ALK-like monooxygenases also possess amine demethylation activity. This could be tested through sequential deletion of all seven members of CYP families CYP52, CYP501 and CYP5217 in Sc. stipitis. However, the current limitation in genetic tools for this Sc. stipitis makes this approach impractical at present. In summary, this study demonstrates the substantial gap that remains in functional annotation of genomes from so-called non-conventional yeasts. The common baker's yeast Sa. cerevisiae has long been the dominant system for the application of reverse genetics to the study of yeast metabolism. However, the limited number of nitrogen substrates assimilated by Sa. cerevisiae makes it unsuited to the study of poorly characterised nitrogen assimilation pathways (Large 1986;Linder 2014). This study focused specifically on the yeast choline catabolic pathway, which has previously been studied using predominantly biochemical methods (for example Large 1983, 1984;Mori et al. 1988;Fattakhova et al. 1991). A complementary reverse genetics approach was used in this study and the resulting data can be condensed into three general observations. The first general observation is that there appeared to be a consistent difference between growth patterns on methylamine versus dimethylamine, trimethylamine and choline in Sc. stipitis. Methylamine was the only methylated amine substrate where a growth defect was observed in a Damo2 strain (Fig. 2). Conversely, methylamine was the only methylated amine substrate where a growth defect was not observed in a Dfgh1 strain (Fig. 3b) as well as the only methylated amine substrate where the growth dynamics of the Damo1 and Dfgh1 strains deviated significantly from each other (Fig. S3). One hypothesis to explain this observation is that cell compartmentalisation distinguishes methylamine provided externally in the growth medium versus methylamine synthesised internally from the demethylation of dimethylamine, trimethylamine and choline (Fig. 5). External methylamine would be expected to enter directly into the cytosol with some portion further transported into the peroxisome. This could explain how both amine oxidase mutants displayed an extended lag before initiating growth (Fig. 2). Internally generated methylamine is expected to originate from the endoplasmic reticulum as this is the reported localisation of di-and trimethylamine wildtype Δpicst_49761 Fig. 4 The requirement for the putative cytochrome P450 (CYP) monooxygenase genes PICST_49761 and PICST_63000 for the utilisation of methylated amines as sole nitrogen sources. Sc. stipitis strains TLSS001 (wildtype control), TLSS009 (Dpicst_49761) and TLSS010 (Dpicst_63000) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD 600 0.005). Samples were incubated in a shaker set at 30°C, 200 r.p.m., and OD 600 was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation monooxygenases in yeast (Green and Large 1983). The resulting methylamine is then transported into the peroxisome for the final demethylation step catalysed by Amo1. The cytosolic Amo2 would therefore play little or no role in the demethylation of internally generated methylamine in Sc. stipitis due to spatial separation between internally generated methylamine in the endoplasmic reticulum and the cytosolic Amo2 enzyme (Fig. 5) (Zwart et al. 1980(Zwart et al. , 1983Green and Large 1984). In the proposed model, extracellular methylamine is demethylated by both Amo1 and Amo2 while methylamine produced through the catabolism of choline, trimethylamine or dimethylamine is predominantly demethylated by Amo1. The intracellular localisation of the Cmo1 choline monooxygenase has not yet been established and its placement in the peroxisome in the current diagram should be considered entirely speculative (see text for details) detectable when cells had been cultivated on dimethylamine, trimethylamine or choline (Haywood and Large 1981), which would support such a hypothesis. Expression analyses of individual genes in Sc. stipitis were beyond the scope of the current study but may be pursued in future studies. The intracellular localisation of the choline monooxygenase Cmo1 has not yet been established but the presence of a conserved C-terminus specific to fungal members of this protein family (Linder 2014) could indicate the presence of a noncanonical type 1 peroxisomal targeting signal (PTS1; Brocard and Hartig 2006). However, the current placement of Cmo1 in the peroxisome by the author as shown in Fig. 5 should be considered entirely speculative. The second general observation is that budding yeasts appear to have at least one additional, as-yet unidentified enzyme capable of converting S-formylglutathione into glutathione (Fig. 3a). Weak but detectable growth in a Dfgh1 background has now been demonstrated both in Sc. stipitis (this study; Fig. 3b) and in the methylotrophic yeast O. boidinii (Yurimoto et al. 2003). The fact that SFA1 is still essential for growth on all tested methylated amines in Sc. stipitis (Fig. 3b) would argue against an entire pathway that is functionally redundant with the cyclic glutathione-dependent formaldehyde oxidation pathway in this yeast. However, this might not be the case in other yeasts such as O. boidinii where there is still detectable growth in a Dsfa1 background (Yurimoto et al. 2003). The third and final observation is that the demethylation of di-and trimethylamine does not appear to involve the CYP protein family in Sc. stipitis. Whether or not this also the case in other budding yeasts remains to be established. Previous studies on the catabolism of secondary and tertiary amines have established that these enzymatic activities are localised to the endoplasmic reticulum in yeast and are sensitive to heme-binding inhibitors such carbon monoxide and cyanide Large 1983, 1984;Fattakhova et al. 1991). One possibility is that a non-CYP family heme-containing enzyme catalyses the demethylation of di-and trimethylamine. The author has so far been unsuccessful in identifying any other likely gene candidates based on sequence similarity and gene annotation data alone. A more straightforward strategy to identify the genes encoding the enzymes responsible for these demethylation reactions would be comparative expression analysis of Sc. stipitis cultivated on di-or trimethylamine versus a non-methylated reference nitrogen substrate. The author would like to add that to the best of his knowledge, the deletion mutants of PICST_49761 and PICST_63000 generated in the present study represents the first attempt at phenotypic characterisation of CYP families CYP5217 and CYP501 in yeast. In conclusion, the yeast Sc. stipitis has shown itself a promising system for the investigation of metabolic pathways not found in Sa. cerevisiae. The fairly recent development of genetic tools for targeted gene deletion in this species now enables more comprehensive reverse genetics studies in this yeast (Maassen et al. 2008;Linder 2014). As the present study demonstrates, there still remains much to be learned about the assimilation of alternative nitrogen sources in budding yeasts.	v3-fos-license
2019-04-08T13:02:39.621Z	2016-02-12T00:00:00.000	99953786	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "http://www.esciencecentral.org/journals/synthesis-characterization-and-electrochemical-properties-of-compositemembrane-by-an-aqueous-solgel-method-2329-6798-1000174.pdf", "pdf_hash": "6bd67786469e15a7ddce62b2d10a19c46f7fb111", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1716", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "5604c02409a76be55c52e636d4651db68dfb70fb", "year": 2016 }	pes2o/s2orc	Synthesis, Characterization and Electrochemical Properties of Composite Membrane by an Aqueous Sol-Gel Method The composite membranes were prepared by sol-gel process, and the membrane potential has been measured for characterizing the ion-transport phenomena across a charged membrane using electrolytes (KCl, NaCl and LiCl). The membrane potential offered by the electrolytes is in the order of LiCl>NaCl>KCl. The results have been used to estimate fixed-charge density, distribution coefficient, charge effectiveness and transport properties of electrolytes of this membrane. The fixed-charge density is the most important parameter, governing transport phenomena in membranes. It is estimated by the TMS method; it is dependent on the feed composition due to the preferential adsorption of some ions. The results indicate that the applied pressure is also an important variable to modify the charge density and, in turn, the performance of membrane. The experimental results for membrane potential are quite consistent with the theoretical prediction. The morphology of the membrane surface is studied by Scanning Electron Micrographs (SEM). Introduction Ion-exchange membranes (IEM) carry the fixed positive or negative charges (called anion-exchange membranes, AEM or cation-exchange membranes, CEM, respectively). They are generally used in the treatment of ionic aqueous solutions, e.g., electrodialytic concentration of seawater, desalination of saline water, demineralization process, acid and alkali recovery and others [1][2][3][4]. Ion-exchange charged membranes, which are now extensively utilized in industries, have attracted considerable attentions due to their extraordinary properties and practical demands and thus a large number of researchers have concentrated on these investigations for many years [5]. With the rapid development of industry and population explosion throughout the world, the demand for fresh water has become increasingly urgent due to the scarcity of drinking water resource and the contamination of industry to environment. Thus, the treatment of industrial wastewater is becoming imperative; while innovative technologies, which are used to prepare fresh water such as the desalination of brackish water and to treat the industrial refuses, have attracted numerous researchers. Among these novel methods, ion-exchange membrane-based technologies have been regarded as both effective and economical due to its lower operation expense and secure process, etc. [6][7][8]. Composite membranes have high thermal and chemical stability, long life and good defouling properties in their application, and they can have catalytic properties [9]. These properties have made these membranes desirable for industrial applications in the food, pharmaceutical and electronic industries. The sol-gel technique is an extremely flexible method to produce inorganic materials with highly homogeneous and controlled morphology [10][11][12]. Recently, due to the mild reaction conditions that can be used, the great potential of sol-gel processes, both hydrolytic and non-hydrolytic, has been extensively investigated for the synthesis of organic/inorganic materials [13]. A potential difference can be observed and measured, at least partly ionically perm selective, membrane in contact with two solutions at following cases: (1) same electrolyte of different concentration; and (2) same ionic strength but different counter-ions or co-ions. The former is called concentration potential and the latter bi-co-ionic/bicounter-ionic potential [14,15]. These potentials are of great interest in connection with the analysis of effective charge density, ionic transport number, and selectivity as well as interaction between charged species and membranes in both single charged membrane and bipolar membranes and thus caused great attention for many years [16][17][18]. For this purpose, a potential model correlating the intrinsic parameters of the membrane and ionic species with transport properties is actually needed and a body of such models has been obtained for single charged membranes and bipolar membranes [19][20][21][22]. It is now recognized that the electrical charge on the pore wall of membranes plays an important role in its separation performance and fouling behavior [23][24][25]. The choice of a membrane with suitable charge or electrical potential property can lead to optimization of existing processes or allow selective separations. For these reasons there is much interest in characterizing the charge or potential property of membranes. The electrical potential difference which is generated when an electrolyte solution flows across a charged membrane under a concentration gradient is among the most convenient experimental techniques for studying such electrical potential properties of porous membranes [26]. In the present investigation, a composite titanium-vanadium phosphate membrane is developed by sol-gel process using polystyrene as a binder. Fixed-charge density, the most effective parameter, has been evaluated and utilized to calculate membrane potentials for different electrolyte concentrations using TMS method [27][28][29][30][31]. In addition to the fixed-charge density, distribution coefficient, transport numbers, mobility, charge effectiveness and other related parameters were calculated for characterizing the composite membrane. Preparation of membrane Titanium-Vanadium phosphate precipitate was prepared by mixing a 0.2 mol titanium (III) chloride (Otto Kemi, India with 99.989% purity) and vanadium (III) chloride (Merck, Germany with 99.989% purity) with 0.2 mol tri-sodium phosphate (E. Merck, India with 99.90% purity) solutions. The precipitate was washed properly with deionized water to remove free electrolytes and then dried at 100°C. The precipitate was ground into fine powder and was sieved through 200 mesh (granule size <0.07 mm). Pure crystalline polystyrene (Otto Kemi, India, AR) was also ground and sieved through 200 mesh. The titanium-vanadium phosphate along with appropriate amount of polystyrene powder was mixed thoroughly using mortar and pestle. The mixture was then kept into a cast die having a diameter of 2.45 cm and placed in an oven maintained at 300°C for about an hour to equilibrate the reaction mixture [32]. The die containing the mixture was then transferred to a pressure device (SL-89, UK), and various pressures such as 80, 100, 120, 140 and 160 MPa were applied during the fabrication of the membranes. As a result titanium-vanadium phosphate membrane of approximate thicknesses 0.095, 0.090, 0.085, 0.080 and 0.075 cm were obtained, respectively. The membranes prepared by embedding 25% of polystyrene by weight were suitable, and the greater or lesser than this weight did not show reproducible results and appeared to be unstable. Membranes prepared in this way were stable and further subjected to microscopic and electrochemical examinations for cracks and homogeneity of the surface. Scanning electron microscopy (SEM) The prepared samples at various pressures was heated in the tabular furnace for 3 hours and then cooled. A very thin transparent polymer glue tape was applied on the sample and then placed on an aluminum stub of 15 mm diameter. Thereafter, the sample was kept in a chamber at a very low pressure where the entire plastic foil containing the sample was coated with gold (60 µm thickness) for 5 minutes. The scanning electron micrograph of gold coated specimen was recorded, operating at an accelerating voltage of 10 kV using the scanning electron microscope (GEOL JSM-840). Measurement of membrane potential The freshly prepared charged membrane was installed at the center of the measuring cell, which had two glass containers, one on either side of the membrane. Both collared glass containers are having a hole for introducing the electrolyte solution and Saturated Calomel Electrodes (SCEs). The half-cell contained 40 ml of the electrolyte solutions. Electrochemical cells of the type C 1 SCE Solution and C 2 Membrane Solution SCE were used for measuring membrane potential using Osaw Vernier Potentiometer. In all measurements, the electrolyte concentration ratio across the membrane was taken as C 2 /C 1 =10. All solutions were prepared by using Analytical Reagent (AR) grade chemicals and ultra-pure distilled water. The electrodes used were saturated calomel electrode and were connected to a galvanometer. The solutions in both containers were stirred by a magnetic stirrer to minimize the effects of boundary layers on the membrane potential. The pressure and temperature were kept constant throughout the experiment and the potentials were measured at 25°C. Results and Discussion The composite membranes using polystyrene as a binder were prepared by sol-gel process. The membranes were found to have the following properties: • They were thermally stable up to 500°C. • They were resistant to compaction. • They were inert to harsh chemical (K 2 Cr 2 O 7 , H 2 O 2 , HNO 3 , H 2 SO 4 , etc.) as they did not decompose in their presence. • They did not show any swelling. • They were stable after long usage, i.e., they were durable. The characterization of membrane morphology has been studied by using SEM [33]. The information obtained from SEM images have provided guidance in the preparation of well-ordered precipitates, composite pore structure, micro/macro porosity, homogeneity, thickness, surface texture and crack-free membranes [34]. The SEM surface images of the composite membranes were taken at different applied pressure and are presented in Figure 1. Inorganic composite membranes have the ability to generate potential when two electrolyte solutions of unequal concentration are separated by a membrane and driven by different chemical potential acting across the membrane [35]. The electrical character of the membrane regulates the migration of charged species, and diffusion of electrolytes from higher to lower concentration takes place through the charged membrane. The values of membrane potential m Ψ ∆ measured across membranes in contact with various 1:1 electrolytes (KCl, NaCl and LiCl) were dependent on concentration of electrolytes present on both sides of the membrane at 25 ± 1°C are given in Table 1. The observed potential was low (mV, +ve). It was found to increase on decreasing the concentration of electrolytes (KCl, NaCl and LiCl), which is a usual behavior of inorganic membranes. The selectivity character of ion-exchange membranes were reported on the basis of membrane potential values, performed on uni-uni and multi-uni valents electrolytes as 1:1, 2:1 and 3:1. The reversal in sign from positive to negative values of membrane potential occurred with the 2:1 and 3:1 electrolytes. This is evidently due to the adsorption of multivalent ions, which led to a state where the net positive charge left on the membrane surface made the anion selective with 2:1 or 3:1 electrolytes. The membrane potential was also seen to be largely dependent on the pressure applied during the membrane fabrication. Application of higher pressure at composite membranes led to reduction in their thicknesses, contraction in pore volume and consequently offered a progressively higher fixed-charge density [36]. The charge property of the membrane matrix greatly influences the counter-ion than co-ion as well as the transport phenomena in the solutions. The surface charge concept of the TMS model for charged membrane is an appropriate starting point for the investigations of actual mechanisms of ionic or molecular processes which occur in membrane phase [27][28][29][30][31]. The TMS model assumes uniform distribution of surface charge and consists of Donnan potential and diffusion potential. According to the TMS, the membrane potential m Ψ ∆ is applicable to an idealized system and is given by Where v and v are the ionic mobilities (m 2 /V/s), of cation and anion, respectively, in the membrane phase. The charge densities of inorganic membranes were estimated from the membrane potential measurement and can also be estimated from the transport number. From the plots in Figure 2, the charge density parameters can be evaluated for a membrane carrying various charge densities, D ≤ 1 for different 1:1 electrolytes systems. The theoretical and observed potentials were plotted as a function of -logC 2 as shown in Figure 2. Thus, the coinciding curve for various electrolytes system gave the value for the charge density D within the membrane phase. Therefore, the increase in the values of D with higher applied pressure is due to successive increase of charge per unit volume as well as the modification in the surface microstructure of the membrane. The plot of charge density D of the membrane for 1:1 electrolytes (KCl, NaCl and LiCl) versus pressures is shown in Figure 3. The order of charge density of various electrolytes is found to be KCl>NaCl>LiCl throughout the range of applied pressure at which the membranes were prepared. The surface charge model may work as a tool to improve the performance of the membrane filtration process. Since, the charge density is an important parameter governing transport phenomena and the charge property of the membrane dominates the electrostatics interaction between the membrane and particles in the feed solution due to the prefential adsorption of some ions. Therefore, by controlling the solution physico-chemistry, the optimum charge property of the membrane can be obtained as desired. The TMS equation (1) The R, T and F have their usual significance; ± ′ ′ γ and ± ′ ′ γ are the mean ionic activity coefficients; v u = ω is the mobility ratio of the cation to the anion in the membrane phase and + 2 C and + 2 C are the cation concentrations in the membrane phase first and second, respectively. The cation concentration is given by the equation Here V k and V x refer to the valency of cation and fixed-charge group on the membrane matrix, q is the charge effectiveness of the membrane and is defined by the equation Where K ± is the distribution coefficient. It is expressed as Where i C the i th ion concentration in the membrane is phase and C i is the i th ion concentration of the external solution. The transport properties of the membrane in various electrolyte solutions are important parameters to further investigate the membrane phenomena as shown in Eq. (7) Equation (7) was first used to calculate the values of transport numbers t + , mobility ratio v u = ω and finally Ū as given in Table 2. The values of mobility ω of the electrolytes in the membrane phase were found to be high at lower concentration of all the electrolytes (KCl, NaCl and LiCl). Further increase in concentration of the electrolytes led to a sharp drop in the values of ω as given in Table 2. The high mobility is attributed to higher transport number of comparatively free cations of electrolytes and also be similar trend as the mobility in least concentrated solution. The values of the parameters K + , q and + C derived for the system have also been included in Table 2. Using Eq. (6) it was found that the values of distribution coefficients increased at lower concentration of electrolytes. As the concentration of electrolytes increased, the values of distribution coefficients sharply dropped and, thereafter, a stable trend was observed as shown in Figure 4. The large deviation in the value of K ± at the lower concentration of electrolytes was attributed to the high mobility of comparatively free charges of the strong electrolyte and thus, reached into the membrane phase easily compared to higher concentrated electrolytes solution. In order to interpret the variation of the charge effectiveness depending on those values, that the ion-pairing effect causes the difference between the effective charge density and the fixed-charge density in membrane phase. In our membrane, counter ion Cl − is same for 1-1 electrolytes therefore, the variation in the q values are follow the similar trend and the order is LiCl>NaCl>KCl up to the C 2 =0.01 mol/l and then drop in the q values were analyzed from Figure 5. When, the external electrolyte concentration is higher or lower, a number of counter ions go into the membrane due to imbalance in the counter ion concentration of external electrolyte and fixed charged group in the membrane phase. Therefore, the ion association with the fixed charged group and counter ions in the membrane is enhanced as a result the charge effectiveness has a lower value whereas in the moderate concentration region the counter ion concentration in the external electrolyte and the fixed-charge density in the membrane are comparable. Therefore, a less number of ion pair formation and consequently higher values of the charged effectiveness, the optimum value of charge effectiveness are obtained at C 2 =0.01 mol/l and then decreased steeply. The order of the charge effectiveness of 1-1 electrolytes may depend on increasing ionic charge density of co-ion adsorption on the charged membranes. The membrane potential derived in this way (theoretical) and the experimentally obtained membrane potentials at different concentrations for various electrolytes systems have been compared and provided in Figure 6. It may be noted that the experimental data follow the theoretical curve quite well. However, some deviations may be due to various non ideal effects, such as swelling effect and osmotic effects. These effects are often simultaneously present in the charged membranes. Conclusion In the present study, the composite membranes were prepared by sol-gel process, and results indicate that the sol-gel approach is appropriate for composite membrane synthesis. The sol-gel technique is an extremely flexible method to produce inorganic materials with highly homogeneous and controlled morphology. The experimental results were analyzed on the basis of the TMS approach, and it was found that the calculated values agree well with the experimental results. The fixed-charge density is the central parameter governing transport phenomena in membranes. The electrical charge on the pore wall of membranes plays an important role in its separation performance and fouling behavior and it depends upon the feed composition and applied pressure due to the prefential adsorption of some ions. The charge effectiveness of membrane is greatly influenced form applied pressure and increase in adsorption of co-ions on charged membrane, order is KCl<NaCl<LiCl. Thus, this membrane can be suited for commercial application.	v3-fos-license
2019-03-30T13:37:32.577Z	2019-03-29T00:00:00.000	85566731	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10295-019-02165-7.pdf", "pdf_hash": "49669092c477de338d002db363e54e4dcf704cf7", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1718", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "sha1": "f93eb4a8a2b00d158e2c6f0050604662dde7ed42", "year": 2019 }	pes2o/s2orc	Towards sustainable bioplastic production using the photoautotrophic bacterium Rhodopseudomonas palustris TIE-1 Bacterial synthesis of polyhydroxybutyrates (PHBs) is a potential approach for producing biodegradable plastics. This study assessed the ability of Rhodopseudomonas palustris TIE-1 to produce PHBs under various conditions. We focused on photoautotrophy using a poised electrode (photoelectroautotrophy) or ferrous iron (photoferroautotrophy) as electron donors. Growth conditions were tested with either ammonium chloride or dinitrogen gas as the nitrogen source. Although TIE-1’s capacity to produce PHBs varied fairly under different conditions, photoelectroautotrophy and photoferroautotrophy showed the highest PHB electron yield and the highest specific PHB productivity, respectively. Gene expression analysis showed that there was no differential expression in PHB biosynthesis genes. This suggests that the variations in PHB accumulation might be post-transcriptionally regulated. This is the first study to systematically quantify the amount of PHB produced by a microbe via photoelectroautotrophy and photoferroautotrophy. This work could lead to sustainable bioproduction using abundant resources such as light, electricity, iron, and carbon dioxide. Electronic supplementary material The online version of this article (10.1007/s10295-019-02165-7) contains supplementary material, which is available to authorized users. Introduction Polyhydroxybutyrates (PHBs) are the most well-studied members of the polyhydroxyalkanoates (PHAs), which is a family of biodegradable intracellular polyesters produced by several bacteria [38,45,67,79,85]. Due to its thermoresistance, moldability, and biodegradability, PHB is a promising substitute for conventional petroleum-derived plastics [11]. Because of its biocompatibility, PHB is also used in many medical applications such as drug delivery, reconstructive surgery and bone tissue scaffolding [48]. However, its production is currently underexploited due to high feedstock costs [67]. Heterotrophic microbes can be promising PHB producers as they can use low-cost carbon sources including food wastes such as sugar beet, soy, and palm oil molasses [69]. However, the requirement for a continuous supply of food wastes makes them an infeasible source of carbon. Additional challenges of using food wastes are its sorting, transport, and pre-treatment prior to utilization [42,54]. Lignocellulose from food [54] and forestry industries [41] or glycerol wastes from biofuel production have also been explored in heterotrophic PHB production [4,75]. Some studies have used pure substrates such as glucose, acetate, and ethanol [79]. However, due to the requirement of arable land, and direct competition with human food consumption, using these substrates for PHB production is not desirable [17]. These potential limitations of using heterotrophs eventually led to the investigation of autotrophs for PHB production [38]. A handful of studies have demonstrated autotrophs as efficient PHB producers over heterotrophs [30]. A chemoautotrophic hydrogen-oxidizing bacterium Ideonella spp. strain O-1 has been shown to produce PHB using industrial exhaust gas containing hydrogen (H 2 ), carbon dioxide (CO 2 ), and carbon monoxide (CO). The exceptional ability of strain O-1 to grow even at CO concentration of 70% (v/v) without suppression of PHB production made it an attractive Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1029 5-019-02165 -7) contains supplementary material, which is available to authorized users. 1 3 candidate to produce PHB using industrial exhaust gas (rich in CO) [76]. However, this route of PHB production may not be ideal because of the high cost of operation, and the risk of explosion associated with the use of H 2 [14]. A sulfatereducing bacterium Desulfococcus multivorans has also been shown to produce PHB [26], but its slow growth rate can make this process inefficient. To make PHB bioproduction more efficient, economically viable and sustainable, research on autotrophic PHB production was further extended to photoautotrophs. The ability of photoautotrophs to use solar energy and CO 2 for biosynthesis makes them unique candidates for efficient PHB synthesis [19,34,74]. Under photoautotrophic growth conditions, CO 2 is fixed using energy harvested from light to generate ATP [9]. The fixed carbon can be used for the biosynthesis of acetyl-CoA, a substrate for PHB synthesis. In addition, photoautotrophs capable of fixing dinitrogen gas (N 2 ) using ATP generated by photosynthesis are even more desirable. Moreover, nitrogen limitation has been reported to increase PHB accumulation [34]. Indeed, a recent study reported the suitability of the photoautotrophic organism Synechocystis sp. PCC 6714 as a potential host strain for PHB production [34]. However, the PHB amount based on cell mass and volumetric productivity was very low [19]. To produce higher PHB with greater efficiency, research on bacterial PHB synthesis was further expanded to microbial electrosynthesis (MES). This approach is based on the ability of some autotrophs (also called electroautotrophs) to acquire electrons from solid-phase conductive substances (SPCSs) such as electrodes using them as electron donors. This process of using SPCSs as electron donors or acceptors is termed "extracellular electron transfer (EET)" [20,23,36,66]. When microbes use SPCSs as electron donors, the form of EET they use is also called microbial electron uptake (EU). This capability of electroautotrophs has been leveraged to produce value-added multi-carbon products via MES by reducing CO 2 via either indirect or direct EU. In direct EU, microorganisms attach to the electrode and directly take up electrons from them [3,6,25,52,53,65,70,78,87,88], whereas indirect EU involves transport of electrons by diffusible electron careers such as H 2 , formate or ammonia (either produced electrochemically or added to the reactors) from the electrode to microbes [13,18,28,33,39,43,58,62,68,72,80]. Indirect EU has been successfully used for PHB bioproduction by the chemoautotroph Cupriavidus necator (previously named Ralstonia eutropha). Nishio et al. reported that PHB productivity in C. necator was enhanced by EET using a biocompatible mediator (2-poly (2-methacryloyloxyethyl phosphorylcholine-co-vinylferrocene) (PMF) in an electrochemical system with an anode that was poised at + 0.6 V vs. the standard hydrogen electrode (SHE). Here, the anode served as an additional electron acceptor for microbial metabolism, resulting in acceleration of glycolysis and hence PHB synthesis [55]. Indirect MES of PHB by C. necator using formate as an electron carrier has also been reported recently [12]. To enhance CO 2 assimilation by C. necator, a formate dehydrogenase (FDH)-assisted MES system was constructed, in which FDH catalyzed the reduction of CO 2 to formate in the cathodic chamber. Formate served as the electron carrier to transfer electrons into C. necator generating PHBs [12]. The involvement of mediators in indirect EU lowers the efficiency of product formation. Direct EU in the context of MES is desirable because it omits the extra steps involved in indirect EU [12,55]. Although a thermodynamic evaluation of bacterial PHB production via MES was proposed nearly a decade ago [64], no pure microbial culture has been known to produce PHBs using MES via direct EU (i.e., without the use of mediators). Due to the abundance of iron on earth [24], PHB production linked to autotrophy using ferrous iron, Fe(II), as an electron donor could be leveraged for sustainable PHB production. Some Fe(II)-oxidizing chemoautotrophs such as Gallionella ferruginea have been reported to accumulate PHBs intracellularly. However, quantitative measurements on PHB production have not been reported for this organism [27,46,83]. The use of oxygen as the terminal electron acceptor by these organisms is a challenge because oxygen reacts readily with Fe(II) to oxidize and precipitates it to Fe(III). Therefore, G. ferruginea can only oxidize iron under low-oxygen concentrations [46]. Photoautotrophs such as purple bacteria and green sulfur bacteria have been shown to oxidize Fe(II) while fixing CO 2 using light via a process called photoferroautotrophy [21,73]. Photoferroautotrophs are more attractive for PHB synthesis because they oxidize Fe(II) in the absence of oxygen. However, thus far PHB accumulation has not been demonstrated during photoferroautotrophic growth. Here, Rhodopseudomonas palustris TIE-1 was chosen as a platform for PHB bioproduction because it demonstrates extraordinary metabolic versatility [6,32]. TIE-1 can grow chemoheterotrophically in rich medium as well as photoheterotrophically using various organic carbon sources [32]. It can also use several inorganic electron donors such as H 2 and thiosulfate for photoautotrophic growth [32]. More importantly, TIE-1 is the only genetically tractable bacterium that has the ability to perform photoautotrophy using inorganic electron donors such as Fe(II) (photoferroautotrophy) and a poised electrode (photoelectroautotrophy) [6,32]. TIE-1 performs direct EU to support photoelectrotrophy [6,65]. These exceptional abilities make TIE-1 a very promising candidate to study photoautotrophic PHB production under different growth conditions. We assessed PHB production quantitatively on several growth conditions and found that TIE-1 can produce PHBs both photoautotrophically and photoheterotrophically. Among the photoautotrophic growth conditions, the highest PHB electron yield [percentage of the electron (mol) from the substrate that was converted into PHBs] was obtained under photoelectroautotrophy, and the highest specific PHB productivity was obtained under photoferroautotrophy. These novel routes of PHB synthesis by TIE-1 can potentially serve as a stepping stone for future bioengineering efforts towards sustainable PHB bioproduction. Bacterial strain, media, and growth conditions Rhodopseudomonas palustris TIE-1 was originally isolated by Jiao et al. and has been used throughout this study [32]. For aerobic chemoheterotrophic growth, TIE-1 was routinely grown in 0.3% yeast extract and 0.3% peptone (YP) medium, with 10 mM MOPS [3-N (morpholino) propanesulfonic acid] at pH 7 in the dark at 30 °C with shaking at 250 rpm. For growth on solid medium, YP medium was solidified with 1.5% agar supplemented with 10 mM MOPS and 10 mM sodium succinate. For anaerobic photoautotrophic growth, TIE-1 was grown in anaerobic bicarbonatebuffered freshwater (FW) medium [21] supplemented with ammonium chloride (NH 4 Cl) (5.61 mM) or mixed N 2 /CO 2 (80%/20%) gas at a pressure of 34.5 kPa as the sources of nitrogen. For anaerobic photoheterotrophic growth, 10 mL of FW medium was supplemented with anoxic 1 M stocks of sodium succinate, sodium butyrate and sodium 3-hydroxybutyrate to a final concentration of 1 mM in Balch tubes. However, to have higher biomass required for PHB, RNA and protein extraction, substrate concentrations were increased to 10 mM. A pre-grown TIE-1 culture with optical density (OD 660 ) of 1 was inoculated with a final OD 660 of 0.01 (100 × dilution) followed by incubation at 30°C in an environmental chamber fitted with infrared LED (880 nm). Time-course cell growth was monitored using Spectronic 200 (Thermo Fisher Scientific, USA). For photoautotrophic growth with H 2 and Fe(II), TIE-1 was adapted to photoautotrophic growth using H 2 as the sole electron donor as described previously [7]. For growth with Fe(II), 50 mL of FW medium was prepared under the flow of 34.5 kPa N 2 / CO 2 (80%/20%) and dispensed into pre-sterilized serum bottles purged with 34.5 kPa N 2 /CO 2 (80%/20%). The bottles were then sealed using sterile butyl rubber stoppers with aluminum crimp followed by the addition of anoxic sterile stocks of FeCl 2 and nitrilotriacetic acid (NTA) to a final concentration of 5 mM and 10 mM, respectively. All sample manipulations were performed inside an anaerobic chamber with 5% H 2 /75% N 2 /20% CO 2 (Coy laboratory, Grass Lake) [7]. The bacterial generation time was determined as described previously [77]. Lag time (lag) was determined as a period that precedes the exponential phase [47]. Cell enumeration Samples were fixed with paraformaldehyde (20% v/v), transferred into Amicon centrifuge filters (Amicon Ultracel 100 k, regenerated cellulose membrane, Millipore, Carrigtwohill, CO, Ireland) and centrifuged for 10 min at 1000×g. The pellets were resuspended and washed twice in PBS (phosphate-buffered saline). The cells were recovered by centrifugation at 3000×g for 15 min. After the addition of PicoGreen ® (Quant-iT PicoGreen ® dsDNA, Life Technologies, Grand Island, NY, USA), the cells were counted in 96-well plates along with 50 μL of Sphero™ AccuCount blank beads (Spherotech, Lake Forest, IL, USA). Cell density was estimated with an LSRII flow cytometer (BD, Sparks, MD, USA) using a 488-nm laser. A calibration curve relating the ratio of cell events to bead events and the cell density was constructed using a serial dilution of a cell sample. Density was then determined by microscopy (Helber Bacteria Cell counting chamber with Thoma ruling, Hawksley, Lancing, Sussex, UK). The OD 660 of TIE-1 cells vs. cell numbers were plotted to obtain a standard curve. Bioelectrochemical setup and growth conditions All photoelectroautotrophic experiments were performed using a three-electrode configured seal-type bioelectrochemical cell (BEC, C001 Seal Electrolytic cell, Xi'an Yima Opto-electrical Technology Com., Ltd, China). The three electrodes were configured as the working electrode (graphite rod, 3.2 cm 2 ), reference electrode (Ag/AgCl in 3 M KCl) and counter electrode (Pt foil, 5 cm 2 ). 70 mL of FW medium was dispensed into sterile BECs and made completely anaerobic by N 2 /CO 2 (80%/20%) bubbling for 60 min with the final pressure maintained at ~ 50 kPa. 10 mL of TIE-1 cells (OD 660 ~ 2.4) pre-grown in FW with H 2 was then inoculated with a starting OD 660 ~ 0.3 as described previously [65]. The OD 660 of the inoculated BECs was monitored with a BugLab Handheld OD Scanner (Applikon Biotechnology, Inc., Foster City, CA). To evaluate the influence of NH 4 Cl and N 2 gas as the nitrogen sources on PHB biosynthesis via photoelectroautotrophy, the BECs were operated simultaneously (c = 3 biological replicates) with NH 4 Cl and N 2 gas as nitrogen sources with negative controls: open-circuit (OC) control (no current) and abiotic controls. The graphite electrode was constantly poised at a potential of + 100 mV vs. standard hydrogen electrode (SHE) for 130 h using a multichannel potentiostat (Interface 1000E, Gamry Multichannel Potentiostat, USA). All photoelectroautotrophic experiments were performed at 26 °C under continuous infrared light (880 nm) unless noted otherwise. At the end of the bioelectrochemical experiment, samples were immediately collected from the BEC reactors for RNA extraction and PHB production analysis as mentioned above. Analytical procedures PHB measurement From all the growth conditions tested, 10 mL of bacterial samples at an OD 660 0.7 (unless stated otherwise) was pelleted at 8000×g for 10 min and stored at − 80 °C until PHB extraction and analysis were performed. 1 mL of water (LC-MS grade) and 600 µL of methanol (HPLC grade) were added to arrest metabolic activity of TIE-1. 10 mg/mL of poly[(R)-3-hydroxybutyric acid] (Sigma-Aldrich, USA) was used as a PHB standard. Extraction of PHB was followed by its conversion to crotonic acid. The concentration of crotonic acid was measured using an Agilent Technologies 6420 Triple Quad LC/MS as follows: using Hypercarb column, particle 5 µm, 100 × 2.1 mm (Thermo Fisher Scientific, USA) as stationary phase; water with 0.1% (v/v) formic acid as phase A; acetonitrile and 1% (v/v) formic acid as phase B. The injection volume was 5 µL; the flow rate was set at 500 µL min −1 ; the column temperature was set at 15 °C and the gas temperature was 300 °C [31]. PHB was detected as crotonic acid with mass to charge ratio (m/z) = 87 which was normalized to bacterial cell number. Details on PHB extraction, PHB carbon yield, and PHB electron yield calculations are described in supplemental methods. H 2 and CO 2 measurement Time-course H 2 and CO 2 from photoautotrophic conditions were analyzed using gas chromatography (Shimadzu BID 2010-plus, equipped with Rt ® -Silica BOND PLOT Column: 30 m × 0.32 mm; Restek, USA) with helium as a carrier gas. At each time point, 10 µL of gas was sampled from the headspace of the serum bottles using a Hamilton™ gas-tight syringe and injected into the column. To quantify dissolved CO 2 , 1 mL of filtered (using 0.22 µm PES membrane filter) aqueous samples from each reactor was collected and injected into helium-evacuated 12-mL septum-capped glass vials (Exetainer, Labco, Houston, TX, USA) containing 1 mL of 85% phosphoric acid. The concentration of the dissolved CO 2 was then measured by injecting 10 µL of evolved CO 2 in the headspace into the column. The total CO 2 in the reactors was calculated as described previously [50]. Organic acid measurement Time-course consumption of organic acids such as sodium succinate, sodium butyrate, and sodium 3-hydroxybutyrate under photoheterotrophic conditions were quantified using an Ion Chromatography Metrohm 881 Compact Pro using a Metrosep organic acid column (250 mm length). 0.5 mM H 2 SO 4 with 15% acetone was used as eluent at a flow rate of 0.4 mL min −1 with suppression (10 mM LiCl regenerant). Fe(II) measurement Time-course Fe(II) concentration was measured using the Ferrozine Assay as described previously [7]. Total protein measurement Total protein during photoferroautotrophy was measured using trichloroacetic acid (TCA) precipitation as follows: total protein from 2 mL culture (at time point zero and at 192 h for the growth with NH 4 Cl and 360 h with N 2 gas) in microcentrifuge tube was precipitated using 500 µL 100% TCA. This mixture was incubated for 10 min at 4 °C and centrifuged at 18,000×g for 30 min at 4 °C. The pellet was washed with 200 µL cold acetone and centrifuged at 18,000×g for 10 min at 4 °C. The pellet was then dried at 95 °C for 10 min to remove any residual acetone and resuspended in 50 µL HCl buffered with 100 mM Tris-Cl, pH 8.0. The BCA (bicinchoninic acid) Protein Assay Kit was employed using the microtiter plate method for protein estimation as specified by the manufacturer's protocol following TCA precipitation (Thermo Scientific, Waltham, MA). Total protein was measured at an absorbance of 562 nm using the Biotek Synergy HTXmicrotiter plate reader [7]. For a total protein to OD 660 conversion, total protein of known OD 660 values of TIE-1 cells was quantified. A standard curve was obtained by plotting OD 660 vs. total protein measured. RNA extraction and sequencing 5 mL of bacterial culture were collected at an OD 660 ~ 0.7. The RNA was stabilized using 5 mL RNAlater (Qiagen, USA) (buffer that stabilizes and protects RNA from degradation) and incubated at room temperature for 10 min. Bacterial cells were centrifuged at 5000 × g for 10 min and pellets were stored at − 80 °C until RNA extraction was performed. RNA extraction was performed using the RNeasy Mini Kit (Qiagen, USA) following the manufacturer's protocol. DNA removal was performed using the Turbo DNA-free Treatment and Removal Kit (Ambion, USA). DNA contamination was tested using PCR using the primers listed in Table S1 as previously described [6,7]. Illumina unpaired 150-bp libraries were prepared and sequenced at the Genome Technology Access Center, Washington University on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Trimmomatic (version 0.36) was used to remove Illumina sequencing adapters, quality trim deteriorating bases (threshold = 20), and length filter (min = 60 bp) [5]. Preprocessed RNA-seq reads were mapped to the published R. palustris TIE-1 genome using TopHat2 (version 2.1.1) (https ://genom ebiol ogy.biome dcent ral.com/artic les/10.1186/gb-2013-14-4r36) and the gff3 annotation file as a guide for sequence alignment. Bowtie 2 (version 2.3.3.1). (https ://www.ncbi.nlm.nih.gov/pmc/artic les/PMC33 22381 /) was used to index the reference genome FASTA file. The number of reads mapping to each feature was counted by HTSeq (version 0.9.1). Differentially expressed genes were predicted in DESEQ 2 (version 1.16.1) using the HTSeq (https ://www.ncbi.nlm.nih.gov/pubme d/25260 700) read counts and an adjusted p value cutoff of 0.05. Heat maps were drawn in R using ggplot2 [44,82]. Reverse transcription quantitative PCR analysis (RT-qPCR) cDNA template was synthesized using the purified RNA samples using the iScript cDNA Synthesis Kit (Biorad, USA). Primers listed in (Table S2) were designed using primer3 software (http://bioin fo.ut.ee/prime r3/). RT-qPCR was performed using Biorad CFX connect Real-Time System Model # Optics ModuleA using the following thermal cycling conditions: 1 cycle at 95 °C for 3 min and 30 cycles of 95 °C for 3 s, 60 °C for 3 min, and 65 °C for 5 s according to the manufacturer's protocol. Fold change comparison and standard deviation calculations were performed as described previously [2]. Identification of PHB cycle genes of TIE-1 The available TIE-1 genome in the JGI Genome Portal (https ://genom e.jgi.doe.gov/) was used to search for homolog genes involved in the PHB cycle using Blast search. Scanning transmission electron microscopyelectron energy loss spectroscopy (STEM-EELS) TIE-1 grown under sodium butyrate, Fe(II)-NTA and poised graphite electrode was used as representative samples for STEM-EELS. Briefly, 5 mL planktonic cell suspensions were centrifuged at 6000×g for 5 min. followed primary fixation by resuspending the cells pellets in 2% formaldehyde and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for ~ 45 min at room temperature. After agar encapsulation followed by primary fixation for ~ 20 min, agar cubes were subjected to secondary fixation for ~ 5 h followed by acetone dehydration and resin infiltration. Ultrathin sections (~ 50-60 nm) were obtained using Reichert Ultracut UCT ultramicrotome (Donald Danforth Plant Science Center, Saint Louis, MO), then mounted directly on amorphous-carbon film-coated TEM Cu-grids. Intracellularly localized PHB granules were characterized using a JEOL JEM-2100F field emission scanning transmission electron microscopy (FE-STEM) with an accelerating voltage of 200 keV (Institute of Material Science and Engineering, WUSTL); the microscope is attached with a Gatan 805 BF/DF detector, Gatan 806 HAADF detector and Gatan 863 Tridiem imaging filter (GIF) system. Images were obtained in STEM mode using HAADF detector and BF detector. EELS spectral images were acquired through working HAADF and GIF jointly. Carbon-K edge and nitrogen-K edge elemental maps were retrieved from STEM-EELS spectral images. Statistical analysis The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment. For the pairwise test with Bonferroni adjustment, the cutoff P value is equal to 0.025 Nucleotide sequence accession numbers All RNAseq datasets have been deposited in NCBI under BioProject accession number PRJNA417278. Results and discussion Rhodopseudomonas palustris TIE-1 possesses putative PHB cycle genes PHB production has been previously reported by several R. palustris strains [15,51,84]. However, the PHB cycle genes have not been explored thus far. This lack of information critically limits the potential prospects for future bioengineering efforts for PHB bioproduction. Availability of the TIE-1 genome allowed us to identify the genes that are homologous to the PHB cycle genes of C. necator [59,60,81], an H 2 -oxidizing betaproteobacterium that is known to produce and sequester PHAs intracellularly [8]. Reconstruction of the PHB cycle in E. coli using PHB genes from C. necator has been previously used to elucidate the biochemical pathway of PHB production and its subsequent metabolism [81]. Briefly, the pathway starts with the condensation of two acetyl-CoAs into acetoacetyl-CoA, a reaction driven by the β-ketothiolase enzyme, PhaA [81] (Fig. 1a). Acetoacetyl-CoA gets reduced to (R)-3-hydroxybutyryl-CoA by the enzyme acetoacetyl-CoA reductase, PhaB. Eventually, PHB polymerization is achieved by the PHB polymerase, PhaC (Fig. 1a). Interestingly, PHB can also serve as an important source of carbon and energy during environmental stress, where PHB molecules are catabolized by the PHB depolymerase, PhaZ (Fig. 1a) [81]. In Bradyrhizobium diazoefficiens, a nitrogen-fixing symbiont closely related to TIE-1, PhaR represses the expression of phaC 1 and phaC 2 . In addition, PhaR regulates PhaP, the phasin protein that binds to and controls the number and size of the PHB granules. PhaR also binds to PHB granules and dissociates from it as the granule size grows [63]. TIE-1 encodes one phaR gene (Rpal_0531); one phaZ gene (Rpal_0578); multiple copies of phaA and phaB; two copies of phaC (Rpal_2780 and Rpal_4722); and three genes for phaP 2 (Rpal_4291, 4616 and 4617) (Fig. 1a, b). A phaB gene (Rpal_0533) is located next to the phaA gene (Rpal_0532) (Fig. 1b) forming a putative operon (Fig. 1b). The gene for phaR (Rpal_0531) 1 3 is positioned next to the phaA (Rpal_0532) gene in this operon but expressed from the opposite strand. This shows that TIE-1 possesses all the necessary genes for both PHB biosynthesis, polymerization and depolymerization. TIE-1 produces PHB under photoautotrophic conditions using different electron donors Photoautotrophic PHB production using H 2 as an electron donor TIE-1 was grown with H 2 as the sole electron donor with N 2 and NH 4 Cl as fixed nitrogen sources. We call the N 2 fixing conditions as the electron donor-N 2 system and the NH 4 Cl conditions as the electron donor-NH 4 Cl system throughout. TIE-1 showed a higher maximum OD 660 of 1.16 (P ≤ 0.001, Table 1) with NH 4 Cl (H 2 -NH 4 Cl system) compared to OD 660 of 0.54 with N 2 (H 2 -N 2 system). This growth defect observed in H 2 -N 2 was also reflected in the longer generation time of 41 h in the H 2 -N 2 system compared to 34 h in the H 2 -NH 4 Cl system (P =0.005, Table 1). Slow growth under N 2 fixing conditions was previously observed in R. palustris strain 42 OL [15] and is likely due to the high-energy requirement of this process. TIE-1 showed a lower PHB carbon yield [percentage of carbon (mol) from the substrate that was converted into PHBs] of 2.55% in the H 2 -N 2 system compared to the ~ 3 times higher yield of 7.23% in the H 2 -NH 4 Cl system. In contrast, no significant difference was observed in PHB electron yield [percentage of the electron (mol) from the substrate that was converted into PHBs] between the H 2 -N 2 system and the H 2 -NH 4 Cl system (Fig. 2a, Table 2). Interestingly, the specific PHB productivity almost doubled in the H 2 -N 2 system compared to the H 2 -NH 4 Cl system (from 1.30 × 10 −14 to 3.08 × 10 −14 mg/L/Cell/h) ( Table 2). The higher specific productivity under N 2 fixing conditions and the growth defect (Table 1) indicate a direct impact of stress caused by the high-energy-consuming N 2 fixation process. This stress might have induced TIE-1 to accumulate intracellular PHBs as a carbon and energy reserve. Similar observations have been reported for other purple non-sulfur bacteria [29] grown under nitrogen-limited conditions, including R. palustris strains grown in nitrogen-deprived conditions [51]. Increased accumulation of PHB to over 30% of the dry cell weight was observed previously when R. palustris CGA009 cells were N 2 starved [51]. Because H 2 is known to be produced during N 2 deprivation by CGA009 [51], this H 2 could serve as an additional electron donor for photoautotrophy, accounting for additional PHB accumulation. Further studies will be required to test whether this is happening in CGA009 and TIE-1. Although the growth with NH 4 Cl had N 2 gas in the headspace under most conditions in our experiment, the presence of NH 4 Cl has been known to inhibit nitrogenase gene expression, preventing N 2 fixation from occurring in the presence of NH 4 Cl [40]. Photoautotrophic PHB production using a poised electrode as an electron donor To evaluate PHB production under photoelectroautotrophy using poised electrodes as the sole electron donor, graphite electrodes were poised at a potential + 100 mV vs. SHE to mimic the Fe(OH) 3 /Fe 2+ redox couple. Although the electrode-NH 4 Cl system resulted in higher maximum OD 660 compared to the electrode-N 2 system (0.73 vs. 0.61, respectively) ( Table 1), there was no significant difference in the generation time of TIE-1 between these two conditions ( Fig. 3a; Table 1). Nonetheless, after 96 h of incubation, EU by TIE-1 in the electrode-NH 4 Cl system was 1.92 µA/cm 2 , about double of that obtained in the electrode-N 2 with an EU of 0.93 µA/cm 2 (Fig. 3b, Table S6, P ≤ 0.01). Despite the difference in the total EU, no notable change was observed in the PHB carbon yield, PHB electron yield and specific PHB productivity between the electrode-NH 4 Cl system and the electrode-N 2 system ( Fig. 2a; Table 2). This result could be due to the continuous supply of electrons from a poised electrode, which did not seem to directly impact PHB biosynthesis. As expected, no cell growth was observed in the biotic reactors with unpoised electrodes (open circuit, OC) with both NH 4 Cl and N 2 gas (Fig. 3a). Abiotic controls did not show any EU (Fig. 3b). Bioelectrosynthesis of PHB has been recently reported via a method using enzymatic and electrochemical approaches. A modified electrode poised at − 386 mV vs. SHE was used to synthesize NADH in the presence of enzymes of the PHB cycle to convert acetate to PHB. The amount of PHB produced was 0.3 mg/L (under a maximum current density (J max ) of 27.9 ± 1.3 μA cm −2 ) [1]. In another study, 226 ± 6 mg/L of PHB was produced via indirect EU using formate as a mediator by C. necator (Ralstonia eutropha) at − 395 mV vs. SHE (~ J max 213 μA cm −2 ) [12]. In addition, overexpression of the ruBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) gene was performed to increase the CO 2 fixation [12]. As a result, PHB production was enhanced to 485 mg/L [12]. However, both of these approaches involve multiple steps including enzyme purification. They also operate at a higher reduction potential than what we use to grow TIE-1 for PHB production (+ 100 mV vs. SHE, J max of only<2 μA cm −2 ). Although our results show a low (~ 5-6 mg/L) amount of PHB production under photoelectroautotrophy via direct MES, our approach offers numerous advantages: (1) using direct EU, we minimize the complexity of the MES system; (2) TIE-1 grows at a lower reduction potential than that used in the studies above. This lower reduction potential ultimately saves electrical energy; and (3) TIE-1 is a photoautotroph and, therefore, can use the energy of light to make excess ATP for biosynthesis. The photoautotrophic ability of TIE-1 makes is especially attractive for sustainable PHB bioproduction because light is an abundant resource. The major hurdle for using TIE-1 for bioproduction is the low electron uptake it demonstrates from graphite electrodes. This is reflected in the lower maximum current density (J max ) values that were observed here in our study. Improving electron uptake would increase J max values, which would also increase bioproduction [65]. A previous study from our laboratory has shown that inexpensive electrode modifications such as coating the electrodes with Prussian Blue can enhance electron uptake in the absence of a mediator via direct EU [65]. We are pursuing reactor design and electrode modifications further to enhance direct EU by TIE-1 because that will ultimately improve product formation. Photoautotrophic PHB production using ferrous iron as an electron donor Specific PHB productivity was higher in the H 2 -N 2 compared to the H 2 -NH 4 Cl systems (Table 2) but no difference was observed under photoelectroautotrophy. However, under photoferroautotrophy, the specific PHB productivity was higher in the Fe(II)-NH 4 Cl system compared to the Fe(II)-N 2 ( Table 2). In addition, PHB carbon yield decreased in the Fe(II)-N 2 system compared to the Fe(II)-NH 4 Cl system ( Fig. 2a; Table 2). By carefully examining Fe(II) oxidation in Fe(II)-NH 4 Cl system, a significant drop in Fe(II) concentration via microbial Fe(II) oxidation was observed during the first 96 h, and Fe(II) was completely oxidized by 384 h (Fig. 4a). In contrast, the Fe(II)-N 2 system showed a very slow Fe(II) oxidation where a significant drop in Fe(II) concentration was observed only after 360 h (Fig. 4b). In addition, there was a decrease in the maximum OD 660 in the Fe(II)-N 2 system compared to the Fe(II)-NH 4 Cl system (Table 1). Additionally, total protein concentration with the Fe(II)-NH 4 Cl nearly doubled after 192 h whereas, with the Fe(II)-N 2 , the total protein concentration doubled only after 360 h (Fig. 4a, b). Based on these results, it is plausible that the growth defect in the Fe(II)-N 2 system is a consequence of the high-energy demand during N 2 fixation (Table 1; Fig. 4). Comparison of PHB production under photoautotrophic conditions Among the three electron donors tested, photoautotrophic growth with H 2 showed the highest maximum OD 660 when grown with NH 4 Cl ( Table 1, P ≤ 0.001). Although the Fe(II)-NH 4 Cl and the Fe(II)-N 2 conditions resulted in the lowest maximum OD 660 (Table 1, P ≤ 0.001), the . Error bars are from the standard deviations calculated using three biological replicates. b Current density during photoelectroautotrophy. Current density (µA/cm 2 ) from TIE-1 grown with freshwater medium with NH 4 Cl as a nitrogen source or with N 2 gas as a nitrogen source using a poised electrode at a potential of + 100 mV vs. Standard hydrogen electrode (SHE) and the associated abiotic control. The negative sign on the Y-axis indicates current uptake. Error bars are the standard deviations calculated using two biological replicates. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment; ns not significant. For a pairwise test, the cutoff P value is 0.025. P values are indicated in Table S5 Fig . 4 Growth of TIE-1 under photoferroautotrophic conditions. Fe(II) oxidation and protein concentration measured during photoferroautotrophy a with NH 4 Cl as a nitrogen source, and b with N 2 gas as a nitrogen source. Error bars are from the standard deviations calcu-lated using three biological replicates. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment; ns not significant. For a pairwise test, the cutoff P value is 0.025). P values are indicated in Table S5 PHB carbon yield under the Fe(II)-NH 4 Cl is comparable to that obtained from photoautotrophy in the H 2 -NH 4 Cl system (Table 2, P =0.0613) and higher than under the electrode-NH 4 Cl ( Table 2, P = 0.028). Moreover, the specific PHB productivity obtained in the Fe(II)-NH 4 Cl is the highest amongst all the photoautotrophic conditions with NH 4 Cl ( Table 2, P ≤ 0.001). The electrode-N 2 system showed the longest generation times amongst all the photoautotrophic growth conditions (Table 1, P ≤ 0.001). Photoelectroautotrophy showed the lowest PHB carbon yield both with NH 4 Cl and N 2 (Fig. 2a, P = 0.0045). Nevertheless, TIE-1 showed the highest efficiency in converting electrons to PHB in the electrode-NH 4 Cl system (4.39% PHB electron yield) and in the electrode-N 2 system (7.34% PHB electron yield) ( Table 2, P ≤ 0.001). These results indicate that although the growth of TIE-1 during photoelectroautotrophy was slow, this condition was the most efficient at converting electrons obtained from a poised electrode into PHB. A previous comparative growth study of TIE-1 using H 2 and soluble Fe(II) as electron donors revealed H 2 as a preferred electron donor over Fe(II) [32]. This preference was reflected in the lower PHB carbon yield during photoferroautotrophy vs. photoautotrophy with H 2 (Fig. 2; Table 2). Effect of N 2 fixation on photoautotrophic PHB production The effect of N 2 fixation was clearly observed in both H 2 and Fe(II) systems, where cell growth and the PHB carbon yield was significantly reduced under N 2 fixing conditions (Tables 1, 2). Based on previous studies, it is likely that the stress caused by the high-energy N 2 fixation process led to the accumulation of PHB by TIE-1 [29,51]. However, N 2 fixation did not have a significant impact on the growth of TIE-1, the specific PHB productivity and the PHB carbon yield under photoelectroautotrophy (Table 2). A continuous electron supply might have allowed TIE-1 to fix N 2 to ammonium without affecting the supply of electrons to produce PHBs. Interestingly, PHB biosynthesis was proposed to be a potential electron sink when Rhodopseudomonas palustris CGA009 was incubated in the presence of argon (nitrogen deprived) [51]. However, our results show that only a small percentage of electrons go to PHB biosynthesis under all N 2 fixing conditions ( Table 2). In the electrode-N 2 system, where the maximum PHB electron yield was obtained, only 7.34% of the available electrons contributed to PHB biosynthesis. Chemoheterotrophic PHB production To further investigate the effect of different media on PHB production, TIE-1 was grown chemoheterotrophically (aerobic) with rich media containing yeast extract and peptone (YP). Aerobic growth of TIE-1 on YP resulted in the longest generation time (g = 11.18 h) compared to the generation time observed from all the other photoheterotrophic conditions (Table 1, P ≤ 0.001). YP-grown cells produced the highest specific PHB productivity of 24.60 × 10 −14 mg/L/Cell/h compared to all the conditions tested ( Table 2, P ≤ 0.001). It is likely that the amino acids provided by peptone contributed to the increased PHB production by TIE-1 under this condition. The availability of amino acids precludes their de novo biosynthesis, and peptone has been previously reported to increase PHB production in Azotobacter vinelandii [56]. Photoheterotrophic PHB production with succinate To test the effect of different carbon sources with different oxidation/reduction values (Supplemental Table S4) on PHB production, TIE-1 was further grown photoheterotrophically (anaerobic) using three different substrates: succinate, butyrate, and 3-hydroxybutyrate with NH 4 Cl and N 2 gas. Similar to photoautotrophy, use of N 2 gas as the source of nitrogen resulted in longer lag time, longer generation time and a longer time to reach maximum OD 660 compared to NH 4 Cl as the nitrogen source (Table 1). However, there was no significant difference in specific PHB productivity between the succinate-N 2 system and the succinate-NH 4 Cl system ( Table 2). Rather there was an increase in the PHB carbon yield and PHB electron yield in the succinate-N 2 system compared to the succinate-NH 4 Cl system ( Fig. 2b; Table 2) indicating that there was no significant effect of N 2 fixation on PHB productivity (Table 2). Photoheterotrophic PHB production with butyrate and 3-hydroxybutyrate Interestingly, TIE-1 grown photoheterotrophically with the less oxidized substrate, butyrate, showed a decrease in maximum OD 660 (0.42) in the butyrate-NH 4 Cl compared to 0.69 with the butyrate-N 2 systems (Table 1). An increase of about ninefold in PHB carbon yield was obtained in the butyrate-N 2 system compared to the butyrate-NH 4 Cl system ( Fig. 2b; Table 2). Similarly, specific PHB productivity in the butyrate-N 2 system was more than three times higher compared to the butyrate-NH 4 Cl system ( Table 2). The highest PHB production (~ 17.1 mg/L), as well as the highest PHB carbon yield, was obtained in the butyrate-NH 4 Cl system ( Table 2). Although this PHB production is lower than the PHB production previously reported by another photosynthetic purple bacterium, Rhodobacter sphaeroides (60 mg/L) grown in olive mill wastewater under N 2 -limited conditions [22], TIE-1's ability to produce PHB under various conditions such as photoautotrophy offers an obvious advantage when considering mixotrophic (photoheterotrophy and photoautotrophy) growth conditions for PHB production. Butyrate has been previously reported to be a preferred substrate over acetate in a PHB-producing mixed culture dominated by Plasticicumulans acidivorans due to the lower ATP need for PHB production using butyrate [49]. In addition, a study performed on C. necator has shown that butyrate is metabolized into 3-hydroxybutyryl-CoA via the beta-oxidation pathway. 3-Hydroxybutyryl-CoA is a direct precursor for PHB biosynthesis [16]. This shorter pathway might explain the higher PHB production along with faster generation time and a higher maximum OD 660 obtained from butyrate compared to succinate and 3-hydroxybutyrate ( Table 1; Table 2). Moreover, the higher numbers of electrons in butyrate compared to succinate and 3-hydroxybutyrate could contribute to higher PHB production in TIE-1 [29,51]. A previous study by Shi et al. also reported similar results using metabolic flux balance analysis of PHB biosynthesis by C. necator under nitrogen-limited conditions using butyrate [71]. When TIE-1 was grown photoheterotrophically in 3-hydroxybutyrate, shorter lag time was observed in the 3-hydroxybutyrate-NH 4 Cl compared to the 3-hydroxybutyrate-N 2 ( Table 1). Although the maximum OD 660 values were similar, N 2 fixing conditions significantly increased the time to reach the maximum OD 660 values (Table 1). A decrease in PHB electron yield was observed when TIE-1 was grown in the 3-hydroxybutyrate-NH 4 Cl system compared to the 3-hydroxybutyrate-N 2 ( Table 2). However, no significant differences were observed in the PHB carbon yield and specific productivity under these conditions ( Table 2). These results indicate that N 2 fixation slowed the growth of TIE-1 in 3-hydroxybutyrate but increased the PHB electron yield similar to the results obtained under other heterotrophic growth conditions (Table 1; Table 2). Effect of N 2 fixation on photoheterotrophic PHB production Overall, N 2 fixing conditions during photoheterotrophy delayed cell growth and resulted in a longer lag time as well as a longer time to achieve maximum OD 660 (two times or longer) ( Table 1). In contrast, PHB carbon yield under photoheterotrophy under N 2 fixing conditions was higher than with NH 4 Cl ( Table 2). This increase in PHB production under N 2 fixing or N 2 deprivation conditions is consistent with previous findings [29,51]. This trend was not observed under photoautotrophic conditions. Interestingly, our results show that only a small percentage of carbon from the different substrates contributes to PHB synthesis by TIE-1. For example, the maximum PHB carbon yield during photoheteroautotrophic growth with butyrate was 8.81%. This indicates that the remaining carbon is likely used for biomass production. A previous report has shown that R. palustris grown on acetate converts 93% of its carbon into biomass [50]. STEM-EELS confirms intracellular accumulation of PHB granules in TIE-1 Because the LC-MS method used for PHB quantification involves digestion of the PHB polymer into crotonic acid [35], it was necessary to confirm the presence of intracellular PHB granules in TIE-1 using an additional technique. Nile red staining has previously been used to screen for PHB and fatty acid esters in bacteria [61]. However, this staining technique was ineffective in showing any intracellular inclusion bodies in TIE-1 possibly due to its small size. Hence, scanning transmission electron microscopy-electron energy loss spectroscopy (STEM-EELS) under conditions that produced the highest accumulation of PHB (mg/L/Cell) was performed. All the conditions imaged contained NH 4 Cl. The intracellular localization of PHB granules was confirmed by the carbon and nitrogen maps (Fig. 5). The nitrogen signal is likely from the phasin protein that is known to bind PHB granules [63]. PHB mostly aggregated as small multiple granules under photoelectroautotrophy compared to larger granules under photoferroautotrophy and photoheterotrophy with butyrate (Fig. 5). A change in the number and morphology of PHB granules was also observed previously in an anoxygenic phototrophic bacteria Dinoroseobacter sp. JL1447 when it was grown with different carbon sources such as sodium acetate, glucose, sodium glutamate, sodium pyruvate, and trisodium citrate [85]. Moreover, in a study on a purple non-sulfur bacterium Rhodovulum visakhapatnamense, a change in size and an increase in the number of PHB granules were also observed under nitrogen stress [29]. PHB cycle genes are not differentially expressed Our data show that there is significant variation in PHB production by TIE-1 under different growth conditions ( Fig. 2; Table 2). To determine if genes involved in PHB production are transcriptionally regulated, transcript levels of the genes involved in the PHB cycle was assessed using RNA-Seq and reverse transcription quantitative PCR (RT-qPCR). PHB cycle genes were chosen and are summarized in Fig. 1a. RNA-Seq analysis showed that the PHB cycle genes were not differentially expressed with respect to growth conditions or levels of PHB (values having p > 0.05 are statistically not significant) (Fig. 6, Tables S7-S10). RT-QPCR was performed to corroborate these data. For this analysis, we chose one phaA isozyme (Rpal_0532) of many because it showed the highest expression in the RNAseq data. This gene exists in an operon with a phaB homolog (Rpal_0533) (Fig. 1b), which was the only phaB homolog that was chosen for RT-qPCR analysis. The other PHB cycle genes only had 1-2 representatives in the TIE-1 chromosome and these were all analyzed using RT-qPCR. RNAseq analysis combined with RT-qPCR analysis of these phaAB homologs (Table S11) revealed no significant changes in gene expression ( Figure S1; Tables S8, S9). Moreover, the analysis of phaC 1 and phaC 2 polymerase genes did not show any significant differential expression (Fig. 6, Tables S7, S10). McKinlay et al. reported that R. palustris CG009 when incubated photoheterotrophically with acetate, under N 2 deprivation (with argon), accumulated PHBs with no change in transcript levels of genes known to be involved in PHB biosynthesis [51]. Phasin proteins have been reported to play a significant role in the PHB cycle [86]. Deletion of the phaP gene reduced PHB production significantly in C. necator [86]. Here, no significant upregulation of phaP 2 genes was observed using RNAseq even during photoheterotrophic growth with the butyrate-N 2 system, where the highest PHB production in mg/L/cell was obtained (Supplemental Table S7). Although phaP 2a appears to show a slight upregulation of 2.33-fold change under photoelectroautotrophy with N 2 fixing conditions, the P value was more than 0.05, rendering it not significant (Fig. 6, Table S7). Gene expression analysis results during photoautotrophy and photoheterotrophy using both RNAseq and RT-qPCR show that there is no differential expression in the genes involved in the PHB cycle. This could suggest that in TIE-1 (and perhaps even CGA009), the variation in PHB accumulation might be regulated post-transcriptionally. These findings are useful for the further optimization of PHB production using TIE-1 [45]. Conclusions and future perspectives Our study demonstrates the ability of a metabolically versatile photoautotroph Rhodopseudomonas palustris TIE-1 to produce PHB intracellularly under various growth conditions using different electron donors. The TIE-1 grown photoheterotrophically with butyrate, photoferroautotrophically with Fe(II) and photoelectroautotrophically with a poised electrode. From top to bottom panel: bright-field image, carbon, and nitrogen map, and the composite images. Bright areas represent the dominance of the corresponding element (carbon, and nitrogen). The red background in the composite images is due to carbon signals from Spurr's resin used for embedding the cells during sample preparation. The scale bars are 0.2 μm novel photoautotrophic metabolism using a poised electrode as the source of electrons produced the highest PHB electron yield. Another key discovery of this study is the ability of TIE-1 to yield the highest specific PHB productivity using Fe(II) as an electron donor for photoautotrophy. In summary, these newly described routes can serve as potential substitutes for PHB bioproduction. The application of these novel approaches can be especially important in areas where organic carbon sources are limited while resources such as light, CO 2 [37], iron, and electricity [57] are abundant. TIE-1's ability to fix N 2 gas photoautotrophically makes it a more attractive biocatalyst for many applications including PHB biosynthesis. The extreme metabolic versatility of TIE-1 can also be considered for waste management efforts combined with MES (for example for PHB biosynthesis). This approach can be further scaled up using underwater tubular photobioreactors that have been used previously to investigate the photosynthetic efficiency of R. palustris 42OL [10]. The future of using biocatalysts like TIE-1 via direct EU for bioproduction needs further consideration. We are pursuing modifications of electrodes and changes in reactor design to improve direct EU by TIE-1 as this represents the first major hurdle in the application of such microbes for bioproduction [65]. Using TIE-1 in the context of photoferroautotrophy also needs further investigation as our data support the idea that biomolecule production can be linked to this process. Fig. 6 Heat map showing log 2 fold change in the expression of PHB genes from RNA sequencing analysis (RNASeq). Results are from TIE-1 grown in freshwater (FW) medium photoheterotrophically with butyrate, photoautotrophically with H 2 , photoferroautotrophically with Fe(II), and photoelectroautotrophically using a poised electrode. Growth with NH 4 Cl is indicated by NH 4 Cl whereas growth under N 2 fixing conditions is indicated by N 2 . Error bars are the standard deviations calculated using three biological replicates. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment; ns not significant. For a pairwise test, the cutoff P value is 0.025). P values are indicated in Table S7-9	v3-fos-license
2021-08-02T00:05:34.729Z	2021-05-14T00:00:00.000	236570447	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.ajol.info/index.php/tjpr/article/download/207132/195274", "pdf_hash": "223638c481c98f13dd059cc0f79e0fed210a34a0", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1742", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "sha1": "328313179dff14c123ecff22831d93228d076753", "year": 2021 }	pes2o/s2orc	Effects of (-)-epigallocatechin gallate and quercetin on the activity and structure of α-amylase Purpose: To investigate the effects of (-)-epigallocatechin gallate (EGCG) and quercetin on the activity and structure of α-amylase. Methods: The inhibitory effects of 7 functional factors were compared by measuring half maximal inhibitory concentration (IC50) values. Lineweaver-Burk plots were used to determine the type of inhibition exerted by EGCG and quercetin against α-amylase. The effect of EGCG and quercetin on the conformation of α-amylase was investigated using fluorescence spectroscopy. Results: Quercetin and EGCG inhibited α-amylase with IC50 values of 1.36 and 0.31 mg/mL, respectively, which were much lower than the IC50 values of the other compounds (puerarin, paeonol, konjac glucomannan and polygonatum odoratum polysaccharide). The Lineweaver−Burk plots indicated that EGCG and quercetin inhibited α-amylase competitively, with ki values of 0.23 and 1.28 mg/mL, respectively. Fluorescence spectroscopy revealed that treatment with EGCG and quercetin led to formation of a loosely-structured hydrophobic hydration layer. Conclusion: This study has unraveled the mechanism underlying the inhibition of α-amylase activity by EGCG and quercetin in vitro. This should make for better understanding of the mechanisms that underlie the antidiabetic effects of EGCG and quercetin in vivo. INTRODUCTION Diabetes mellitus is a chronic metabolic disorder characterized by high level of fasting blood glucose. One therapeutic approach for diabetes is to decrease postprandial hyperglycemia by the inhibition of carbohydrate-hydrolyzing enzymes such as α-amylase and α-glucosidase [1]. α-Amylase (α-1,4-glucan-4-glucanohydrolase) catalyzes the hydrolysis of internal α-1,4-glucosidic linkage in starch, releasing glucose, maltose and maltotriose [2]. The control of carbohydrate digestion and monosaccharide absorption is beneficial for avoiding complications of diabetes. Acarbose, a fermentation product of actinoplanes species, has been shown to inhibit α-amylase competitively [3]. Studies have been carried out to identify inhibitors of α-amylase from natural sources so as to develop physiologically functional foods for treating diabetes [4,5]. Studies have shown that tea polyphenols and flavonoids effectively inhibit the activity of αamylase [6,7]. Tea catechins include EGCG, (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC). In recent studies, it was shown that EGCG treatment ameliorated free fatty acid-induced peripheral insulin resistance through decrease in oxidative stress, activation of the AMPK pathway and improvement of the insulin signaling pathway in vivo [8]. Although the prevention and treatment of type 2 diabetes mellitus have been investigated using EGCG supplementation [9], the effect of EGCG on the secondary and tertiary structures of α-amylase have not been investigated. Based on previous reports, dietary polyphenols have considerable potential for reducing the risk of diabetes. Epidemiological studies have also shown that the intake of certain types of flavonoids, including quercetin and myricetin is inversely associated with the risk of type 2 diabetes [10]. Flavonoids are beneficial for reducing the risk of metabolic syndrome. In addition to their antioxidant effects, flavonoids have been reported to prevent diabetes in vivo [11]. Studies on the inhibitory effects of isolated flavonoid compounds against α-glucosidase and α-amylase revealed that quercetin inhibited αamylase with IC 50 of 4.8mM [6,7]. However, the effect of quercetin on α-amylase conformation has not been demonstrated. The objectives of the present study were to evaluate in vitro pancreatic α-amylase-inhibitory activities of 7 functional factors, and the mechanism underlying the inhibition of αamylase by EGCG and quercetin. Furthermore, fluorescence measurements were applied to analyze changes in the tertiary structure of αamylase due to interaction of the enzyme with EGCG and quercetin. EXPERIMENTAL Materials α-Amylase was purchased from Sigma Aldrich (St. Louis, MO, USA). (-)-Epigallocatechin gallate (EGCG), quercetin, puerarin, paeonol, sulfated konjac glucomannan (SKGM) and Polygonatum odoratum polysaccharide (PoPs) (> 98 % purity) were purchased from Jingzhu Biotechnology Co. Ltd (Nanjing, China). Enzymatic assays were carried out using a UNIC-2100 visible spectrum. α-Amylase inhibition assay The inhibition of α-amylase was assayed according to the procedure of Song Liu [8]. Sample solution (50 µL) and 50 µL of 20 mM phosphate buffer (pH 6.9) containing 0.006 M sodium chloride and α-amylase solution (15 u/mL) were incubated at 37 °C for 10 min. The reaction was initiated by adding 600 µL of 1.5 % starch solution in 0.02 M sodium phosphate buffer, pH 6.9, and the mixture was incubated for 5 min at 37 °C, followed by the addition of 1 mL dinitrosalicylic acid. The reaction mixture was then placed in a boiling water bath for 5 min, and thereafter cooled to room temperature. The absorbance was measured at 540 nm in a UVvisible spectrophotometer (Shimadzu UV-1700, Japan). Acarbose was used as a positive control. Inhibition was calculated using Eq 1. (1) where Abs1 and Abs2 represent absorbance at 540 nm without and with inhibitor, respectively. Determination of inhibition mechanism and V max and K m values The mechanisms of the inhibitory effect of EGCG and quercetin against α-amylase, and values of maximum velocity (V max ) and Michaelis constant (K m ) were determined using the Lineweaver-Burk plot [11]. Substrate solutions at concentrations of 6.0, 8.0, 10.0, 12.0, 14.0, and 16.0 mg/mL were reacted with α-amylase, with or without inhibitor. The concentrations of α-amylase and inhibitor were 0.4 and 0.2 mg/mL, respectively, while distilled water was used as control. The V max and K m values were obtained from the least-squares regression lines of the double reciprocal plots of the tested sample (inhibitor) concentration (1/[S]) against the reciprocal of reaction rate (1/v). Half -maximal inhibitory concentration (IC 50 ) was calculated from inhibition curve. V max and K m values were obtained from the least-squares regression lines of the double reciprocal plots of the tested sample (inhibitor) concentration versus the reciprocal of reaction rate. Fluorescence measurements All fluorescent spectra measurements on the potential interaction between α-amylase, EGCG and quercetin were carried out on an F-7000 fluorescence spectrophotometer (HITACHI, F-7000, Japan). To each of a series of 5-mL test tubes was successively added 0.3 mL buffer solution (pH 7.4), 0.2 mL α-amylase (1 mg/mL), and varying amounts of EGCG and quercetin. After equilibration for 5 min, fluorescence spectra were measured at excitation wavelength of 280 nm, and emission wavelengths of 300 -480 nm. The slit width was set at 3 nm, and the scan speed was 12000 nm/min. Statistical analysis The results obtained were analyzed with SPSS version 16.0 (SPSS Inc, Chicago, IL, USA). Significant differences were determined by Student t-test. P-<0.05 was considered statistically significant. The inhibitory effects of seven functional factors on α-amylase activities In this study, the inhibitory effects of seven functional factors against α-amylase were evaluated, with acarbose as control. As shown in Figure 1, the IC 50 values for α-amylase inhibition by EGCG, quercetin and acarbose (as the positive control) were 0.31, 1.36, 0.45 mg/mL, respectively. The IC 50 value of EGCG (0.31mg/mL) was much lower than that of acarbose (0.45 mg/mL), indicating that EGCG strongly suppressed α-amylase activity, indicating that it could possibly be utilized for controlling postprandial hyperglycemia. Quercetin (IC 50 =1.36 mg/mL) had a stronger inhibitory effect on α-amylase activity than puerarin, paeonol, SKGM, and PoPs. It has been reported that quercetin significantly and dose-dependently decreased plasma glucose level of streptozotocin-induced diabetic rats [12]. In this study, quercetin inhibited αamylase activity in a dose-dependent manner, indicating that quercetin inhibition may effectively reduce plasma glucose level. Puerarin and paeonol showed weaker α-amylase inhibitory activities, while PoPS and SKGM had little inhibitory activities against α-amylase. Determination of inhibition types and V max and K m Values To investigate the inhibition characteristics of EGCG and quercetin against α-amylase, the kinetics of α-amylase reaction was investigated at different substrate concentrations. The Lineweaver -Burk plots for EGCG (Figure 2 A) and quercetin (Figure 2 B) showed the same intersection on Y-axis, indicating that the mode of inhibition of α-amylase by EGCG and quercetin was competitive. As the dose of EGCG increased in Figure 2 A, the K m value for α-amylase increased, while the value of V max remained unchanged. Such results are consistent with competitive inhibition characteristics. The K i values for EGCG and quercetin were 0.23 and 1.28 mg/mL, respectively. The smaller the K i , the higher the affinity of the inhibitor for α-amylase and the higher is the inhibition. It appears therefore that the inhibition of starch hydrolysis was significantly higher with EGCG than with quercetin. Effects of EGCG and quercetin on the tertiary structure of α-amylase To monitor the changes in the microenvironment of aromatic amino acid residues of α-amylase in response to EGCG and quercetin treatment, intrinsic fluorescence spectra of the enzyme were recorded in the range of 300 -500 nm. As shown in Figure 3, the relative fluorescence quantum yields of EGCG-and quercetin-treated α-amylase exhibited obvious decreases. A blue shift in the maximum peak wavelength was observed with increasing concentrations of EGCG and quercetin. The intrinsic fluorescence of α-amylase was quenched by EGCG and quercetin. Compared to quercetin, the addition of increasing concentrations of EGCG caused more progressive reductions in fluorescence intensity. The reduction in fluorescence intensity indicated that EGCG and quercetin treatment induced disruption of hydrophobic bonds, thereby exposing the nonpolar amino acid residues (e.g., tryptophan) to a more polar environment. It also caused the formation of a loosely structured hydrophobic hydration layer, and the fluorescence was quenched by that environment. DISCUSSION It has been suggested that the inhibition of αamylase and other carbohydrate-hydrolyzing enzymes is a potential way of controlling postprandial blood glucose levels. Thus, the search for effective and non-toxic inhibitors of αamylase has important significance for the prevention and treatment of diabetes. Radovanović has assessed the antioxidant and antimicrobial activities of polyphenolic extracts of three wild berry fruit species from Southeast Serbia [13]. The anti-glycemic and hypolipidemic potential of polyphenols from Zingiber officinale in streptozotocin-induced diabetic rats have been reported [14]. Previous studies have shown that polyphenols and flavonoids inhibit or activate enzymes in vitro [15]. In a study by Kalita et al, it was reported that potato polyphenolic compounds inhibited pancreatic α-amylase in vitro [16]. Radovanović have assessed the antioxidant and antimicrobial activities of polyphenolic extracts of three wild berry fruit species from Southeast Serbia [13]. The antiglycation and hypolipidemic potential of polyphenols from Zingiber officinale in streptozotocin-induced diabetic rats have been verified [14]. Previous research have shown that polyphenol and flavonoids have the ability to inhibit or activate enzymes in vitro [15]; Kalita discovered that potato polyphenolic compounds have the ability to inhibit pancreatic α-amylase in vitro [16]. Recent findings showed that Qingzhuan tea extracts exerted potent inhibitory effects on αamylase [17]. In addition, tea polyphenols composed of EGCG, EGC, ECG and EC inhibited α-amylase with an IC 50 of 0.41 mg/mL. In the study, EGCG which appeared to be one of the main components of tea polyphenols, exhibited the most effective inhibition of αamylase, with IC 50 value of 0.31 mg/mL. It has been reported that quercetin significantly and dose-dependently decreased the plasma glucose level of streptozotocin-induced diabetic rats [12]. In this study, the inhibition of α-amylase by quercetin was dose-dependent, with IC 50 value 1.36 mg/mL, indicating that the inhibition may be an effective approach towards decreasing plasma glucose level. Overall, the findings suggest that EGCG and quercetin may limit the release of simple sugars from the gut, thereby alleviating postprandial hyperglycemia. The fluorescence spectrum was associated with polarity of the environment of the tryptophan and tyrosine residues. The decreases in fluorescence quantum yield may be due to the interaction of chromophores with quenching agents. Changes in intrinsic fluorescence emission have been attributed to the changes in protein tertiary structure [18]. Molecular interactions between pancreatic lipase and EGCG have been studied [19]. It has been shown that the α-helix content of pancreatic lipase secondary structure decreased as a function of EGCG concentration, and that static fluorescence quenching occurred as a result of EGCG treatment [20]. Tryptophan fluorescence is considered a very reliable index of conformational changes in proteins [21]. Thus, it was used to investigate the effect of EGCG and quercetin on the tertiary structure of α-amylase in this study. The fluorescence intensity of α-amylase decreased with increasing concentrations of EGCG and quercetin. This implies that the binding of EGCG and quercetin to α-amylase caused microenvironment changes in α-amylase. Inhibitors of α-amylase may directly interact with the side chains of Asp197, Glu233, and Asp300: substitution of these residues lead to a considerable drop in catalytic activity of the enzyme [22]. The inhibitory activity of EGCG on α-amylase led to the formation of soluble or insoluble complexes. The hydrogen bonds between the hydroxyl groups of EGCG and the catalytic residues of the binding site stabilized the interaction with active site [23]. Some researchers have used molecular docking to study the structure-activity relationship in the binding of flavonols to α-amylase and the possible mechanisms involved. Molecular modeling studies revealed that salivary αamylase inhibitors occupied a docking mode that allowed for H-bonds between the enzyme Asp197 side chain carboxyl oxygen atom and the hydroxyl groups in ring B of the flavonoid skeleton [24]. Thus, the hydrogen bond formed between the quercetin hydroxyl groups and the binding site of the catalytic residue accounts for the inhibition of α-amylase by quercetin. CONCLUSION The results of this study indicate that EGCG and quercetin inhibit α-amylase activity in a dosedependent manner. Lineweaver−Burk plots demonstrate that inhibition of α-amylase by EGCG and quercetin are competitive. Furthermore quenching of fluorescence ofαamylase induced by EGCG and quercetin suggest possible changes in the conformation of α-amylase which decreased enzyme catalytic activity. If this antidiabetic function is confirmed after clinical studies in type 2 diabetic patients, EGCG and quercetin should be beneficial in the treatment of hyperglycemia.	v3-fos-license
2019-03-27T20:57:50.107Z	2018-12-18T00:00:00.000	86848539	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.eurasianjournals.com/pdf-101785-33256?filename=Environmentally%20Friendly.pdf", "pdf_hash": "505fba327f83c7705359a10c03a1eab48f6343e0", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1751", "s2fieldsofstudy": [ "Chemistry", "Engineering" ], "sha1": "505fba327f83c7705359a10c03a1eab48f6343e0", "year": 2018 }	pes2o/s2orc	Environmentally Friendly Preparation of Zinc Oxide , Study Catalytic Performance of Photodegradation by Sunlight for Rhodamine B Dye This study includes the photocatalytic degradation of Rhodamine B (Rh.B) employing a heterogeneous photocatalytic process by using ZnO nanoparticles that was prepared by green sol-gel process. The structural, morphological, and its optical properties of ZnO Photocatalyst was studied using different characterization techniques such as Xray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), The influencing factors studied were the amount of the catalyst, the concentration of dye and pH on photocatalytic degradation of Rhodamine B. The experiments were carried out by irradiating the aqueous solutions of dyes containing photocatalysts with Sunlight. The rate of decolorization was estimated from residual concentration spectrophotometrically. Similar experiments were carried out by varying pH (3–11), amount of catalyst (0.25–2.0 g/L) and initial concentration of dye (5–50 mg/L). The experimental results indicated that the maximum degradation (71%) of dyes was achieved using ZnO photocatalyst at pH 10 after 240 min. INTRODUCTION Pollution problem is getting worse day by day, so Researchers are looking for ways to get rid of this problem.Among the most common contaminants are dissolved dyes in industrial wastewater from textile [1] and paper mills [2].Synthetic dyes are extensively used for dyeing and printing in textile industries.Over 10,000 dyes with an once a year production over 10 5 metric tons worldwide are commercially available and about 50% among them are azo dyes [3].It is estimated that approximately 15% of the dyestuffs are lost in the industrial effluents during manufacturing and processing operations [4].Color is usually the first contaminant to be recognized in wastewater.Many dyes may be decomposed into potential oncogenic amines under anaerobic conditions in the environment [5].There are many ways for pollutant elimination such as adsorption on activated carbon [6], reverse osmosis [7], ultrafiltration [8], and ozonation [9] etc. Photodegradation is also one of the most important technologies used in the disposal of pollutants in industrial wastewater.Researchers want to use natural resources available to obtain the energy needed for the degradation of dyes in industrial wastewater, the most important source of natural energy is sunlight that consists of about 5-7% UV light, 46% visible light and 47% infrared radiation [10].Photocatalytic oxidation of various harmful organic dyes in industrial wastewater has been carried over ZnO semiconductor oxides under UV light irradiation [11].Research is now focused on to achieve high photocatalytic efficiency with ZnO [12] especially with sunlight.Zinc oxide can be prepared in more than one way, each method has its own conditions that determine product characteristics, preferably use environmentally friendly methods.Green chemistry is generally accepted as "the design, development, and implementation of chemical processes and products to reduce or eliminate substances hazardous to human health and the environment" [13].There has been an explosive growth in the field of green chemistry both in preparing green Nanocatalysts [14] as well as green conditions during catalysis of industrially important reactions.Preparing green Nanocatalysts refers to manufacturing the nanocatalysts using green solvents or processing the nanocatalysts so that they are finally Al-Bedairy & Alshamsi / Environmentally Friendly Preparation of Zinc Oxide, Study Catalytic Performance … 2 / 9 dispersed in green solvents.Green nanocatalysis refers to doing the catalytic reaction in green solvents and rather by the use of green nanocatalysts for these reactions [15].According to the fourth principle of Anastas' and Warner's 12 principles of green chemistry "Chemical products should be designed to preserve efficacy of function while reducing toxicity," [16], should of course also be applied to the synthesis of Nanocatalysts [17].This is typified by the synthesis of nontoxic ZnO nanoparticle catalysts [18,19]. Materials Zinc acetate, potassium hydroxide were obtained from B.D.H Company, Rhodamine B dye, were supplied from Sigma-Aldrich.All materials were used directly without further purification. Instruments UV-vis spectrophotometer double beam PC 1650 SHIMADZU, UV-vis spectrophotometer 780 Sunny China, The crystalline character of the solid has been identified by X-ray diffraction (XRD) analysis using a D/Max 2,550 V diffractometer with Cu Kα radiation (λ = 1.54056Å) ( Japan), and the XRD data were collected at a scanning rate of 0.03 s -1 for 2θ in a range from 10° to 80°, The morphology of prepared materials was noted by field emission scanning electron microscopy (FE-SEM ) with (MIRA3 TESCAN -Czech), The optical band gap Eg was projected from the UV-Vis-NIR diffuse reflectance spectroscopic (UV-Vis-NIR DRS) determined in a wavelength range from (200 -1100) nm with UV-1800 UV-VIS Spectrophotometer from SHIMADZU, pH meter (Sartorius, Germany), 100 mL Teflon-lined autoclave. Preparation of ZnO In a typical synthesis, of ZnO nanoparticles is carried out by sol-gel process, at 80-90C o .Solution of zinc acetate Zn(CH3COO)2 was prepared by dissolving 2.195 g of zinc acetate in 100ml distilled water, and stirred in ambient atmosphere.Potassium hydroxide KOH 1.122g is dissolved in 10 ml distilled water and was added to the above solution drop wise under continuous stirring.After few minutes solution turn into jelly form and a milky white solution was obtained, the mixture was then further heated for 3 h at 80-90C o without stirring.The resulting suspension was centrifuged to retrieve the product, and the mixture was washed with distilled water and then the powder was dried at 70 C o overnight and determined in terms of their structural, morphology and optical properties [20,21]. Photo-catalytic Degradation Experiment ZnO was added to 100 ml of Rhodamine-B dye solution and it is undergo irradiation by sunlight.After adding the catalyst and stirrer it at a constant speed.The samples were taken at different times.Before the irradiation, the dye catalyst suspension was kept in the dark with steering for 90 min to ensure an adsorption-desorption equilibrium.The solution was separated from the catalyst by the centrifuge.Measure the absorbance of each sample, absorption spectra were recorded and rate of decolorization was seen as far as change in power at λmax of the colors.The decolonization efficiency has been ascertained as equation 1: Where Co is the initial concentration of dye and C is the concentration of dye after photo irradiation.Similar experiments were carried out by varying the pH of the solution (pH 3-11), concentration of dye (10 -100 mg/L) and catalyst loading (0.25-2.0 g/L), pH of aqueous solution was adjusted with 0.1M H2SO4 or 0.1M NaOH. FE-SEM The surface morphology of catalyst is one of the important parameters that impact on the photocatalytic efficiency.the nanoparticles were investigated by FE-SEM image as shown in Figure 2 The FE-SEM images shows that the ZnO particles were formed in a very uniform manner in the form of cubes and a few of them appeared spherically. UV-vis Diffuse Reflectance Spectra (DRS) The properties of semiconductor nanoparticles are strongly size dependent.It is well known that the nano-scale systems show interesting properties, for example, increasing of the semiconductor band gap due to electron confinement [24].The UV-vis diffuse reflectance, (Tauc's plot) of synthesized ZnO was shown in Figure 3, the calculated band gap energy for synthesized ZnO is 3.39 eV, the determination of optical band gap is obtained by Tauc's equation [25]. Adsorption of the dyes on ZnO-photocatalyst Degradation of the dyes occurs predominantly on the photocatalyst surface [26].In order to investigate the adsorption behavior of Rhodamine B, the suspension was prepared by mixing 100 ml of dye solutions 20 mg/L with fixed photocatalyst ZnO amount (1 g/L) at 35 C°, natural pH of Rhodamine B. The suspensions were kept for different times in the dark under shaking for 120 min.The absorbance measured at the max553 nm to determine the concentration of dyes.The experimental results are shown in Figure 4 from the results, it was noticed that the adsorption equilibrium under 20 mg/L initial concentration was reached at about 60 min of equilibration time. Photocatalyst loading The experiments were carried out by varying ZnO photocatalyst amount from 0.25 to 2.0 g/L for dye solutions of 20 mg/L at natural pH of Rhodamine B. The decolorization efficiency for various photocatalysts amount for Rhodamine B has been depicted in Figure 5.It is observed that rate increases with increase in catalyst amount and becomes constant above a certain level then will be decreased.The optimum photocatalyst amount for Eurasian J Anal Chem 5 / 9 decolorization efficiency of dye is 0.8 g/L.The reasons for this decrease in decolorization efficiency at given time it aggregation of catalyst particles at high concentrations causing a decrease in the number of surface active sites [27,28] and increase in light scattering of catalyst particles to decrease in the passage of irradiation through the sample [29,30]. Effect of pH Wastewater containing dyes at different pH therefore it is important to study the effect of pH on degradation efficiency of dye [29], the amphoteric behavior of most semiconductor oxides influences the surface charge of the photocatalyst [31].The effect of pH values on the degradation efficiency is studied in the pH range 3-11 at the dye concentration 20 mg/L and catalyst amount 0.8 g/L, 35C°.Figure 6 illustrated the results of the decolorization efficiency after 160 min irradiation with different pH values (3, 5, 7, 9 and 11) It can be observe increase in the decolorization efficiency of Rhodamine B with increase of the pH value from 3 up to 11, exhibiting maximum efficiency at pH 10, This behavior could be explained by pHpzc of the material, as well as the molecular nature of the dyes, the zero point charge of ZnO equal to 9.0, Therefore photocatalysts surface is positively charged below pHzpc, whereas it is negatively charged when pH > pHzp. Effect of initial dye concentration The results reported that the initial dye concentration effects the degradation efficiency strictly.With the increase of initial dye concentration, the degradation efficiency decreases remarkably [27].The negative effects of the initial dye concentration are attributed to the competency between dye and OH − ion adsorption on the surface of catalyst.The adsorption of dye reduces the OH − ion adsorption, which results in the reduction on the formation of hydroxyl radicals [32].The rate of degradation relates to formation of OH radicals, which is the critical species in the degradation process.At the same time, as the initial dye concentration increases, the path length of photons entering the solution decreases [33].Hence in the solution with constant catalyst concentration, the formation of hydroxyl radicals that can attack the pollutants decreases, thus leading to the lower decolorization efficiency.Figure 7 shows the effect of initial dye concentration on degradation efficiency by varying the initial concentration from 5 to 50 mg/L with the constant ZnO catalyst loading (0.8 g/L) and pH 10. Kinetic of photodegradation Figure 8 shows a typical UV-Vis spectrum of Rhodamine B solution during photo-irradiation time (dye conc.10 ppm, pH10, catalyst amount 0.8 g/L, T= 308K°).The absorption peaks, corresponding to dye, diminished and finally disappeared under reaction which indicated that the dye had been degraded.No new absorption bands appear in the visible region.The spectrum of Rhodamine B in the visible region exhibits a main band with a maximum at 553 nm. Figure 9 shows the kinetics of disappearance of Rhodamine B for an initial concentration of 20 ppm at pH10, catalyst amount 0.8 g/L.The results show that the photodegradation of the dye in aqueous ZnO can be described by the first order kinetic model, ln(C0/C) = kt, where C0 is the initial concentration and C is the concentration at any time, t.The semi logarithmic plots of the concentration data give a straight line. CONCLUSION In this study, Photocatalytic degradation of Rhodamine B dye has been investigated using ZnO photocatalyst.The following conclusions have been obtained about photodegradation: • Photocatalytic degradation process is more suitable for low concentration of the pollutants. • Photocatalytic degradation process is more suitable with low concentration of photocatalyst. • Photocatalytic degradation process is more suitable with pH more than 9.	v3-fos-license
2020-02-13T09:22:14.176Z	2020-02-01T00:00:00.000	211099756	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1660-4601/17/3/1103/pdf", "pdf_hash": "be9d50fc4c0e5d3ae8d858ef461ea622f4a4536f", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1791", "s2fieldsofstudy": [ "Engineering" ], "sha1": "dfb1d25c60e2d93e4c0833d9e05db9da26e32891", "year": 2020 }	pes2o/s2orc	Assessment of the Nutrient Removal Potential of Floating Native and Exotic Aquatic Macrophytes Cultured in Swine Manure Wastewater Although eutrophication and biological invasion have caused serious harm to aquatic ecosystems, exotic and even invasive plants have been used extensively in phytoremediation water systems in China. To identify native aquatic plants with excellent water restoration potential, two representative native floating aquatic plants from Guangdong Province, namely Ludwigia adscendens (PL) and Trapa natans (PT), were selected, with Eichhornia crassipes as a control, to study their growth status, adaptability, and nutrient removal potentials in swine manure wastewater. The results demonstrated that the two native plants offered greater advantages than E. crassipes in water restoration. Within 60 days, PL and PT exhibited excellent growth statuses, and their net biomass growth rates were 539.8% and 385.9%, respectively, but the E. crassipes decayed and died with an increasing HRT (hydraulic retention time). The PL and PT could adjust the pH of the wastewater, improve the dissolved oxygen and oxidation-reduction potential, and reduce the electrical conductivity value. The removal rates of NH4+–N, NO3−–N, NO2−–N, total nitrogen, total phosphorus, chemical oxygen demand (COD), and Chl-a in the PL group reached 98.67%, 64.83%, 26.35%, 79.30%, 95.90%, 69.62%, and 92.23%, respectively; those in the PT group reached 99.47%, 95.83%, 85.17%, 83.73%, 88.72%, 75.06%, and 91.55%, respectively. The absorption contribution rates of total nitrogen (TN) and total phosphorus (TP) in the PL group were 40.6% and 43.5%, respectively, while those in the PT group were 36.9% and 34.5%, respectively. The results indicated that L. adscendens and T. natans are both promising aquatic plants for application to the restoration of swine manure wastewater in subtropical areas. Introduction Eutrophication and biological invasion are widespread problems in rivers, lakes, and coastal oceans, caused by over enrichment with nitrogen and phosphorus and invasion of alien organisms [1,2], which seriously degrades aquatic ecosystems and impairs the use of water for drinking, industry, Wastewater samples were collected from each tank at various intervals (typically 0, 15, 30, 45, and 60 days), by dipping a 500 mL graduated cylinder at three locations across the container surface and combining these during mid-morning. Care was taken to minimize disturbance of the plants. The aliquots of each sample were filtered, and both the filtered and unfiltered portions were immediately stored at 4 °C. The parameters measured included T, pH, DO, ORP, EC, TN, NH4 + -N, NO3 − -N, NO2 − -N, TP, Chl-a, and COD. Losses in the culture volume owing to evapotranspiration were countered by the addition of deionized water to the original level every second day. Water sampling was performed on the second day following the volume adjustment, so that the deionized water additions had a minimal impact on the measurements. Aquatic plants were sampled from the treatment plots. The roots, shoots, and fruit of the tested plants were separated, washed three times with deionized water, briefly treated at 105 °C, and then dried to a constant weight at 65 °C. The dried plant material was ground, and the N and P contents were measured. At the end of the experiment, all plants were removed from their tanks and weighed. In addition to the regular sampling, the plants were also periodically harvested to maintain optimal plant density. In each harvest, the total fresh weight of the plants was recorded, the plant moisture was determined, and the total quantity of dry plant biomass yield was calculated for each plot. The total amounts of N and P removed from the water by the harvested plants were quantified by multiplying the amounts of plant biomass by the N and P concentrations in the plant. The plant N concentration was determined using a CN analyzer (vario Max CN, Elemental Analysensystem GmbH, Hanau, Germany). Sub-samples (each 0.400 g) of the plant material were digested with 5 mL of concentrated HNO3 in a digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Hornibrook, Australia), and the P concentration in the digester was determined using inductively coupled plasma atomic emission spectroscopy. Wastewater samples were collected from each tank at various intervals (typically 0, 15,30,45, and 60 days), by dipping a 500 mL graduated cylinder at three locations across the container surface and combining these during mid-morning. Care was taken to minimize disturbance of the plants. The aliquots of each sample were filtered, and both the filtered and unfiltered portions were immediately stored at 4 • C. The parameters measured included T, pH, DO, ORP, EC, TN, NH 4 + -N, Chl-a, and COD. Losses in the culture volume owing to evapotranspiration were countered by the addition of deionized water to the original level every second day. Water sampling was performed on the second day following the volume adjustment, so that the deionized water additions had a minimal impact on the measurements. Aquatic plants were sampled from the treatment plots. The roots, shoots, and fruit of the tested plants were separated, washed three times with deionized water, briefly treated at 105 • C, and then dried to a constant weight at 65 • C. The dried plant material was ground, and the N and P contents were measured. At the end of the experiment, all plants were removed from their tanks and weighed. In addition to the regular sampling, the plants were also periodically harvested to maintain optimal plant density. In each harvest, the total fresh weight of the plants was recorded, the plant moisture was determined, and the total quantity of dry plant biomass yield was calculated for each plot. The total amounts of N and P removed from the water by the harvested plants were quantified by multiplying the amounts of plant biomass by the N and P concentrations in the plant. Chemical Analysis The TN was determined by a TOC analyzer (TOC-L, SHIMADZU (Hong Kong) Limited, Kyoto, Japan). The NH 4 + -N, NO 3 − -N, and NO 2 − -N were determined by a flow injection analyzer (QC8000, LACHAT, Milwaukee, WI, USA). The TP was determined using the ammonium molybdate spectrophotometric method (GB11893-1989). The COD was determined using a spectrophotometer (DRB 200, Hach, Loveland, CO, USA). The Chl-a was determined spectrophotometrically (HJ 897-2017). The other physical and chemical characteristics, including T, pH, ORP, DO, and EC, were obtained using a portable multimeter (EXO2, YSI, Ohio, OH, USA). The plant N concentration was determined using a CN analyzer (vario Max CN, Elemental Analysensystem GmbH, Hanau, Germany). Sub-samples (each 0.400 g) of the plant material were digested with 5 mL of concentrated HNO 3 in a digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Hornibrook, Australia), and the P concentration in the digester was determined using inductively coupled plasma atomic emission spectroscopy. Data Analysis In this study, all data were statistically analyzed by one-way ANOVA using the R-3.4.3 software (https://cran.r-project.org/bin/windows/base/old/3.4.3/), and significant differences were tested using the least significant difference and Duncan multiple comparisons (p = 0.05). In this case, R 1 represents the total removal rate of nutrients in the wastewater; R 2 represents the absorption contribution rate of the plants for the nutrient removal in the wastewater; and R 3 represents the relative growth rate of the plant biomass in each group. The computation formulae are expressed as follows: (1) where C i (mg/L) represents the initial concentration of nutrients in each tank; C r (mg/L) represents the residual concentration; P i and P f (mg) represent the initial and final N or P contents of the aquatic macrophytes, respectively; and W i and W f (mg) represent the initial and final fresh weights of the plants, respectively. Visual Observations and Biomass Production All of the plants selected for the experiment were collected locally and had few roots at the beginning. We selected young and thriving individuals. During the test, as illustrated in Figure 2, the L. adscendens grew vigorously and its biomass increased significantly. Its floating stems grew rapidly, from 15 to 200 cm, with numerous branched, new creeping, rooting, and white spindle-shaped pneumatophores in clusters at the nodes. Moreover, as illustrated in Figure 3, T. natans was rapidly established in the wastewater, with the surface area of the containers being completely covered. Its leaves and roots grew rapidly; the diameter of its leaves reached at least twice the initial diameter, at approximately 20 to 25 cm; and the length of its root system reached 0.5 m, at least 20 times that of the initial length. The leaves and roots of the E. crassipes grew normally, and the biomass nearly doubled on the 15th day of the experiment. Thereafter, an abnormality appeared: from the 20th day, the stems, leaves, and roots gradually whitened and further decayed and died, as illustrated in Figure 4. The net biomass growth rates of the PL, PT, and PE at the end of the experiment are presented in Table 1, which indicates that the two native aquatic plants exhibited higher growth rates and superior adaptability in the swine manure wastewater compared to the E. crassipes. Nutrient Concentrations and Absorption Capacities of the Plants During the experiment, the TN and TP concentrations in the L. adscendens and T. natans plant tissues all decreased continuously with an increasing HRT (hydraulic retention time) ( Table 2 and 3). The E. crassipes is not discussed here because of its death. The absorption contribution rate of plants (R2) refers to the proportion of N and P contents removed by the plants through absorption to the total amount removed in the water. In this experiment, the R2 values of the TN in the PL and PT groups were 40.6% and 36.9%, respectively; the R2 values of the TP were 43.5% and 34.5%, respectively. The results indicated that L. adscendens and T. natans exhibited a significant enrichment ability for N and P in the swine manure wastewater. Nutrient Concentrations and Absorption Capacities of the Plants During the experiment, the TN and TP concentrations in the L. adscendens and T. natans plant tissues all decreased continuously with an increasing HRT (hydraulic retention time) ( Table 2 and 3). The E. crassipes is not discussed here because of its death. The absorption contribution rate of plants (R2) refers to the proportion of N and P contents removed by the plants through absorption to the total amount removed in the water. In this experiment, the R2 values of the TN in the PL and PT groups were 40.6% and 36.9%, respectively; the R2 values of the TP were 43.5% and 34.5%, respectively. The results indicated that L. adscendens and T. natans exhibited a significant enrichment ability for N and P in the swine manure wastewater. Nutrient Concentrations and Absorption Capacities of the Plants During the experiment, the TN and TP concentrations in the L. adscendens and T. natans plant tissues all decreased continuously with an increasing HRT (hydraulic retention time) (Tables 2 and 3). The E. crassipes is not discussed here because of its death. The absorption contribution rate of plants (R 2 ) refers to the proportion of N and P contents removed by the plants through absorption to the total amount removed in the water. In this experiment, the R 2 values of the TN in the PL and PT groups were 40.6% and 36.9%, respectively; the R 2 values of the TP were 43.5% and 34.5%, respectively. The results indicated that L. adscendens and T. natans exhibited a significant enrichment ability for N and P in the swine manure wastewater. Variations in pH, DO, ORP, and EC in Wastewater The values of the pH, DO, ORP, and EC in the wastewater with an increasing HRT are illustrated in Figure 5. The initial pH value of the tested water was 7.71 ± 0.19, which is weakly alkaline. During the experiment, the pH values in the PL and PT groups were reduced to neutral on the 15th day, and then remained stable; the pH values in the PE and BC groups fluctuated significantly and eventually reached 8. Nutrient Reduction in Wastewater As a major nutrient for aquatic ecology, excess N can lead to eutrophication of surface waters [14]. The concentration and removal efficiencies of NH4 + , NO3 − , and NO2 − , varying with an increasing The C i value of the DO in the wastewater was 1.46 ± 1.12 mg/L. During the first 15 days, the DO concentrations in the three plant groups all increased rapidly, while that in the BC group decreased, indicating that the aquatic plants could improve the DO concentration in the wastewater. On the 60th day of the experiment, the DO concentrations in the four groups all exceeded 5 mg/L, with the exception of PT (3.59 ± 0.07 mg/L). The change trend of the ORP exhibited a similar pattern to that of the DO. At the end, the average ORP values in the PL, PE, BC, and PT groups were 307.03, 279.70, 277.57, and 203.00 mV, respectively, all substantially higher than the initial value (120.11 ± 6.02 mV). The initial EC value in the wastewater was 432.0 ± 2.0 uS/cm. The EC values of all treatments decreased and the water quality improved with an increasing HRT. At the end of the experiment, the EC values of the PL, PT, BC, and PE groups decreased to 79.8, 123.4, 263.2, and 304.3 uS/cm, respectively, which demonstrated that the two native floating aquatic plants were effective at reducing the EC value in the wastewater. Nutrient Reduction in Wastewater As a major nutrient for aquatic ecology, excess N can lead to eutrophication of surface waters [14]. The concentration and removal efficiencies of NH 4 + , NO 3 − , and NO 2 − , varying with an increasing HRT during the experiment, are illustrated in Figure 6. The C i value of the NH 4 + was 7.83 ± 0.36 mg/L, which decreased rapidly with an increasing HRT. On the 15th day of the experiment, the R 1 values in the PT, PE, PL, and BC groups were 94.98%, 92.59%, 91.61%, and 78.75%, respectively. On the 60th day, the C r values of the NH 4 + in the four groups were all less than 0.1 mg/L (R 1 > 98%). During the experiment, the R 1 value of the NH 4 + in the BC group was high, but lower than that of the plant groups at the beginning, and even reached the same level as the plant groups in the end, although the change trend was slower. The varied little with an increasing HRT, but those in BC and PE increased rapidly. At the end of the experiment, the R1 values of the TN in the PT, PL, PE, and BC groups were 84.05%, 79.04%, 77.84%, and 72.98%, respectively, while the Cr values of the TN were 3.80, 5.01, 5.51, and 5.80 mg/L, respectively. P is an important nutrient in the composition of biological life, and a highly significant limiting factor for water eutrophication [14]. In this experiment, the Ci value of the TP was 5.39 ± 0.66 mg/L. The variations in the TP concentration and the homologous removal rate are illustrated in Figure 7. During the first 15 days, the Cr values in the PL and PT groups decreased rapidly (Cr = 0.79 ± 0.03; Cr = 2.92 ± 0.08 mg L −1 ), and the R1 values of the TP were 85.11% and 51.66%, respectively, which were significantly higher than those of the BC (13.20%) and PE (12.77%) groups (p < 0.05). Subsequently, the R1 values of the TP in the PL, PT, and BC groups all increased slightly with an increasing HRT, while that in PE began to decrease from the 45th day. At the end, the R1 values of the TP in the PL, PT, BC, and PE groups were 95.90%, 88.72%, 38.32%, and 26.82%, respectively. The results P is an important nutrient in the composition of biological life, and a highly significant limiting factor for water eutrophication [14]. In this experiment, the C i value of the TP was 5.39 ± 0.66 mg/L. The variations in the TP concentration and the homologous removal rate are illustrated in Figure 7. During the first 15 days, the C r values in the PL and PT groups decreased rapidly (C r = 0.79 ± 0.03; C r = 2.92 ± 0.08 mg L −1 ), and the R 1 values of the TP were 85.11% and 51.66%, respectively, which were significantly higher than those of the BC (13.20%) and PE (12.77%) groups (p < 0.05). Subsequently, the R 1 values of the TP in the PL, PT, and BC groups all increased slightly with an increasing HRT, while that in PE began to decrease from the 45th day. At the end, the R 1 values of the TP in the PL, PT, BC, and PE groups were 95.90%, 88.72%, 38.32%, and 26.82%, respectively. The results demonstrated that the two native floating aquatic plants exhibited significant advantages in TP removal compared to the blank control and E. crassipes. Chl-a and COD Reduction In this experiment, the removal efficiency and concentration of the COD and Chl-a exhibited a similar pattern to that of the nutrients, which can be observed in Figure 8 and 9, respectively. The most notable difference was in the BC group, where the Cr value of the COD experienced significant negative growth. The Ci value of the Chl-a was 364.04 ± 39.12 mg/L, which decreased rapidly with an increasing HRT. On the 15th day, the R1 values in the PT, PE, PL, and BC groups were 93.08%, 84.61%, 82.71%, and 68.60%, respectively. On the 60th day, the R1 values of the Chl-a in the three plant groups were all above 90%-significantly higher than that in group BC (80.72%) (p < 0.05). The results indicated that the plants played an important role in the removal of Chl-a in the wastewater. Chl-a and COD Reduction In this experiment, the removal efficiency and concentration of the COD and Chl-a exhibited a similar pattern to that of the nutrients, which can be observed in Figures 8 and 9, respectively. The most notable difference was in the BC group, where the C r value of the COD experienced significant negative growth. The C i value of the Chl-a was 364.04 ± 39.12 mg/L, which decreased rapidly with an increasing HRT. On the 15th day, the R 1 values in the PT, PE, PL, and BC groups were 93.08%, 84.61%, 82.71%, and 68.60%, respectively. On the 60th day, the R 1 values of the Chl-a in the three plant groups were all above 90%-significantly higher than that in group BC (80.72%) (p < 0.05). The results indicated that the plants played an important role in the removal of Chl-a in the wastewater. The C i value of the COD was 103.6 ± 3.4 mg/L. On the 15th day, the COD concentration in the PL and PT groups decreased rapidly, and the R 1 values were 72.40% and 75.59%, respectively, which were significantly greater than those in the PE (46.38%) and BC (−18.41%) groups. This result indicated that the aquatic plants could effectively reduce the COD value of wastewater; the two native aquatic plants offered greater advantages than the E. crassipes in COD purification. Thereafter, the R 1 values of the COD in the PL and PT groups remained relatively stable, while those in the PE and BC groups fluctuated significantly, indicating that the strong plant growth was conducive to the water stability. On the 60th day, the R 1 values in the three plant groups were close to 70%, which was significantly higher than that in the BC group (−33%) (p < 0.05). The Ci value of the COD was 103.6 ± 3.4 mg/L. On the 15th day, the COD concentration in the PL and PT groups decreased rapidly, and the R1 values were 72.40% and 75.59%, respectively, which were significantly greater than those in the PE (46.38%) and BC (−18.41%) groups. This result indicated that the aquatic plants could effectively reduce the COD value of wastewater; the two native aquatic plants offered greater advantages than the E. crassipes in COD purification. Thereafter, the R1 values of the COD in the PL and PT groups remained relatively stable, while those in the PE and BC groups fluctuated significantly, indicating that the strong plant growth was conducive to the water stability. On the 60th day, the R1 values in the three plant groups were close to 70%, which was significantly higher than that in the BC group (−33%) (p < 0.05). Visual Observations In this study, the native aquatic plants both grew well, but the exotic plant E. crassipes decayed and died. The R1 values of the TN and TP in the PE group were −3.50% and −7.40%, respectively, which aggravated water pollution. Many researchers have reported the growth restriction and death Visual Observations In this study, the native aquatic plants both grew well, but the exotic plant E. crassipes decayed and died. The R 1 values of the TN and TP in the PE group were −3.50% and −7.40%, respectively, which aggravated water pollution. Many researchers have reported the growth restriction and death phenomena of E. crassipes in animal wastewater [20,21]. However, the phenomenon that stems, leaves, and roots of E. crassipes gradually whitened, rotted, and eventually died in the wastewater was first observed and reported in the present study, which deserves further investigation. Sooknah and Wilkie [21] suggested that high salinity was the principal reason for the inhibited phenomenon of E. crassipes. Haller et al. [20] reported that seawater with salt concentrations of 2500 mg/kg had toxic effects on E. crassipes, which was equivalent to a conductivity of 4040 µS/cm, using a conversion factor of 1000 mg/kg = 1616 µS/cm. But the highest EC value in the present study was just 434 µS/cm. Gerendás et al. [22] reported that numerous aquatic plants exhibit reduced growth under strict NH 4 + nutrition, and develop NH 4 + toxicity symptoms, including chlorosis of the leaves, overall growth suppression, and reduced root-to-shoot ratios. Ammonium levels of 188 mg/L were considered high N levels in constructed wetland cells [23]. Moreover, the unionized form of ammonia, namely, NH 3 , may contribute to the death of plants, the concentration of which depends on the ammonium ions, NH 4 + , and pH [24]. Reddy et al. [25] pointed out that the content of NH 3 in wastewater is negligible if the pH is below 8.0. In the present study, the highest concentration of NH 4 + -N in all treatments was below 8.00 mg/L, and the pH was between 7.00 and 8.00 in the E. crassipes group, indicating that NH 3 and NH 4 + were not the principal reasons for the death. Marschner [26] reported that a surplus uptake of NO 3 − can be stored in the vacuoles of plant cells without harmful effects. The change trend of the NO 3 − -N in the PE group was totally different from the others, but normal. Its concentration increased to 9.85 mg/L from below 0.1 mg/L, which was similar to that in the study of Sooknah and Wilkie [21], and suggested that a certain amount of nitrification occurred in the PE group. Moreover, the NO 2 − -N increased to 6.82 mg/L, nearly 34,764 times the initial value. It is worth investigating whether this was the reason for the reduced growth and death of the E. crassipes. Furthermore, Wan et al. [27] demonstrated that the lethal TN and TP concentrations for E. crassipes were 1514.26 mg/L and 200.4 mg/L, respectively, substantially higher values than those in our experiment. Because a wide range of soluble organic compounds has been reported to be toxic to plants [28], the nature of the uncharacterized organic matter in the wastewater could be another explanation for the death in this study [29]. Plant invaders can significantly diminish the abundance or survival of native species, and may completely alter the native ecosystem in terrestrial and freshwater habitats [30]. As an invasive aquatic plant that is commonly used in water restoration engineering, the ecological risks of E. crassipes require further assessment. The majority of experimental studies on the effects of plant purification have been carried out in manually disposed wastewater, which differs from an in-situ water body. Moreover, as the water environment is relatively complex, when selecting aquatic plants in engineering practice, it is necessary to conduct a pre-test on the water body to be repaired, consult the literature, and draw lessons from previous experience, which can prevent secondary pollution caused by plant growth discomfort and large-scale death to a certain extent. Nutrient Concentration and Absorption Capacity of Plants It has been established that the N and P concentrations in aquatic plant tissue gradually decrease during the active growth period, while the nutrients begin to accumulate after the active growth period [31]. In this study, the TN and TP concentrations in the L. adscendens and T. natans plant tissues all decreased continuously with an increasing HRT, which was related to the rapid reduction in the inorganic N concentration in the wastewater and the rapid growth of the plant biomass in the earlier period, resulting in the decrease of nutrient elements per unit of biomass, which was similar to the findings of Debusk et al. [29]. During the process of purifying eutrophic water by means of aquatic plants, the pollutants in the water can be transferred to the plants through plant absorption, and then removed from the water by harvesting the plants. Numerous studies have demonstrated that the absorption of nutrients by aquatic plants contributes little to the removal of nutrients in wastewater, at only approximately 2% to 6% [32,33]. However, Jiang et al. [34] reported that the uptake of N and P by 17 plants accounted for 46.8% and 51.0% of the total removal of water pollutants, respectively. In this study, the absorption contribution rates (R 2 ) of the TN and TP in the two native plants were between 34.5% and 43.5%, suggesting that R 2 was affected by the plant species and water pollution degree. As reported by Brix [35] and Peterson and Teal [36], the uptake of N and P by plants contributes significantly to the removal of nutrients in low-load constructed wetlands, and plays a limited role in high-load systems. Therefore, it is ineffective to evaluate the absorptive capacity of plants without considering the research background. Jiang et al. [34] demonstrated that suitable water purification plants can be selected directly by means of the biomass index. In this study, more rapid plant biomass growth resulted in a superior purification effect of the water quality. Within 60 days, the biomass of L. adscendens increased 5.4 times, and the removal rates of the TN and TP in the water body were higher than 87.0%. This indicated that, with a large biomass and rapid growth capacity, the native aquatic plants L. adscendens and T. natans can be favored in selection for the relevant engineering practice of water ecological restoration. Changes in pH, DO, ORP, and EC in Wastewater pH is an important factor affecting nutrient removal; excessive acidity and alkalinity of the water body may aggravate the release of N and P in the sediment, which is the smallest when the water pH is neutral [37,38]. Moreover, a change in the water pH will affect the growth of cyanobacteria; a higher pH can promote the growth of algae cells, while a lower pH has the opposite effect [39]. In this study, the pH values in the PL and PT groups were reduced to neutral, which indicates that planting L. adscendens and T. natans in an alkaline environment can effectively reduce the pH value of the water body, and subsequently inhibit the release of N and P nutrients in the sediment and the growth of algae. In this study, the phenomenon whereby that the DO concentration in the PT group was relatively low compared to that of other groups could be explained by the reduced oxygen diffusion from the atmosphere into the water column, owing to the plant cover, higher root respiration rates, and oxygen uptake by the microorganisms attached to the roots [21]. Therefore, for effective water purification, the grown biomass of aquatic macrophytes must be removed from water bodies to maintain an optimal plant density and permit increased oxygen exchange. The EC represents the ability of a solution to conduct current, and indirectly infers the total ion concentration in the water. In this experiment, the EC values in the PL, PT, and BC groups exhibited a downward trend with the removal of N and P in the wastewater. However, the EC value in the PE group did not exhibit the same downward trend with the relatively fast removal rate of the nutrients, which could be owed to the growth stopping, the color change from purple to white, and the gradual decay of its roots. It has been established that the purification effects of aquatic plants on wastewater are closely related to their root systems. The allelochemicals secreted by the huge root systems of plants may influence the water's EC values, which requires further study [40]. Nutrient Removal Efficiency by Aquatic Plants In the process of using aquatic plants to repair eutrophic water bodies, the coupling effects of the microorganisms and plants play an important role in nutrient removal from the wastewater. The roots of floating aquatic plants provide substrates for microbial communities and aerobic microsites in a generally anaerobic environment [41]. Microbial communities promote nutrient assimilation by plant roots and largely aid in chemical transformations, including nitrification and denitrification [36,42,43]. N has a complex biogeochemical cycle, with multiple biotic/abiotic transformations involving seven valence states (+5 to −3) [14]. Effective means of N removal in wastewater include assimilation and absorption by plants, volatilization of NH 3 , nitrification/denitrification, entrapment of particulate matter (organic nitrogen) by the extensive root systems, and settling [21]. In this study, the rapid increases in the NO 3 − -N and NO 2 − -N in the PE group indicated that nitrification occurred extensively, whereby NH 4 + -N was oxidized into NO 2 − -N and NO 3 − -N by nitrifying bacteria, while the reduction in the NO 3 − -N and NO 2 − -N could be owed to the plant uptake and denitrification. The NH 4 + in wastewater may be lost from the system through volatilization, taken up by plants and microbes, or oxidized into nitrate during the nitrification process. Kronzucker et al. [44] reported that NH 4 + is the predominant form of inorganic N available for plant uptake. In the beginning of the present study, the removal of NH 4 + in the plant groups was significantly higher than that in the BC group, indicating that the aquatic plants played a significant role in the removal of NH 4 + . Reddy et al. [25] pointed out that losses of NH 3 through volatilization are insignificant if the pH value is below 7.5, and the losses are very often not serious if the pH is below 8.0. This suggests that the NH 4 + -N removal in the plant cultures may have been primarily owed to the plant uptake and nitrification, along with a lesser level of volatilization, given that these systems had less alkaline pH in this study. The P cycle is fundamentally different from the N cycle [14]. P cannot leave the water through gaseous volatilization, and the majority of P exists in the form of insoluble phosphate [45]. In this study, the TP removal rates of the two native plants in the wastewater were between 88.72% and 95.90%, which were substantially higher than those of the blank control (38.32%) and E. crassipes (26.82%) groups. The TP absorption contribution rates of the native plants were between 34.5% and 43.5%. The results indicated that plants play an important role in the removal of TP in wastewater; the uptake of aquatic plants contributes significantly to the reduction; and the other processes, such as desorption, precipitation, dissolution, microbial uptake, fragmentation, leaching, mineralization, sedimentation, and burial, work together. In this study, a major part of the degradation of pollutants (COD) in the wastewater could be attributed to the microorganisms around the roots, which may establish a symbiotic relationship with the plants. As an important indicator for evaluating the eutrophication level, Chl-a is significantly corrected with the biomass of phytoplankton. While competing with algae for nutrients and living space, many aquatic plants can produce allelochemicals, which are considered to play a role in regulating the distribution of the phytoplankton population, and may become an important means of controlling algal blooms [46]. In this study, the R 1 values of the Chl-a in the three plant groups were all significantly higher than that in the BC group, indicating that the aquatic plants could inhibit the growth of algae in the wastewater. Overall, the existence of aquatic plants can enrich the water biodiversity, improve the stability of the aquatic ecosystem resistance, improve the quality of the water environment, and promote the removal of N and P by other factors. For example, the oxygen secretion function of aquatic plant roots can promote the growth and metabolism of nitrifying bacteria, denitrifying bacteria and other rhizosphere microorganisms, and accelerate the decomposition of pollutants. The introduction of appropriate aquatic plants into eutrophic water bodies may facilitate long-term improvement in the water quality. Conclusions Floating aquatic plants can play a significant role in purifying eutrophic water. The present results show that L. adscendens and T. natans are both promising native aquatic plants to be applied to the restoration of swine manure wastewater; plants can adjust the pH of the wastewater, improve the DO and ORP, reduce the EC value and COD, and inhibit the growth of algae; the main removal pathways of N and P include the plant uptake and the synergistic effects of plant roots and microorganisms, revealing that the suitable water purification plants can be selected directly by means of the large biomass, rapid growth capacity, and developed root system index; for effective water purification, the grown biomass of aquatic macrophytes must be removed from water bodies to maintain an optimal plant density and permit increased oxygen exchange; as an invasive aquatic plant that is commonly used in water restoration engineering, the ecological risks of E. crassipes require further assessment. In conclusion, our results have indicated that the native aquatic plants have great potential in the ecological restoration of water bodies.	v3-fos-license
2019-06-20T13:11:19.919Z	2019-06-01T00:00:00.000	195067265	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/2073-4409/8/6/574/pdf", "pdf_hash": "1cff417b1e35b9820c1828a7cde5b865c47fc993", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1810", "s2fieldsofstudy": [ "Biology" ], "sha1": "1cff417b1e35b9820c1828a7cde5b865c47fc993", "year": 2019 }	pes2o/s2orc	A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism. Introduction Selenocysteine (Sec) is the 21st proteinogenic amino acid and it was the first addition to the genetic code deciphered in the 1960s. Selenocysteine, a cysteine analogue with selenium replacing sulphur, is co-translationally inserted in the protein sequence via an unusual translational mechanism that consists of the recoding of an UGA codon which is normally read as a stop codon for other cellular mRNAs [1][2][3][4][5][6][7]. In human, 25 selenoprotein genes have been identified [8], in which the termination signal is often one of the two other stop codons, namely UAA or UAG. This UGA/Sec recoding process is possible due to two dedicated RNA components and their interacting partners. First, the selenocysteine insertion sequence (SECIS) is a stem-loop structure of approximately 100 nucleotides that is located in the 3 UTR of all selenoprotein mRNAs [9,10]. The SECIS is necessary and sufficient to drive the efficient recoding of an in-frame UGA codon. This feature has been particularly convenient when performing a structure-function analysis of the SECIS element in heterologous gene systems, such as luciferase reporter constructs containing the SECIS in the 3 UTR [9,[11][12][13][14][15]. The SECIS element serves as a dynamic platform to recruit the SECIS binding protein 2 (SECISBP2) and other Sec dedicated recoding factors [14,[16][17][18][19][20]. The second key RNA component of Sec insertion machinery is the Sec-tRNA [Ser]Sec ( Figure 1A) which associates with a selenocysteine-specific elongation factor (EFSec) [17,18]. One Sec-tRNA [Ser]Sec gene is present in the human genome in chromosome 19 (TRU-TCA1-1 or TRNAU1) [21]. A pseudogene is also found in chromosome 22 (TRU-TCA2-1 or TRNAU2) and contains mutations leading to an inactive acceptor arm (see Figure 1C). In addition, TRNAU2 does not seem to be transcribed by RNA polymerase (Pol) III [21]. Strikingly, the Sec-tRNA [Ser]Sec is the only known tRNA that governs by itself the expression of an entire group of proteins, the selenoproteome, which is composed by 25 selenoprotein genes [2,22]. Therefore, in contrast to other cellular tRNAs, the inactivation of the tRNA [Ser]Sec could be achieved by only one gene disruption. In mice, its gene inactivation (Trsp∆) leads to embryonic lethality at stage E6.5, indicating the essentiality of at least one selenoprotein in mammalian development [23]. To study the role of selenoproteins in various tissues, the inactivation of selenocysteine insertion is achieved by crossing mice carrying a conditional allele of Sec-tRNA [Ser]Sec (Trsp fl/fl ) with tissue-specific Cre strains [24]. To date, the removal of mouse Trsp was reported in mammary glands, liver, kidney, heart, thyroid, skeletal muscle, prostate, skin, endothelial cells, T-cells, macrophages, osteo-chondroprogenitors, and neurons with different phenotypes (as reviewed in Reference [24]). The Sec-tRNA [Ser]Sec harbors many different features in terms of size, structure, transcription, modification, aminoacylation, and transport [1][2][3]22] that make it unique in comparison with the other cytoplasmic tRNAs. First, with 96 nucleotides in length, it is by far the largest tRNA in eukaryotes. Then, the relative ratio between the acceptor arm size (expressed in base pairs (bp)) versus TψC arm is distinct from canonical tRNAs. The Sec-tRNA [Ser]Sec folds in a 9/4 secondary structure instead of 7/5 in other cellular tRNAs (see Figure 1A,B). In addition, the variable arm is particularly large with 16 nucleotides folded in a stem loop. These features not only prevent it from interacting with the elongation factor EF-1A but they are also used to specifically interact with EFSec. The transcription of TRNAU1 gene in pre-tRNA [Ser]Sec by RNA Pol III is also singular. Instead of having the two intragenic Box A and B sequences, the tRNA [Ser]Sec gene has three upstream promoters: a TATA box, a proximal sequence element (PSE) and a distal sequence element (DSE); and one intragenic Box B as illustrated in Figure 2A. Interestingly, this unusual transcription causes a 5 leaderless pre-tRNA [Ser]Sec with only the 3 -end to be processed into a mature tRNA. In terms of post-transcriptional modifications, only four modified bases are found in Sec-tRNA [Ser]Sec which is in the lower range for tRNAs ( Figure 2A). Methyladenosine (m 1 A) at position 58 and pseudouridine (ψ) at position 55 are both important for the tRNA folding [25,26]. In the anticodon loop, one finds two other modified bases that are critical for UGA recoding, namely the 5-methoxycarbonylmethyl-uridine (mcm 5 U) at position 34 and N 6 -isopentenyladenosine (i 6 A) at position 37. Interestingly the mcm 5 U34 base, which is in the wobble position in tRNA [Ser]Sec can be further methylated into 5-methoxycarbonylmethyluridine-2 -O-methylribose (mcm 5 Um). Since this latter methylation reaction is not complete, two isoforms (mcm 5 U34 vs mcm 5 Um34) co-exist in the cytoplasm, the methylated form being stimulated by selenium supplementation both in cell and animal models [27,28]. Interestingly, mouse models missing mcm 5 Um34 are unable to synthesize several selenoproteins including Gpx1, SelenoW, and Msrb1 [29]. In contrast to other proteogenic amino acids, selenocysteine is not charged as such on its dedicated tRNA but it is instead synthesized onto the tRNA from the amino acid serine, its oxygen analog, and hydrogen selenide (HSe − ) as the selenium donor. Therefore, the aminoacylation of Sec-tRNA [Ser]Sec involves four enzymes rather than only the amino acid-tRNA synthetase (aaRS) for other tRNAs [1][2][3]22]. Namely, the seryl-tRNA synthetase (SerRS), the phosphoseryl-tRNA kinase (PSKT), Sec synthase (SepSecS), and selenophosphate 2 synthetase (Sephs2) are required for the charging of a serine amino acid which is further transformed into a selenocysteine. Finally, concerning its transport, EFSec has evolved from EF1A to form a complex with the charged Sec-tRNA [Ser]Sec . In one of the current models for selenocysteine insertion, the EFSec/GTP/Sec-tRNA [Ser]Sec ternary complex is recruited by the SECISBPP2/SECIS complex in the 3 UTR of selenoprotein mRNAs to deliver the tRNA to the ribosome when an UGA codon occupies the A site [1,3]. Nevertheless, the sequence of events leading to selenocysteine insertion awaits further analysis, notably at the level of a three-dimensional structure and mRNP complexes characterization. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized the research in life science by allowing simple and versatile gene engineering [30]. CRISPR is a two component system composed of the bacterial derived nuclease CRISPR-associated protein 9 (Cas9) associated with the single-guide RNA (sgRNA) designed to target the complementary DNA sequence in genomic DNA (gDNA). This complex induces a double strand DNA (dsDNA) break at the specific locus, which is then repaired by the cell using the non-homologous end-joining (NHEJ) machinery. In most cases, the resulting gDNA is different from the original sequence with several nucleotides insertion or deletion (Indel) at the cutting site. CRISPR strategies result in either knock-out or knock-down depending on the experimental design [31]. Our lab has recently developed a protein-delivery vector, named Nanoblades, which is a viral-like particle (VLP) based on the murine leukemia virus (MLV) which allows the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) to various cells [32]. These Nanoblades are devoid of genetic material and lead to a rapid, transient, and efficient action of the Cas9-sgRNA RNPs on the gDNA, even in cells that are technically difficult to transfect. Table S1. Nucleotide insertion or deletion (Indel) can occur at the cutting site due to imperfect repair by NHEJ. This Indel can impact the tRNA [Ser]Sec expression at three levels: (1) The efficiency of transcription by altering the B box and/or the termination site, (2) the removal of the 3'-trailer and the CCA-addition at the 3 -end of the tRNA by altering the recognition of the tRNA by the respective enzymes (tRNA endonuclease and tRNA nucleotidyltransferase), and (3) the modifications of the tRNA. In any case, the action of Cas9-sgRNA at this site leads to impaired and/or reduced levels of tRNA [Ser]Sec . The tRNA sequence and the important regions for the transcription are represented in blue and green, respectively. DSE, distal sequence element; PSE, proximal sequence element; TATA, TATA box; polyT, transcription stop site. Here, we have engineered a novel method allowing a robust reduction of selenoproteins in various cell lines by targeting the 3 -side of the acceptor arm of Sec-tRNA [Ser]Sec using CRISPR-Cas9-VLPs. We showed an efficient Indel score at the specified gDNA locus in six different cell lines, namely Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP. We observed an efficient down-regulation of the Sec-tRNA [Ser]Sec levels in response to genomic alteration which provokes a decrease in selenoprotein expression. In summary, we report a novel experimental strategy to study selenoprotein deficient cell lines without affecting selenium levels. As such, it was designed to knock-down Sec-tRNA [Ser]Sec expression to a level that was sufficient to drastically down-regulate selenoprotein synthesis without affecting cell viability. Materials and Methods This manuscript adopts the new systematic nomenclature of selenoprotein names [33]. Production of Viral-Like-Particles sgRNA coding sequence was cloned into pBlade plasmid as previously described [32]. One microliter of each primer (10 pmol/µL) 5'-caccgCTTAGTTACTACCGCCCGAA-3' (TRNAU1) and 5'-aaacTTCGGGCGGTAGTAACTAAGc-3' (EMX-1) were hybridized into 50 µl of 1X New England Biolabs Buffer 2. The mixture was heated at 94 • C for 2 min and slowly decreased to 40 • C. One microliter of the hybridized mix was used in a ligation reaction (New England Biolabs, Ipswitch, MA, USA) containing 200ng of pBlade digested by BsmBI and 1 µL of T4DNA-Ligase. This operation resulted in the insertion of the gRNA sequence upstream of the pBlade U6-promoter to drive tRNA-gRNA expression in transfected cells. Plasmid transformation was performed into 5-alpha Competent E.coli (New England Biolabs) according to the manufacturer's instructions. To quantify the amount of Cas9 packaged into particles, VLPs or recombinant Cas9 (New England Biolabs) were diluted in Reporter Lysis 0,5× Buffer (Promega, Madison, WI, USA) and serial dilutions were spotted onto a nitrocellulose membrane. After incubation with a blocking buffer (nonfat milk 5% w/v in Tris buffer saline with 0.1% Tween20 (TBST)), the membrane was incubated with a Cas9 antibody coupled with HRP (7A9-3A3 clone, Cell signaling Technology, Danvers, MA, USA). Cas9 spots were revealed with ECL Select reagent by the Chemidoc touch imaging system (Biorad, Hercules, CA, USA) and analyzed with ImageLab Software (Biorad, Version 6.0.1). Transduction Procedure Cells were platted in a minimal volume to optimize cell/particles interactions for at least 2 h before supplementation with fresh medium. The indicated amount of VLP preparation was added to the medium for 2 × 10 5 adherent cells. Cells were harvested after 4 days for genomic DNA extraction or grown for another 4 days in medium supplemented or not with 100 nM sodium selenite for protein and RNA extractions. For the rescue experiment, cells were transfected with the TRNAU1 containing plasmid using TurboFect Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions 4 days after transduction. Genomic DNA Extraction and Analysis Genomic DNA was extracted from VLP-treated cells using the Nucleospin gDNA extraction kit (Macherey-Nagel). One hundred fifty nanograms of genomic DNA was then used for polymerase chain reaction (PCR) amplification with primers: Fw: 5 -CAGGGCTGTCACCCACCGCTGCGTCCTC-3 Rev: 5 -GTCAACCATCTCACACCTTTCCAAAGG-3 . For the T7 endonuclease1 mismatch assay, PCR products were diluted twice and complemented with Buffer 2 (New England Biolabs). Diluted PCR amplicons were then denatured at 95 • C and cooled down to 20 • C with a 0.1 • C/s ramp. Heteroduplexes were incubated for 15 min at 37 • C with T7 endonuclease 1 (New England Biolabs). Samples were run on a 2.5% agarose gel stained with ethidium bromide. Quantifications were performed with ImageLab software (Bio Rad). To analyze the mutants generated by Cas9, the region encompassing the tRNA gene was amplified by PCR from the gDNA extracted from cells treated with tRNA VLPs using the forward (5 -GGGGCCAGGGT GAATCAGACTC-3 ) and reverse primers (5 -TCCGGAGGGGGAAATAAGTAACG-3 ) with the Gotaq polymerase (Thermo Fisher Scientific). The PCR products from Hek293 and HAP1 gDNA were cloned into a pCRII TA cloning vector according to manufacturer's instructions. The plasmid DNAs from a total of 57 and 58 colonies were extracted and sequenced for Hek293 and HAP1 cells, respectively. RNAs were reverse transcribed using qScript cDNA Synthesis kit (Quanta Bio, Beverly, MA, USA) according to the manufacturer's instructions. Real time PCR was performed in triplicate using FastStart Universal SYBR ® Green master (Roche Applied Science, Penzberg, Germany) on a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers used are described in Reference [36] and listed in Table S1. Serial dilutions of a cDNA mixture were used to create a standard curve and determine the efficiency of the amplification for each couple of primers. Protein Extraction and Analysis by Western Blot Cellular protein extracts were harvested, from 6-well plates, with 150 µL passive lysis buffer containing 25 mM Tris phosphate, pH 7.8, 2 mM DTT, 2 mM EDTA, 1% Triton X-100 and 10% glycerol. Next, protein concentrations were measured using the DC kit protein assay kit (Biorad) in microplate assays. Design of a CRISPR-Cas9-Viral-Like-Particles Targeting the Sec-tRNA [Ser]Sec (TRNAU1) Gene The Sec-tRNA [Ser]Sec gene is essential in mice and conditional knock-out mice have a peculiar phenotype in all the tissues studied [2,23]. Therefore, a complete knock-out of this gene in human cell lines would probably result in lethality or the strong perturbation of cell growth. Thus, we aimed at altering the Sec-tRNA [Ser]Sec gene to decrease overall selenoprotein expression without affecting cell viability. As illustrated in Figure 2B, we designed a CRISPR strategy targeting the 3 -side of the acceptor arm of Sec-tRNA [Ser]Sec . After cutting the gDNA at this specific locus, nucleotide insertion or deletion were generated by the NHEJ pathway leading to either a less functional tRNA and/or a decreased expression of the tRNA. Another reason for targeting the acceptor arm was that, in human genome, there are two encoding Sec-tRNA [Ser]Sec genes, TRNAU1 and TRNAU2 (in chromosome 19 and 22, respectively) which mostly differs in this region, as illustrated in Figure 1C. Even though TRNAU2 is a pseudogene that does not express any Sec-tRNA [Ser]Sec in cells [21], we thought that is was wise to design a CRISPR specific of TRNAU1 gene. In addition, we calculated the MIT guide specificity score of our sgRNA targeting the TRNAU1 gene (Position: chr19:45478589-45478611). The obtained MIT value of 99 in a 0-100 scale indicates a particularly low expected off-target effect. Another specificity of our experimental design consisted in delivering Cas9-sgRNA RNP using CRISPR-Cas9-Viral-Like-Particles, referred to as Nanoblades [32]. In this system, different components from various viruses are dismantled in several pieces and are co-expressed in 293T producer cells by co-transfection of the respective plasmids. The stoichiometry between the five plasmids has been optimized [32] to produce large quantities of efficient CRISPR-VLPs. In these VLPs, the Cas9 is fused to the MLV Gag gene via a proteolytic linker. Therefore, the Cas9-sgRNA is released from the viral Gag protein within the particle and therefore delivered to the transduced cells. The pseudotyping with envelope proteins VSV-G and BaEVRless provides a wide tropism for the entry of the VLPs. Here, two different CRISPR-VLPs were produced with distinct sgRNAs, one targeting the TRNAU1 gene and the other the EMX-1 endogenous cellular gene as an internal control [32]; this was referred to as tRNA and EMX VLPs, respectively. CRISPR-Cas9-VLPs Induced Mutations in TRNAU1 Gene with High Efficiency in Various Cell Lines In the present study, we selected five cell lines that are commonly used in selenium biology, Hek293, HepG2, HaCaT, HeLa, and LNCaP, which originate from kidney, liver, skin, ovary and prostate, respectively. The other one (HAP1) that is derived from a patient with chronic myeloid leukemia is haploid and therefore widely used in CRISPR experiments. The Cas9 concentration was measured in the produced tRNA and EMX VLPs ( Figure S1). The six cell lines were transduced identically using increasing amounts of tRNA or the control EMX VLPs. Four days after transduction, the cells were harvested and their gDNA was purified and analyzed either by T7 endonuclease 1 mismatch detection assay ( Figure 3 and Reference [32]) or by Sanger sequencing followed by TIDE analysis (Figure 4 and Reference [34]). As illustrated in Figure 3, the T7 endonuclease 1 mismatch detection assay is commonly used to evidence Cas9 induced mutations in gDNA. Although mostly qualitative, it allows the comparison of different experimental parameters such as the quantity of VLPs used for transduction. When the NHEJ repair system modifies the gDNA by inserting or deleting nucleotides at the Cas9 cutting locus, it generates a cutting site for the T7 endonuclease 1. Therefore, the efficiency of cleavage on the re-hybridized PCR product is directly correlated to the number of Cas9-induced mutations that have occurred. As observed in Figure 3B, at the highest concentration of tRNA VLPs, the cleavage efficiency of the 600 bp DNA into a 150 and a 450 bp fragments was higher than 80% in all of the six cell lines analyzed. As a control experiment, the transduction of EMX VLPs did not induce mutations at the TRNAU1 locus of the gDNA in any of the cell lines studied. In order to determine the amount of VLPs required to reach the maximum Indel scores in different cell lines, we performed a dose-response analysis. Importantly, even with the highest amount of VLPs delivered, we did not observe any adverse effects on the growth and viability of the cell lines studied, as revealed by cell counting. In four cases, including HepG2, HAP1, HeLa, and HaCaT, we noticed an increase in cleavage efficiency in response to the amount of tRNA VLPs used for transduction. In the other two, namely Hek293 and LNCaP, the cleavage efficiency reached a plateau at the lowest concentration of tRNA VLPs added. However, due to the characteristics of the T7 endonuclease 1 assay, we could not compare the genome editing efficiencies between these different cell lines. We then performed the analysis of the gDNA from the TRNAU1 gene by TIDE software, which provides more quantitative results [34]. Based on the comparison of two Sanger sequences, the software performs a sequence trace decomposition from which the efficiency of genome editing can be estimated. Thus, the Indel score is inferred by the resulting percentage of differences from the wild-type (WT) sequence. A typical analysis is shown in Figure 4A,B for Hek293 cells in which different amounts of VLPs were transduced. When compared to the non-transduced cell line, the percentage of the Indel score increased with the quantity of Cas9-sgRNA in a dose-dependent manner to reach a maximal value of almost 100% in Hek293 cells. This result was highly specific since the EMX VLPs gave a background Indel of only 0,9% in a similar experiment. This analysis by TIDE software was applied to every cell lines and the Indel scores were plotted relative to the Cas9 concentration delivered (see Figure 4). This reflects that the optimal VLP amount might vary from one cell line to another. For the highest amount of Cas9 used (i.e., 20 pmol), the efficiencies of genome editing ranged as follows: Hek293 > HeLa, HAP1, LNCaP > HaCaT > HepG2. Taken together, our results provide evidence that the sgRNA chosen here is particularly efficient to trigger mutations of the gDNA at the TRNAU1 locus to reach an average of approximately 80% of genome editing. In addition, our data indicate that the VLPs have a broad tropism of transfection to deliver Cas9 in many cell lines. Comparison of TIDE Analysis with the Sequencing of Indel in the Hek293 and HAP1 Cell Lines When comparing results from Figures 3 and 4, it appears that the analyses by TIDE software provide more quantitative results than those obtained with the T7 endonuclease 1 mismatch detection assay. However, it has been reported that TIDE analysis slightly underestimates the actual genome editing as revealed by targeted next-generation sequencing (NGS). To receive further insight into the efficiency of our CRISPR design, we cloned the PCR products encompassing the TRNAU1 gene in a TA cloning vector and sequenced the plasmids purified from isolated colonies. The cloning was performed after delivering the highest dose of tRNA VLPs in Hek293 and HAP1 cells and a total of 57 and 58 inserts were sequenced. In Hek293 cells, we inferred an Indel score of 100% since none of the WT sequence was retrieved in the sequenced clones and this was in agreement with the Indel score of 96.7% given by TIDE analysis (see Figure 5A). A similar set of results was also obtained with HAP1 (see Figure 5C,D), with the noticeable difference that several WT sequences were recovered (8.4%). Therefore, the Indel score deducted from sequencing (91,4%) was moderately higher than the one obtained from TIDE analysis (76.5%). Taken together, our data indicate that the genuine efficiency of genome editing is slightly underestimated by TIDE analysis and they validate accurately the correct design of our CRISPR strategy ( Figure 5A,C). In both cases, when looking at individual clones, the repartition of Indel was strongly in favor of deletions rather than insertions (see Figure 5B,D and S2). None of our clones had a single point mutation at the cleavage site. Interestingly, a significant number of our clones (7.0% in Hek2393, 24.1% in HAP1) have the same +1G insertion ( Figure S2). This unexpected over representation may result from either a preferred NHEJ repair or a cellular selection for mutated tRNAs that could still be functional. Selenium Levels and tRNA VLP Treatment Altered the Levels of tRNA [Ser]Sec The aim of this study was to modulate selenoprotein expression by either down-regulating the expression levels of tRNA [Ser]Sec or to affect its function by modifying the 3 -side of its acceptor arm. To investigate this issue, we performed northern blot analysis with total RNAs extracted from Hek293 and HAP1 cells treated with tRNA or EMX VLPs. Infrared fluorescent oligonucleotides were used to probe the tRNA [Ser]Sec (IR800) and the tRNA Ser (IR700) represented as green and red in Figure 5, respectively. The expression level of tRNA [Ser]Sec was normalized to the level of tRNA Ser . It should be noted that in these experiments, the concentration of selenium was adjusted to 100 nM and compared with unsupplemented medium. With both Hek293 and HAP1 cell lines, we observed an increase of tRNA [Ser]Sec abundance in response to selenium supplementation. This induction of steady state levels of tRNA [Ser]Sec by selenium supplementation was previously observed in human myeloid leukemia (HL-60) cells and rat mammary tumor (RMT) with 127 (10 ng/mL) and 109 nM (8.5 ng/mL) of selenium added, respectively [28]. However, the transduction of the tRNA VLPs triggered a decrease in tRNA [Ser]Sec levels of 50 and 70% in Hek293 and HAP1 cells, respectively (see Figure 6). Figure 5. Analysis of the Cas9 induced mutations found in Hek293 and HAP1 cell lines. Genomic DNAs extracted from Hek293 and HAP1 cells treated with 20 pmol of Cas9 tRNA VLPs were used as a template for PCR amplification and cloning into a PCRII cloning vector. The sequences of the plasmid DNAs obtained from 57 and 58 colonies (Hek293 et HAP1, respectively) were used to calculate total genome editing efficiency (deletion plus insertion) at the TRNAU1 locus. This efficiency was compared to the one obtained by TIDE in Figure 4, for Hek293 (A) and HAP1 (C) cells. After alignment with the WT sequence, the size of the deleted (red) or inserted (blue) nucleotides are indicated in a histogram for Hek293 (B) and HAP1 (D) cells. In Hek293 cells, the efficiency of genome editing was close to 100%, indicating that the remaining 50% of the tRNA [Ser]Sec detected by northern blot should bear mutations. These mutations in the TRNAU1 gene do not seem to inhibit the transcription nor the maturation of tRNA [Ser]Sec but they could affect the stability of the tRNA. Indeed, it has been reported that the removal of 3 -trailer and the addition of CCA end are important steps in the quality control stage of tRNAs [37,38]. In any case, our data demonstrated that our Cas9-sgRNA design leads to a strong reduction of tRNA [Ser]Sec levels in Hek293 cells. Interestingly, a more pronounced decrease in tRNA [Ser]Sec levels was observed in HAP1 than in Hek293 cells ( Figure 6), even though genome editing efficiency was slightly lower in HAP1 (Indel score of 91,4%). This data suggest that the effects of genome editing could vary amongst different cell lines and this can be explained by several features. First, HAP1 cell lines are haploids while Hek293 cells have between four to five copies of chromosome 19, the chromosome which bears the TRNAU1 gene [39] as many immortalized or cancerous cell lines used in research laboratories contain numerous chromosomal copies. Secondly, the endogenous concentration of the tRNA [Ser]Sec and the requirement for selenoprotein activities may greatly vary between cell lines. As a consequence, the balance between the modifications of the tRNA [Ser]Sec gene and the maintenance of cell viability should be specific to every cell line. Selenoprotein Levels Are Differently Affected by CRISPR-Cas9-VLPs Amongst Cell Lines Next, we looked at the expression of ubiquitous selenoproteins, Gpx1, Gpx4, and Txnrd1, which ranked differently in the selenoprotein hierarchy in response to selenium fluctuation. As reported in [36] and shown in Figure 7, the sensitivity to selenium supplementation ranged as follows: Gpx1 > Gpx4 > Txnrd1 in most cells. In several cell lines, such as HeLa, Gpx4 was slightly more sensitive to selenium variation than Gpx1. Thus, we investigated whether the down-regulation of tRNA [Ser]Sec could affect the expression of these well-characterized selenoproteins in response to selenium supplementation and in different cellular models ( Figure 7A). Interestingly, we observed that in every cell line tested here, the down-regulation of tRNA [Ser]Sec had a similar effect on selenoprotein expression, with Gpx1 being more sensitive than Gpx4, and Txnrd1 being the least responsive ( Figure 7B). Importantly, even though Txnrd1 was poorly regulated by selenium supplementation, its expression was significantly affected by the VLP treatment in the majority of the cell lines tested. gives an non-specific band (indicated by an asterisk) below the correct one. Gpx4 has two isoforms, mitochondrial and cytoplasmic, respectively. Depending on cell lines, Txnrd1 has several isoforms. The quantifications were normalized to the intensity of the signal for actin and expressed relative to the non-VLP treated condition in control medium, set as 1. (B) The relative decrease of Gpx1, Gpx4, and Txnrd1 protein abundance in response to 20 pmol of Cas9 tRNA VLPs treatment and in presence of 100 nM Se was plotted for the six different cell lines. The results obtained with Hek293 cells were particularly informative, since the genome editing reached almost 100% at the TRNAU1 locus. The observation that these cells are still able to insert a selenocysteine and produce selenoproteins at significant levels indicate that the tRNA [Ser]Sec was still functional, at least partially, with mutations in the acceptor stem. Changes in selenium levels induce a prioritized synthesis of selenoprotein, and this phenomenon is linked to changes in tRNA [Ser]Sec levels [28]. Therefore, it seems rational that reducing tRNA [Ser]Sec levels and/or its activity would induce a similar hierarchy than the decrease of selenium levels. Taken together, our data provide evidence that selenoprotein expression can be significantly decreased by targeting the TRNAU1 gene in different cell lines. Selenoprotein mRNA Levels Are Not Affected by tRNA [Ser]Sec Down-Regulation in Hek293 and HAP1 When the UGA-recoding efficiency is altered, the UGA codon can then be seen as a premature stop codon by the nonsense mediated decay (NMD). For example, the knockdown of SECISBP2 was shown to reduce the levels of several selenoprotein mRNAs, including SelenoH, SelenoT, Txnrd2, and Gpx1 [40]. Additionally, in cells from patients with mutant forms of SECISBP2, several transcripts were significantly reduced (SelenoH, SelenoT, and SelenoW) while others were preserved or even upregulated (SelenoO, SelenoI, SelenoM, and SelenoK) [41]. Since we observed a decrease in selenoprotein levels, it was therefore important to verify whether the reduced levels of tRNA [Ser]Sec could have an impact on the landscape of selenoprotein transcripts. As illustrated in Figure 8, total RNAs extracted from Hek293 and HAP1 treated, or not, with tRNA or EMX VLPs, and supplemented, or not, with 100 nM selenium, were evaluated for the expression levels of the 25 selenoprotein mRNAs. These data were normalized to the levels of five housekeeping transcripts (HspcB, rPS13,18S rRNA, Hprt, Gapdh) used as a reference. First, we confirmed that in Hek293 and HAP1 cell lines, selenium supplementation did not dramatically alter the level of expression of selenoprotein transcripts. Only SelenoH and SelenoW were significantly different between supplemented and unsupplemented extracts in Hek293 cells ( Figure 8B, left panel). These data are in agreement with our recently published work [36], where we reported only marginal variations of selenoprotein mRNAs in response to selenium supplementation in various cell lines, including Hek293, HepG2, HaCaT, and LNCaP. In the present work, we further evaluated whether the selenoprotein transcripts were affected by the tRNA VLP treatments. We found no significant variation of these transcripts in response to VLP treatments (EMX or tRNA) indicating that selenoprotein transcripts were not targeted by the NMD following the decrease in levels of tRNA [Ser]Sec and this situation occurred independently from the addition of selenium in the medium. The Overexpression of tRNA [Ser]Sec Allowed the Recovery of Selenoprotein Expression in VLP Treated Cells In order to validate the design of our experimental strategy and rule out a potential off-target effect, it was important to confirm that the decrease of tRNA [Ser]Sec is actually directly responsible for the down-regulation of selenoprotein levels. Therefore, we performed rescue experiments where the wild-type TRNAU1 gene was transfected in VLP-treated cells. It has been previously shown that tRNA [Ser]Sec overexpression had a moderate stimulatory effect on selenoprotein levels in wild-type cells [42,43]. As illustrated in Figure 9, when we increased the levels of tRNA [Ser]Sec in Hek293 cells treated with the control VLPs, this led to higher expression of Gpx1, Gpx4, and Txnrd1. In cells treated with the tRNA VLPs, even though we had a lower transfection efficiency (data not shown), the overexpression of the tRNA led to an efficient recovery of selenoprotein expression to a level similar to the wild type ( Figure 9A). By performing northern blotting experiments, we confirmed that the expression of the tRNA [Ser]Sec was restored ( Figure 9B). Taken together, these data confirmed that the effects observed with the VLPs are indeed due to Cas9 induced mutations (knock-out and mutations in the 3' side of the acceptor arm) of the TRNAU1 gene. In the future, it will be important to determine the molecular mechanism put in place and, especially, how these mutant forms of the tRNA can still be able to be matured, modified, and charged with selenocysteine. Figure 6 were also used for RT-qPCR analysis to measure selenoprotein and housekeeping mRNA levels. The geometrical mean of five housekeeping genes (Hpcb, Rps13, rRNA 18S, Hrpt, and Gapdh) was used to normalize mRNA abundance. Selenoprotein mRNA levels are represented in logarithmic scale in a heatmap. The values are given in Table S2 and Table S3 Development of a Novel Method to Produce Cells Lines With Reduced Selenoprotein Levels Changing the selenium concentration of the culture medium is the most common method to modulate the expression of the selenoproteome. This results in significant differences in selenoprotein production with some being more expressed than others both in cultured cells and animals; this is referred to as the selenoprotein hierarchy [1,11,36,44,45]. However, changes in selenium levels can have other indirect cellular consequences, such as modulating potential trace element signaling pathways. Therefore, in order to precisely understand the function of selenoproteins in various mechanisms, there is certainly a need to develop alternative methods to modulate selenoprotein expression without changing the intracellular concentration of selenium. This was the global aim of this study, in which we describe a novel strategy to target the 3 -side of tRNA acceptor stem of the tRNA [Ser]Sec by using a CRISPR/Cas9 approach based on delivery via a retroviral VLP. The Cas9 induced mutations in the gDNA locus induced functional disruptions that significantly altered tRNA expression and therefore reduced selenoprotein levels. The use of a VLP system allowed the efficient delivery of its contents to virtually any human cell line, as observed here and reported for other cell lines [32]. The tRNA [Ser]Sec is one of the key components of the UGA recoding machinery by the ribosome. Present in only one copy in the human genome and required for the synthesis of all of the 25 selenoproteins, it is the ideal target to control selenoproteome expression. However, there is a fine balance between the modification of tRNA concentration or alteration of its function and the maintenance of cell viability. Indeed, the complete inactivation of the tRNA [Ser]Sec gene is lethal in mice and even a conditional knockout in mice liver, endothelial cells, heart and skeletal muscle, and skin leads to premature animal death [24]. Next, in most of other animal models with tissue selective inactivation of the tRNA [Ser]Sec gene, a strong phenotype was also observed, even though the mice remained viable. These data obtained in animals suggested that our first goal should be to reduce the tRNA [Ser]Sec concentration to a level which is compatible with cell survival. This is why we have induced Cas9 mutations at the site near the 3 -end of the tRNA, as it seemed less detrimental for cell viability than in another region. On the other hand, it appeared that the level of genome editing efficiency did not completely match with the phenotype in all cell lines. However, the phenotype at the selenoprotein level seemed to be specific for every cell line tested and thus needed to be characterized every time in response to VLP-CRISPR transduction. In our hands, we were able to reduce between 50% and 90% of the production of Gpx1 and between 40% and 85% of that of Gpx4 in the six cell lines studied (Figure 7). Importantly, in every case, we observed that the expression of all selenoproteins retained their ability to be stimulated by selenium induction. A novelty of our experimental approach lies in the delivery mode of Cas9-sgRNA RNP by viral pseudoparticles instead of transfections or infection with CRISPR-coding viral vectors. In comparison with these commonly used methods, our novel strategy allows to fine tune the level of genome editing with minimal off-target cleavages of gDNA [32]. We validated the proof of concept of this method in six human cell lines by applying the same protocol of transduction. Indeed, the current version of the VLP-CRISPR has a wide tropism due to the presence of the two envelope proteins VSV-G and BaEVRless. Overall, we provide a versatile strategy to significantly reduce the levels of expression of selenoproteins in various human cells without altering their ability to respond to selenium concentrations in the medium. Another conceivable strategy for down-regulating tRNA [Ser]Sec levels is the use of small hairpin RNAs (shRNAs) gene silencing. To our knowledge, only one successful example of tRNA down-regulation by shRNAs has been reported in mammals and this was achieved by targeting the tRNA Thr (CGU) that pairs to the ACG codon which is one of the rarest tRNAs in HeLa cells [46]. However, reduction of the tRNA Thr (CGU) concentration remained of limited extent reaching only between a 25 to 40% decrease, depending on the nature of the shRNAs used. Moreover, the treatment with shRNAs targeting the anticodon loop of tRNA Thr (CGU) was only effective for a limited period of time (48 h). In our case, the editing on the gDNA is a definite genetic event which allows the selection by cellular sub-cloning. Another major difference between a shRNA and CRISPR strategy to down-regulate tRNAs, lies in the fact that shRNA only reduces the concentration of the endogenous tRNA target whilst Cas9 induces multiple mutations in the gene locus that result in alterations of gene expression. As such, the targeting of the 3 -end of the tRNA [Ser]Sec can induce changes at the level of RNA-PolIII transcription, 3 -trailer sequence removal, 3 -end CCA addition, post-transcriptional modifications and cloverleaf folding. Modifications of all of these steps will have effects on the ability of the tRNA to recode UGA into selenocysteine and its stability. Our data show that we obtained the right balance between modifications of the levels and function of the tRNA [Ser]Sec gene and the maintenance of cell viability in our experimental design. The Cas9-Induced Mutations in TRNAU1 Gene Lead to a Selective Down-Regulation of Selenoproteins to a Level Which Is Similar to Selenium Deficiency As stated before, an increase in selenium concentration in the culture medium results in the activation of selenoproteins but to a level that varies greatly across them. Although well described, this so-called hierarchy is only partially understood at the molecular level. It is proposed that the tRNA [Ser]Sec , the SECIS and SECISBP2 are involved in this phenomenon [1,3]. Concerning the tRNA, its steady state level and modification at the wobble position U34 are sensitive to selenium changes. To our knowledge, this is the first report of tRNA modification by a CRISPR-Cas9 experimental strategy. Obviously, the number of copies of tRNA genes represents a major hurdle for this strategy as tRNAs are often coded by several genes. However, in our case, the tRNA [Ser]Sec was present at only one functional copy in the human genome with the other copy having evolved towards a pseudogene. As observed by sequencing the Cas9-induced mutations of the gDNA at the TRNAU1 locus, the extent of genome editing ranged between 38-nt deletion and 171-nt insertion in Hek293 and between 32-nt deletion and 231-nt insertion in HAP1 cells. In Hek293 cells, genome editing attained nearly 100% efficiency, this means that every tRNA gene was expected to be hit by Cas9-sgRNA cleavage. However, the analysis by northern blot indicated that a significant level of full-length tRNA remained expressed in this Hek293 population. Therefore, this suggests that some mutations in the 3 -end of the tRNA can be tolerated without a significant loss of function. This was confirmed by the fact that these mutants were still responsive to changes in selenium concentration. Another interesting finding was that the selenoproteins are differently affected by the reduced levels and mutations of the tRNA [Ser]Sec . As such, the expression of Gpx1 was more sensitive than Gpx4 and Txnrd1 was the least responsive one. Thus, the hierarchy in selenoprotein expression is maintained after the transduction of tRNA VLPs and is similar in all points to what is observed upon changes in selenium concentration. These data validate nicely our experimental system as a novel alternative to study the regulation of the selenoproteome. Since HAP1 cells are haploid, they represent a very convenient model for genome editing. In this cell line, all chromosomes are expected to be present in only one copy. Therefore, mutations and/or large deletions are expected to induce strong phenotypes on selenoprotein synthesis. In our hands, these deletions were viable in HAP1 suggesting that either selenoproteins are not essential in this cell line or that these mutants have retained a residual functional activity to maintain cell viability. Structure-function analyses of the TRNAU1 gene will now be possible with the many Cas9-induced mutants generated by our Cas9-sgRNA design. It should be noted that one mutation is overrepresented in HAP1 and it consists in a +1 insertion of a G at the Cas9 cutting site. This mutation is expected to change the 3 -side of the acceptor arm from CUUUCGGGCGCCA into CUUUCGGGGCCCA (underlined is the changed sequence, and in bold the inserted G). It will be of great interest to determine whether or not this mutation can affect any of the transcription, maturation, aminoacylation or stability of the tRNA [Ser]Sec . In any case, if a positive selection has occurred for this mutation over others in HAP1 cells, it certainly suggests that this modification is either silent or less detrimental than others. Alternatively, this could also be due to a repair bias in the insertion of a G rather than any another nucleotide by the NHEJ machinery. Since this machinery is different from one cell line to another, the Cas9-induced mutations are expected to be different from one model to another. Conclusions In this manuscript, we designed a novel method to reduce selenoprotein expression without changing the selenium concentration by targeting the human tRNA [Ser]Sec . This was rendered possible by the development of a CRISPR-Cas9-VLP that induced mutation at the 3 -end of the tRNA. This Cas9-sgRNA design was able to knock-down tRNA [Ser]Sec expression to a level that was sufficient to significantly reduce selenoprotein levels while maintaining cell viability. Proof of concept of this method was validated in six human cell lines, namely Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP. Overall, this method offers an original, novel alternative to study variations in the expression of the selenoproteome without affecting the intracellular level of selenium concentration. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/6/574/s1, Figure S1: Evaluation of Cas9 content in tRNA and EMX VLPs, Figure S2: Sequence alignment of the PCRII inserts shown in Figure 5B,D for Hek293 and HAP1 cell lines, respectively, Table S1: List of qPCR primers used in this study, Table S2: Values of Selenoprotein mRNA levels in response to the CRISPR-Cas9-VLP treatments and/or addition of selenium (100 nM) in HAP1 cell lines obtained by RT-qPCR, Table S3: Values of selenoprotein mRNA levels in response to the CRISPR-Cas9-VLP treatments and/or addition of selenium (100 nM) in Hek293 cell lines obtained by RT-qPCR.	v3-fos-license
2020-04-16T09:03:16.396Z	2020-04-01T00:00:00.000	215771463	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1422-0067/21/8/2666/pdf", "pdf_hash": "f1237914e8a42b43e3d87b1290c2d75012853b56", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1876", "s2fieldsofstudy": [ "Biology", "Engineering" ], "sha1": "837c96fdab77b4a3b364dc0c0125f71f98166ea4", "year": 2020 }	pes2o/s2orc	Transcriptional Profiling of the Probiotic Escherichia coli Nissle 1917 Strain under Simulated Microgravity. Long-term space missions affect the gut microbiome of astronauts, especially the viability of some pathogens. Probiotics may be an effective solution for the management of gut microbiomes, but there is a lack of studies regarding the physiology of probiotics in microgravity. Here, we investigated the effects of microgravity on the probiotic Escherichia coli Nissle 1917 (EcN) by comparing transcriptomic data during exponential and stationary growth phases under simulated microgravity and normal gravity. Microgravity conditions affected several physiological features of EcN, including its growth profile, biofilm formation, stress responses, metal ion transport/utilization, and response to carbon starvation. We found that some changes, such as decreased adhesion ability and acid resistance, may be disadvantageous to EcN relative to gut pathogens under microgravity, indicating the need to develop probiotics optimized for space flight. Introduction The recent surge of long-term space exploration necessitates an increased attention on the health risks associated with space travel. Studies have shown that astronauts who stay in space for extended time undergo changes in their microbiome environment. A study conducted by Garrett-Bakelman et al., at the National Aeronautics and Space Administration (NASA), has shown that an astronaut who underwent a year-long space mission experienced a change in his gut microbiome composition, compared to that of his earthbound twin sibling [1]. Furthermore, astronauts who have spent six to twelve months at the International Space Station (ISS) have also undergone changes in their gut microbiomes [2]. These changes include the increase of Parasutterella, which is associated with chronic intestinal inflammation with inflammatory bowel disease (IBD) and the reduction of intestinal Fusicatenibacter, Pseudobutyrivibrio, and Akkermansia, which have anti-inflammatory properties [2]. These results demonstrate that some factors during spaceflight can be critical to the gut microbiome of astronauts. These factors may affect the growth of both beneficial and detrimental microbial strains in the gut and thereby increase the possibility of the development of IBD. Multiple studies have indicated that microgravity (MG) conditions induce several physiological changes. Studies conducted at the ISS primarily targeted pathogens such as Salmonella enterica serovar Growth and Transcriptomic Profiling of EcN under Simulated MG and NG To culture EcN under simulated MG and NG conditions, we used a clinostat with different directions of rotation ( Figure 1A). MG conditions were simulated by rotating the system around the horizontal axis and manipulating the rotation speed of the vessel to reduce shear stress [19]. NG conditions were maintain by rotating the system around the vertical axis [9]. When EcN growth in glucose M9 minimal media was measured under MG (µ MG ) and NG (µ NG ) conditions, the maximum specific growth rates were 0.36 and 0.44 h −1 , respectively ( Figure 1B). In contrast to other studies that used E. coli K-12 MG1655 [7,9], the growth of EcN was slower under MG than NG. The growth difference under MG compared to that under NG may have been due to the sum effect of several factors involved with cell fitness. To reveal the metabolic changes of EcN in response to decreased gravity, we performed comparative transcriptomic profiling (RNA-seq) during exponential and stationary growth phases under MG and NG conditions. Differentially expressed genes (DEGs) were selected using the criteria of ≥2-fold changes (P ≤ 0.01) in fragments per kilobase of exon per million fragments mapped (FPKM) values. As many genes in the EcN reference strain CP007799.1 from the National Center for Biotechnology Information (NCBI) database were unannotated, we identified these unlabeled genes using Basic Local Alignment Search Tool (BLAST) software (blastp) [20] and publicly available web-based databases, such as the EcoCyc E. coli database (http://ecocyc.org/) [21] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [22]. A total of 86 genes were differentially expressed during the exponential growth phase and 224 genes were differentially expressed during the stationary growth phase ( Figure 1C). Among these DEGs, 11 were common to both growth phases ( Figure 1C). As shown in Table 1, the majority of common DEGs maintained their tendencies of being either up-regulated or down-regulated in both phases. Interestingly, some genes, such as cytochrome bo 3 ubiquinol oxidase (cyoA), cold shock protein (cspA), and cysteine transporter (tcyP), exhibited changes in their direction of regulation. Among the DEGs in the exponential and stationary growth phases, 38 and 43 genes were up-regulated, respectively, while 48 and 181 genes were down-regulated, respectively (Tables S1 and S2). Some were represented according to functional categories of hierarchical clusters diagrams based on expression ratios of MG to NG ( Figure 2). These categories included biofilm formation, stress responses, metal ion transport/utilization, and carbon metabolism. Growth and Transcriptomic Profiling of EcN under Simulated MG and NG To culture EcN under simulated MG and NG conditions, we used a clinostat with different directions of rotation ( Figure 1A). MG conditions were simulated by rotating the system around the horizontal axis and manipulating the rotation speed of the vessel to reduce shear stress [19]. NG conditions were maintain by rotating the system around the vertical axis [9]. When EcN growth in glucose M9 minimal media was measured under MG (μMG) and NG (μNG) conditions, the maximum specific growth rates were 0.36 and 0.44 h −1 , respectively ( Figure 1B). In contrast to other studies that used E. coli K-12 MG1655 [7,9], the growth of EcN was slower under MG than NG. The growth difference under MG compared to that under NG may have been due to the sum effect of several factors involved with cell fitness. Effect of MG on EcN Biofilm Formation Cell surface components, such as type I curli fimbriae and lipopolysaccharide (LPS), are important for the formation of complex biofilm structures, which protect cells from exterior factors [23][24][25]. Previous research has shown that MG can induce changes in gene expression related to Effect of MG on EcN Biofilm Formation Cell surface components, such as type I curli fimbriae and lipopolysaccharide (LPS), are important for the formation of complex biofilm structures, which protect cells from exterior factors [23][24][25]. Previous research has shown that MG can induce changes in gene expression related to biofilm formation [4,9,12,[26][27][28][29][30]. Similar to previous studies, most EcN genes related to type I fimbriae synthesis (fimA, fimI, fimC, fimD, fimG), flagella motility regulation (ycgR), and flagella synthesis (fliC) were up-regulated (2.18-4.14-fold) under MG during the exponential growth, which allowed for increased biofilm formation ( Figure 2A and Table S1). However, these genes were not differentially expressed between conditions of MG and NG during stationary growth. Since oxygen availability is lower in MG than NG [31], cells may offset this difference by forming thicker and more structured biofilm architecture from the onset, regardless of growth phase [28]. Unlike other bacteria, EcN under MG during stationary growth phase repressed genes related to major structural subunit of curli (csgB, csgA; 5.19-6.55-fold), LPS core synthesis (rfaQ, rfaG, rfaP; 2.03-2.39-fold), and lipid A (lpxP; 2.25-fold) [32]. CsgC, which functions as an inhibitor of csgA expression was highly expressed under MG conditions ( Figure 2A and Table S2) [33]. In addition to the structural robustness of the cell itself, the adhesion ability of EcN appeared to be the opposite of that reported for other gut microbiome species. For instance, F1C fimbriae, E. coli common pili (ECP), and K5 capsule play key roles in the adhesion of EcN to gut epithelial cells in [34]. Under MG, focA, which encodes the major F1C fimbriae subunit, was repressed during the exponential growth phase (2.28-fold) and ecpC, ecpB, ecpA, which encode ECP, and ecpR, which encodes the transcriptional regulator of ECP synthesis, were repressed (2.48-7.36-fold) during entire cell growth. Additionally, multiple copies of kfiA and kfiB encoding K5 capsule biosynthesis components were up-regulated (2.25-2.61-fold) under the same conditions (Tables S1 and S2). When kfiB is knocked-out, EcN attachment to Caco-2 cells is known to increase [35]. Collectively, the shifts in expression we observed may interfere with EcN adhesion to epithelial cells and prevent intestinal colonization. Additional experimental validation may need to be performed outside Earth in the future. Considering that cell adhesion of pathogens is increased under MG conditions [2], these results suggest that EcN may be less competitive in MG, resulting in pathogens causing an imbalance of the gut microbiome during spaceflight [34]. Effect of MG on EcN Stress Responses Exposure to MG can result in several changes outside of cell membrane, including convectional thermal loss and reductions in shear force. Cells must adapt to new conditions under MG by expressing resistance-related proteins [36]. In previous studies, bacteria in MG showed increased resistance to heat, osmotic pressure, and acidic conditions [6,8,9,12,37]. EcN for the most part maintained these general stress response tendencies ( Figures 2B and 3). During the exponential growth phase, EcN in MG displayed higher expression levels of heat shock protein (htpG; 2.06-fold), cold shock protein (cspA; 2.38-fold) compared to those under NG conditions (Table S1). Expression of heat shock proteins (hspQ, htpX, and ibpAB) and membrane protein involved in stress response (bhsA) were increased (2.21-7.96-fold) in the stationary growth phase. Meanwhile, cspA, which was induced during exponential growth, was down-regulated (2.24-fold) during the stationary growth phase (Table S2). While cspA has a role as a global regulator [38], it is unclear why its expression tendency differed under the two growth phases. Interestingly, EcN exhibited different trends in acidic resistance during the exponential growth phase under MG compared to that of other bacteria, with the repression of acid resistant chaperones (hdeBA and hdeD) being 2.38-2.75-fold [39] and glutamate decarboxylases (gadB and gadA) being 2.36-2.48-fold (Table S1). Gad encoded proteins are known as the components of the glutamate-dependent acid resistance (AR) system, which is an efficient AR system called AR2 [40]. During the stationary growth phase, glutamate decarboxylase (gadA) remained repressed (2.27-fold), along with arginine decarboxylase (adiA; 2.30-fold), which is another AR system known as AR3 (Table S2) [41]. These findings suggest that MG conditions may retard the growth of EcN by inducing more acidic conditions compared to that of NG, especially during the exponential growth phase [42]. Lower resistance to acidic stress by EcN under conditions of MG indicates that EcN may not function efficiently as a probiotic during spaceflight when administered orally, as it must pass through the gastrointestinal tract. cold shock protein (cspA; 2.38-fold) compared to those under NG conditions (Table S1). Expression of heat shock proteins (hspQ, htpX, and ibpAB) and membrane protein involved in stress response (bhsA) were increased (2.21-7.96-fold) in the stationary growth phase. Meanwhile, cspA, which was induced during exponential growth, was down-regulated (2.24-fold) during the stationary growth phase (Table S2). While cspA has a role as a global regulator [38], it is unclear why its expression tendency differed under the two growth phases. The tendency to lower free metal ion concentrations in the cytoplasm of cells was observed according to the utilization of iron ions. Intracellular iron ions may remain as free cations or form complexes, such as iron-sulfur (Fe-S) clusters or heme groups [55,56]. By forming Fe-S complexes, imported free iron ions associate with sulfate ions to alleviate the exposure of cells to hydroxide radicals that result from the presence of excessive free iron ions [57]. During the exponential growth phase, genes related to Fe-S assembly, such as iron-sulfur cluster assembly protein synthesis (iscR, iscS, iscUA) and iron-sulfur cluster biosynthesis chaperone (hscA), were up-regulated 2.05-3.31-fold. To supply sulfur for combining with iron ions, cysteine can be broken down by cysteine desulfurase [58,59]. Therefore, elevated assembly of the Fe-S clusters may induce sulfur and cysteine starvation inside the cell, resulting in the activation of sulfate transporter (sbp), which was up-regulated 2.32-fold, and cysteine transporter (tcyP), which was up-regulated 2.27-fold ( Figure 2C and Table S2) [59,60]. However, during the stationary growth phase, the direction of regulation of genes related to iron utilization was the opposite of that during exponential growth. For instance, sulfur metabolism was generally down-regulated. As shown in Figure 2C and Table S2, repressed genes included transcription factor cbl (2.07-fold), cysteine transporter (tcyP, tcyN, tcyJ; 2.30-2.69-fold), cysteine synthase (cysM; 2.13-fold), and genes related to sulfate assimilation (cysDNC and cysJIH; 2.61-4.63fold) [61]. These genes are known to be activated by transcription factor cysB when induced by Oacetylserine (OAS) [59]. Furthermore, sulfide and thiosulfate act in a competitive manner with OAS as anti-inducers [62]. These results indicate that the intracellular availability of sulfide and thiosulfate during stationary growth of the EcN under MG conditions may be the opposite to that during exponential growth. Effect of MG on EcN Carbon Metabolism MG is able to induce a lack of convective mixing, resulting in the creation of what have been referred to as "depletion zones" [63]. Many studies have reported that MG induces carbon starvation stress due to nutrient depletion [7,9,26]. Under conditions of carbon starvation, carbohydrate catabolism was altered, especially the sequentially inhibition of glycolysis and the pentose phosphate (PP) pathway [64]. Consistent with this, we found that the glycolysis enzymes 6-phosphofructokinase (pfkA) and glyceraldehyde-3-phosphate dehydrogenase (gapA) were repressed (2.27-3.68-fold) during the exponential growth phase, while genes related to the pentose phosphate (PP) pathway (pgl, tktB, ribB, prsA, and hisG) and Entner-Doudoroff (ED) pathway (gntK and edd) [65] were However, during the stationary growth phase, the direction of regulation of genes related to iron utilization was the opposite of that during exponential growth. For instance, sulfur metabolism was generally down-regulated. As shown in Figure 2C and Table S2, repressed genes included transcription factor cbl (2.07-fold), cysteine transporter (tcyP, tcyN, tcyJ; 2.30-2.69-fold), cysteine synthase (cysM; 2.13-fold), and genes related to sulfate assimilation (cysDNC and cysJIH; 2.61-4.63-fold) [61]. These genes are known to be activated by transcription factor cysB when induced by O-acetylserine (OAS) [59]. Furthermore, sulfide and thiosulfate act in a competitive manner with OAS as anti-inducers [62]. These results indicate that the intracellular availability of sulfide and thiosulfate during stationary growth of the EcN under MG conditions may be the opposite to that during exponential growth. Effect of MG on EcN Carbon Metabolism MG is able to induce a lack of convective mixing, resulting in the creation of what have been referred to as "depletion zones" [63]. Many studies have reported that MG induces carbon starvation stress due to nutrient depletion [7,9,26]. Under conditions of carbon starvation, carbohydrate catabolism was altered, especially the sequentially inhibition of glycolysis and the pentose phosphate (PP) pathway [64]. Consistent with this, we found that the glycolysis enzymes 6-phosphofructokinase (pfkA) and glyceraldehyde-3-phosphate dehydrogenase (gapA) were repressed (2.27-3.68-fold) during the exponential growth phase, while genes related to the pentose phosphate (PP) pathway (pgl, tktB, ribB, prsA, and hisG) and Entner-Doudoroff (ED) pathway (gntK and edd) [65] were repressed (2.17-14.89-fold) during the stationary growth phase ( Figure 5, Tables S1 and S2). Repression of pfkA at the exponential growth phase can induce the higher flux of glucose to the PP or ED pathway. Activation of these pathways may help protect cells from oxidative stress by production of NADPH [66,67]. By the way, regulation of the tricarboxylic acid (TCA) cycle can burden oxidative stress to the cell. Repressed glycolytic metabolism due to carbon starvation may also affect the tricarboxylic acid (TCA) cycle [68]. During the exponential growth phase, succinate:quinone oxidoreductase (sdhA) and malate:quinone oxidoreductase (mqo) were up-regulated 2.76-3.24-fold, concurrently with the repression of fumarate hydratase (fumB) 2.68-fold and fumarate reductases 2.77-3.03-fold ( Figure 5, Tables S1 and S2). The reduction of quinone by increased expression of mqo may be especially detrimental to EcN as the imbalance between quinone and quinol can induce oxidative stress [69]. In addition, there is a possibility of fumarate accumulation due to the activation of sdhA, which may consequently result in oxidative stress [70,71]. repressed (2.17-14.89-fold) during the stationary growth phase ( Figure 5, Tables S1 and S2). Repression of pfkA at the exponential growth phase can induce the higher flux of glucose to the PP or ED pathway. Activation of these pathways may help protect cells from oxidative stress by production of NADPH [66,67]. By the way, regulation of the tricarboxylic acid (TCA) cycle can burden oxidative stress to the cell. Repressed glycolytic metabolism due to carbon starvation may also affect the tricarboxylic acid (TCA) cycle [68]. During the exponential growth phase, succinate:quinone oxidoreductase (sdhA) and malate:quinone oxidoreductase (mqo) were up-regulated 2.76-3.24-fold, concurrently with the repression of fumarate hydratase (fumB) 2.68-fold and fumarate reductases 2.77-3.03-fold ( Figure 5, Tables S1 and S2). The reduction of quinone by increased expression of mqo may be especially detrimental to EcN as the imbalance between quinone and quinol can induce oxidative stress [69]. In addition, there is a possibility of fumarate accumulation due to the activation of sdhA, which may consequently result in oxidative stress [70,71]. Other genes related to energy metabolism were also differentially regulated. During the exponential phase, vitamin synthesis (cobW) and biotin synthesis (bioD) genes were down-regulated 2.96-4.17-fold (Table S1). Since biotin is an essential cofactor for fatty acid metabolism [72], a shortage of biotin under MG may hamper cell growth. During the stationary growth phase, pyrimidine synthesis enzymes (carA and carB) were repressed 2.27-2.65-fold. Furthermore, the γ-aminobutyric Other genes related to energy metabolism were also differentially regulated. During the exponential phase, vitamin synthesis (cobW) and biotin synthesis (bioD) genes were down-regulated 2.96-4.17-fold (Table S1). Since biotin is an essential cofactor for fatty acid metabolism [72], a shortage of biotin under MG may hamper cell growth. During the stationary growth phase, pyrimidine synthesis enzymes (carA and carB) were repressed 2.27-2.65-fold. Furthermore, the γ-aminobutyric acid (GABA) shunt pathway was also repressed, along with the acid resistance system ( Figure 5) [73,74]. Finally, gadA and gadB were repressed 2.36-2.48-fold during the exponential growth phase while gadA and gdhA were repressed 2.27-2.28-fold during the stationary growth phase ( Figure 2D, Tables S1 and S2). Conclusions Probiotics, including EcN, may be an effective solution in the management of the gut microbiome of astronauts as they can protect the gut microbiome from pathogens during the space flight. We investigated the effects of simulated MG on EcN using comparative transcriptomic analysis during exponential and stationary growth phases. Some changes identified were disadvantageous to EcN when competing against other pathogenic microbes. EcN tended to develop lower relative abilities for acidic resistance and adhesion under MG conditions, which may make it more difficult for it to survive as it passes through the acidic conditions of stomach and to attach to intestinal cells. Additionally, EcN exhibited altered metal ion transport/utilization and repressed energy and carbon metabolism in response to oxidative stress and nutrient depletion, respectively. Collectively, these results demonstrate the need for further studies that will be essential for the development of effective probiotics that are able to function under MG as guardians of the gut microbiome. Overall Work Flow of This Study The total work flow of our study was listed in the following order. Bacterial Strain and Growth Conditions The strain used in this study was EcN, which was cultured in M9 minimal media (47.8 mM Na 2 HPO 4 ·7H 2 O, 22 mM KH 2 PO 4 , 8.6 mM NaCl, 18.7 mM NH 4 Cl, 2 mM MgSO 4 , and 0.1 mM CaCl 2 ) supplemented with 0.4% glucose (w/v). To develop the inoculum, a single colony of EcN grown on a Luria-Broth (LB) broth agar plate was inoculated into M9 media with 0.4% glucose and incubated with shaking at 37 • C. The cultures were incubated overnight to allow the cells to enter the stationary growth phase and then used as inocula for the clinostat system. Growth of EcN in a Clinostat After culturing overnight, and the EcN cells entering the stationary growth phase, a sample of the culture was diluted 10 −3 and added to clinostat vessels (Synthecon, Inc., Houston, TX, USA). The vessels (d = 12.5 cm) were installed either perpendicularly or horizontally to simulate MG and NG conditions, respectively. To generate conditions of low shear stress, samples were incubated in vessels rotating < 25 rpm. The optical density at 600 nm (OD 600 ) was checked to monitor the growth profile of EcN for each condition. Briefly, the clinostat vessel was detached from the instrument every hour and the OD 600 of the culture was determined to generate the growth profiles of the EcN. Based on the growth curves, we determined the points of exponential and stationary growth. For RNA extraction, 1 mL of EcN was harvested at two points of cell growth, which included the exponential growth phase (OD 600 0.6-0.8) and stationary growth phase (OD 600 1.2). Each experimental condition was performed in duplicate. RNA Extraction, cDNA Synthesis, and Sequencing Total RNA was extracted from 3 mL of cultured EcN samples at each growth point for each gravitational condition. Before RNA extraction, harvested samples were treated with RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). Then, we used Qiagen RNeasy Plus Mini kit (Qiagen, Hilden, Germany) for RNA extractions. First, 6 mL of RNA protect reagent was added to each culture sample. After 5 min incubation, the samples were pelleted and resuspended in 400 µL Tris-EDTA pH 8.0 (TE) buffer (Sigma-Aldrich, Saint Louis, MO, USA). After a second round of pelleting and washing, the sample was eluted with 200 µL TE buffer. Then 1 µL SUPERase•In RNase inhibitor (Invitrogen, Carlsbad, CA, USA), 3 µL of 10 M units of Ready-Lyse Lysozyme solution (Lucigen, Middleton, WI, USA), 1 µL of 20 mg/mL Proteinase K (Invitrogen, Thermo Fisher Scientific Inc, Waltham, MA, USA), and 3 µL of 20% sodium dodecyl sulfate (Sigma-Aldrich, Saint Louis, MO, USA) were added and incubated for 30 min in ice. Then, 700 µL RLT-β-ME buffer, which was prepared with addition of 10 µL of β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, USA) to 1 mL of RLT Buffer, was added to make visible particle. The samples were centrifuged and the supernatants purified using gDNA Eliminator Mini Spin Columns (Qiagen, Hilden, Germany). The filtered samples then had 500 µL of 100% EtOH added and purified using RNeasy Mini Spin Columns (Qiagen, Hilden, Germany). Using 350 µL RW1 and 1 mL RPE buffer (Qiagen, Hilden, Germany), the RNA samples were washed and eluted with RNase-free deionized distilled water. Purified RNA samples were analyzed using an RNA 6000 Pico Kit on an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). The RNA integrity number (RIN) ranged from 2.6 to 9.9. To remove any ribosomal RNA (rRNA) from the RNA stock, a Ribo-Zero rRNA Removal Kit for Gram-negative bacteria (Illumina, San Diego, CA, USA) was used. The RNA solutions were treated with 2 µL Ribo-Zero Reaction Buffer (Illumina, San Diego, CA, USA) and 5 µL Ribo-Zero Removal Solution (Illumina, San Diego, CA, USA). Magnetic beads were prepared by washing and then resuspending them in 30 µL AMPure XP magnetic bead resuspension solution (Beckman Coulter, Fullerton, CA, USA) and 3 µL of RiboGuard RNase Inhibitor (Illumina, San Diego, CA, USA). The prepared magnetic beads were then added to hybridized the RNA sample and purified using RNeasy MinElute spin columns (Qiagen, Hilden, Germany). Removal of the rRNA was confirmed using a Qubit Fluorometer (Invitrogen, Thermo Fisher Scientific Inc, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The extracted RNA samples with the rRNA removed were then used for cDNA synthesis using a KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA, USA). Samples were fragmented and each strand synthesized into cDNA. After bead-based purification, the cDNA strands were A-tailed for adapter ligation. After the adaptor ligation incubation, the samples were purified using 20% PEG 8000/2.5 M NaCl solution (Kapa Biosystems, Inc., Wilmington, MA, USA). The cDNA strands of sizes consistent with the target range were selected and then amplified by real-time PCR. Overall, eight samples including duplicate samples from the two growth phases and two culture condition were quality-checked (9.86-64.4 ng/µL) using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and sequenced using Illumina HiSeq 4000 System (Macrogen, Seoul, Korea). All raw data for RNA-seq has been deposited into the Gene Expression Omnibus at the NCBI under GSE147272. Read Processing, Alignment, and Quantification Sequenced reads from the RNA-seq analysis showed low proportions of rRNA, which indicated that the rRNA was successfully removed. The RNA-seq reads were then mapped based on the EcN reference genome (CP007799) using bowtie and cuffdiff tools. The calculated FPKM values from cuffdiff were used to identify the DEGs. DEGs with log 2 -fold expression changes ≥ 1.0 and false discovery rates (FDR) ≥ 0.01 were selected. Differential Expression and Functional Analyses Unidentified DEGs from the reference genome were compared with genes from other E. coli strains using blastp BLAST software and the EcoCyc database (www.ecocyc.org). The function of each DEG was then analyzed based on the KEGG and EcoCyc gene operon online databases.	v3-fos-license
2018-12-13T14:05:40.469Z	2018-12-13T00:00:00.000	54474901	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2018.03109/pdf", "pdf_hash": "b093ed3b4391787efb654c40250bf2818681773d", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1880", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "b093ed3b4391787efb654c40250bf2818681773d", "year": 2018 }	pes2o/s2orc	Electron and Proton Flux for Carbon Dioxide Reduction in Methanosarcina barkeri During Direct Interspecies Electron Transfer Direct interspecies electron transfer (DIET) is important in diverse methanogenic environments, but how methanogens participate in DIET is poorly understood. Therefore, the transcriptome of Methanosarcina barkeri grown via DIET in co-culture with Geobacter metallireducens was compared with its transcriptome when grown via H2 interspecies transfer (HIT) with Pelobacter carbinolicus. Notably, transcripts for the F420H2 dehydrogenase, Fpo, and the heterodisulfide reductase, HdrABC, were more abundant during growth on DIET. A model for CO2 reduction was developed from these results in which electrons delivered to methanophenazine in the cell membrane are transferred to Fpo. The external proton gradient necessary to drive the otherwise thermodynamically unfavorable reverse electron transport for Fpo-catalyzed F420 reduction is derived from protons released from G. metallireducens metabolism. Reduced F420 is a direct electron donor in the carbon dioxide reduction pathway and also serves as the electron donor for the proposed HdrABC-catalyzed electron bifurcation reaction in which reduced ferredoxin (also required for carbon dioxide reduction) is generated with simultaneous reduction of CoM-S-S-CoB. Expression of genes for putative redox-active proteins predicted to be localized on the outer cell surface was higher during growth on DIET, but further analysis will be required to identify the electron transfer route to methanophenazine. The results indicate that the pathways for electron and proton flux for CO2 reduction during DIET are substantially different than for HIT and suggest that gene expression patterns may also be useful for determining whether Methanosarcina are directly accepting electrons from other extracellular electron donors, such as corroding metals or electrodes. INTRODUCTION The mechanisms by which methanogens conserve energy to support growth during direct interspecies electron transfer (DIET) are of interest because it is becoming increasingly apparent that DIET may be an important alternative to hydrogen interspecies transfer (HIT) for methane production in anaerobic digesters as well as methanogenic soils and sediments (Shrestha and Rotaru, 2014;Dubé and Guiot, 2015;Cheng and Call, 2016;Lovley, 2017c). A better understanding of DIET could help with the development of molecular approaches that can be used to detect DIET in methanogenic environments (Rotaru et al., 2014b;Holmes et al., 2017) and might lead to new approaches for promoting DIET to accelerate and stabilize anaerobic digestion (Cheng and Call, 2016;Barua and Dhar, 2017;Lovley, 2017b,c;Baek et al., 2018;Park et al., 2018). Physiological studies of DIET require defined co-cultures. Geobacter metallireducens is an environmentally relevant pure culture model for electron-donating partners for DIET because Geobacter species function as the electron-donating partner in important methanogenic environments such as anaerobic digesters (Morita et al., 2011;Rotaru et al., 2014b) and terrestrial wetlands (Holmes et al., 2017). Studies with defined co-cultures in which G. metallireducens was the electron-donating partner for DIET (Shrestha et al., 2013;Rotaru et al., 2014a;Ueki et al., 2018) have suggested that c-type cytochromes and electrically conductive pili [e-pili] (Lovley, 2017a) facilitate electron transport from G. metallireducens to the electron accepting partner. Outer-surface c-type cytochromes and e-pili are also involved in electron uptake by G. sulfurreducens when it is the electron-accepting partner in DIET-based co-cultures with G. metallireducens (Summers et al., 2010;Shrestha et al., 2013;Ueki et al., 2018). However, Methanosarcina barkeri and Methanothrix (formerly Methanosaeta) harundinacea, the only methanogens definitively shown to participate in DIET (Rotaru et al., 2014a,b), do not possess outer-surface c-type cytochromes or e-pili. Their outer surface electrical contacts for DIET are unknown. The basic physiology and biochemistry of M. barkeri are much better understood than for Mt. harundinacea (Thauer et al., 2008;Gonnerman et al., 2013;Welte and Deppenmeier, 2014;Boone and Mah, 2015;Kulkarni et al., 2018;Mand et al., 2018). This makes M. barkeri the organism of choice for initial DIET mechanistic studies. Another advantage is that methods are available for genetic manipulation of M. barkeri (Kohler and Metcalf, 2012), but not Mt. harundinacea. However, one caveat for the study of DIET is that M. barkeri mutants have been previously constructed in a strain adapted to grow in high salt concentrations to prevent cell aggregation (Kohler and Metcalf, 2012). G. metallireducens, the only known electron-donating partner for M. barkeri, has yet to be adapted to grow at such high salt conditions. Thus, at least at present, alternative approaches to evaluating the physiology of M. barkeri during DIET are required. Comparing the transcriptome of cells grown via DIET versus cells grown via HIT clearly reflected differences in electron uptake mechanisms in studies in which G. sulfurreducens functioned as the electron-accepting partner (Shrestha et al., 2013). G. sulfurreducens was grown by DIET with G. metallireducens as the electron-donating partner, or by HIT in co-culture with Pelobacter carbinolicus a microorganism closely related to G. metallireducens, but which is incapable of DIET (Shrestha et al., 2013). The G. sulfurreducens transcriptome demonstrated that cells were poised for growth on H 2 when G. sulfurreducens was grown with P. carbinolicus and expressed genes for outer-surface proteins involved in direct uptake of electrons during DIET-based growth with G. metallireducens (Shrestha et al., 2013). M. barkeri can also be grown in co-culture with either G. metallireducens or P. carbinolicus (Rotaru et al., 2014a), providing an opportunity to compare M. barkeri gene expression patterns during growth via DIET and HIT. Any model describing how the electron-accepting partner utilizes electrons derived from DIET must account for the uncoupling of the routes for interspecies electron and proton flux (Figure 1). e-Pili only transport electrons. Protons move between DIET partners by diffusion. This uncoupled transport of electrons and protons is in stark contrast to HIT in which H 2 simultaneously transports both electrons and protons as the H 2 diffuses between the two partners. When the H 2 is oxidized in the cytoplasm with electron transfer to an electron acceptor, protons are also released and are immediately available to balance the negative charge transferred to the electron acceptor. This maintains charge balance within the cell (Figure 1). In contrast, in DIET, e-pili and associated electron transport proteins deliver electrons to cytoplasmic electron acceptors. Protons have to be translocated into the cytoplasm for charge balance (Figure 1). This proton consumption also prevents acidification of the extracellular matrix of the DIET aggregates. Thus, proposed mechanisms for electron uptake during DIET need to include an explanation for how protons are translocated into the cytoplasm of the electron-accepting partner. Here we report transcriptomic data from M. barkeri grown via DIET and HIT. The results suggest a mechanism for M. barkeri to utilize electrons and protons, derived from the electron-donating partner during DIET, to conserve energy to support growth from the reduction of carbon dioxide to methane. Co-culture Incubation and mRNA Extraction Triplicate replicates of co-cultures of G. metallireducens/M. barkeri and P. carbinolicus/M. barkeri were grown under strict anaerobic conditions as previously described (Rotaru et al., 2014a). Cultures were harvested during the exponential phase of growth and mRNA was isolated as previously described (Shrestha et al., 2013). Illumina Sequencing and Assembly of Reads Directional multiplex libraries were prepared with the ScriptSeq TM v2 RNA-Seq library preparation kit (Epicentre). Single end sequencing was performed with a Hi-Seq 2000 platform at the Deep Sequencing Core Facility at the University of Massachusetts Medical School in Worcester, MA, United States. All raw data generated by Illumina sequencing were quality checked by visualization of base quality scores and nucleotide distributions with FASTQC 1 . Initial raw non-filtered forward and reverse sequencing libraries contained an average of 3892089 ± 134932 reads that were ∼100 basepairs long. Sequences from all of the libraries were trimmed and filtered with trimmomatic (Bolger et al., 2014) with the sliding window approach set to trim bases with quality scores lower than 3, strings of 3+N's, and reads with a mean quality score lower than 20. Bases were also cut from the start and end of reads that fell below a threshold quality of 3, and any reads smaller than 50 bp were eliminated from the library. These parameters yielded an average of 2732020 ± 217212 quality reads per RNA-Seq library. SortMeRNA (Kopylova et al., 2012) was then used to separate all ribosomal RNA (rRNA) reads from the libraries. Databases used by SortMeRNA to identify all rRNA sequences included Rfam 5.8S Eukarya, Rfam 5S Archaea/Bacteria, SILVA 16S Archaea, SILVA 16S Bacteria, SILVA 23S Bacteria, SILVA 18S Eukarya, and SILVA 28S Eukarya (Burge et al., 2013;Quast et al., 2013). Mapping of mRNA Reads Trimmed and filtered mRNA reads from the triplicate samples for the two different co-culture conditions were mapped against the genome of M. barkeri strain MS DSM 800 downloaded from IMG/MER 2 . Mapped reads were normalized with the RPKM (reads assigned per kilobase of target per million mapped reads) method (Mortazavi et al., 2008;Klevebring et al., 2010) using ArrayStar software (DNAStar). Graphical analysis of reads from all three biological replicates for each condition demonstrated that the results were highly reproducible. Therefore, all reported values were obtained after merging and averaging replicates. Expression levels were considered significant only when the log 2 RPKM value was higher than that of the median RPKM value. Out of the 3,809 predicted protein-coding genes in the M. barkeri MS genome, 1,912 and 1,909 genes had expression levels that were higher than the median in DIET-and HIT-grown cells, respectively. Genome Data Analysis Sequence data for all of the bacterial genomes was acquired from the U.S. Department of Energy Joint Genome Institute 3 or from GenBank at the National Center for Biotechnology Information (NCBI) 4 . Initial analyses were done with analysis tools available on the Integrated Microbial Genomes (IMG) website (see text footnote 2). Some protein domains were identified with NCBI conserved domain search (Marchler-Bauer et al., 2015) and Pfam search (Finn et al., 2016) functions. Transmembrane helices were predicted with TMpred (Hofmann and Stoffel, 1993), TMHMM (Krogh et al., 2001), and HMMTOP (Tusnady and Simon, 2001) and signal peptides were identified with PSORTb v. 3.0.2 (Yu et al., 2010) and Signal P v. 4.1 (Petersen et al., 2011). Accession Number Illumina sequence reads have been submitted to the NCBI database under project number PRJNA501858 and accession SAMN10346831-SAMN10346836. RESULTS AND DISCUSSION As previously described (Rotaru et al., 2014a) co-cultures of G. metallireducens and M. barkeri that were well-adapted for growth via DIET required ca. 25 days to metabolize the 20 mM ethanol provided as substrate whereas P. carbinolicus/M. barkeri co-cultures required ca. 15 days. Both G. metallireducens and P. carbinolicus metabolized ethanol to acetate with either the production of H 2 (P. carbinolicus) or extracellular electron transfer (G. metallireducens). M. barkeri metabolized acetate in both co-cultures, but in the initial growth phases of the cultures acetate production was faster than consumption, resulting in an accumulation of acetate (Rotaru et al., 2014a). Transcriptome Reflects Faster Growth During HIT and Possible Greater Importance of Membrane and Outer-Surface Proteins During DIET Transcript abundances for M. barkeri genes involved in amino acid biosynthesis, protein synthesis, and enzymes in the methane production pathways from both carbon dioxide and acetate were generally higher in the P. carbinolicus/M. barkeri co-cultures than in the G. metallireducens/M. barkeri co-cultures (Figure 2 and Supplementary Tables S1, S2). This is consistent with the faster growth of the P. carbinolicus/M. barkeri co-cultures. The highest proportion of genes that were more highly expressed during DIET-based growth were genes for proteins predicted to be associated with the membrane or cell surface (Figure 2). Genes for all three functional M. barkeri hydrogenases [Ech, Frh, and Vht ] were more abundant during HIT-based growth (Table 1). However, the results suggest that it will not be possible to use hydrogenase gene transcript levels to diagnose whether M. barkeri is participating in HIT or DIET in microbial communities. The increase in hydrogenase gene expression in HIT-grown cells was comparable to the general increase in expression of genes for other methanogenesis enzymes, such as Mcr (Table 1 and Supplementary Table S2), suggesting that there was not a specific upregulation of hydrogenase genes in response to growth via HIT. Considering that gene expression for many metabolic genes was generally lower in DIET-grown cells, any genes for which transcript abundance was higher during DIET, or even comparable to HIT-grown cells, are of considerable interest. In the following sections, genes with higher expression during growth on DIET are examined further. The results are placed in the context of a working model (Figure 3) for generating the reduced co-factors required for carbon dioxide reduction to methane (F 420 H 2 , reduced ferredoxin) while also providing a mechanism for CoM-S-S-CoB reduction and a chemiosmotic potential to provide energy to support DIET-based growth. Proton-Driven Reverse Electron Transport to Reduce F 420 With Fpo Transcripts for genes for most of the subunits for the membranebound F 420 H 2 dehydrogenase, Fpo, were higher in DIET-grown cells ( Table 2). Considering that transcripts for most genes for methanogenesis were more abundant in HIT-grown cells, these The log 2 RPKM median for HIT-grown M. barkeri cells was 7.5. The log 2 RPKM median for DIET-grown M. barkeri cells was 7.9. * Transcripts with values below the log 2 RPKM median. ND, no significant difference in transcription. FIGURE 3 \| Model for electron and proton flux for carbon dioxide reduction to methane in Methanosarcina barkeri during DIET-based growth. Each two moles of ethanol oxidized to acetate by G. metallireducens releases eight electrons and eight protons. Electrons delivered to methanophenazine in the cell membrane are transferred to Fpo. Proton translocation drives Fpo-catalyzed reduction of F 420 to F 420 H 2 . Half of the F 420 H 2 produced serves as a reductant in the carbon dioxide reduction pathway. The remaining F 420 H 2 is the electron donor for HdrABC, which reduces ferredoxin and CoM-S-S-CoB in an electron bifurcation reaction. The steps in carbon dioxide reduction, including the role of reduced ferredoxin, CoM-SH, and CoB-SH, as well as sodium pumping with Mtr, are as previously described (Thauer et al., 2008) for M. barkeri. The log 2 RPKM median for HIT-grown M. barkeri cells was 7.5. The log 2 RPKM median for DIET-grown M. barkeri cells was 7.9. * Transcripts with values below the log 2 RPKM median. results suggest that Fpo plays a key role in electron transport for carbon dioxide reduction to methane during DIET. During methylotrophic methanogenesis Fpo oxidizes F 420 H 2 with the reduction of methanophenazine in the membrane, coupled with vectorial proton translocation to the outside of the membrane (Welte and Deppenmeier, 2014;Kulkarni et al., 2018;Mand et al., 2018). However, under some conditions Fpo may catalyze the reverse reaction in which reduced methanophenazine serves as the electron donor for the reduction of F 420 . In this direction, proton translocation through Fpo into the cytoplasm is required in order to make the reaction thermodynamically favorable. Therefore, it is proposed that electrons derived from DIET reduce methanophenazine in the oxidized state (MP) to MPH 2 and that MPH 2 is the electron donor for Fpo to reduce F 420 in the cytoplasm (Figure 3). A proton gradient to drive the reaction is available from the protons released into the extracellular matrix from G. metallireducens metabolism in direct proportion to electrons transported from G. metallireducens through e-pili. The proton flux through Fpo does not acidify the cytoplasm because an equivalent number of protons are consumed from the cytoplasm when MP is reduced to MPH 2 (Figure 3). The protons required to produce MPH 2 are transferred to F 420 during the Fpo-catalyzed reaction MPH 2 + F 420 → MP + F 420 H 2 . In this way electron transfer through methanophenazine to F 420 is achieved with charge balance. Possible Increased Methanophenazine Production to Support DIET The proposed generation of F 420 H 2 by Fpo with electrons derived from DIET requires an abundance of reduced methanophenazine (Figure 3). The pathway involved in biosynthesis of methanophenazine has not been identified, however, it is likely to resemble those of respiratory quinones because both have a polyprenyl side-chain connected to a redox-active moiety. In fact, studies have shown that a farnesylgeranyl pyrophosphate synthetase from the terpenoid backbone biosynthesis pathway is required for methanophenazine biosynthesis in M. mazei (Ogawa et al., 2010). Nine genes predicted to code for proteins involved in ubiquinone/menaquinone biosynthesis; six UbiE methyltransferase proteins, UbiA prenyltransferase, phenylacrylic acid decarboxylase (UbiD), and a ubiquinone biosynthesis protein (UbiB) were >2 fold more highly expressed in DIET-grown cells and another 11 putative ubiquinone biosynthesis genes were >1.5 fold up in DIET grown cells ( Table 3). Given that M. barkeri does not contain ubiquinone or menaquinone, it seems likely that these genes code for enzymes involved in methanophenazine synthesis. It has been calculated that the concentration of methanophenazine in membranes of M. acetivorans grown on methanol is sufficient to convert the membrane into an "electrically quantitized" conductive material (Duszenko and Buan, 2017). Methanophenazine concentrations in membranes of methanol-grown M. barkeri were too low for this effect (Duszenko and Buan, 2017). However, increased methanophenazine synthesis during growth via DIET might also yield an electrically quantitized membrane in M. barkeri, alleviating the need for redox-active proteins to aid in electron transport through the membrane during DIET. Generating Reduced Ferredoxin and Reducing CoM-S-S-CoB With HdrABC In addition to F 420 H 2 , M. barkeri needs to generate reduced ferredoxin during DIET. It is required for the first step in carbon dioxide reduction to methane (Thauer et al., 2008). One of the few soluble protein complexes with higher gene transcript abundance during DIET is the heterodisulfide reductase HdrA1B1C1 (Table 4), suggesting that it is important for DIET. Transcript Negative values in the column "fold up-regulated in DIET" indicate that transcripts were more abundant in HIT-grown cells. The log 2 RPKM median for HIT-grown M. barkeri cells was 7.5. The log 2 RPKM median for DIET-grown M. barkeri cells was 7.9. * Transcripts with values below the log 2 RPKM median. The log 2 RPKM median for HIT grown M. barkeri cells was 7.5. The log 2 RPKM median for DIET grown M. barkeri cells was 7.9. Negative values in the "Fold up in DIET" column indicate that genes were more highly transcribed in HIT-grown cells. * Transcripts with values below the log 2 RPKM median. abundance for the genes for subunits of the homologous HdrA2B2C2 was more comparable to that during growth on HIT, with the transcripts for the genes of the A2 and B2 slightly higher during DIET and lower transcripts for the C2 subunit. When the general pattern of higher gene transcript abundance for soluble proteins in HIT-grown cells is considered, these results suggest that HdrA2B2C2 might also be important in DIET. In vitro purified HdrA2B2C2 from M. acetivorans oxidizes F 420 H 2 with the reduction of ferredoxin and CoB-S-S-CoM through flavin-based electron bifurcation (Yan et al., 2017). The phenotypes for various Methanosarcina mutants have suggested that HdrA1B1C1 can couple the oxidation of reduced ferredoxin with the reduction of both F 420 and CoB-S-S-CoM (Buan and Metcalf, 2010;Gonnerman et al., 2013). However, this reaction has not been verified biochemically (Yan and Ferry, 2018) and the direction of electron flow for the HdrA1B1C1 complex could be similar to that demonstrated for the HdrA2B2C2 complex, especially under conditions in which there is substantial production of reduced F 420 and limited routes for generating reduced ferredoxin. An electron bifurcation reaction in this direction would also be consistent with the electron bifurcation from flavin with the reduction of CoB-S-S-CoM and ferredoxin associated with the MvhADG/HdrABC complexes found in methanogens that specialize in growth with H 2 /CO 2 (Kaster et al., 2011). The Completed Pathway for Energy Conservation During DIET Therefore, it is proposed that half of the F 420 H 2 generated with Fpo is the electron donor for HdrABC (one or both homologs) to produce reduced ferredoxin with the simultaneous reduction of CoM-S-S-CoB (Figure 3). In this way the coupled activity of Fpo-and HdrABC-catalyzed reactions deliver the eight moles of electrons derived from the oxidation of two moles of ethanol to each required step in the carbon dioxide reduction pathway (Figure 3). As noted above, the Fpo-catalyzed reduction of F 420 is proton balanced. Protons are released from F 420 H 2 oxidation by HdrABC, but an equivalent number of protons are consumed in other reactions in the carbon dioxide reduction pathway (Figure 3). Thus, the model also balances proton flux. The proposed model generates a chemiosmotic gradient to produce ATP through the activity of the Mtr complex that is known to pump sodium across the membrane during methyl transfer in the carbon dioxide reduction pathway (Thauer et al., 2008). There are two possibilities for ATP generation from the sodium gradient. Genes for both the A 1 A 0 ATP synthase and the F 1 F 0 ATP synthase were expressed during DIET (Supplementary Table S3). The available evidence suggests that both can translocate sodium (Schlegel and Muller, 2013). Genes for several components of the F 1 F 0 ATP synthase were more highly expressed during growth on DIET and others were expressed at levels comparable to HITgrown cells (Supplementary Table S3). This suggests that the F 1 F 0 ATP synthase may play a more important role during growth on DIET, but at present there is not enough information on F 1 F 0 ATP function in M. barkeri to speculate why. Transcriptomics Suggests Potential Outer Surface Electrical Contacts A number of genes predicted to encode redox active proteins expected to be associated with the M. barkeri membrane and/or cell surface were more highly expressed in cells grown via DIET (Table 5). However, it is premature to speculate on their possible role in mediating electron transfer into the cell in the absence of further biochemical characterization to determine whether important characteristics, such as redox potential and cellular localization, are appropriate for proposed roles. For example, a gene putatively encoding a membranebound protein with a cupredoxin domain (Ga0072459_111371) was highly expressed specifically during DIET ( Table 5). The cupredoxins rusticyanin and sulfocyanin play important roles in electron transfer into cells of Acidithiobacillus and Sulfolobus species during Fe(II) and S 0 oxidation (Komorowski and Schafer, 2001;Dennison, 2005) and thus might play a similar role in electron transport into M. barkeri. The mid-point potentials of known cupredoxins (150 to 680 mV) are more positive than that expected for an electron carrier involved in electron transport to methanophenazine [mid-point potential of −165 mV (Tietze et al., 2003)]. However, modifications in cupredoxin structure and environment may greatly influence their mid-point potential (Marshall et al., 2009) and thus a role in electron transport into the cell is conceivable. In a similar manner, genes encoding proteins that putatively incorporate pyrroloquinoline quinone (PQQ) as a co-factor were highly expressed during growth via DIET ( Table 5). Like rusticyanin and sulfocyanin, proteins with PQQ-binding domains are involved in electron transport into the cell during oxidation of Fe(II) or Mn(II) (Croal et al., 2007;Johnson and Tebo, 2008). The mid-point potential of proteins with PQQ domains (∼90-100 mV) is too positive to play a role in electron transfer to methanophenazine. However, genes for PQQ biosynthesis were not found in the M. barkeri genome. Thus, it is possible that these proteins with predicted PQQ domains may incorporate another co-factor. Methanophenazine is one possibility. Further analysis of these proteins and others with higher gene expression during DIET (Table 5) is warranted. The expression of genes for a number of soluble electron carriers/co-factors were higher in DIET-grown cells, further suggesting differences in electron flux during DIET (Supplementary Table S4), but more analysis will be required to evaluate their role/significance. IMPLICATIONS The results suggest a pathway for electron and proton flux in M. barkeri during DIET that is significantly different than during HIT-based growth. The increased expression of genes for key components, including Fpo and HdrABC, and considerations of electron and proton transport during DIET, suggest an electronand proton-balanced model in which the required electron donors are generated for each of the reductive steps of carbon dioxide reduction to methane while conserving energy to support growth (Figure 3). This model provides hypotheses that can be further evaluated experimentally with the appropriate M. barkeri mutants. However, as noted in the Introduction, this will require the discovery or development of an electron-donating strain capable of growing in the high salt medium that is used to generate M. barkeri mutants (Kohler and Metcalf, 2012). An alternative approach might be to adapt M. barkeri mutants to lower salt conditions, but this would require a time-consuming, labor-intensive adaption of each M. barkeri mutant strain with These genes were at least two fold more highly expressed in M. barkeri cells grown via DIET than M. barkeri cells grown via HIT. PS51527: prokaryotic lipoprotein attachment site; pfam08309: LVIVD repeat found in bacterial and archaeal surface proteins. Cell localization predictions were made with PSORTb v3.0.2 software. * Transcripts with values below the log 2 RPKM median (7.5 for HIT and 7.9 for DIET). the risk of additional mutations arising during the adaption process. The DIET transcriptome did not conclusively identify electrical contacts for DIET beyond the cell membrane. One potential reason for this is that M. barkeri might constitutively express these contacts. It is difficult to envision how Geobacter or other electron-donating partners could make electrical contacts with the outer surface of M. barkeri unless those contacts were expressed in advance of the initial Geobacter-M. barkeri electrical interaction. M. barkeri's low affinity for H 2 makes it a poor competitor for H 2 in many environments (Thauer et al., 2008). Constitutive production of outer surface electrical contacts could poise M. barkeri for DIET and provide a competitive advantage in utilizing this alternative source of electrons for carbon dioxide reduction. Elucidating the role of Methanosarcina species in DIET in complex natural environments is complicated by the possibility that H 2 must also be considered as a potential electron donor for carbon dioxide reduction (Holmes et al., 2017). The differences in gene expression patterns between DIET-and HIT-grown cells suggest that metatranscriptional analysis is a route to better characterize the extent to which Methanosarcina are involved in DIET. It has been suggested that M. barkeri as well as other methanogens, can directly accept electrons from other extracellular sources such as electrodes, conductive carbon materials, and metals, but it has been difficult to rule out the possibility that H 2 might be an intermediary electron carrier (Cheng and Call, 2016;Blasco-Gomez et al., 2017;Lovley, 2017b,c). The finding that transcriptome patterns in cells directly accepting electrons from an external source differ substantially from cells utilizing H 2 as an electron donor suggests that the transcriptomic analysis approach described here could also help resolve this question. AUTHOR CONTRIBUTIONS DH, A-ER, PS, and DL conceived the study. A-ER grew the co-cultures. PS extracted and processed the nucleic acids for sequences. DH re-annotated the genome as necessary and analyzed the transcriptome data. DH and DL wrote the initial version of the manuscript. All authors made important modifications and additions.	v3-fos-license
2015-09-18T23:22:04.000Z	2015-03-01T00:00:00.000	3197951	{ "extfieldsofstudy": [ "Chemistry", "Computer Science", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1424-8220/15/3/5884/pdf", "pdf_hash": "312c78c7fabbfa59d6cdf6f74e3deeaf30938a02", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1884", "s2fieldsofstudy": [ "Medicine" ], "sha1": "312c78c7fabbfa59d6cdf6f74e3deeaf30938a02", "year": 2015 }	pes2o/s2orc	Study of the Interaction of Trastuzumab and SKOV3 Epithelial Cancer Cells Using a Quartz Crystal Microbalance Sensor Analytical methods founded upon whole cell-based assays are of importance in early stage drug development and in fundamental studies of biomolecular recognition. Here we have studied the binding of the monoclonal antibody trastuzumab to human epidermal growth factor receptor 2 (HER2) on human ovary adenocarcinoma epithelial cancer cells (SKOV3) using quartz crystal microbalance (QCM) technology. An optimized procedure for immobilizing the cells on the chip surface was established with respect to fixation procedure and seeding density. Trastuzumab binding to the cell decorated sensor surface was studied, revealing a mean dissociation constant, KD, value of 7 ± 1 nM (standard error of the mean). This study provides a new perspective on the affinity of the antibody-receptor complex presented a more natural context compared to purified receptors. These results demonstrate the potential for using whole cell-based QCM assay in drug development, the screening of HER2 selective antibody-based drug candidates, and for the study of biomolecular recognition. This real time, label free approach for studying interactions with target receptors present in their natural environment afforded sensitive and detailed kinetic information about the binding of the analyte to the target. Introduction Cell-based assays are interesting for drug development and diagnostics. Even though the standard methods for early drug development are based on in vitro-assays, cell-based assays offer extended possibilities for understanding the biology including in vivo-effects [1,2]. Traditional methods for binding studies between cell membrane-incorporated receptors and corresponding antibodies are often based on purified receptors or cell lysate. Immunoassays, e.g., ELISA, can be time consuming, expensive and for some assays, also not sufficiently sensitive [3]. Moreover, fluorescence labeling of antibodies to intact cells (immunocytochemistry) and flow cytometry have limitations in quantification and qualification of the binding, and for cell-based systems in general there is a need to develop protocols that take into account the stability of the cells. Additionally, labeling can affect the interaction properties and increase the non-specific binding [4], which highlights the fundamental benefits offered by label-free analytical tools. The affinity between drug and target, including on-and off-rates, are evaluated for drug development [5,6] which increases the need of a tool for proper affinity data in its natural environment. Biosensors can offer a time efficient, label-free assay for studying binding with affinity features in real time. Limitations of traditional biosensors are the need for isolated and purified target molecules immobilized on the sensor surface where the target is not present in its natural context, possibly resulting in undesirable conformation alterations of the target molecule [7]. To overcome that limitation and offer a target binding assay in a more biological context, cell-based biosensors have been explored and shown to be promising tools [8][9][10][11][12]. Here we have addressed the use of the human ovary adenocarcinoma epithelial cancer (SKOV3) cell line, with an overexpression of the breast cancer-related human epidermal growth factor receptor 2 (HER2), as a means for studying the interaction with the monoclonal antibody-based drug trastuzumab, and with the long term goal of establishing methods for the rapid screening of new antibody-based candidate drugs. The quartz crystal microbalance (QCM) is a piezoelectricity-based biosensor technology for quantitative and qualitative measurements of binding affinity. Due to its robustness, low-cost and ease-of-use it has become a powerful label-free tool to identify binding in real time [9,[13][14][15][16][17] suitable for a wide range of molecules, from small organic compounds to large proteins. Like most biosensors, the QCM technique has traditionally been used mostly for studying the interaction between purified antibodies and antigens or related substances. The first immunosensor based on piezoelectric detection was reported in 1972 by Shons and co-workers [18][19][20]. The QCM technique has since been undergone significant development, whereby today it is possible to use synthetic polymer-based antibody mimics [21,22] and even cells as sensor recognition elements [7,[23][24][25][26][27][28]. The initial development of applications based upon cells attached to QCM surfaces have included the monitoring of cell adhesion [9], the effects of anti-cancer treatments on cells [29,30], the detection of cancer cells [31,32] and the affinity of antibodies for a cell membrane receptor [33]. The possibility of studying the binding of an analyte to cells attached to resonator surfaces allows for the interaction to be examined in a more natural environment, i.e., together with other cell membrane components that may affect the binding properties [7,[23][24][25]. In this work we have studied the binding of the monoclonal antibody trastuzumab (commercially known as Herceptin™) to the receptor HER2 on SKOV3 epithelial cancer cells using quartz crystal microbalance studies (Figure 1). Overexpressed HER2 are found in aggressive forms of breast cancer and are therefore considered an important target for diagnosis and treatment [34]. The humanized anti-HER2 antibody trastuzumab is widely used for clinical diagnosis and treatment of these cancer forms since it has found to bind to the HER2 and induce apoptosis. We have, in this study, developed a system for measuring the interaction to HER2 on SKOV3 epithelial cancer cells attached to a COP-1 QCM chip. The HER2 is overexpressed in this cell line. Since cell adhesion is cell dependent [9,35] and may affect the measured frequency shift [36] it was important to study the impact of preparation procedure in order to acquire accurate binding data. The SKOV3 cell line was attached and fixed to COP-1 chips and trastuzumab was passed over the surface allowing binding to the HER2 on the cell membrane. Preparation of Cell Chips Human ovary adenocarcinoma cells (SKOV3) were cultured in RPMI 1640 (1×) medium, complemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured in T flasks in a humidified incubator with 5% CO2/95% air atmosphere at 37 °C. Before adhesion of cells on sensor chip the cells were washed in PBS and trypsinized with 0.25% trypsin-EDTA mixture for 5-10 min at 37 °C. After addition of at least an equal amount of media the cell suspension was transferred into a falcon tube and centrifuged at 1000 rpm for 5 min to obtain a pellet. The pellet was resuspended in an appropriate amount of media and mixed carefully to obtain a homogenous suspension. Cell chips were prepared by incubating aliquots of cell suspension on COP-1 chip in a humidified incubator. After 24 h of incubation, the cell suspension was gently removed and the chips were washed with cold PBS. The cells were then fixed using either formaldehyde in PBS or glutaraldehyde in water at 4 °C for 10 min followed by soaking with PBS (3 × 5 min). Finally, the chips were stained by addition of 50 μL of DAPI (0.6 nM in PBS) to each chip, incubation for 4 min at room temperature in the dark followed by rinsing in PBS. Cell immobilization was confirmed using an epifluorescence microscope (Eclipse E400, Nikon, Tokyo, Japan) equipped with an appropriate DAPI filter and a digital camera (Nikon DS-U1). The cell chips were stored in PBS at 4 °C in the dark until use. Quartz Crystal Microbalance Studies QCM studies were conducted under flow injection analysis (FIA) conditions using an automated Attana Cell 200 instrument (Attana AB, Stockholm, Sweden). Typically, a SKOV3 coated chip was placed in a chip holder with a chamber volume of 1.46 μL and docked in the instrument. The temperature was set to 22 °C and the flow rate to 20 μL/min. Running buffer, PBS (pH 7.4), was passed over the chip until stabilization of baseline (frequency change ≤0.5 Hz over 600 s). The binding of trastuzumab was studied by 35 μL injections (105 s) followed by dissociation in PBS for 300 s. For removal of any remaining analyte, 12.5 μL glycine (10 mM in water, pH 2) was injected (38 s) followed by continuous PBS flow until stabilization of baseline before next injection. Data was collected using and analyzed with Attester software (Attana AB). Results and Discussion SKOV3 cancer cell line was cultivated in T flasks until 80% of confluence, moved to suspension and cultivated for 24 h on top of COP-1 chip, optimized for cell attachment. To ensure accurate binding data, the preparation procedure was optimized before using the COP-1 chips as sensor resonators. Trastuzumab was passed over the cell chip allowing the antibody to bind with its target receptor, HER2, present in the SKOV3 cell membrane. Fixation of SKOV3 Cells to COP-1 Chips There is no doubt that good fixation should preserve the cells without affecting their components [37]. It is crucial however that when studying interactions to whole cells that the membrane proteins stay intact without changing epitopes. The outcome is dependent upon several factors such as fixation agent, concentration and exposure time. The most common fixation strategies use aldehydes, which form cross-linkages between proteins to preserve the cells. The binding properties of the SKOV3 cell chips were studied after fixation using two of the most widely used aldehydes, formaldehyde (FA) and glutaraldehyde (GA), in different concentrations. After incubating chips in cell suspension (corresponding to 4 × 10 4 cells per sensor surface), the cells were fixed with aldehyde followed by staining using DAPI to visualize the nuclei of the cells attached to the sensor surface. As shown in Figure 2, cells treated with FA exhibited increased densities on the chips with higher concentrations of aldehyde. GA, on the other hand, showed similar or even less coverage at higher concentrations. Preliminary binding studies were performed using two chips, one fixed with 3.7% FA and one with 0.5% GA. The chip treated with 3.7% FA showed a binding curve with both association and dissociation evident, indicating specific binding of trastuzumab to the cells attached to the chip (Figure 3a). On the other hand, the maximum frequency shift (−Δf) was significantly higher for the 0.5% GA fixed cell chip and the profile of the binding curve showed a strong and rapid decrease in frequency during injection, followed by a fast return to baseline after injection (Figure 3b). This observation indicated a greater degree of weak or non-specific binding to this chip, as reflected in the immediate commencement of return to baseline after completion of injection non-specific binding. GA is widely used because of its efficiency in crosslinking but the effect is also cell dependent and can cause cell damage even if a low concentration is used [38]. There are also known problems with residual unreacted aldehyde functionalities, which can contribute to non-specific binding of antibodies, thus giving a false positive result [39]. Since the GA fixed cell chip showed high cell coverage, the non-specific binding could result from a negatively affected HER2, binding to free aldehydes or even cell lysis due to the impact of the fixation solution. Following these preliminary studies a more extensive study of trastuzumab (10-80 μg/mL) binding to the FA fixed cell chips was conducted (Figure 3c). For chips fixed using 3.7% and 5% FA, the maximum frequency response indicated a concentration dependent binding to the cell chips. For the 2% FA fixed cell chips the maximum frequency response did not follow the same trend, moreover, the response showed a much wider distribution between different chips (n = 3). This could indicate non-specific binding, which supports the results from the DAPI staining where this chip showed a lower cell density on the chip surface. Additionally, the cell chips fixed with 2% FA showed a different binding curve profile than those fixed with 3.7% FA, with a rapid frequency increase during injection and rapid decrease after injection stop, indicating non-specific binding (not shown). The 5% FA fixed cell chips also showed a slightly wider distribution of maximum frequency responses (Figure 3c) as compared to the chip fixed with 3.7% FA. Considering the high cell density on these chips, which also indicates non-specific binding, this could be caused by a toxic effect from this high concentration of FA. Seeding Density To examine how seeding density affects the binding capacity of trastuzumab, COP-1 chips were incubated in cell suspensions with different concentrations corresponding to seeding densities of 2 × 10 4 , 4 × 10 4 and 8 × 10 4 cells per sensor surface (15.9 mm 2 ). After incubation (24 h), the chips were fixed with 3.7% formaldehyde, stained and examined using fluorescence microscopy ( Figure 4a). All chips showed well-distributed cells with no indication of cell cluster growth. As expected, cell density increased if more cells were added. Next, the binding properties of the chips were examined using trastuzumab injections (5-80 μg/mL) under FIA conditions (Figure 4b). The maximum frequency response increased with increasing seeding density for all trastuzumab concentrations. For the lowest seeding density, 2 × 10 4 cells/chip, very low binding with no concentration dependence was observed, most likely due to the low surface coverage (Figure 4a). For the higher seeding densities, the maximum frequency responses were higher and concentration dependent, reflecting the higher surface coverage of these chips. To verify that the frequency shifts resulted from the specific binding of trastuzumab, the sensorgrams were examined in more detail (Figure 4c) using 20 μg/mL injections. The binding curves showed association and dissociation curvature for all seeding densities. The injection part of the curves for the cell chips seeded with corresponding to 2 × 10 4 showed saturation and 4 × 10 4 cells/surface approached saturation, reaching its maximum frequency response quickly, while for chips seeded with 8 × 10 4 cells/surface showed no saturation using this trastuzumab concentration. Figure 4. (a) Fluorescence micrographs of DAPI stained SKOV3 cells on COP-1 chips prepared using increasing seeding density. Scale bars 200 μm; (b) Maximum frequency response for different concentrations of trastuzumab to 2 × 10 4 (red), 4 × 10 4 (blue) and 8 × 10 4 (black) cells per chip. Error bars represent SD for triplicate injections on three different chips; Representative sensorgrams after injection of (c) 20 μg/mL trastuzumab to chips prepared using increasing seeding density and (d) trastuzumab binding (black curves) to cell chip seeded with corresponding to 8 × 10 4 cells/surface. Red curves represent theoretical curve fitting using a mass transport limited model. The mean KD value was calculated to 7 ± 1 nM (standard error of the mean). Data from two cell chips, with four analyte concentrations. Kinetic Evaluation of Trastuzumab Binding to SKOV3 Cell Chips In order to determine the affinity for trastuzumab binding to the SKOV3 cell chip, the dissociation constant (KD) was calculated using binding data from two prepared chips using a seeding density of 8 × 10 4 cells/chip and fixed using 3.7% FA (Figure 4d). Sensorgrams from different concentrations of the analyte were fitted to a mass transport limited model. The diffusion rate of the analyte, which is carried form the bulk solution to the sensor surface needs to be considered. Mass transport limitations occur when the association rate is fast and the diffusion of the analyte becomes limiting for the interaction [6,40]. Mass transport limitations can be recognized in the sensorgram by a linear appearance in the early stages of the association phase and non-exponential dissociation phase. Such effects can be avoided by using lower surface densities, by increasing the flow rate during analysis, or by using a calculation model for mass transport limitation, as here. KD values were calculated resulting in a mean KD value of 7 ± 1 nM (standard error of the mean). This value is strongly correlated to other published data for the interaction using other cell-based assays (KD = 5 nM according to the manufacturer of Herceptin TM ). Other reported studies of the kinetics of this interaction, Carter et al. and Bostrom et al. have reported dissociation constants of 0.1 nM [41] and 0.5 nM [42] for trastuzumab binding to immobilized HER2 (extra cellular domain) using ELISA and SPR technology, respectively. Accordingly, these studies of antibody-HER2 interactions provide a new perspective on the affinity in a more natural environment. Moreover, this study demonstrates the potential for using whole cell-based QCM studies in drug development and for the study of biomolecular recognition. Clinical tests for diagnostics are often time-consuming and not sufficiently sensitive, resulting in false-positive or negative outcomes. Improving assay reliability while retaining high sensitivity is therefore of great importance, in particular for the early stage detection of diseases such as various forms of cancer. Furthermore, for drug development; speed, quality and cost are in focus and time-effective high throughput tools are important [5]. Antibody-mediated diagnostics and treatments have proven of value in the diagnosis and treatment of several diseases, e.g., cancer, though the development of new sensitive tools remains an important challenge for researchers in this field. The sensitivity of the technique was sufficient to be able to determined data on the affinity of trastuzumab for the target receptor that were comparable with those previously reported, and the technique also provided access to kinetic data for this antibody-cell surface interaction, highlighting its potential for use in further studies of this and other cell-surface mediated interactions. Conclusions/Outlook In this work, we have studied the interaction between the antibody trastuzumab to SKOV3 cells, a human ovarian cancer cell line, fixed to COP-1 QCM sensor chips. We have also shown that the cell chip preparation (e.g., fixation procedure and seeding density) affects the cell conditions and binding response. This real time study, involving the direct measurement of interaction with intact cellular receptors provided quantitative binding data regarding both affinity and kinetics of interactions, in contrast with that generally derived from immunohistochemical assays. This approach offers potential for the screening of antibody derived HER2-directed candidate drugs and as a proof of concept for other binding studies involving membrane bound targets. These real time, label-free measurements, give sensitive and detailed kinetic information about the binding of the analyte to the cell-associated target as compared to label-dependent assays such as flow cytometry, fluorescence microscopy or standard microplate, i.e., ELISA. Moreover, we have shown that this QCM cell biosensor can be used to provide information of biomolecular recognition processes in environments more like the natural environments of the target structures. Author Contributions Cell chip preparations and QCM studies were performed by L.E. Kinetic calculations were performed by L.E and C.K. All authors (L.E., C.K., T.A., I.A.N.) contributed to the design of experiments and drafting of the manuscript.	v3-fos-license
2018-04-03T02:21:21.261Z	2002-05-10T00:00:00.000	23010190	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/277/19/17147.full.pdf", "pdf_hash": "be09569e5468c61a6b2be4df5f65c58c68ec60f2", "pdf_src": "Highwire", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1907", "s2fieldsofstudy": [ "Biology", "Medicine" ], "sha1": "cb475d755c5734e51db264f89ae8cce3588bd8db", "year": 2002 }	pes2o/s2orc	Impaired Stratum Corneum Hydration in Mice Lacking Epidermal Water Channel Aquaporin-3* The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouse skin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wild-type and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a >4-fold reduced osmotic water permeability and >2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165 ± 10 versus 269 ± 12 microsiemens (μS), p < 0.001), as well as in a CD1 genetic background (209 ± 21 versus 469 ± 11 μS). Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels (90–100 μS) after a 24-h exposure to a dry atmosphere, but the difference was increased when surface evaporation was prevented by occlusion or exposure to a humidified atmosphere (179 ± 13versus 441 ± 34 μS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC in AQP3 null mice. Water sorption-desorption experiments indicated reduced water holding capacity in the SC of AQP3 null mice. The impaired skin hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Modulation of AQP3 expression or function may thus alter epidermal moisture content and water loss in skin diseases. The water content of the stratum corneum is an important determinant of the appearance, physical properties, and bar-rier function of the skin (1)(2)(3). The stratum corneum, the most superficial layer of skin, consists of layers of flattened corneocytes (dead epidermal cells) embedded in a lipid-rich matrix containing specialized proteins and lipids (4). Abnormalities of stratum corneum hydration are seen in a variety of hereditary and acquired skin diseases such as atopic dermatitis (5), eczema (6), psoriasis (7), senile xerosis (8), and hereditary ichthyosis (9). Hydration of the stratum corneum could in principle be determined by a number of factors including the concentration of water-retaining osmolytes, the water and solute transporting properties of the underlying layers of viable epidermal keratinocytes, and the barrier properties of the stratum corneum. There is evidence for a high concentration of solutes (Na ϩ , K ϩ , and Cl Ϫ ) and a low concentration of water Ref. 10) in the superficial stratum corneum, producing in the steady-state gradients of both solutes and water from the skin surface to the viable epidermal keratinocytes (11)(12)(13). Although transepithelial fluid transporting properties have been studied extensively in various mammalian epithelia, the molecular mechanisms of fluid transport across epidermal keratinocyte layers remain poorly understood, as is the relationship between keratinocyte fluid transport and stratum corneum hydration. It has been proposed that aquaporin-3 (AQP3) 1 might facilitate transepidermal water permeability to protect the stratum corneum against desiccation by evaporative water loss from the skin surface and/or to dissipate water gradients in the epidermal keratinocyte cell layer (14). The integral membrane protein AQP3 has been proposed to be a potentially important transporter of water and solutes across epidermal keratinocytes (14 -16). AQP3 was initially cloned from rat kidney (17,18) and is a member of a family of homologous aquaporin water channels expressed widely in mammalian epithelia and endothelia that facilitate fluid transport. Phenotype studies in aquaporin knockout mice have implicated the involvement of aquaporins in the urinary concentrating mechanism in kidney (19), water movement in lung (20,21), cerebral water balance (22), exocrine gland secretion (23,24), and mechano-electric signal transduction (25). AQP3 is a member of a subclass of aquaporins, called aquaglyceroporins, which transport not only water but also glycerol and possibly other small solutes. AQP3-mediated water permeability was reported to be pH-dependent, decreasing at low pH (26). The physiological role of AQP3-mediated glycerol transport has been the subject of considerable speculation but remains unknown. We first reported by immunocytochemistry the expression of AQP3 protein in epidermal keratinocytes in rat skin (15), and subsequently AQP3 was localized in human keratinocytes (14), and in moist noncornified barriers such as the mucous membranes in the mouth (16). It was reported recently that water permeability of human epidermal keratinocytes was inhibited by mercurials and low pH, consistent with the involvement of AQP3 (14). Additional indirect evidence supporting a role for AQP3 in skin physiology includes the regulation of epidermal cell AQP3 expression by extracellular osmolality, barrier perturbation, and exposure to a dry atmosphere (27,28). The purpose of this study was to investigate whether AQP3 is involved in stratum corneum hydration. Because humans with AQP3 deficiency have not been identified and nontoxic inhibitors of AQP3 are not yet available, we assessed skin phenotype in transgenic mice lacking AQP3. For studies in skin, the AQP3 null genotype, originally created in a CD1 genetic background (29), was transferred to the hairless SKH1 background. We found remarkably reduced stratum corneum water content in the skin of AQP3 null mice and investigated possible mechanisms for this defect. An interesting and unexpected finding was that AQP3 deletion remarkably impaired the ability of stratum corneum to become hydrated even when water loss from the skin surface was prevented by occlusion or exposure to a humidified atmosphere. EXPERIMENTAL PROCEDURES Mice-The AQP3 null genotype originally generated in CD1 genetic background (29) was transferred to a hairless background by back-cross breeding of heterozygous AQP3 CD1 mice with SKH1 hairless mice. Hairless AQP3 heterozygous founder mice were bred to generate wildtype, heterozygous, and AQP3 null hairless mice. The mice were maintained in air-filtered cages and fed normal mouse chow in the University of California, San Francisco, Animal Care Facility. All procedures were approved by the UCSF Committee on Animal Research. Urine Output, Osmolarity, and Serum Chemistries-24-h urine output was measured using metabolic cages (Harvard Apparatus). Urine osmolality was measured by freezing-point osmometry (Precision Systems Inc.). Serum chemistries were measured by the UCSF Clinical Laboratory. Immunofluorescence, Light Microscopy, and RT-PCR-Immunofluorescence was done on frozen sections of mouse skin fixed in PBS containing 2% paraformaldehyde using a 1:200 dilution of affinity-purified anti-AQP3 rabbit polyclonal antibody. Slides were washed three times in PBS and incubated for 40 min at room temperature in PBS/bovine serum albumin containing fluorescein isothiocyanate-coupled anti-rabbit antibody (10 g/ml). Micrographs were obtained with a cooled CCD camera in an Olympus fluorescence microscope. For morphological examination by light microscopy, skin was fixed in PBS containing 4% paraformaldehyde, and 2 m plastic sections were stained with toluidine blue. For RT-PCR analysis, total RNA was isolated from the epidermis of wild-type and AQP3 null mice. RT-PCR was performed with sequence-specific sense and antisense oligonucleotide primers for mouse aquaporins 1-9 as described previously (23). Electron Microscopy-Skin samples were fixed in 2% glutaraldehyde and embedded in Epon. 70-nm thick sections were cut on a Reichert-E ultramicrotome and collected on Formvar-coated electron microscopy grids. Sections were stained in 5% uranyl acetate for 3 min and then in lead citrate for 1 min, dried, and observed by electron microscopy (Philips CM 400). For immunogold labeling, skin samples were postfixed in 2% paraformaldehyde ϩ 0.1% glutaraldehyde and embedded in Unicryl. 70-nm thick sections were preincubated in 20 mM Tris buffer (pH 7.4) containing 0.1% bovine serum albumin, 0.1% fish gelatin, and 0.05% Tween 20 (TBuffer) followed by a 1-h incubation in affinitypurified anti-AQP3 antibody (1:50 dilution) in TBuffer. Grids were incubated in a 1:25 dilution of 10-nm gold-coupled anti-rabbit antibody (Amersham Biosciences) for 1 h, washed six times, stained in 5% uranyl acetate for 3 min and then in lead citrate for 1 min, dried, and observed by electron microscopy. Water and Glycerol Permeability Measurements-For measurement of water permeability, epidermal sheets were freshly isolated from 6 -8-week-old wild-type and AQP3 null mice by digesting full thickness skin fragments in 50 units/ml dispase solution (BD Biosciences) at room temperature for 1 h. After washing in cold PBS, the epidermis was peeled off the dermis. Small fragments of the epidermal sheets (ϳ12-mm diameter) were immobilized (surface facing downward) on 18-mm-diameter round coverglasses using superglue. The coverglass was mounted in a perfusion chamber (exchange time Ͻ 0.2 s) for measurement of osmotic water permeability by spatial filtering microscopy as described previously for studies in cultured cell layers and bladder sheets (30) and in kidney tubules (29). The time course of transmitted light intensity was measured in response to changing between perfusate osmolalities of 300 (PBS) and 600 mosM (PBS containing 300 mM sucrose). For measurement of glycerol permeability, viable keratinocytes were isolated from epidermal sheets (prepared as above) by digestion in 0.5% trypsin solution for 10 min at room temperature with gentle shaking. After neutralizing with fetal bovine serum (10% final concentration), the cell suspension was centrifuged three times for 10 min at 800 ϫ g. More than 95% of the cells were viable as judged by trypan blue exclusion. The keratinocyte cell suspension (ϳ10 6 cells/ml) was incubated for specified times with PBS containing tracer quantities of [ 3 H]glycerol (Amersham Biosciences) at room temperature. After separating on glass fiber filters and washing three times with ice-cold PBS in a suction filtration apparatus, cells were disrupted with 1 M NaOH. Cell-associated radioactivity was determined by scintillation counting. Protein concentration was measured using a Bio-Rad DC protein assay kit. Skin Conductance Measurements-Stratum corneum hydration was determined by high frequency electrical conductance using a Skicon-200 skin surface hygrometer (IBS). Three independent measurements from the same area of skin were averaged for each value. Measurements were performed on mice under normal conditions (external temperature 22 Ϯ 2°C, humidity 40 Ϯ 3%) or on mice treated with various maneuvers including exposure to low (10%) and high (90%) external humidity, tape stripping, and topical occlusion. Water sorption-desorption studies were done as described (7,31), where conductance was measured before and at 30-s intervals after pipetting 20 l of distilled water onto the skin and blotting after 10 s. In some experiments, conductance measurements were conducted on shaved skin of wild-type and AQP3 null mice in the (hairy) CD1 genetic background. Analysis of Stratum Corneum Protein Content-Stratum corneum layers were stripped and collected using Scotch cellophane tape. The stripped stratum corneum layers on the tape were dissolved in 1 M NaOH for 1 h. After neutralization with 1 M HCl, total protein concentration was measured using a Bio-Rad DC protein assay kit (Richmond, CA). RESULTS To study skin phenotype, the AQP3 null mutation generated in CD1 mice was transferred to a SKH1 hairless genetic background by back-cross breeding. The AQP3 null hairless mice generated by breeding of heterozygous founder mice had normal perinatal survival and growth compared with wild-type littermates. However, the daily urine output of the AQP3 null mice was ϳ10-fold greater than in wild-type mice ( Fig. 1, A, top, and B). Urine osmolality in the AQP3 null hairless mice was very low (132 Ϯ 22 versus 1974 Ϯ 123 mosM in wild-type mice) when given free access to water (Fig., 1A, bottom). After a 24-h water deprivation, urine osmolality increased submaximally (481 Ϯ 11 versus 3792 Ϯ 204 mosM in wild-type mice) (Fig. 1A, bottom), indicating a urinary concentrating defect as found for AQP3 null mice in a CD1 genetic background (29). Analysis of serum chemistries showed no differences in electrolyte concentrations, creatinine, and liver function tests, except for mild elevation in blood urea nitrogen (45 Ϯ 4 versus 29 Ϯ 3 mg/dl) and decreased triglyceride concentration (110 Ϯ 12 versus 166 Ϯ 16 mg/dl). Immunofluorescence showed strong AQP3 protein expression in wild-type mice in a membrane pattern in the keratinocyte layer just below the stratum corneum ( Fig. 2A). Immunogold electron microscopy indicated plasma membrane localization of AQP3 protein in epidermal keratinocytes (Fig. 2B). Antibody staining was absent in AQP3 null mice. RT-PCR analysis of mRNA isolated from epidermis using primers specific for aquaporins 0 -9 revealed only AQP3 transcript in wildtype mice and no aquaporins in AQP3 null mice (Fig. 2C). The general structure of the epidermis was studied by light and electron microscopy. Light microscopy of plastic-embedded sections of epidermis stained with toluidine blue revealed sim-ilar thickness and structure of the stratum corneum and epidermal cell layers (Fig. 2D). Thickness measurements on a series of sections showed no significant differences in the stratum corneum, epidermal, or dermal layers (Fig. 2E). The thickness of the innermost fat layer was reduced in the AQP3 null mice (p Ͻ 0.01), which may be related to the relative serum hypotriglyceridemia. Transmission electron microscopy showed no apparent differences in the structures of the stratum corneum and keratinocytes in wild-type (Fig. 3, A and C) and AQP3 null (Fig. 3, B and D) mice. At the apex of the epidermis (Fig. 3, A and B), the stratum corneum contained five layers in both wild-type and AQP3 null mice, with each layer of a similar thickness. Keratohyalin granules seen in the superficial epidermal cells were of similar size in wild-type and AQP3 null mice. As expected, keratinocytes at the base of the epidermis (Fig. 3, C and D) were taller but showed no differences between genotypes. Despite the possible consequences of AQP3 deficiency on water movement, intercellular spaces were not dilated in AQP3 null mice from the base to the surface of the epidermis. Osmotic water and glycerol permeability were measured to investigate the functionality of plasma membrane AQP3 in epidermal keratinocytes. Osmotic water permeability was measured by spatial filtering light microscopy using fragments of epidermis (isolated by dispase digestion), which were mounted in a perfusion chamber with epidermal keratinocytes facing upward. Fig. 4A (left) shows the reversible time course of keratinocyte layer shrinking and swelling in response to the changing of perfusate osmolality between 300 and 600 mosM. Fitted reciprocal exponential time constants (proportional to water permeability; Fig. 4A, right) were significantly reduced by ϳ4-fold in epidermis of AQP3 null mice. Because of the possible unstirred layer effects in the multilayered epidermis, which limits observed water permeability, the 4-fold difference formally represents a lower limit to the difference in epidermal water permeability in wild-type versus AQP3 null mice. Glycerol permeability was measured from the uptake of radiolabeled [ 3 H]glycerol in suspensions of freshly isolated keratinocytes. Measurements in keratinocytes from wild-type mice indicated approximately linear [ 3 H]glycerol uptake for 10 min (not shown). Fig. 4B shows that the average [ 3 H]glycerol uptake at 90 s (after subtraction of bound [ 3 H]glycerol uptake by 0 time measurement) was significantly reduced in keratinocytes from AQP3 null mice. The ϳ2-fold reduction in glycerol permeability represents a lower limit to the difference in epidermal glycerol permeability in wild-type versus AQP3 null mice because of possible effects of the protease treatment (needed to release keratinocytes) on AQP3 function. These results establish the functionality of AQP3 as a water/glycerol transporter in mouse epidermis. Stratum corneum water content was measured using a well established surface conductance method (32). Surface electrical conductance is approximately linearly related to percentage water content of the outer stratum corneum (33). Fig. 5A shows remarkably reduced skin conductance in different areas of the skin in adult hairless AQP3 null mice measured under standard atmospheric conditions (22°C, 40% relative humidity). The greatest differences was found in the upper abdominal skin, with ϳ50% reduced conductance in the AQP3 null mice. To confirm that these observations are not unique to the hairless genetic background, skin conductance was measured at 1 day after shaving the mid-back skin of CD1 mice. Although skin conductance was greater in the CD1 than the hairless background, there remained a large effect of AQP3 deletion (209 Ϯ 21 versus 469 Ϯ 11 S in wild-type mice). Skin conductance was measured in mice of different ages to determine when the defect in stratum corneum hydration is first manifest. As summarized in Fig. 5B, skin conductance was low and similar at 1 and 2 days after birth but was significantly lower by day 3 in AQP3 null mice compared with wild-type mice. Skin conductance increased substantially during the first month in wild-type mice, to a much greater extent than in AQP3 null mice. To test the hypothesis that decreased stratum corneum hydration in AQP3 null mice is due to impaired replacement of surface evaporative losses by water transport across epidermal keratinocytes, conductance measurements were performed after subjecting mice to different external humidity conditions and after skin surface occlusion. Exposure to 10% humidity increases evaporative water loss, whereas exposure to 90% humidity or surface occlusion prevents evaporative water loss. The prediction is that the defective stratum corneum hydration in AQP3 null mice would be corrected by preventing evaporation but exaggerated by exposure to a 10% humidity atmosphere. Fig. 5C summarizes conductance measurements. Exposure to 90% humidity or occlusion for 24 h resulted in increased stratum corneum hydration in wild-type mice, but contrary to expectations, the impaired hydration in AQP3 null mice was not corrected. Also, contrary to expectations, skin conductance of the wild-type and AQP3 null mice became similar after exposure to 10% humidity. Fig. 5D shows the full time courses of skin conductance after changing external humidity. In both cases the new steady-state conductance was achieved in 2-4 h but with a small undershoot for exposure to 10% humidity. To investigate whether the impaired hydration occurs in deeper layers of the stratum corneum of AQP3 null mice, conductance measurements were made after layers of the stratum corneum were removed progressively by cellophane tape (tape stripping). Fig. 6A shows that the accumulated total protein was approximately linear with the number of tape strippings and that the amount of protein removed was similar in wildtype and AQP3 null mice. Fig. 6B shows the correlation between skin conductance and total accumulated protein for a series of wild-type and AQP3 null mice subjected to serial tape stripping. Hydration increased progressively as deeper layers of the stratum corneum were exposed by tape stripping, which supports the notion of a water gradient from deep to superficial stratum corneum (12,34). Skin conductance was lower in AQP3 null mice, but the steepness of the water gradient was increased. These results are consistent with the notion that a water gradient is established from the well hydrated epidermal keratinocytes through the relatively water-poor and watertight stratum corneum. The results shown in Figs. 5 and 6 provide evidence against the hypothesis that impaired hydration in the stratum corneum of AQP3 null mice results from decreased epidermal water permeability. We tested whether the stratum corneum of AQP3 null mice has an intrinsic defect in its ability to be hydrated and in its "water holding capacity," as found in diseases associated with dry skin. An established sorption-desorption test was used in which skin conductance is measured before and at different times after a 10-s exposure of the skin to distilled water (32). Fig. 6C shows that skin conductance increased immediately after water exposure and then recovered over a few min. The initial increase in skin conductance, which was significantly greater in wild-type mice, has been taken as a measure of the ability of the stratum corneum to become hydrated. More importantly, the recovery after hydration has been taken as a measure of the water holding capacity of the stratum corneum. The area under the recovery curve (after subtraction of prehydration conductance) has been used as a single parameter describing water holding capacity (10). The recovery area parameter was remarkably reduced in AQP3 null mice (8.2 Ϯ 1.7 ϫ 10 4 versus 4.5 Ϯ 1.9 ϫ 10 4 S⅐s, p Ͻ 0.005) (see "Discussion"). DISCUSSION The goal of this study was to determine whether AQP3 plays a role in skin physiology. As discussed in the Introduction, previous findings of AQP3 expression and regulation in epidermal keratinocytes provided indirect evidence for a role of AQP3 in skin function. To facilitate the study of stratum corneum hydration, the AQP3 null genotype was transferred to a SKH1 hairless background. The hairless AQP3 null mice manifested a urinary concentrating defect with polyuria and reduced urinary osmolality, as found previously for AQP3 null mice in the CD1 background (29). AQP3 null CD1 mice develop progressive renal failure with dilatation of the kidneys and urinary bladder by 8 weeks of life (35) C, RT-PCR analysis of aquaporin expression in epidermis isolated from wild-type and AQP3 null mice. Transcripts for portions of the coding sequences of the indicated mouse aquaporins were PCR-amplified using specific primers. Rows labeled "ϩ/ϩ" and "Ϫ/Ϫ" correspond to amplifications done using epidermal cDNA as template, and the row labeled "control " corresponds to amplifications done using a mixture of cDNAs from brain, lung, liver, and kidney, which contain all mouse aquaporins. D, toluidine blue-stained thin plastic sections from two wild-type and two AQP3 null hairless mice. Scale bar, 20 m. E, thickness of indicated skin layers (means Ϯ S.E.) measured in plastic sections from n ϭ 4 mice. Differences in thickness of the stratum corneum, epidermis, and dermis not significant. less AQP3 null mice had normal perinatal survival, growth, and serum chemistries, except for a mild elevation in blood urea nitrogen and a decrease in serum triglyceride concentration. We do not believe that these mild alterations in blood chemistry cause the abnormalities in stratum corneum hydration in AQP3 null mice. The decreased stratum corneum hydration was found in AQP3 null mice at 4 weeks of age before changes in blood urea nitrogen were detected, and AQP1 null mice have an even greater degree of serum hypotriglyceridemia (37) but do not manifest altered stratum corneum hydration. Adequate stratum corneum hydration is important in maintaining skin plasticity and barrier integrity. The stratum corneum gains water from underlying viable layers of epidermis and dermis to maintain its proper hydration status in the relatively dry external environment. There are several possible mechanisms by which AQP3 deletion might affect stratum corneum hydration. Reduced epidermal water permeability might impair water transport into the stratum corneum, which would lead to decreased stratum corneum water content when surface evaporation occurs but not when the skin surface is occluded or exposed to a humidified environment. Reduced permeability of the epidermal cell layer to glycerol or other small solutes might produce alternations in the composition and/or structure of the stratum corneum that alter its ability to hold water even when surface evaporation is blocked. High aquaporin-dependent water permeability in the epidermal cell layer might prevent water gradients within the viable keratinocytes, preserving their normal biosynthetic functions. However, it seems unlikely that water gradients are present in the layer of viable keratinocytes because water permeability of the stratum corneum is orders of magnitude lower than in the keratinocyte layer (38). Transport measurements showed significantly reduced water and glycerol permeability in AQP3 null mice. These results are consistent with localization of AQP3 in the plasma membrane of keratinocytes, as shown by immunofluorescence and immunogold electron microscopy, and with the absence of the compensatory expression of another aquaporin in epidermis of AQP3 null mice, as shown by RT-PCR. The finding of functional expression of AQP3 in mouse epidermis agrees with the conclusion of a recent study showing that apparent water permeability in human epidermis was inhibited by mercurials and low pH (14). Utilizing a spatial filtering approach to measure osmotic water permeability in freshly isolated, intact epidermal sheets, we found that AQP3 deletion produced an ϳ4-fold reduction in water permeability. This approach preserves the normal epidermal anatomy, and unlike keratinocyte isolation procedures, the isolation of epidermal sheets does not require digestion with proteases. In other studies we have found up to 10-fold reductions in water permeability after aquaporin deletion, for example in erythrocytes (35), the proximal tubule (19), and the thin descending limb of Henle (39) of AQP1 null mice FIG. 5. Decreased stratum corneum hydration in AQP3 null mice. A, skin surface conductance (proportional to stratum corneum water content) in indicated areas of hairless wild-type and AQP3 null mice (means Ϯ S.E., 10 -14 mice/group). , p Ͻ 0.001. B, age dependence of skin conductance (means Ϯ S.E., 25 mice/group). C, skin conductance (back skin) measured after a 24-h exposure to atmosphere (room temperature) at relative humidity of 10, 40, or 90%; "occluded " indicates an occlusion dressing (means Ϯ S.E., 5 mice/group). , p Ͻ 0.01. D, time course of skin conductance after exposure (at time 0) to a 90% (left) or 10% (right) humidified atmosphere. and in the lungs of AQP1 and AQP5 null mice (20,21). The relatively small reduction in epidermal water permeability in AQP3 null mice may be related to the relatively weak intrinsic water transporting activity of AQP3 compared with other aquaporins (40). In addition, unstirred layer effects in the multilayered epidermal sheet would decrease apparent osmotic water permeability and blunt the effects of AQP3 deletion. Nevertheless, the results shown here prove that AQP3 is functional as a plasma membrane water/glycerol transporter in the epidermis, but they provide only a lower limit to the contribution of AQP3 to total water permeability. Conductance measurements showed remarkably reduced stratum corneum water content in AQP3 null mice in most areas of the skin, despite grossly normal morphology by light and electron microscopy. Reduced stratum corneum hydration was also seen in AQP3 null mice in the CD1 genetic background. The defect in stratum corneum hydration was seen as early as 3 days after birth in the hairless AQP3 null mice, and from tape stripping experiments it appeared to involve the full stratum corneum thickness. Exposure of the skin to moist and dry humidity conditions produced fairly prompt changes in stratum corneum hydration as a new steady state was reached for surface evaporative water loss versus water movement into the stratum corneum through the epidermal cell layer. The surprising observation was that stratum corneum hydration in AQP3 null mice increased little in a humidified environment and with surface occlusion, despite a marked increase in hydration in the wildtype mice. In a dry environment, stratum corneum hydration decreased to comparable levels in wild-type and AQP3 null mice. These results provide evidence against the hypothesis that a major function of epidermal AQP3 is to facilitate water movement into the stratum corneum. Instead, the AQP3 null mice appear to manifest an intrinsic defect in the stratum corneum that limits its ability to accumulate water. Water sorption-desorption studies supported the conclusion that the stratum corneum of AQP3 null mice has an intrinsic defect in its ability to absorb and hold water. The determination of the molecular mechanism by which AQP3 deletion impairs hydration and water holding in the stratum corneum will require analysis of the peptide, lipid, and carbohydrate composition of the stratum corneum and epidermis, as well as high resolution structural studies. In addition, the identification of specific proteins in the stratum corneum and keratinocytes that are up-regulated in AQP3 deficiency may be informative, especially if they are involved in the biosynthetic pathways for generation of water-retaining osmolytes or specialized barrier lipids. Although direct evidence is not yet available, we suspect that defective glycerol/small solute transport in the epidermis of AQP3 null mice is the basis of their abnormal hydration. Studies of non-skin phenotypes in aquaporin-deficient mice support the conclusion that epidermal cell water permeability is probably not a major determinant of stratum corneum hydration. Water movement across the stratum corneum is very slow compared with other tissues, such as the proximal tubule and secretory epithelia, where aquaporins are important. Also, water movement into and through the stratum corneum is likely to be limited by unstirred layers rather than the intrinsic water permeability of the epidermal cell layer. Further studies are needed to determine whether water and/or glycerol transport by AQP3 is essential for normal hydration of the stratum corneum. In summary, the impaired stratum corneum hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Although the exact mechanism for impaired stratum corneum water holding remains to be established, we propose that pharmacological modulation of AQP3 expression or function may alter epidermal moisture content and water loss. Controlled modulation of skin moisture content and barrier function may thus be useful in the treatment of skin disorders associated with abnormally wet, dry, or permeable skin. FIG. 6. Analysis of impaired stratum corneum hydration in AQP3 null mice. A, accumulated total protein content after serial tape-stripping (6 mice/group). B, correlation between skin conductance and accumulated protein content after tape-stripping. See text for further explanations. C, skin conductance before and at indicated times after a 10-s exposure of skin to distilled water (means Ϯ S.E., 6 -8 mice/group).	v3-fos-license
2018-04-03T00:16:07.957Z	2018-02-20T00:00:00.000	3883179	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://downloads.hindawi.com/journals/sci/2018/4098140.pdf", "pdf_hash": "bc8cd909763c68c0a15aca0eaab3a62ee6117a92", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1963", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Medicine" ], "sha1": "ffa0673e2977e35e652673c1739dbba54acfa0a0", "year": 2018 }	pes2o/s2orc	A Nonenzymatic and Automated Closed-Cycle Process for the Isolation of Mesenchymal Stromal Cells in Drug Delivery Applications The adipose tissue is a good source of mesenchymal stromal cells that requires minimally invasive isolation procedures. To ensure reproducibility, efficacy, and safety for clinical uses, these procedures have to be in compliant with good manufacturing practices. Techniques for harvesting and processing human adipose tissue have rapidly evolved in the last years, and Lipogems® represents an innovative approach to obtain microfragmented adipose tissue in a short time, without expansion and/or enzymatic treatment. The aim of this study was to assess the presence of mesenchymal stromal cells in the drain bag of the device by using a prototype Lipogems processor to wash the lipoaspirate in standardized condition. We found that, besides oil and blood residues, the drain bag contained single isolated cells easy to expand and with the typical characteristics of mesenchymal stromal cells that can be loaded with paclitaxel to use for drug-delivery application. Our findings suggest the possibility to replace the drain bag with a “cell culture chamber” obtaining a new integrated device that, without enzymatic treatment, can isolate and expand mesenchymal stromal cells in one step with high good manufacturing practices compliance. This system could be used to obtain mesenchymal stromal cells for regenerative purposes and for drug delivery. Introduction Mesenchymal stromal cells (MSCs) can be easily isolated from several human organs and tissues and, because of their self-renewing capacity and multipotent differentiation properties, they are important tools for treating immune disorder and for tissue repair. In particular, adiposederived MSCs (ASCs) can be harvested in high amounts by minimally invasive procedures with good viability, differentiating potential, and paracrine activity [1][2][3]. These cells can be also used as drug carrier (as already demonstrated for MSCs derived from bone marrow) because, when primed with high doses of the chemotherapeutic drug paclitaxel (PTX), they are capable to uptake the drug and release it in big amounts inhibiting the in vitro proliferation of different tumour cell lines [4,5]. Their definition as advanced-therapy medicinal products (ATMPs) according to the European Medicines Agency (EMA) and the US Food and Drug Administration requirements for their production and use implies the application of production processes that should be in accordance with good manufacturing practices (GMPs). To increase safety and reproducibility and to implement the many clinical trials using ASCs, several methods based on reducing the manipulation of human tissue-based products have been suggested, in particular by using nonenzymatic treatments [6,7]. Among the many, an innovative closed and sterile system (named Lipogems), designed to harvest, process, and transfer a refined and not-expanded adipose tissue characterized by a great regenerative potential and optimal handling, has been developed. By the aid of this technology, and without the addition of enzymes or any other additives, fat tissue is microfragmented in a completely close liquid environment and washed from proinflammatory oil and blood residues [8]. This closed and sterile device reduces the size of the adipose tissue clusters by means of two filters [9]. A drain bag, connected to the second filter of the device, collects the waste fluid containing red blood cells and oil residues. We used a prototype Lipogems processor (PLG-P) to wash and process the lipoaspirate in a standardized condition and we found that, besides oil and blood residues, the drain bag contained single isolated cells easy to expand and with the typical characteristics of ASCs. These cells can be also loaded with paclitaxel to provide a cell-mediated drugdelivering tool. Materials and Methods 2.1. Ethics Statements. Samples from adult donors were collected after signed informed consent of no objection for the use for research of surgical tissues (otherwise destined for destruction) in accordance with the Declaration of Helsinki. The approval for their use was obtained from the Institutional Ethical Committee of Milan University (n.59/15, C.E.UNIMI, 09.1115). Prototype Lipogems Processor (PLG-P). The prototype processor (Figure 1(a)) equipped with the Lipogems device ( Figure 1(b)) is able to guarantee digitally controlled movements according to these main parameters: oscillation amplitude and frequency and pitch movements and saline washing flux. 25 ml of lipoaspirate was loaded in the Lipogems device and washed according to a standardized procedure: process flow 120 ml/min, oscillation frequency 2 Hz, pitch frequency 0.3 Hz, pitch angle 30°, and pitch axis 90°vertical (exit bottom). In a first preliminary experiment, the tissue was processed for 5 minutes and cells were collected one step into 600 ml of saline. In a second experiment, the collection was fractioned and performed at 1 (120 ml), 2.5 (180 ml), and 5 minutes (300 ml). LGP-P shaking transmits a mechanical force to the stainless steel marbles contained in the device that results into a temporary fat emulsion washed by a flux of buffer. This allows the washing buffer to cross a second filter (F2) and accumulate into the drain bag (red circle) together with oil, red blood cells, and mesenchymal stromal cells (ASCs). The pellet obtained after centrifugation was seeded in 25 cm 2 flask in StemMACS medium (Miltenyi Biotec, USA) (see Materials and Methods for details). ASC Isolation from Drain Bag (DB-ASCs) . 100 ml of each sample was centrifuged at 2500 ×g for 15 minutes. Pellet was treated twice with 0.85% ammonium chloride (5 minutes at +4°C) to partially remove red cells. Then, after three washes with PBS 1x through centrifugation (800 ×g, 10 minutes), pellet was plated on 25cm 2 flask (Corning, USA) with 5 ml of Stem MACS MSC Expansion Medium (Miltenyi Biotec, Germany) and incubated at 37°C, 5% CO 2 . The primary culture was expanded with 1 : 2 passages until passage number (pn) 6. At pn 2, the population doubling time (PDT) was calculated as previously described [10]. The clonogenicity of the ASCs was evaluated as colonyforming efficiency (CFE) in multiwell plates (SPL Life Sciences, Korea) by serial dilution (from 50 cells/well to 1 cell/well) in DMEM LG media with 10% fetal bovine serum (EuroClone, UK). After 10 days, cell colonies fixed and labeled with Crystal Violet (0.5%, Fluka, Switzerland) were counted at the microscope (a colony formed by at least 25 cells) and the CFE as the following: CFE = average of colonies formed × 100/number of seeded cells. Immunohistochemical Analysis of DB-MSCs. Mesenchymal cell monolayer (pn 2) was detached with trypsin-EDTA (EuroClone, UK) and 1.7 * 10 6 cells were placed in a 10 ml glass conical tube in 1x PBS and centrifuged at 800 ×g for 10 minutes. Pellet was treated for immunohistochemical characterization as previously reported [11]. Briefly, the centrifuged pellet was treated 20 minutes at room temperature with a solution of 75% methanol, 20% chloroform, and 5% glacial acetic acid. The pellet compacted in a solid mass was placed in an immunohistochemical cassette and immersed in a 10% buffered formaldehyde solution and, after dehydration in ethanol and paraffin fixation, the pellet was cut in sections of 4-5 μm that were stored at −20°C. For phenotypic characterization, monoclonal or polyclonal antibodies were used using the streptavidin-biotin method with diaminobenzidine as chromogen. The following antibodies were used: antismooth muscle actin (SMA), anti-CD14, anti-CD105, and anti-neural/glial antigen 2 (NG2) (Santa Cruz Biotechnology, USA); anti-CD44 (Monosan, Holland); antivascular endothelial growth factor (VEGF), anti-CD90 (Dako, Italy); anti-CD45, anti-CD146, anti-CD31, and anti-CD34 (Leica Biosystems, Germany). The expression of CD73 (CD73-PE Becton Dickinson, USA) was evaluated by flow cytometry (FacsVantage SE, Becton Dickinson, USA). Cells were washed twice in PBS, diluted to a final concentration of 10 6 cells/ml, and incubated with the dark antibody at +4°C for 30 minutes. The cells were then washed with PBS and analysed by cytofluorometry by acquisition of 10,000 events per sample. The results were analysed with CellQuest Pro software (Becton Dickinson, USA). 2.5. DB-ASC Osteo-/Adipo-Differentiation Capacity. DB-ASCs at pn 2 have been evaluated for their osteogenic and adipogenic differentiation using a standardized procedure [12]. As positive control, MSCs obtained from human bone marrow (BM-MSCs) were isolated and expanded in our laboratory as previously reported [5]. As negative control, MSCs were cultured without the addition of supplements. The differentiation was performed by plating cells in 35 mm Petri (Nunc, Germany) at a density of 50 cells/cm 2 in 1 ml of STEM MACS MSC Expansion Medium (Miltenyi Biotec, Germany). After 72 hours of incubation, the cell medium was removed and replaced with a specific medium for cell differentiation. To induce osteogenic differentiation [13], cells were incubated for 14 days in DMEM medium LG + 20% fetal bovine serum in the presence of the following: desametasone 10 nM, glycerol 2-10 mM, and phosphate and ascorbic acid 300 nM (all reagents, Sigma-Aldrich, USA). The differentiation medium was replaced every 3-4 days with fresh medium. Cell monolayer was fixed with cold methanol for 5 minutes at −20°C, then alkaline phosphatase (ALP) was evaluated by SIGMA FAST BCIP/NTB substrate for ALP (Sigma-Aldrich, USA). The colorimetric reaction was detected by optical microscope. The presence of osteoblasts at the mature stage was evaluated by staining with Alizarin Red S (Sigma-Aldrich, USA). To induce adipocytic differentiation, cells were cultured in DMEM LG + 20% fetal bovine serum in the presence of indomethacin 200 μM (Alexis Biochemicals, USA), isobutylmethylxanthine 0.5 mM (AppliChem, Germany), dexamethasone 1 μM, hydrocortisone 1 μM insulin, and 10 μg/ml (all from Sigma-Aldrich, USA). After 10 days of incubation, the cells were fixed with 10% buffered formalin (Sigma-Aldrich, USA) for 30 minutes at room temperature and the adipocyte vacuoles present in the cell cytoplasm were evaluated by using red lipophilic Oil Red O (Sigma-Aldrich, USA). 2.6. Ability of DB-ASCs to Uptake and Release Paclitaxel. The ability to incorporate and release drugs from DB-ASCs has been assessed with a procedure already applied on MSCs obtained from bone marrow, adipose tissue, human gingival tissue, human fibroblasts, and also blood cells [4,5,10,[14][15][16]. The "uptake-release" method was performed using paclitaxel (PTX) that is an anticancer drug with also antiangiogenic activity. Subconfluent ASC cultures (15,000 cells/cm 2 ) were exposed to 2000 ng/ml of PTX (Fresenius Kabi, Italy) dissolved in culture media, and after 24 hours of incubation (37°C, 5% CO 2 ), the cell monolayer was washed with PBS and the cells detached, washed by centrifugation in PBS, and subcultured in a 25 cm 2 flask with fresh culture media. After 48 hours, the conditioned medium (CM) was collected and its antitumour activity was evaluated in vitro. The CM from untreated cells was used as a negative control. tumour cell proliferation has been studied in 96-multiwell plates (Sarstedt, Germany) by using as target pancreatic adenocarcinoma cells (CFPAC-1) [17]. Briefly, 1 : 2 serial dilutions of pure drug or ASC-CM were prepared in 100 μl of culture medium/well and then to each well were added 1000 tumour cells. After 7 days of culture at 37°C and 5% CO 2 , cell growth was evaluated by MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium) as previously described [5,18]. The inhibitory concentrations (IC 50 and IC 90 ) were determined according to the Reed and Muench formula [19]. The antitumor activity of PTX CM was compared to that of pure PTX and expressed as PTX equivalent concentration: PEC (ng/ml) = IC50 PTX × 100/V50 (μl/well); IC50 PTX = the concentration of pure PTX producing 50% of inhibition; V50 is the volume PTX-MSCs-CM able to inhibit cell proliferation by 50%. The PEC referred to a single primed ASC was calculated as ratio between the total amount of PEC (PEC (ng/ml) × CM volume (ml)) and the number of cells seeded: PE release (pg/cell) = PEC (ng tot) * 1000/number of cells seeded. Uptake release experiments were performed in triplicate. 2.9. Statistical Analysis. Experiments were performed using lipoaspirate from three donors and repeated in triplicate. The reported data are expressed as mean ± standard deviation. If necessary, appropriate statistical tests have been performed using GraphPad Software (GraphPad Inc., San Diego, CA, USA). A p value ≤ 0.05 was considered statistically significant. The linearity of response and the correlation were studied using regression analysis, by Excel 2007 software (Microsoft Inc.). DB-ASC Isolation and Expansion. The waste medium recovered from the drain bag contained high amounts of red cells that were removed, only partially, by the ammonium chloride treatment. However, after about 7 days, the adhered ASCs could be clearly identified, by transparency, surrounded by a compact rug of red cells (Figures 2(a)-2(c)). However, the adherent cells (washed every 3 days) improve their growth and after 10 days (early adhesion) or 15 days (late adhesion) of incubation, the cell monolayer (Figures 2(d) and 2(e)) was detached and primary culture (pn 0) was quantified for the number of cells. The MSC primary culture was subsequently expanded by 1 : 2 serial passage until passage number 6. MSC growth was evaluated at pn 2 by evaluating the population doubling Time (PDT) and the colony-forming efficiency (CFE) that resulted, respectively, 55.2 ± 2.3 hours (PDT) and 20.8% ± 10.4% (CFE). DB-ASC Recovery Efficiency. To evaluate the amount of ASCs recovered from the lipoaspirate, the washing fluid was collected from the drain bag by two different ways. In a first experiments (one-step collection), 600 ml of saline was collected after 5 minutes of washing; in a second experiment (fractionated collection), the collection was performed at 1 minute (120 ml), 2.5 (180 ml), and 5 minutes (300 ml) (Box 2F). In the one-step collection, the total recovery resulted 40,000 ± 7000 ASCs/ml of lipoaspirate with a cell viability of 83.9 ± 5.4% as evaluated by trypan blue assay. By fractionated collection, the final recovery at 5 minutes was 46,233 ± 4500 ASCs/ml with a viability of 98.4 ± 1.52. As shown, ASCs accumulate into the drain bag according to a kinetic that collects 13.7% of cells at 1 minute, 54.9% at 2.5 minutes and, at the end of the process, the recovery (100%) is similar to what was observed in the one-step procedure but with a higher percentage of cell viability (Figure 2(g)). 3.3. Immunohistochemical Analysis of DB-ASCs. The immunohistochemistry analysis performed on ASCs showed the high positivity for CD44, CD90, and CD105 (>90%) and the negativity for CD45, CD34, CD31, and CD14 hematopoietic markers (Figure 3(a)). The positivity for CD73 was evaluated by FACS and expressed by the dot-blots (Figure 3(c)). Significant was also the expression of SMA (cytoplasmic and membrane) and VEGF (cytoplasmic). Only a small percentage of cells are positive for CD146, whereas the presence of reactivity for NG2 seems to be mainly expressed at the nuclear level (Figure 3(a)). Figure 4. DB-ASC Osteo-/Adipo-Differentiation Capability. The differentiation capacity towards osteogenic and adipogenic lineages is reported in A remarkable positivity of ALP (Figure 4(d)) confirms the presence of a significant enzymatic activity involved in the mineralization of the bone matrix. The mature stage of osteoblasts is confirmed by the presence of calcium deposits forming complex orange-colored bifurcations, evidenced by the coloration with Alizarin Red S (Figure 4(b)). As shown in Figure 4(f), DB-ASCs are also able to differentiate in adipocytes as evidenced by the red intracellular inclusions. The negative controls (DB-ASCs cultured without the addition of supplements) do not give any evidence of differentiation (Figures 4(a), 4(c), and 4(e)). 3.5. Ability of DB-ASCs to Uptake and Release Paclitaxel. The conditioned media from DB-ASCs, both primed with PTX (CM/PTX) or not (CM CTRL), were tested on the standard laboratory tumour cell line CFPAC-1 ( Figure 5). As expected, the CM CTRL does not inhibit neoplastic growth, which remains stable to 80-100% of proliferation ( Figure 5(a)). On the contrary, CM/PTX affected CFPAC-1 cell growth according to a dose-dependent antiproliferative effect that, even if less effective, reflects the inhibition produced by free PTX (from 0.39 to 50 ng/ml). The inhibitory kinetics analysed by linear regression showed high coefficients of determination (R 2 ) ranging from 0.71 to 0.88 ( Figure 5(b)). The biological dosage of the CM/PTX based on the standard dose-response regression of the pure drugs enabled the estimation of an equivalent paclitaxel concentration (PEC) of 3.94 ± 0.32 ng/ml. The amount of PTX released by a single cell (CPR), expressed as pg/cell, was 0.14 ± 0.01 pg/cell (box 5C). The drug concentrations released by 10 6 DB-ASCs/ PTX were 140 ng of PTX that correspond to values of about 10 times the IC 50 value of the free drug (1.48 ± 0.35 ng/ml). 3.6. Cytokines in DB-ASC Secretome. Among the 40 cytokines analysed in the conditioned medium of DB-ASCs primed or not with PTX (CM DB-ASCs; CM DB-ASCs PTX), only those whose secretion was greater than 1000 pg/ml were considered (IL-1ra, IL-6, IL-8, IL-12 (p70), MCP-1 (MCAF), VEGF, and GROa) and reported in Figure 6. The concentration of these cytokines in the CM was evaluated before and after PTX treatment. As it was shown, the PTX treatment seems to inhibit their secretion that is expressed as percentage ranged from 22.35% (IL12 (p70)) to 70.98% (GROa) with a mean decrease of 37.1 ± 15.7%. Discussion It is known that adipose tissue can be harvested by minimally invasive procedures according to different methodologies and many efforts have been done to reduce the manipulation of the biological material [6]. Lipogems represents a very interesting technique that, without the addition of enzymes or any other additives, microfragments the lipoaspirate in a closed liquid environment that allows, by the protection provided by the liquid (which is noncompressible by definition), to avoid the destruction of the adipocytes. The final product is a nonexpanded and minimally manipulated adipose tissue product suitable for clinical uses because it is in compliant with good manufacturing practices (GMPs). As recently reviewed, Lipogems has been used in many surgical fields such as orthopaedic, reconstructive, and plastic surgery and oncology [20]. In order to obtain more standardized microfragmenting conditions, a prototype apparatus (PLG-P) has been designed that overcoming the manual operations provides a procedure with programmable parameters (angle and frequency of shaking and flux of washing). We mounted a Lipogems device into the PLG-P equipped with a drain bag allowing the collection of high volumes of the washing fluid object of our investigations (Figure 1). Our study demonstrated that the "waste" material contained in the drain bag is surprisingly a rich source of ASCs. In fact, during the long microfragmentation process, the continuous shaking and washing flux allows to detach mechanically many ASCs that accumulate into the drain bag. Of course, the significant amount of single isolated ASCs is diluted into the bag due to the extensive washing volume of buffer used. However, these cells can be easily collected by centrifugation and if cultured, in the presence of contaminant red cells, are able to adhere with a significant CFE and can be easily expanded with a good PDT ( Figure 2). These cells are positive for the expression of CD44, CD73, CD90, CD105, SMA, VEGF, and NG2 and negative for CD14, CD31, CD34, and CD45 and showed osteo-/adipogenic differentiation capability (Figures 3 and 4). Although it is known that the expression of these markers may depend on culture conditions, the pattern clearly confirm the mesenchymal stromal cell type of DB-ASCs. Besides the positivity for the typical MSC markers CD90, CD105, and CD73 [21], DB-ASCs are also positive for NG2 (about 50%) and SMA (90%) that are markers commonly expressed by pericytes in different amount depending from the type of vessel from which they originated (capillaries, venules, or arterioles) [22,23]. Lipoaspirate is very rich in microvessels, and, therefore, a significant amount of ASC progenitors can be obtained with our procedure. Very few cells were positive for CD146, probably because the expression of this marker is reduced in the ASC population washed from lipoaspirate, in agreement with our previous study on MSCs in Lipogems suggesting that CD146 could stain mostly cells of endothelial origin rather than pericytes [24]. The possibility to estimate the cell content and, in particular, the number of ASCs/ml of lipoaspirate give importance to this technique. Our study suggests that washing the lipoaspirate with the PLG-P allows to collect significant amounts of single isolated ASCs with a recovery efficiency, in terms of ASCs per ml of lipoaspirate processed, of 46,233 ± 5500 ASCs/ml that is higher compared to what was reported by other authors [25]. A further important observation is that the expanded ASCs were able to uptake and then release paclitaxel (PTX). Although the PTX treatment modified the amount of some cytokines produced by DB-ASCs (Figure 6), this aspect is not relevant because cells loaded with the drug are able to release PTX in an active form as shown by the in vitro anticancer activity of the conditioned medium ( Figure 5). The uptake-release ability of paclitaxel by DB-MSCs is in line with our previous observations on MSCs from different sources (bone marrow, adipose tissue, human gingival tissue, human fibroblasts, and also blood cells) and confirms that these cells can be considered an important tool for cell-mediated drug delivery [4,5,10,[14][15][16]. Conclusions In conclusion, this system minimally manipulate the adipose tissue thus bypassing the complex requirements of GMP guidelines, with a dramatic reduction of the costs for cellbased therapies on human patients. From a biotechnological point of view, our findings suggest the possible development of a new integrated device that operating without enzymes allows the isolation, expansion, and drug loading of ASCs in one single step. If equipped with selective filters and if the drain bag will be replaced by a "cell culture chamber," the PLG-P + Lipogems device configures a "standardized automated collection system" to isolate mesenchymal cells with a "minimal manipulation" compliant with the GMP standards [26,27]. A system as such has the potential to obtain MSCs not only for regenerative medicine purposes but also for cell-mediated drug delivery. Conflicts of Interest Carlo Tremolada is the president and founder of Lipogems International SpA. Silvia Versari is employed in Lipogems International SpA. All other authors decline any conflict of interests.	v3-fos-license
2019-04-02T13:11:55.534Z	2017-10-24T00:00:00.000	55193349	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://thescipub.com/pdf/10.3844/ajbbsp.2017.176.188", "pdf_hash": "dc90b9ea74b031fc80e212fad7e76b639ff4fb1b", "pdf_src": "MergedPDFExtraction", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:1988", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "464405954b617fe223028e28e3c40c7c72b5023a", "year": 2017 }	pes2o/s2orc	Response Surface Optimized Ultrasonic Assisted Extraction of Total Flavonoids from Walnut Leaves and In Vitro Antibacterial Activities Corresponding Author: Yuanda Song Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, Zibo, Shandong, China Email: [email protected] Abstract: In this paper, the optimum extraction conditions of total flavonoids extracted from walnut leaves subjected to Ultrasonic Assisted Extraction (UAE) were optimized by Response Surface Methodology (RSM). The mathematical model showed the high coefficient of measurement (R = 0.9938) which indicated that this model could be used to guide the response surface methodology. The optimum extraction parameters for extracting flavonoids from walnut leaves determined in this study were extraction temperature 47.73°C, extraction time 30.79 min, ethanol concentration 72.89% (v/v). Under the optimal extraction conditions, the flavonoids yield was about 3.5315%. Statistical analysis of the results showed that extraction temperature, extraction time and ethanol concentration significantly affected the extraction yield of total flavonoids. In addition, the antibacterial activity assays of the flavonoids were carried out and it was demonstrated that the total flavonoids extracted at the optimum conditions had pronounced antibacterial effects against the four bacterial species. Therefore, this study suggested that walnut leaves are promising resources with antibacterial properties for the development of phytomedicines. Introduction Walnut (Juglans regia L.), which belongs to the Juglandaceae family, is a local deciduous tree in northwestern Chain (2012;Moser, 2012). Walnut leaves are known to possess many biological properties and are easily available in abundant amounts (Derebecka et al., 2012). They have been used as a traditional medicine in China and have shown various health benefits for the treatment of skin inflammations, venous insufficiency and ulcers (Cheniany et al., 2013). Moreover, researches in pharmacology and therapeutics have shown that walnut leaves have hypoglycemic, antioxidative, antimicrobial and antihypertensive effects (Gîrzu et al., 1988). In recent years, there has been an in-depth study on substances having considerable antimicrobial properties. It is well known that certain chemicals produced by plants are naturally toxic to bacteria and fungi. Various medicinal plant extracts containing flavonoids are reported to have antimicrobial activity (Basile et al., 1999). Walnut leaves are good sources of flavonoids (Zhao et al., 2014). As natural products, flavonoids exert an extensive biochemical and pharmacological properties. They are described as dietary supplements that promote health, prevention of disease and active cancer preventive agents (Duarte et al., 1993;Hodek et al., 2002). Flavonoids are present in photosynthetic cells and are therefore widespread in the plant kingdom (Manthey et al., 2001). They are common ingredient in the human diet and are found in vegetables and fruits (Xie et al., 2007;Harborne and Baxter, 1999). Flavonoids have been shown to possess a series of important biological activities, including antifungal and antibacterial activities (Galeotti et al., 2008;Kabir et al., 2015;Alarcón et al., 2008). Flavonoids compounds can form complexes with soluble proteins and extracellular matrix and bacterial cell walls, which probably lead to their antibacterial activities (Cushnie and Lamb, 2005). Presence of flavonoids in plants might have some or significant contribution to the antimicrobial activity of plants. Up-to now, several traditional extraction methods have been applied to the extraction of flavonoids from walnut leaves such as Maceration Extraction (ME) (Djozan and Assadi, 1995), Heat Reflux Extraction (HRE) (Zhang and Liu, 2004), soxhlet extraction (Shang and Yuan, 2003) and Microwave-Assisted Extraction (MAE) (Xia et al., 2006). This extraction process usually takes several hours or even days and requires a large amount of solvents, which may result in the damage of flavonoids due to hydrolysis and oxidation (Camel, 2000). Ultrasonic Assisted Extraction (UAE) method can extract bioactive molecules at lower temperature, shorter time and also can relatively reduces the structural damage of compounds in plants than using other traditional extraction (Yuan et al., 2015). Response Surface Methodology (RSM) is a collection of improvement method of optimization mathematical and statistical process (Talebpour et al., 2009). This is a useful tool for studying the mutual effect between various factors on their measurement and quantification of the influence of reaction parameters (Al-Matani et al., 2015;Teng and Choi, 2014). Box-Behnken Design (BBD) is a commonly used process of RSM which make it easier to arrange the experiments result (Borges et al., 2009). Therefore, BBD technology was employed to analyze the influence of various process variables, including extraction temperature, extraction time and ethanol concentration on the yield of the flavonoids extracted from walnut leaves. In the current study, UAE was used to extract flavonoids from walnut leaves using one factor and RSM experimental design to optimize extraction conditions. Furthermore, the antibacterial effects of the total flavonoids extracted at the optimum conditions were determined by using the diffusion methods of agar well. The purpose of this study was to determine the best extraction process parameters for flavonoids extraction from walnut leaves by ultrasonic assisted method and to explore its potential antibacterial properties, so as to establish a scientific basis for the development and utilization of flavonoids. Plant Material The samples of walnut leaves were harvested between June and July at Zibo city, Shandong province of China and the experimental materials were dried in oven and ground to a powder and then filtered using a 10 mesh sieve. Chemicals and Reagents Rutin (purity>98%), sodium hydroxide, aluminum chloride and aluminum nitrate were obtained from Sigma-Aldrich Chemicals Co, Germany and all are analytical grade. The Total Flavonoids Extraction from Walnut Leaves The powders of walnut leaves (1g) were placed in 50 mL −1 centrifuge tubes and mixed with ethanol. After ultrasonic extraction, the samples were centrifuged at 5000 rpm and the supernatant was collected 15 min later. The residue continued to be extracted twice according to the above mentioned conditions, then all supernatants were mixed up and concentrated by a rotary evaporator, then, flavonoids were separated and purified used large hole resin, finally, the collected fractions were freeze dried to powder. Determination of the Content of Total Flavonoids The total flavonoids content in the extracted solution was measured by an aluminum-chloride-colorimetric method (Qadir et al., 2015). In brief, the Rutin standard and the extracted solution with different concentrations were appropriately diluted by 30% ethanol to 5 mL and added 0.3 mL of 5% sodium nitrite solution, placed 6 min then added 0.3 ml of 10% alchlor solution, placed 6 min, then added 4 mL of 5% sodium hydroxide solution. Finally, adjusted the volume of the mixture to 10 mL by 30% ethanol and placed for 15 min. The absorbance of the mixture was measured at 510 nm and distilled water was used as a blank control. The reference standard was Rutin, while the contents of total flavonoids in extracts were presented as Rutin equivalents. All determinations were performed in triplicate. In this work, the total flavonoids of the total extract obtained from the walnut leaves were calculated from the equation of the standard plots ( Fig. 1) as follows: Absorbance = 8.66×total flavonoids + 0.0004 (R 2 = 0.9997) Single Factor Experiments Total flavonoids extraction yield of walnut leaves was influenced by many factors. Therefore, choosing appropriate extraction solvent and extraction method is an important consideration. Based on the preliminary experiments results ethanol and UAE were selected as reasonable options. The experiment used ethanol as the extraction solvent and UAE as extraction method, respectively. The maximum total flavonoids content were determined by single factor experiments. Before RSM analysis, an initial experiment was performed to screen for important factors affecting the experimental responses. Box-Behnken Design (BBD) Optimized UAE Conditions Box-Behnken Design (BBD) is a frequently used method of RSM that is composed of several intermediate points and a central point (Saniah and Samsiah, 2012). BBD was employed to design the experiments, optimize the extraction conditions and analyze the interactions between the above-mentioned parameters. In the present study, three main factors to RSM were used to describe the relationship between responses and variables to obtain the best extraction conditions. Therefore, the influences of three variables X 1 (ethanol concentration, 60 to 80%), X 2 (extraction temperature, 30°C to 50°C) and X 3 (extraction time, 20 to 40 min) were considered (Table 1). The BBD method was consisted of three factors and levels of 17 experimental operations. In the observed response, the experiment was randomized to maximize the effect of unexplained variability. A quadratic equation was used for this model as follows: The leves of independent variable and the term X i , X j and X i 2 represent the interaction and quadratic terms, respectively Bacterial Strains and Cultures Pure bacterial strains used in this study, including Staphylococcus aureus, Escherichia coli, Salmonella typhi and Bacillus subtilis, were obtained from the Department of Microbiology, Agriculture Culture Collection of China. Separate sterile nutrient agar slants were prepared and the bacterial strains were individually inoculated under aseptic conditions and incubated at 37°C for 24 h. Colonies were harvested separately under aseptic condition from the slants and individually inoculated into sterile nutrient broths in separate test tubes and kept in refrigerated condition (Channabasava et al., 2014). Active cultures were achieved by dispensing a tube of cells into 100 mL of nutrient broth and incubating at 37°C for 10 h. The turbidity of the cell suspension was adjusted to the initial concentration 10 8 CFU /mL according to the McFarland standard (Lv et al., 2011). Antimicrobial Assay The effective antibacterial capacity of total flavonoids against bacterial strains was determined by diffusion method of agar well and further confirmed by analyzing the Minimal Inhibitory Concentration (MIC) (Vutuc and Holzer, 2014). The walnut leaves extract was diluted with sterile water to 100 mg/mL. Then pour 100 µL bacteria suspension (108 CFU mL) on the solid medium, evenly distributed. Oxford cup of 5 mm diameter were sterilized, then the cups were set on the medium and different concentration of the extraction (100 µL) were filled respectively. Minimum Inhibitory Concentration (MIC) Determination Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of antibacterial agents that inhibits the proliferation of the bacteria (Doss et al., 2011). For the determination of MIC, 100µL bacteria suspension (10 8 CFU /mL) was added on the medium evenly distributed. Sterilized oxford cup of 5 mm diameter were prepared, then added with 100 µL different concentrations of the extract, which were 100, 50, 25, 12.5, 6.25, 3.125 mg/mL, respectively and kept at 37°C for 24 h. For each microorganism, at least three replicated experiments were carried out for date analysis. Analysis of Statistical Experiments were repeated three times; the mean and standard deviation (X ± SD) of the data were calculated. The statistical analyses were carried out using Design-Expert 8.0, spass 20.0 version and Microsoft Excel program (2007). Single Factor Experiment To evaluate the effect of various factors on the extraction of total flavonoids from walnut leaves and to analyze the influences of different variables we designed the single factor experiments (Wang et al., 2012). Extraction of Total Flavonoids Influenced by Ethanol Concentration A fundamental aspect of solvent selection is 'similar dissolution', which indicated that there is a high degree of solubility in the selected solvents (Mustafa and Turner, 2011). A mixture of ethanol and water is usually used to extract flavonoids from different herbs (Garcia-Castello et al., 2015;Luthria et al., 2007). The main reason is that a large amount of phenolic and flavonoids compounds could be dissolved in water and ethanol mixture (Alothman et al., 2009). In addition, the important aspects of solvent selection are economy, security and sustainability. Due to the volatility of ethanol, it is a better fit polar modifier in the choice of extraction solvent. In addition, ethanol has been considered as one of the most safe and environmentally friendly solvents (Otero-Pareja et al., 2015). To study the influence of ethanol concentration on the total flavonoids extraction from walnut leaves, ethanol concentrations of 30, 40, 50, 60, 70 and 80% were used. Figure 3 indicated the influence of the ethanol concentration on the extraction yield of flavonoids, the extraction yield of flavonoids was not significantly affected by 30-60% ethanol; however, peak extraction of total flavonoids was achieved when the alcohol concentration reached 70%, then the extraction yield decreased with ethanol concentration higher than 70%. Different products are extracted under different conditions. Because of the different chemical structure and polarity of the extracts, solvent has different extraction capacities. Existing studies have shown that the binary solvent system is better than the single-solvent system in extracting flavonoids. It was observed in this study that the optimum yield of flavonoids was obtained at 70% ethanol, which suggested that the flavonoids in walnut leaves were highly soluble in ethanol-water mixture and the yield difference of walnut leaves could be due to different polar and chemical constituents of flavonoids. Extraction of Total Flavonoids Influenced by Extraction Temperature Extraction temperature affects the movement of molecule and heat could promote the dissolution of a large number of compounds (Pompeu et al., 2009). In the present study, the temperatures of 30, 40, 50, 60, 70 and 80°C were selected to study temperature influence on total flavonoids extraction from walnut leaves. Figure 4 presented the influence of the extraction temperature on the extraction yield of flavonoids. When the temperature was increased from 30 to 45°C, the extraction yield increased and then the extraction yield decreased when the extraction temperature was over 45°C. The increase in molecular motion is caused by the increase in temperature, so it accelerating the dissolution of flavonoids from plant cells (Lai et al., 2014). As in this study, the appropriate temperature increase in plant cell decomposition and solubility helping to release flavonoids from the substances. But the temperature was too high; it may also cause the damage of the flavonoids. Similar results have also been reported for total flavonoids extraction from alfalfa (Jing et al., 2015). Therefore, 45°C is selected as the optimal extraction temperature. Extraction of Total Flavonoids Influenced by Extraction Time The time range required of ultrasonic extraction was the third factor investigated, when the other two factors were fixed, e.g., extraction temperature was set at 45°C and ethanol concentration was set at 70%, respectively. As indicated in Fig. 5, the extraction time has a significant effect on total flavonoids and the yield increased with the increase of time and then decreased at long extraction time. The maximum yield was achieved at 30 min. The presence of different degrees of flavonoids polymerization and their interaction, may have caused this phenomenon, as the equilibrium between the bulk solution and the solution in the material being reached at different times) (Lissi et al., 1999). Therefore, the optimum extraction time is 30 min. From the above analysis, we can find that the ethanol concentration, extraction time, extraction temperature are the main factors of the preparation technology and the best extraction conditions were ethanol concentration 70%, extraction time 30 min and extraction temperature 45°C. Data Analysis and Evaluation of RSM Model The experiments for RSM model were conducted based on the design matrix under the defined conditions and the responses from the experimental runs were obtained by using 'design expert' (Table 2). A total of 17 runs of experiments were carried out and three individual parameters that affect the flavonoids extraction yield were optimized. Analysis of variance (ANOVE) and the resulting model regression coefficients were presented in Table 3 which demonstrated the contribution of the variable to the quadratic model. A multivariate regression equation was established and the response variable coding level of the independent variable was analyzed. The quadratic polynomial model of walnut leaves flavonoids was predicted by the least square method and the multiple regression coefficients were determined. The responses of flavonoids extraction ratio of walnut leaves were considered in studying the influence of process variable. The extraction yield of total flavonoids and independent variables of walnut leaves were studied and an empirical model was proposed (Equation 1): Y%=3.7-0.069X 1 +0.059X 2 +0.11X 3 +0.025X 1 X 2 +0.11X 1 X 3 -0.10X 2 X 3 -0.25X 1 The variance analysis of the extraction yields of the total flavonoids from the walnut leaves using Box-Behnken design was shown in Table 3. The determination coefficient (R 2 ) was 0.9938, which is greater than 0.8, indicating a very high correlation (Mirhosseini et al., 2009). The F value and P value were 124.75 and 0.9679, respectively, which indicated the suitability of model that can accurately predict the change of variations. Based on this, the model was used to predict the response. The regression equation coefficients and p-values coefficients for total flavonoids extraction were shown in Table 3. The second-order terms of extraction time, extraction temperature and ethanol concentrations (X 1 2 , X 2 2 , X 3 2 ), one interaction parameters (X 1 X 3 , X 2 X 3 ) and the first-order term of extraction time, extraction temperature and ethanol concentrations(X 1 ,X 2 ,X 3 )were extremely significant with a small P value (p<0.01), whereas parameters (X 1 X 2 ) model term were significant (p<0.05). Response Surface Analysis The three dimensional response surface plots can make predictive model equations more intuitive. Therefore, the surface response plot of the model is established and the influence of independent variables on the dependent variables is visualized through the adjustment of one factor at the same time (Samimi et al., 2015). In the present experiment, Fig. 6 and 7 showed the three-dimensional (3d) curved surface and twodimensional (2d) contour plot of the experiment. The influence of extraction temperature and extraction time on the total flavonoids extract yield was analyzed ( Fig. 6a and 7a). At a definite extraction time, increasing extraction temperature resulted an increase in extraction yield. However, when extraction temperature was higher than45°C the extraction yield was slightly decreased. It was similar for the extraction time. Therefore we can predict that the optimum extraction time is about 30 min and optimum extraction temperature is about 45°C. In addition, the effects of the extraction time and temperature on flavonoids extraction from walnut leaves were very significant. Figure 6b and 7b showed the 3D plot for the extraction yield of flavonoids from walnut leaves with respect to ethanol concentration and reaction temperature. With an increasing extraction temperature the extract yield slightly increased but as the temperature was exceeded 45°C the extract yield decreased extremely. When the extraction temperature was constant, the extraction yield increased with the ethanol concentration, then decreased slightly when the ethanol concentration is too high. The optimum ethanol concentration is about 70%. The influences of extraction time and ethanol concentration on the extraction yield of flavonoids were shown in the Fig. 6c and 7c. The extraction yield increased with the increase in ethanol concentration, however when ethanol concentration was higher than 70%, the extraction yield of flavonoids tend to be stable and slightly decreased. When the concentration of ethanol was constant, the extraction yield of total flavonoids increased with extraction time, as extraction time reached 30 min the best extraction yield was attainted. While extract yield slightly decreased with the extraction time exceeded 30 min. We can conclude that the optimum extraction time is about 30 min and the optimum ethanol concentration is about 70%. The Antibacterial Activity Analysis Antimicrobial activities of the flavonoids extracts from walnut leaves were tested against selected microorganisms (Table 4). The results indicated that the flavonoids obtained from walnut leaves have antibacterial activity at 100mg/mL −1 and the bacterial inhibition ring of flavonoids was measured. It indicated that the flavonoids have best antimicrobial activity against Staphylococcus aureusm, followed by Escherichia coli and then Salmonella typhi. MIC of the extracts recorded was in the range of 12.5-50 mg/mL (Table 5). In this investigation the MIC value of 12.5 mg/mL was recorded for Escherichia coli and Staphylococcus aureus. Whereas, MIC for Bacillus subtilis was 25 mg/mL and for Salmonella typhi was 50mg/mL, indicating that the walnut leave extracts have significant antimicrobial potential. Because the MIC of the extracts were very small, indicating they were highly efficient. An overview of the biological activity data obtained from the current survey can emphasize that the tested extracts have great potential for inhibiting bacteria. Staphylococcus aureus is of considerable importance because it is considered to be one of the main pathogens of many hospitals and community infections. Conclusion Through this study, RSM was applied in optimizing the total flavonoids compounds extraction from walnut leaves and it was also showed that the UAE is a valid method for obtaining flavonoids from walnut leaves. The maximum extraction yield of 3.53% was achieved at temperature of 47.73°C, ethanol concentration of 72.89% and extraction time of 30.79 min. Then, the extracts of flavonoids were used to determine their antibacterial activity. The results showed that the flavonoids extracted from walnut leaves have comparatively significant antibacterial activities with good MIC values. Our results showed that walnut leaves can be a potential source of important bioactive compounds and play an important role in controlling the growth of disease-causing bacteria but further phytochemical analysis is needed to identify the extract types of compounds that presented in walnut leaves.	v3-fos-license
2018-06-21T16:08:09.069Z	2018-06-11T00:00:00.000	47008318	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.7554/elife.35672", "pdf_hash": "f77c8fcaae6270ed6708e88b858733aac84b4ee6", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2007", "s2fieldsofstudy": [ "Biology" ], "sha1": "83b8becc888276400f7a37df8238a1d21a77a7a9", "year": 2018 }	pes2o/s2orc	N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains. Introduction In the human genome,~2% of the genes encode proteases, among which trypsin-like serine proteases are a major group (Overall and Blobel, 2007). Most of the trypsin-like serine proteases act extracellularly to participate in physiological processes, including embryonic development, food digestion, blood coagulation and hormone processing (Ló pez-Otín and Hunter, 2010;Neurath, 1984;Overall and Blobel, 2007;Perona and Craik, 1995;Stroud, 1974). Dysregulated serine protease expression and activity contribute to major health problems such as cardiovascular disease, cancer metastasis, inflammation, and neurological disease (Craik et al., 2011;Gohara and Di Cera, 2011). N-glycosylation is a common post-translational modification in proteins (Eklund and Freeze, 2005;Patterson, 2005;Varki, 1993). About two thirds of the predicted human proteins contain N-glycosylation sites (Apweiler et al., 1999). Consistently, most of the trypsin superfamily members are N-glycosylated proteins (Bolt et al., 2007;Jiang et al., 2014;Liao et al., 2007;Miyake et al., 2010;Wu and Suttie, 1999). Many N-glycosylation sites in these serine proteases, especially those in the protease domain, are highly conserved; that is, a specific N-glycosylation site in a protease is conserved not only in the homologs of different species, but also at the same location in other members of the protease superfamily. Such conservation indicates the functional importance. Indeed, N-glycosylation has been shown to regulate the extracellular expression, secretion and activation of trypsin-like serine proteases, although the underlying mechanisms are not elucidated (Bolt et al., 2007;Gladysheva et al., 2008;Jiang et al., 2014;Lai et al., 2015;Liao et al., 2007;Miyake et al., 2010;Wu and Suttie, 1999). It is unclear if N-glycans at the conserved sites have a general role in the biosynthesis of the trypsin-like serine proteases. Corin is a trypsin-like serine protease that activates natriuretic peptides (Cui et al., 2012;Li et al., 2017;Yan et al., 2000). It consists of a cytoplasmic tail, a transmembrane domain and an extracellular region with multiple protein modules and a C-terminal protease domain (Hooper et al., 2000;Yan et al., 1999). In cells, corin is made as a zymogen and activated on the cell surface by proprotein convertase subtilisin/kexin-6 (PCSK6) 2018). CORIN and PCSK6 variants that impair corin cell surface expression and zymogen activation have been identified in patients with hypertensive diseases Cui et al., 2012;Dong et al., 2013;Dries et al., 2005;Zhang et al., 2014;. Human corin has 19 N-glycosylation sites in its extracellular region (Yan et al., 1999). We and others have shown that N-glycosylation is critical for corin cell surface expression and zymogen activation (Gladysheva et al., 2008;Liao et al., 2007;Wang et al., 2015). Abolishing N-glycosylation sites at Asn80 and Asn231 in the pro-peptide region increased corin shedding on the cell surface, whereas abolishing N-glycosylation site at Asn1022 (N1022), the only N-glycosylation site in the protease domain of human corin, reduced the cell surface expression . To date, how N-glycosylation at N1022 regulates corin cell surface expression remains unknown. In this study, we made membrane-bound and soluble forms of corin with or without the N1022 N-glycosylation site and analyzed the mutant proteins in transfected cells. We also did proteomic analysis to identify intracellular proteins interacting with corin. We verified our findings in enteropeptidase (also called enterokinase, EK), a transmembrane serine protease, and prothrombin, a secreted serine protease. We found that N-glycosylation in the protease domain of corin, EK and prothrombin has a common role in regulating the extracellular expression of these proteases, which involves calnexin-assisted protein folding and ER exiting. Results Glycosylation at N1022 promotes cell surface expression of corin zymogen . To test if the effect is related to zymogen activation, we analyzed corin mutants lacking the activation site (R801A) with or without the N1022 glycosylation site ( Figure 1A). In western blotting of transfected cell lysates, levels of corin zymogen bands (~160-200 kDa) were similar in corin WT and mutants N1022Q, R801A, and R801A/N1022Q ( Figure 1B, left). In corin WT, the cleaved protease domain fragment (Corin-p) migrated as an~40 kDa band under reducing conditions. In the N1022Q mutant, the Corin-p band was lighter and migrated faster, due to the lack of N1022 glycosylation and poor zymogen activation . As expected, no Corin-p band was detected in mutants R801A and R801/N1022Q lacking the activation site. In biotin-labeled cell surface proteins ( Figure 1B, right), levels of corin bands in the N1022Q mutant were 43 ± 9% of that in WT (p=0.002) and levels in the R801A/N1022Q mutant were 41 ± 8% of that in R801A (p=0.027). The total intensity of WT bands (Corin and Corin-p) was similar to that of R801A (Corin band only). The results indicate that lacking N1022 glycosylation reduces corin cell surface expression with or without the activation cleavage at R801. Glycosylation at N1022 promotes soluble corin secretion The cytoplasmic tail was shown to regulate corin intracellular trafficking Qi et al., 2011;Zhang et al., 2014). To test if the cytoplasmic and the transmembrane domains are necessary for the N-glycan-mediated corin expression, we tested soluble corin mutants with the Igk signal peptide with or without mutations at R801 and N1022 ( Figure 1C). In western blotting of transfected cell lysates, sWT and the mutants sN1022Q, sR801A and sR801A/N1022Q appeared as single bands at similar levels ( Figure 1D, left). In the medium ( Figure 1D, right), levels of sWT and sR801A were similar, whereas levels of sN1022Q and sR801A/N1022Q were 11 ± 2 and 9 ± 4% of sWT and sR801A, respectively, indicating that glycosylation at N1022 promotes soluble corin secretion. Glycosylation at N1022 promotes corin exiting from the ER In Western blotting of lysates from cycloheximide (CHX)-treated cells, levels of WT and the N1022Q mutant decreased over time (Figure 2A,B). After 8 hr of CHX treatment, the levels were 7 ± 2% for WT and 32 ± 3% for N1022Q with calculated half-lives of 3.8 ± 0.4 and 6.1 ± 0.3 hr, respectively (p=0.003), indicating that abolishing N1022 glycosylation did not reduce corin protein stability but impaired intracellular trafficking. We digested the proteins with glycosidase Endo H, which removes high-mannose and hybrid N-glycans on proteins in the ER or early Golgi. On western blots, the ratio of Endo H-sensitive vs. resistant corin bands was higher in the N1022Q mutant than WT after CHX treatment for 4 hr ( Figure 2C), indicating that the N1022Q mutant was retained in the ER or early Golgi. We then co-stained corin and protein disulfide isomerase (PDI) in the cells. Without CHX treatment, WT or N1022Q corin and PDI staining mostly overlapped ( Figure 3A) with similar Pearson's correlation coefficients (0.49 ± 0.04 and 0.48 ± 0.06, respectively) ( Figure 3B). After CHX treatment for 4 hr, there was little corin staining in the WT corin-expressing cells, whereas corin staining was strong in the N1022Q-expressing cells ( Figure 3A, corin (red) vs. PDI (green) ratio in two bottom right panels) with Pearson's correlation coefficients of 0.15 ± 0.06 and 0.35 ± 0.05, respectively (p=0.020) ( Figure 3B). In co-staining studies for corin and TGN46, a Golgi marker, WT and N1022Q corin had similar distribution patterns with or without CHX treatment ( Figure 3C,D). These results are consistent with findings from the Endo H experiment, indicating that abolishing N1022 glycosylation prevents corin from exiting the ER. Increased N1022Q binding to calnexin and BiP To identify proteins that interact differentially with corin WT and the N1022Q mutant, we treated the cells with dithiobis succinimidyl propionate (DSP), a protein cross-linker, and did proteomic analysis in samples co-immunoprecipitated with corin. A total of 387 proteins were detected (Supplementary file 1). Among the proteins with 2 fold differences between WT and N1022Q were calnexin and BiP (binding immunoglobulin protein) (Supplementary file 2), two ER proteins in glycoprotein folding and quality control (Hebert et al., 1995;Helenius and Aebi, 2001). Calnexin and BiP levels were 2.1-and 2.0-fold higher, respectively, in N1022Q-derived samples than those in WT (Supplementary file 2). In contrast, the ratio for calreticulin, another ER chaperone in glycoprotein folding (Hebert et al., 1995;Helenius and Aebi, 2001), was 0.88-fold, whereas the ratios for PDI family members A3 and A4 were 1.24-and 1.67-fold, respectively (Supplementary file 1). To show direct interactions between corin and calnexin or BiP, we immunoprecipitated corin in WT-and N1022Q-expressing cells and analyzed co-precipitated proteins by western blotting. Calnexin and BiP levels from N1022Q-expressing cells were 137 ± 9 and 562 ± 82%, respectively, of those from WT ( Figure 4A-C). In contrast, levels of calreticulin, HSP70 and HSP90 (two ER chaperones), and PDI were all similar between WT and N1022Q ( Figure 4A,D). In controls, similar levels between WT and N1022Q were found in V5 pull-down samples and total cell lysates ( Figure 4A). These results indicate that abolishing N1022 glycosylation increases direct corin binding to calnexin and BiP. Effects of glucosidase inhibition on corin binding to calnexin and BiP In calnexin-assisted glycoprotein folding (Caramelo and Parodi, 2008;Helenius and Aebi, 2001), triglucosylated oligosaccharides on nascent proteins are trimmed by a-glucosidases I and II to monoglucosylated oligosaccharides, allowing calnexin binding to N-glycans to assist protein folding ( Figure 5A). Calnexin may bind directly to target proteins via protein-protein interactions, but the functional significance is unclear (Helenius and Aebi, 2001;Ihara et al., 1999). BiP retains poorly folded proteins in the ER ( Figure 5A). We treated the cells expressing WT corin and N1022Q with 1deoxynojirimycin (DNJ), which inhibits glucosidase I and II (Saunier et al., 1982) ( Figure 5A). Without DNJ treatment, calnexin and BiP levels in N1022Q-derived samples were 131 ± 7 and 473 ± 19%, respectively, of WT ( Figure 5B-D). With DNJ treatment, calnexin and BiP levels increased and became similar between the cells expressing WT and the N1022Q mutant ( Figure 5B-D), indicating that inhibiting glucosidase activities blocked calnexin binding to N-glycans at N1022 and other N-glycosylation sites on corin and impaired calnexin-assisted folding, resulting in increased direct corin binding to calnexin and BiP. Effect of N-glycosylation on cell surface expression of chimeric proteins We next made a chimeric protein (CorinEK4N), in which the corin protease domain was replaced by the EK protease domain with four N-glycosylation sites ( Figure 6A), and additional mutants without the four glycosylation sites (CorinEK4Q) and with (CorinEK4Q/N) a new glycosylation site corresponding to N1022 in corin ( Figure 6A). On Western blots, CorinEK4N had two major bands (~190 and~220 kDa) ( Figure 6B). The~220 kDa band (open arrowhead) was on the cell surface and removable by trypsin before the cells were lysed, whereas the~190 kDa band (top black arrowhead) was intracellular and resistant to trypsin. In CorinEK4Q, levels of the cell surface protein were 31 ± 5% of CorinEK4N ( Figure 6B). In CorinEK4Q/N, the level was lower than that in CorinEK4N (49 ± 8%), but higher than that in CorinEK4Q ( Figure 6B). To exclude the possibility that low levels of the cell surface chimeric proteins were due to increased shedding, we examined the shed proteins in the medium. Levels of CorinEK4Q and CorinEK4Q/N were 8 ± 1 and 29 ± 4%, respectively, of that in CorinEK4N ( Figure 6C). These results indicate that the function of N-glycans in the protease domain in promoting cell surface expression is not unique to corin. N-glycosylation in EK and prothrombin protease domains We next studied EK (Kitamoto et al., 1994), a transmembrane serine protease, and prothrombin (Wu et al., 1991), a secreted serine protease. We made EK mutant (EK-4Q) and prothrombin mutant (PT-N416Q) without N-glycosylation sites in the protease domains ( Figure 7A,B). On western blots ( Figure 7C,D), EK-4Q and PT-N416Q bands migrated faster than those in EK-WT and PT-WT. Levels of trypsin-removable EK-4Q band on the cell surface, which migrated much closer to the intracellular band due to the loss of 4 N-glycosylation sites, were 14 ± 1% of EK-WT ( Figure 7C). Levels of PT-WT and PT-N416Q in cell lysates were similar, but the level of PT-N416Q in the medium was 56 ± 30% of PT-WT ( Figure 7D). These results indicate that N-glycans in the protease domain are important for EK cell surface expression or prothrombin secretion. N-glycans in EK and prothrombin protease domains interact with calnexin and BiP In co-immunoprecipitation and western blotting, calnexin levels in EK-4Q-and PT-N416Q-expressing cells were 165 ± 12 and 171 ± 8%, respectively, of those in respective WT controls ( Figure 8A,B). BiP levels were also higher in EK-4Q-and PT-N416Q-expressing cells ( Figure 8A,B). In contrast, calreticulin levels were similar in EK-4Q and PT-N416Q compared with corresponding WT controls. In other controls, EK and PT levels in V5 pull-down samples were similar between the WTs and mutants ( Figure 8A,B). In DNJ inhibition studies ( Figure 8C,D), calnexin and BiP levels increased in all samples. There were no significant differences in calnexin and BiP levels between the DNJ-treated cells expressing ET-WT and EK-4Q or PT-WT and PT-N416Q. These results indicate a general function of N-glycans in the protease domain in trypsin-like proteases in calnexin-assisted protein folding. Effects of DNJ treatment and calnexin knockdown in cardiomyocytes and hepatocytes We verified our findings in murine HL-1 cardiomyocytes and human HepG2 hepatocytes expressing endogenous corin and prothrombin, respectively. In DNJ-treated HL-1 cells, cell surface corin levels were 26 ± 10% of untreated controls, as estimated by western blotting and densitometry ( Figure 9A). In DNJ-treated HepG2 cells, prothrombin levels in lysates were similar to untreated controls, whereas the level in the conditioned medium was~53% of untreated control medium, as measured by ELISA ( Figure 9B). We next knocked down calnexin expression in HL-1 and HepG2 cells using siRNAs targeting murine and human calnexin genes, respectively. Reduced calnexin protein levels in those cells were verified by western blotting (Figure 9C,D). Western blotting and ELISA analyses showed reduced levels of cell surface corin and prothrombin in the conditioned medium, respectively, in HL-1 and HepG2 cells, in which calnexin expression was knocked down ( Figure 9C, D). Discussion N-glycosylation is important in protein expression and function (Dalziel et al., 2014;Hart and Copeland, 2010;Moremen et al., 2012). Previously, N-glycosylation at N1022 was found to be critical for corin cell surface expression, but the underlying mechanism was unknown . In this study, we found that N-glycosylation at this site was important for corin folding and trafficking in the ER. In proteomic analysis, we identified calnexin and BiP, two ER proteins that bound preferably to the N1022Q mutant. Calnexin acts in glycoprotein folding (Caramelo and Parodi, 2008;Helenius and Aebi, 2001). Unlike in heat-shock chaperone-mediated protein folding, which involves direct protein-protein binding, calnexin binds to monoglucosylated oligosaccharides on glycoproteins after triglucosylated N-glycans are trimmed by glucosidases I and II. Calnexin also binds to target proteins via direct hydrophobic interactions (Brockmeier and Williams, 2006). Such interactions alone, however, are insufficient for glycoprotein folding. We found increased binding of the N1022Q mutant to calnexin and BiP, indicating that N-glycans at N1022 on corin is important for calnexin-assisted protein folding and ER exiting. The results led to a working model, in which calnexin first binds to nascent corin through direct protein-protein interactions. Subsequent binding of calnexin to monoglucosylated In CorinEK4N, the corin protease domain (Corin-P) was replaced by the EK protease domain (EK-P). The ADAM10-mediated shedding site is indicated by an arrowhead. In CorinEK4Q, all four N-glycosylation sites in the EK protease domain were mutated by Gln (Q) residues. In CorinEK4Q/N, a new N-glycosylation site corresponding to N1022 in corin was added to CorinEK4Q. (B) Western blotting of CorinEK4N, CorinEK4Q and CorinEK4Q/N in transfected cells treated without (-) or with (+) trypsin before the cells were lysed. GAPDH levels in cell lysates were used to assess amounts of proteins in each sample. (C) Western blotting of shed corin fragments the in medium. Corin levels on the cell surface (B) and in the medium (C) were estimated by densitometric analysis of western blots. In (C), levels of a Coomassie Blue (CB)-stained non-specific protein were used to assess amounts of proteins in each sample. Data are means ± S.E. from at least three independent experiments. p-Values are shown in bar graphs. DOI: https://doi.org/10.7554/eLife.35672.009 N-glycans on corin, at N1022 and other N-glycosylation sites, facilitates corin folding. The resultant conformational change in corin disrupts the interaction with calnexin, allowing corin to exit the ER. Consistent with this model, we showed that the treatment of DNJ, a glucosidase inhibitor, increased the binding of the N1022Q mutant and WT corin to calnexin and BiP to similar levels. The results support the importance of N-glycan-calnexin interactions in corin folding and ER exiting. Moreover, the results indicate that N-glycans at other N-glycosylation sites on corin are also involved in the calnexin interaction. Human corin contains 19 N-glycosylation sites Yan et al., 1999). Among them, N1022 is the only site in the protease domain. Our findings indicate that N-glycosylation in the protease domain is critical for calnexin-assisted folding. In trypsin-like serine proteases, N-glycosylation sites in the protease domain are common. Previously, N-glycosylation in the protease domain of factor VII (FVII) was shown to promote FVII secretion in COS-7 and CHO cells (Bolt et al., 2007). Abolishing the N-glycosylation site in the protease domain of chymotrypsin C reduced the secretion in HEK293 cells (Bence and Sahin-Tó th, 2011). Conversely, overexpression of a mutant chymotrypsin C lacking the N-glycosylation in the protease domain caused ER stress in cancer cells (Bence and Sahin-Tó th, 2011). These data suggest that N-glycosylation in the protease domain of trypsin-like serine proteases has a general role in calnexin binding and protein folding. Consistent with this hypothesis, DNJ treatment and calnexin knockdown decreased corin cell surface expression and prothrombin secretion in cardiomyocytes and hepatocytes, respectively. Moreover, elimination of N-glycosylation sites in the protease domain of EK or prothrombin increased EK and prothrombin binding to calnexin and BiP and decreased EK cell surface expression or prothrombin secretion in HEK293 cells. These results show that in corin, EK and prothrombin, which have distinct protein domain structures and physiological functions, N-glycosylation in their protease domains has a common function in calnexin-assisted folding and extracellular expression. Possibly, this is a general Calreticulin is a soluble calnexin homologous in the ER and acts as a key partner in the calnexincalreticulin cycle (Caramelo and Parodi, 2008;Ellgaard and Helenius, 2001;Helenius and Aebi, 2001). Like calnexin, calreticulin binds to monoglucosylated oligosaccharides on glycoproteins. In a previous study, inhibition of glucosidase II increased calreticulin binding to cruzipain, a protozoan cysteine protease (Labriola et al., 1999). In our study, we found increased binding of N1022Q corin, EK-4Q and PT-N416Q mutants to calnexin but not calreticulin, indicating that calnexin is the primary ER chaperone that interacts with N-glycans in the protease domain of these proteases. If both calnexin and calreticulin recognize similar monoglucosylated N-glycans, how do these proteins distinguish their glycoprotein substrates? Unlike calreticulin, calnexin has a transmembrane domain anchoring calnexin on the ER membrane (Dalziel et al., 2014;Ellgaard and Frickel, 2003). In most trypsin-like serine proteases, the protease domain is C-terminal. Possibly, the membrane-bound calnexin is more accessible to the N-glycans in the C-terminal protease domain, which comes last from the translocon on the ER membrane in protein synthesis. This may explain that despite the 18 N-glycosylation sites in the pro-peptide of corin, N-glycosylation at N1022 in the protease domain is required for optimal corin folding and ER exiting. Consistently, N-glycosylation in the protease domain of the CorinEK4N mutant promotes the cell surface expression of the chimeric protein. These results indicate that N-glycans in the protease domain regulate calnexin-assisted folding in a domain-autonomous and calreticulin-independent manner. The importance of N-glycans in glycoprotein folding varies depending on proteins and cell types (Helenius and Aebi, 2001). In the trypsin-like protease superfamily, not all members are N-glycosylated. Some members have N-glycosylation sites in the pro-peptide but not in the protease domain. It is attempting to postulate that N-glycosylation in the protease domain offers an advantage in protein folding efficiency and hence protein production. The requirement of N-glycosylation in a particular protease may depend on its expression level and specific cell environments. More studies are needed to test the folding efficiencies between the trypsin-like proteases with and without N-glycosylation sites in their protease domains in different cells. As trypsin-like proteases are used as biologics to treat human diseases (Craik et al., 2011), creation of new N-glycosylation sites may also be a strategy to increase the production of recombinant proteases in vitro. In summary, we identify a common mechanism of N-glycosylation in the protease domains of corin, EK and prothrombin in calnexin-mediated folding and ER exiting. This process is calreticulinindependent, operates in a domain-autonomous manner, and involves two steps: direct calnexin binding to the target protein and subsequent calnexin binding to monoglucosylated N-glycans. Our findings suggest that this may be a general mechanism in the trypsin-like proteases with N-glycosylation sites in their protease domains. Naturally-occurring mutations disrupting such N-glycosylation sites may impair the expression and function of the trypsin-like serine proteases. Cell transfection HEK293 cells (ATCC, CRL-1573, authenticated by STR DNA profiling, no mycoplasma contamination) were grown in DMEM with 10% fetal bovine serum at 37˚C in humidified incubators. At 70-80% of confluency, the cells in six-well plates were transfected with the plasmids using Fugene reagents (Promega). To make stable cells expressing recombinant proteins, the transfected cells were cultured with G418 (400 mg/mL, Teknova). After~2 w, G418-resistant cells were selected and analyzed by western blotting. Western blotting Recombinant proteins on the cell surface or in the conditioned media and lysates from the transfected cells were immunoprecipitated with an anti-V5 antibody (Thermo Fisher, R96025) and protein A-Sepharose (Thermo Fisher) for western blotting, as described previously . Antibodies used were against V5 (Thermo Fisher, R96125), BiP (Cell Signaling, 3177T), calnexin (Cell Signaling, 2679T), calreticulin (Cell Signaling, 12238S), HSP70 (Cell Signaling, 4872T), HSP90 (Cell Signaling, 4877T) and PDI (Cell Signaling, 3501T). Horseradish peroxidase-labeled secondary antibodies were used (KPL, 474-1806;474-1516). As a protein loading control for cell lysates, western blots were re-probed with an anti-GAPDH antibody (EMD Millipore, MAB374). As loading controls for cell surface proteins or proteins from conditioned media, eluted biotin-labeled cell surface proteins or total proteins in the conditioned medium were separated by SDS-PAGE followed with Coomassie Blue staining. Levels of prominent non-specific bands were used to assess similar protein amounts in each sample. CHX-based protein chase assay HEK293 cells expressing corin WT or the N1022Q mutant in six-well plates were incubated with or without CHX (Sigma; 100 mg/mL). The cells were lysed at different time points for western blotting, as described above. Endo H digestion Glycosidase Endo H was used to analyze N-glycans on corin in HEK293 cells. The cell lysates were incubated with Endo H (500 U, New England BioLabs) in 50 mM sodium acetate at 37˚C for 1-2 hr. The Endo H-treated proteins were analyzed by Western blotting. Protein cross-linking and proteomic analysis HEK293 cells expressing corin were incubated with dithiobis succinimidyl propionate (DSP) (0.8 mg/ mL; Thermo Fisher) at 4˚C for 30 min. The reaction was stopped with 0.2 M glycine. Cell lysates were analyzed by immunoprecipitation and SDS-PAGE. Proteins on silver-stained gels were analyzed by liquid chromatography-mass spectrum at the Cleveland Clinic Proteomics Core to identify proteins interacting differentially with corin WT and the N1022Q mutant. Glucosidase inhibition Murine HL-1 cardiomyocytes were a generous gift from Dr. William Claycomb (Louisiana State University Medical Center, New Orleans; no established authentication method for this murine cell line, no mycoplasma contamination), as described previously (Wang et al., 2008). Human HepG2 cells were from ATCC (HB-8065, authenticated by STR DNA profiling, no mycoplasma contamination). HL-1, HepG2 and HEK293 cells expressing corin were incubated with 1-deoxynojirimycin (DNJ) (2 mM, Alfa Aesar), which inhibits glucosidases, at 37˚C for 24-48 hr. Corin proteins in HL-1 and transfected HEK293 cells were analyzed by western blotting using an antibody against mouse and human endogenous corin . Prothrombin expression in HepG2 cell lysates and the conditioned medium was analyzed by ELISA (Abcam, ab108909). Trypsin digestion To digest cell surface proteins, HEK293 cells expressing corin or EK were incubated with trypsin (0.05%, AMRESCO) at 37˚C for 10 min. After washing, cell lysates were prepared for western blotting. Effects of calnexin knockdown To examine effects of calnexin knockdown on corin expression in HL-1 and prothrombin expression in HepG2 cells, siRNAs targeting murine and human calnexin genes (Origene, SR417891 and SR300576) and corresponding scrambled control siRNAs (Origene) were transfected using Lipofectamine reagents (Thermo Fisher). After 24-48 hr, the cells were collected. Calnexin, corin and prothrombin proteins were analyzed, as described above.	v3-fos-license
2017-07-17T06:04:20.314Z	2010-12-01T00:00:00.000	40225741	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.scielo.br/j/acb/a/kS7xng83jDx6dFgKPpRhM3n/?format=pdf&lang=en", "pdf_hash": "c3937e560862d371ddea98079ea5d62af6ee8962", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2024", "s2fieldsofstudy": [ "Medicine" ], "sha1": "c3937e560862d371ddea98079ea5d62af6ee8962", "year": 2010 }	pes2o/s2orc	Ingestion of polydextrose increase the iron absorption in rats submitted to partial gastrectomy Purpose: To investigate whether polydextrose stimulates iron absorption in rats submitted to partial gastrectomy and sham operated. Methods: The rats were submitted to partial gastrectomy (Billroth II) or laparotomy (sham-operated control), in groups of 20 and 20 each respectively. The animals were fed with a control diet (AIN-93M) without polydextrose or a diet containing polydextrose (50g/Kg of diet) for eight weeks. They were divided into four subgroups: sham-operated and Billroth II gastrectomy and with or without polydextrose. Two animals died during the experiment. All rats submitted to gastrectomy received B-12 vitamin (intramuscular) each two weeks. The hematocrit and hemoglobin concentration were measured at the start and on day 30 and 56 after the beginning of the experimental period. At the end of the study, the blood was collected for determination of serum iron concentration. Results: The diet with polydextrose reduced the excretion of iron. Apparent iron absorption was higher in the polydextrose fed groups than in the control group. The haematocrit and haemoglobin concentration were lower after Billroth II gastrectomy rats fed the control diet as compared to the polydextrose diet groups. Conclusion: Polydextrose increase iron absorption and prevents postgastrectomy anemia. Introduction The iron is a main element in the terrestrial crust and it is an essential nutrient for all living organisms.However, in spite of it is abundance, it is poorly and biologically available, except in acid solution 1 . The total or partial gastrectomy is indicated in the surgical treatment of gastroduodenal diseases, including the gastric cancer.The gastrointestinal transit after the partial gastrectomy is recovered through anastomosis with the duodenum (Billroth I-BI) or the jejunum (Billroth II-BII).The partial or total resection of the stomach includes the removal of the whole antrum decreasing the production of hydrochloric acid (HCl), intrinsic factor and changes the gastric and duodenal function, lowering the absorption of the iron 2 . Prebiotics are compounds which are not hydrolyzed nor absorbed in the upper gastrointestinal tract.They reach the large intestine intact, where they serve as substrate for bacterial metabolism 3 , promoting the proliferation of among others bifidobacteria and the synthesis of short chain fatty acids 4 . The polydextrose is not hydrolyzed in the intestine after oral administration 5 .It is fermented in the large intestine producing short chain fatty acids and CO 2 and part is excreted in the feces 6 .Polydextrose increases the volume of fecal mass, reduces the intestinal transit time, softens the stool and reduces fecal pH.The fermentation leads to symbiotic the growth of favorable members of the microbiota, and suppresses the production of carcinogenic metabolites 7 . The reduction of the faecal pH and the observation that prebiotics can improve the absorption of minerals such as calcium and magnesium led us to hypothesize that ingestion of prebiotic polydextrose could increase the bioavailability of dietary iron. This hypothesis was assessed by investigated the effect of the polydextrose supplementation on the absorption of iron in rats submitted to partial gastrectomy. Methods Forty male Wistar rats (Cemib/Unicamp, Campinas, Brazil) 250.0 ± 5g of body weight were kept in collective cages in a room with controlled temperature (22 ± 1ºC), humidity (60-70%), cycle of 12 hours day-night (lights on at 7:00 am), with diet and deionized water ad libitum.The animals were randomly assigned to two groups of 20 animals each.Twenty animals were submitted to anterior truncal vagotomy and to partial gastrectomy (Billroth II).The sham group (twenty animals) were submitted to the same surgical stress, where the abdominal cavity was maintained open for approximately 45 minutes, which it is the duration of a gastrectomy.The rats were anesthetized with sodic thiopental (25mg/Kg body weight, intravenous).The experimental protocol was previously approved by the Committee of Ethics in Animal Experimentation (CEEA) of the State University of Campinas -UNICAMP (record nº.839-1, 08/06/2005). Experimental groups and diets After 15 days of the procedure, the rats were divided randomly into four experimental subgroups (sham-operated vs. gastrectomy, control vs. polydextrose diet), (Sham/Control: n=10; Sham/PDX: n=10; Gastrectomy/Control: n=9; Gastrectomy/ PDX: n=9).Two rats of the groups gastrectomy died during the experiment.They were fed the assigned experimental diets for eight weeks.The control and experimental were prepared according to the AIN-93M formulation by eight weeks 8 .Polydextrose (Litesse Ultra, Danisco Brazil Ltda, Cotia, Sao Paulo, Brazil) was added at 50g/Kg diet by replacing sucrose in the control diet. Table 1 shows the composition of the two experimental diets (control and PDX).One-half of the rats submitted to gastrectomy and sham-operated rats were fed the control diet, and the remaining rats were fed the polydextrose diet.The animals were allowed free access to deionized water throughout the experimental period.For the prevention of the megaloblastic anemia, the rats received supplements of vitamin B12 (Cianocobalamin/0.5mg/Kg/intramuscular)(Cianotrat 5000-Institute Therapeutic Delta Ltda, Indaiatuba-SP, Brazil) every two weeks, beginning one week after surgery.In the Sham group received chloride of the sodium 0.9% (Sanobiol Ltda, Pouso Alegre-MG, Brazil). For feces collection, the animals were placed into individual metabolic cages, for three days in three periods at 15 th , 35 th and 55 th day of the experiment 9 . The weight gain and the consumption of the diet of the animals were monitored three times a week, for eight weeks. Analytical methods The blood of the anesthetized animals was collected by retroocular vein puncture every fourth week during the experimental period.In the beginning of the experiments, the blood was collected from randomized animals before the surgery procedure.Blood samples were analyzed to determine the hematocrit and hemoglobin concentration using a hematological analyzer (Advia TM 120, Bayer ® , Ireland). On the final day of the experiment, all rats were anesthetized with sodic thiopental (25mg/Kg body weight).Whole blood was collected by cardiac puncture, and the animals were sacrified. The serum iron contents were determined by a commercially available colorimetric method (Laborlab, Guarulhos-SP, Brazil). Freeze-dried feces were weighed and milled.Diets and the powdered feces were dry-ashed at linearly increased temperatures up to 550 o C for 6 h and then at 550 o C for 18h by a muffle furnace (Fornitec Industry and Trade Ltda, Sao Paulo, Brazil).Samples were heated with 0.5mL concentrate HNO 3 65% and 0.15mL (30%) H 2 O 2 (Merck Brazil, Sao Paulo, Brazil) in closed pressurized Hostaflon tubes heated in microwave (DGT 100 Plus-Provecto).The determinations of fecal iron and diet were performed in an Optic Emission IRIS-AP (Thermo Jarrell Ash) at the specialized Laboratory of Biominerals Chemical Analyses, Campinas, Sao Paulo -Brazil and the calculations were: Apparent iron absorption (mg/day) = iron intake (mg/day) -fecal iron excretion (mg/day). Statistical analysis The results were submitted to analysis of variance (ANOVA), with the use of Duncan's test for the comparison of the averages.The data were analyzed by two-way (treatment and diet) or three-way (treatment, diet and time).Differences were considered significant at p<0.05.Data are expressed as means and standard error of mean (SEM).STATISTICA Ver 6.0 ® (Statsoft, Inc.Tulsa, USA) for Windows 10 . Body weight and food intake Initial body weight in both sham-operated feeding groups was significantly higher than those in the corresponding rats with gastrectomy (p<0.05).Final body weights in both gastrectomy rat groups were significantly lower than those in sham-operated rats (P < 0.05).However, total body weight gain (results not shown) was not significantly different between the sham and groups with gastrectomy (p>0.05).Food intake in rats submitted to gastrectomy eating the control diet was significantly lower compared to the other three groups (p<0.05). Each value in the Humid and dry weight of feces The results are showed in the Table 3: In sham-operated and rats with gastrectomy, the mean wet and dry weight of the feces was significantly higher in rats that received the polydextrose diet then in rats that received the control diet (p<0.05).Each value in the Table 3 represents a mean ± SEM.Values in a column not sharing a superscript letters were significantly different, p<0.05 (Duncan's multiple range test). Hematocrit and hemoglobin concentration The starting hematocrit levels were the same for the sham and animals with gastrectomy (Figure 1). After four weeks in the experiment, the sham-operated rats showed higher hematocrit levels as compared to the animals with gastrectomy.In the eighth week, the hematocrit level of the these animals receiving polydextrose-diet, was higher than, as compared to the Billroth II gastrectomy receiving controlled-diet, not different from the sham-operated rats which received diet with or without polydextrose (p<0.05).In the fourth and eighth week of the experiment, the hemoglobin was higher in the sham-operated rats with control and polydextrose diet as compared to animals with gastrectomy (p<0.05),An increase was observed in the rats with gastrectomy receiving polydextrose enriched-diet as compared to the rats with gastrectomy and with control diet, this did, however, not reach statistical significance. Serum iron concentration The concentration of iron in the serum of sham-operated rats fed either control or polydextrose diet did not differ from each other but was significantly higher than those rats with gastrectomy fed either diet (p<0.05).Serum iron in rats fed the polydextrose was higher than that in rats with gastrectomy fed the control diet, however this did not reach statistical significance (Figure 3). The starting hemoglobin levels were the same for the rats submitted to gastrectomy and sham-operated rats (Figure 2). The concentration of iron in feces in the sham-operated rats and with gastrectomy receiving polydextrose enriched-diet was lower (P < 0.05) as compared to the control diet (Figure 4). Discussion The rat digestive system exhibits certain similarities to that of humans.Good correlations have been observed between work with rats as experimental animals and their comparison to humans [11][12][13][14] .The present study confirms observations on partial gastrectomy in adult rats where less feed consumption and smaller weight in animals with gastrectomy was reported 15 .That smaller weight gain of the rats with gastrectomy can be associated to the smaller feed consumption due to a smaller size of the stomach (Table 2).The fecal production increased significantly in rats fed with polydextrose (Table 3).An earlier study with rats submitted to gastrectomy found that after the procedure, metabolic changes occur that reduce the absorption of nutrients, causing a delay in growth of the animals 16 . In rats with gastrectomy the hematocrit and hemoglobin were significantly lower than in the sham-operated rats (Figures 1 and 2).Similar results have been reported before, after gastrectomy 15,17,18 .Starting in the 4 th week of supplementation, the rats with gastrectomy that receiving polydextrose enriched-diet presented hematocrit concentration and hemoglobin levels significantly higher compared to the rats with gastrectomy that received the control diet (Figures 1 and 2).The apparent absorption of iron by the sham-operated and rats with gastrectomy receiving polydextrose enriched-diet was higher as compared to the animals with normal diet (Figure 5).The sham-operated rats showed higher apparent absorption compared to the rats with gastrectomy, both receiving polydextrose or control-diet (p<0.05). Fecal iron and apparent iron absorption The polydextrose enriched-diet prevented the development of the anemia.The final concentration of hematocrit (eight weeks) in the rats with gastrectomy was not different from the sham-operated rats fed with any one of the diets (Figure 1).Similar effects have also shown by other researchers for other prebiotics [19][20][21][22] . The concentration of serum iron was decreased after the partial gastrectomy (Figure 3).The rats with gastrectomy fed with polydextrose presented the higher concentration of serum iron as compared to the rats that receiving control diet, however this did not reach statistical significance (Figure 3).In this respect, it is important to point out that there is a substantial positive correlation between the concentration of serum iron and the absorption of iron.Studies with fructooligosaccharides also presented an increased absorption of iron in deficient rats iron deficiency 19 , suggesting that this diet could prevent the anemia. The excretion of iron in the feces was significantly higher in the sham-operated rats and in the animals with gastrectomy receiving controlled diet (without polydextrose) (Figure 4).The apparent absorption was lower after the partial gastrectomy (Figure 5).However, the sham-operated rats and rats with gastrectomy that received the polydextrose supplemented diet exhibited a significantly larger apparent iron absorption then their respective counter parts receiving the control (Figure 5).Our results suggests that the ingestion of polydextrose may reduce the risk for anemia due the increased the absorption of iron, since ingestion of polydextrose by both the sham-operated rats and rats with gastrectomy presented smaller excretion of iron in the feces and larger apparent absorption (Figures 4 and 5).Similar results for calcium have also been reported in rats submitted to the total gastrectomy 16 . The results showed that the gastrectomy induced a severe deficiency of iron in the rats.The primary cause of this anemia was considered to be the blood lost during the surgical procedure, followed by the impaired absorption of iron (Figure 5) in rats with gastrectomy fed control diet, where the hemoglobin concentration decreased during the experimental period (Figure 2).In the present study rats with gastrectomy received B12 vitamin to prevent the pernicious anemia 18 .These results indicate that the anemia after gastrectomy observed in this study is anemia caused by iron deficiency. Many authors have postulated that the reduced absorption of iron is caused by the lower intake after the gastrectomy 15,23,24 .The absorption of the iron occurs in all parts of the small intestine, but, mainly in the duodenum and proximal jejunum 25,26 .In agreement, iron deficiency is observed in up to 70% of the patients with gastrectomy 22,26 .This fact added to the reduced gastric capacity and the absence hydrochloric acid increases the iron and other nutrients deficiencies 15 .Previous studies showed that the partial gastrectomy associated to anterior truncal vagotomy were a good experimental model to study the iron metabolism 27 and calcium metabolism 28 . The absorption of iron seems to occur not only in the small intestine, the large intestine appears to contribute to this absorption as well.However, the sufficient iron has been reported to be absorbed in the large intestine for recovery from anemia in iron deficient rats 29,30 .It has been showed that the proximal colon, can be an important site of iron absorption when the absorption in the small intestine is insufficient 25,29,31 , such as after the gastrectomy 32 .Furthermore, animal studies have shown that the short chain fatty acids, in particular propionate seem to increase the absorption of iron in the colon proximal.This may indicate a potential mechanism whereby ingestion of prebiotics increases the bioavailability of dietary iron 31 . An in vitro experimental model employing colonic fermentation showed that polydextrose administration increases the level of bifidobacteria and the level of short chain organic acids, in particular acetic acid.Furthermore, polydextrose reduced the levels of potentially pathogenic bacteria in the proximal colon 32 .Consequently, we speculated that in our study the effect of the polydextrose in increasing the absorption of iron in rats after gastrectomy happened in the large intestine. Conclusion Polydextrose increase iron absorption and prevented anemia after partial gastrectomy in rats. TABLE 1 - Composition of experimental diet TABLE 2 - Initial and final body weights and food intake of shamoperated and rats with gastrectomy fed diets with polydextrose (PDX) or without polydextrose (control) Table 2 represents a mean ± SEM.Values in a column not sharing a superscript letters were significantly different, p<0.05 (Duncan's multiple range test). TABLE 3 - Weight wet and dry of the feces (g) collected in 3 times (15, 35 and 55 days) of the experimental phase, for three days each period of the groups sham-operated and rats with gastrectomy fed diets with polydextrose (PDX) or without polydextrose (control)	v3-fos-license
2021-07-26T00:06:38.910Z	2021-06-03T00:00:00.000	236284504	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1420-3049/26/15/4599/pdf", "pdf_hash": "adda18d47c5f5fa76f57b460251759258abd517d", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2095", "s2fieldsofstudy": [ "Chemistry", "Biology" ], "sha1": "42cf6aa72c002468d02a77a16cd742f34ba9488d", "year": 2021 }	pes2o/s2orc	Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/β (GSK-3 α/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia. Introduction Terrestrial plants are a crucial source of medicines, especially in developing countries. According to the WHO, about 80% of the world's population depends on plant-derived medicines for their health care [1,2]. Secondary metabolites present in natural extracts purified from plants and microorganisms are known to possess multiple bioactivities (ranging from cytotoxic to cytoprotective) [3]. Accordingly, intensive study has been devoted to the purification and chemical characterization of active constituents that could possibly yield a novel chemical compound suitable for drug development [4,5]. Human protein kinases represent the third largest enzyme class and are responsible for modifying up to one-third of the human proteome. Over 518+ protein kinases, including serine/threonine and tyrosine kinases, are encoded by the human genome [6]. Dysregulation of kinase function (e.g., by hyperactivation, or mutation) plays an important role in many diseases, such as cancer [7], neurodegenerative disorders, inflammation, and diabetes, thereby making protein kinases attractive targets for the pharmaceutical industry [8]. From 20 to 33% of current drug discovery efforts worldwide are focused on the protein kinases [9]. Consequently, the FDA in the United States has already approved 65 small molecule protein kinase inhibitors, as of July 2021 [10][11][12]. At least 18 of these kinase inhibitors inhibit a number of kinases; that is, they are multi-target inhibitors [9]. Metabolites from plants are known to be a rich source of putative protein kinase inhibitors (e.g., flavonoid compounds that function as competitive inhibitors of ATP binding [13]). The genus Acacia belongs to the family Fabaceae and includes about 1400 species of trees and shrubs widespread throughout warm and semiarid regions of the world including subtropical and tropical Africa (e.g., Nigeria, Senegal, Egypt, and Mozambique) [14]. Within this vast genus, Acacia auriculiformis, commonly referred to as Black Wattle, is an important medicinal plant. The Ibibio community of Niger Delta region in Nigeria uses this plant as antimalarial [15]. Moreover, an infusion of the bark of this plant is used to treat inflammation among the aborigines of Australia [16]. Several Acacia species including Black Wattle are also known to contain components that inhibit tumor growth, and thus understanding the mechanism for the reported activity is of great interest [17]. In addition, the antimutagenic and chemoprotective activities of Acacia auriculiformis, particularly the tannins contained in the bark, as well as the ability of its ethyl acetate and acetone extracts to scavenge free radicals have been reported [18][19][20]. Recent publications have shown the purification of one new triterpenoid trisaccharide and three new triterpenoids, and have demonstrated antimicrobial activity of acaciaside a and acaciaside b [21,22]. The mosquito larvicidal activity of the fruit extracts and the protein kinase inhibitory activity of a tetrahydroxy flavone isolated from the stem bark of Acacia auriculiformis have also been reported [23,24]. In the present study, we report the isolation of the triterpenoid betulin and the investigation of this compound's activity against a panel of disease-related kinases. We also demonstrate the effect of betulin on the viability of doxorubicin-resistant and -sensitive human leukemia cell lines. Purification of Betulin from Acacia Auriculiformis Stem Bark and Evaluation of Its Biological Activity against Disease-Related Protein Kinases Preliminary kinase-based screening was carried out using Acacia auriculiformis stem bark extracts, and it was discovered that the ethyl acetate soluble fraction was the most active among the three fractions investigated, namely chloroform, ethyl acetate, and Nbutanol [23]. Chromatographic purification of the compound(s) that might be responsible for the kinase inhibition from the ethyl acetate soluble fraction led to the isolation of a 13 C-NMR (DEPT) and proton NMR spectra of the betulin purified from Acacia) consistent with literature data for betulin (3-lup-20(29)-ene-3β,28-diol) [25]. Acacia auriculiformis stem bark was found to contain about 0.002% of betulin by dry weight. The chemical structure of betulin is depicted on Figure 1. Molecules 2021, 26, x FOR PEER REVIEW for the kinase inhibition from the ethyl acetate soluble fraction led to the isolat compound as a white amorphous solid. This compound displayed spectral prope and 13 C, see Figures S1-S6 for 13 C-NMR (DEPT) and proton NMR spectra of the purified from Acacia) consistent with literature data for betulin (3-lup-20(29)-en diol) [25]. Acacia auriculiformis stem bark was found to contain about 0.002% of be dry weight. The chemical structure of betulin is depicted on Figure 1. As triterpenoids have already been shown to inhibit protein kinases, we te inhibitory effect of betulin on a panel of protein kinases (PKs). Eight disease-rela man PKs were tested including cyclin-dependent kinases (CDK5/p25 and CDK9/C Haspin, proto-oncogene proviral integration site for moloney murine leukemia (Pim1), glycogen synthase kinase-3 beta (GSK-3β), casein kinase 1 epsilon (CK1ε kinase 3 (JAK3), and Abelson murine leukemia viral oncogene homolog 1 (ABL1) shows the results of the primary screening. Betulin showed weak activity again i.e., at 10 µg/mL, it showed only 10% inhibition of kinase activity. This contraste edly with ABL1, whose kinase activity was inhibited by 79%. JAK3 and GSK showed inhibition by betulin. Betulin was next tested against a larger panel of 16 protein kinases, as rep Figure 2, using a range of betulin concentrations. IC50 values were determined f dose-response curves for the target kinases most potently inhibited by betulin (dis more than ~45% of inhibition at 10 µM of betulin). Results in Figure 2 revealed that gave the highest inhibitory activity against GSK-3α, with IC50 of 0.72 µM; follo ABL1, with IC50 of 0.93 µM (see Figure 3a for dose-response curve for ABL1); G with IC50 of 1.06 µM; JAK3 kinase with IC50 of 1.08 µM; CK1ε, with IC50 of 2.11 µ cular endothelial growth factor receptor 2 (VEGFR2), with IC50 of 2.45 µM; and Related Kinase 6 (NEK6), with IC50 of 3.02 µM. Given these results and the we lished role of BCR-ABL1 in chronic myelogenous leukemia (CML), we next focu work on the inhibition of ABL1 by betulin. As triterpenoids have already been shown to inhibit protein kinases, we tested the inhibitory effect of betulin on a panel of protein kinases (PKs). Eight disease-related human PKs were tested including cyclin-dependent kinases (CDK5/p25 and CDK9/CyclinT), Haspin, proto-oncogene proviral integration site for moloney murine leukemia virus-1 (Pim1), glycogen synthase kinase-3 beta (GSK-3β), casein kinase 1 epsilon (CK1ε), Janus kinase 3 (JAK3), and Abelson murine leukemia viral oncogene homolog 1 (ABL1). Table 1 shows the results of the primary screening. Betulin showed weak activity against Pim1; i.e., at 10 µg/mL, it showed only 10% inhibition of kinase activity. This contrasted markedly with ABL1, whose kinase activity was inhibited by 79%. JAK3 and GSK-3β also showed inhibition by betulin. Table represent the % of kinase activity that remained following treatment of each kinase with 10 or 1 µg/mL of betulin, normalized to control (that is, kinase activity in the presence of the vehicle, DMSO, only). ATP concentration used in the kinase assays was 10 µM, and values given represent mean (n = 2). A value ≥100 indicates that the compound does not detectably inhibit the enzymatic activity at the tested concentration. Betulin was next tested against a larger panel of 16 protein kinases, as reported in Figure 2, using a range of betulin concentrations. IC 50 values were determined from the dose-response curves for the target kinases most potently inhibited by betulin (displaying more than~45% of inhibition at 10 µM of betulin). Results in Figure 2 revealed that betulin gave the highest inhibitory activity against GSK-3α, with IC 50 of 0.72 µM; followed by ABL1, with IC 50 of 0.93 µM (see Figure 3a for dose-response curve for ABL1); GSK-3β, with IC 50 of 1.06 µM; JAK3 kinase with IC 50 of 1.08 µM; CK1ε, with IC 50 of 2.11 µM; vascular endothelial growth factor receptor 2 (VEGFR2), with IC 50 of 2.45 µM; and NIMA Related Kinase 6 (NEK6), with IC 50 of 3.02 µM. Given these results and the well-established role of BCR-ABL1 in chronic myelogenous leukemia (CML), we next focused our work on the inhibition of ABL1 by betulin. Molecular Mechanism of ABL1 Inhibition by Betulin To test the hypothesis that kinase inhibition by betulin might be the driver of its cellular effects, we explored the binding mode of betulin to ABL1, using ATP competition assays. Accordingly, we measured % of maximal activity (relative to a DMSO control) remaining in the presence of betulin, at ATP concentrations of 10, 50, and 100 µM. As shown in Figure 3, the results obtained strongly suggest competitive inhibition of ATPbinding to ABL1 by betulin. The inhibition of the ABL1 activity by 10 µM betulin was significantly decreased in the presence of a high concentration of ATP (100 µM). We note here that other triterpenoids, for example those extracted from the dry infructescences of Liquidambaris Fructus (also called Lu Lu Tong when used in Traditional Chinese medicine to treat some breast disease) have also been implicated as putative ATP competitors [26]. Molecular Mechanism of ABL1 Inhibition by Betulin To test the hypothesis that kinase inhibition by betulin might be the driver of its cellular effects, we explored the binding mode of betulin to ABL1, using ATP competition assays. Accordingly, we measured % of maximal activity (relative to a DMSO control) remaining in the presence of betulin, at ATP concentrations of 10, 50, and 100 µM. As shown in Figure 3, the results obtained strongly suggest competitive inhibition of ATP-binding to ABL1 by betulin. The inhibition of the ABL1 activity by 10 µM betulin was significantly decreased in the presence of a high concentration of ATP (100 µM). We note here that other triterpenoids, for example those extracted from the dry infructescences of Liquidambaris Fructus (also called Lu Lu Tong when used in Traditional Chinese medicine to treat some breast disease) have also been implicated as putative ATP competitors [26]. Molecules 2021, 26, x FOR PEER REVIEW 5 of 14 Molecular Modeling of the ABL1-Betulin Complex To gain further insight, we investigated the interaction of betulin with the ATP binding site of ABL1 tyrosine kinase by molecular docking. To accomplish this, we used the crystal structure of ABL1 tyrosine kinase complexed with the established inhibitor, imatinib, as an adduct, and carried out docking with Discovery Studio 3.1 and AutoDock Vina [27,28] software. The accuracy of the docking procedure was evaluated by docking imatinib back into its established binding site. The root mean square deviation (RMSD) of the highest-ranked orientation from the position of the imatinib in the crystal structure was found to be 1.01 Å (Figure 4). We note that RMSD values <1.5 Å are considered to indicate successful molecular docking [29]. The results show that betulin fits within the ATP binding site of ABL1 tyrosine kinase, in a position that overlaps with the methylpiperazine ring of imatinib ( Figure 4). The positioning of betulin is similar to that predicted recently for a pentacyclic triterpenoid gypsogenin derivative in a recent report [30]. In contrast to imatinib, the large size of the betulin molecule sterically hinders it from binding deep within the binding pocket. The secondary alcohol extends towards the exterior of the protein while the hydroxymethyl and vinyl substituents are directed towards the interior of the protein. While imatinib undergoes extensive hydrogen bonding, the lipophilic structure of betulin undergoes mostly van der Waals interactions with the surrounding amino acid residues (e.g., Glu286, Met290, Ile293, Val298, Leu354, Ile360, His361, Arg362, and Asp381). This computational approach suggests that betulin is located far from amino acid residue Thr315, a residue of particular interest as the T315I mutation of ABL1 kinase attenuates inhibition by imatinib. To test this notion, we assayed betulin effects on ABL1 kinase, bearing the T315I mutation in its kinase domain. As shown in Figure S7, betulin at a concentration of 1µM, i.e., a dose similar to the IC50 value of betulin against wild type ABL1, retains its capacity to inhibit the enzymatic activity of T315I mutant of ABL1. As expected, inhibition of T315I mutant of ABL1 kinase by imatinib is strongly reduced. These results support the putative binding mode of betulin determined by molecular docking (Figure 4). Molecular Modeling of the ABL1-Betulin Complex To gain further insight, we investigated the interaction of betulin with the ATP binding site of ABL1 tyrosine kinase by molecular docking. To accomplish this, we used the crystal structure of ABL1 tyrosine kinase complexed with the established inhibitor, imatinib, as an adduct, and carried out docking with Discovery Studio 3.1 and AutoDock Vina [27,28] software. The accuracy of the docking procedure was evaluated by docking imatinib back into its established binding site. The root mean square deviation (RMSD) of the highestranked orientation from the position of the imatinib in the crystal structure was found to be 1.01 Å (Figure 4). We note that RMSD values <1.5 Å are considered to indicate successful molecular docking [29]. Betulin Selectively Inhibits Proliferation of Human Leukemic Cells The anticancer and chemoprotective potential of betulin has already been reported (see [31] for a table reporting the in vitro antiproliferative effect of betulin on >40 cancer The results show that betulin fits within the ATP binding site of ABL1 tyrosine kinase, in a position that overlaps with the methylpiperazine ring of imatinib ( Figure 4). The positioning of betulin is similar to that predicted recently for a pentacyclic triterpenoid gypsogenin derivative in a recent report [30]. In contrast to imatinib, the large size of the betulin molecule sterically hinders it from binding deep within the binding pocket. The secondary alcohol extends towards the exterior of the protein while the hydroxymethyl and vinyl substituents are directed towards the interior of the protein. While imatinib undergoes extensive hydrogen bonding, the lipophilic structure of betulin undergoes mostly van der Waals interactions with the surrounding amino acid residues (e.g., Glu286, Met290, Ile293, Val298, Leu354, Ile360, His361, Arg362, and Asp381). This computational approach suggests that betulin is located far from amino acid residue Thr315, a residue of particular interest as the T315I mutation of ABL1 kinase attenuates inhibition by imatinib. To test this notion, we assayed betulin effects on ABL1 kinase, bearing the T315I mutation in its kinase domain. As shown in Figure S7, betulin at a concentration of 1µM, i.e., a dose similar to the IC 50 value of betulin against wild type ABL1, retains its capacity to inhibit the enzymatic activity of T315I mutant of ABL1. As expected, inhibition of T315I mutant of ABL1 kinase by imatinib is strongly reduced. These results support the putative binding mode of betulin determined by molecular docking (Figure 4). Betulin Selectively Inhibits Proliferation of Human Leukemic Cells The anticancer and chemoprotective potential of betulin has already been reported (see [31] for a table reporting the in vitro antiproliferative effect of betulin on >40 cancer cell lines). Because betulin inhibits ABL1, a kinase shown to cause chronic myelogenous leukemia (CML) when deregulated by fusion with BCR, we next used the human K562 CML cell line to test the effects of betulin in cultured cells (Figure 5a). Although the effects of betulin on K562 cells has been reported in the literature, the results seem to be quite variable: for half-maximal inhibition of cell growth, IC 50 from 14.5 µM to >200 µM has been reported [31]. Using cell viability to evaluate the betulin activity in K562 cells, we determined that: (i) betulin decreased the viability of K562 leukemic cells in a 48-h assay, with an IC 50 of 16.5 µM; (ii) leukemia cells resistant to treatment with doxorubicin (a chemotherapeutic drug marketed as Adriamycin ® , see Figure S8) were equally sensitive to treatment with betulin (IC 50 of 13.5 µM) as doxorubicin-sensitive cells; in contrast, the doxorubicin-resistant cells were less sensitive to imatinib mesylate (Figure 5b). Note here that the effects of betulin on cell viability were not significantly altered when cells were treated with 20 µM z-VAD-fmk, a pan-inhibitor of caspases ( Figure S9). The efficacy of cancer chemotherapy is critically dependent upon tumor cell selectivity. We next tested the effect of betulin on human peripheral blood lymphocytes (hPBLs) purified from four healthy donors. As shown in Figure 5c, treatment with betulin ≤ 100 µM did not induce a significant decrease of the viability of hPBLs. This result indicates an acceptable level of selectivity of betulin against cancer cells. Effects of Betulin on the MAPK/ERK Signaling Pathway The chimeric protein BCR-ABL1 was previously shown to drive neoplastic transformation of hematopoietic stem cells in chronic myelogenous leukemia [32]. ABL1 is the kinase portion of the BCR-ABL1 oncogene. In the BCR-ABL1 fusion protein, ABL1 tyrosine kinase activity is constitutively activated to interact in various signaling pathways including most notably the mitogen activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) pathway that increases cellular proliferation [30]. The K562 CML cell line is known to express the bcr-abl fusion gene [33]. Accordingly we examined phosphorylation of ERK kinases in doxorubicin-sensitive K562 (K562S) cells treated with 50 or 100 µM betulin or 20 µM imatinib mesylate (the latter dose has already been shown to be effective on ERK phosphorylation [30]). All of the treatments tested were shown to decrease the viability of K562S cells after a 48 h treatment (see Figure 5a,b). In Figure 6, we prepared extracts of treated cells after 6 h treatment, so we decided to use a higher dose of each compound compared to that necessary to affect cell viability (~3-6 times more than the IC 50 of betulin reported in Figure 5a). This strategy was employed to obtain a test for effects on the signaling pathway at the shorter treatment interval. We conducted immunoblot analysis on the protein extracts using anti-phospho-ERK1/2 antibody. As shown in Figure 6 and Figure S10, betulin treatment of cells inhibited the phosphorylation of ERK in a dosedependent manner. As a control, imatinib mesylate showed a stronger effect and almost completely abrogated the phosphorylation of ERK (Figure 6b). This result demonstrates that betulin markedly decreases the downstream signaling of BCR-ABL oncoprotein. Effects of Betulin on the MAPK/ERK Signaling Pathway The chimeric protein BCR-ABL1 was previously shown to drive neoplastic transf mation of hematopoietic stem cells in chronic myelogenous leukemia [32]. ABL1 is kinase portion of the BCR-ABL1 oncogene. In the BCR-ABL1 fusion protein, ABL1 ty shown in Figure 6 and Figure S10, betulin treatment of cells inhibited the phosphorylation of ERK in a dose-dependent manner. As a control, imatinib mesylate showed a stronger effect and almost completely abrogated the phosphorylation of ERK (Figure 6b). This result demonstrates that betulin markedly decreases the downstream signaling of BCR-ABL oncoprotein. Figure 6. Effects of betulin on extracellular signal-regulated kinase (ERK) signaling. (a) K562S CML cells that were untreated (K562S) or treated with 1% DMSO, 50 or 100 µM betulin, or 20 µM of imatinib mesylate for 6 h were subjected to an immunoblot analysis as described in the Methods section. Briefly, extracts of treated K562S cells were analyzed by SDS-PAGE, followed by Western blotting with antibodies directed against phospho-ERK1/2 (Thr202/Tyr204) and α-Tubulin (as a loading control). (b) The open source image processing program "ImageJ" was used to quantify the intensity of each band. Discussion Betulin is a pentacyclic triterpenoid of lupane type found in plants, and it is naturally abundant in many species of trees in northern Europe. In some birch tree species, the quantity of betulin can be over 50% of the dry weight of the bark [31,34]. In this study, we isolated betulin for the first time from Acacia auriculiformis stem bark, in which it is four orders of magnitude less abundant. Betulin and its derivatives were intensively studied and found to exhibit a broad spectrum of pharmacological activities, including anti-cancer, anti-viral, anti-microbial, anti-inflammatory, and anti-fibrotic effects [35,36]. Moreover, betulin-containing extracts from birch bark were formulated as an oleogel (Episalvan ® , also known as Oleogel-S10), in which betulin was shown to be the active pharmaceutical ingredient. Oleogel-S10 was approved in 2016 by the European Medicines Agency (EMA) for treatment of partial thickness wounds in adults and was in a Phase III efficacy and safety study since 2017, in patients with inherited epidermolysis bullosa (NCT03068780) [37]. Despite growing interest Figure 6. Effects of betulin on extracellular signal-regulated kinase (ERK) signaling. (a) K562S CML cells that were untreated (K562S) or treated with 1% DMSO, 50 or 100 µM betulin, or 20 µM of imatinib mesylate for 6 h were subjected to an immunoblot analysis as described in the Methods section. Briefly, extracts of treated K562S cells were analyzed by SDS-PAGE, followed by Western blotting with antibodies directed against phospho-ERK1/2 (Thr202/Tyr204) and α-Tubulin (as a loading control). (b) The open source image processing program "ImageJ" was used to quantify the intensity of each band. Discussion Betulin is a pentacyclic triterpenoid of lupane type found in plants, and it is naturally abundant in many species of trees in northern Europe. In some birch tree species, the quantity of betulin can be over 50% of the dry weight of the bark [31,34]. In this study, we isolated betulin for the first time from Acacia auriculiformis stem bark, in which it is four orders of magnitude less abundant. Betulin and its derivatives were intensively studied and found to exhibit a broad spectrum of pharmacological activities, including anti-cancer, anti-viral, anti-microbial, anti-inflammatory, and anti-fibrotic effects [35,36]. Moreover, betulin-containing extracts from birch bark were formulated as an oleogel (Episalvan ® , also known as Oleogel-S10), in which betulin was shown to be the active pharmaceutical ingredient. Oleogel-S10 was approved in 2016 by the European Medicines Agency (EMA) for treatment of partial thickness wounds in adults and was in a Phase III efficacy and safety study since 2017, in patients with inherited epidermolysis bullosa (NCT03068780) [37]. Despite growing interest in therapeutic use of betulin, notably for cancer treatment, the molecular mechanism of action of betulin is not well understood [31]. As previously described, triterpenoids were shown to inhibit various protein kinases: e.g., ursolic acid has been reported to inhibit tyrosine kinase activity [38,39], and plant-derived pentacyclic triterpenoid gypsogenin and derivatives were reported by Ciftci et al. to show activity against myelogenous leukemia by virtue of their inhibition of ABL1 kinase [30]. We thus tested betulin against a panel of disease-related human kinases. Betulin was shown to inhibit several kinases in the panel, with activity in the micromolar range, including ABL1, CK1ε, GSK-3α/β, JAK3, NEK6, and VEGFR2. Especially notable amongst our results is the inhibitory activity of betulin against ABL1 kinase (IC 50 of 0.93 µM). ABL1 kinase, a member of the Abelson kinase family that also includes ABL2, has been implicated in cancer, particularly in hematological malignancies such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and lymphoblastic leukemia [3]. At present, ABL1 kinase is among the most common drug targets of approved therapeutic kinase inhibitors (ABL1 is the target of five molecular entities approved by FDA for cancer therapy) [12,40]. The activated chimeric BCR-ABL tyrosine kinase is the key biochemical defect that causes Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) [40]. In our study, we showed that betulin inhibits the enzymatic activity of ABL1 and perturbs the MAPK/ERK signaling pathway in chronic myelogenous leukemia cells. Since the antineoplastic mechanism of action of betulin is not yet known, our results raise the possibility that its antitumor mechanism may be at least partially explained by its inhibition of kinases. In future studies, we will increase the panel of kinases, in order to explore more deeply the kinome and to test whether any other kinases are more potently inhibited by betulin than those we found already. Multidrug resistance (MDR) mediated by the drug efflux protein, P-glycoprotein (P-gp), is one of the major obstacles to successful cancer chemotherapy [41]. As an example, cancer cells use P-gp to escape cell death induced by doxorubicin chemotherapeutic agent [42]. We showed here that contrary to doxorubicin, K562 doxorubicin-resistant cells retained an undiminished sensitivity to betulin. This result indicates that betulin is probably not a substrate for the P-glycoprotein, a crucial factor in considering its potential as part of a treatment strategy to combat human MDR cancers. The results obtained in this study shed light on a putative mechanism of action of betulin that may drive its known effects on cancer cells. Indeed, betulin was shown to have a multi-pharmacological profile, affecting notably ABL1, JAK3, and GSK-3α/β. These results support the notion that betulin could be used alone or in combination with other anticancer drugs as a putative natural product-based therapeutic for the treatment of haematological malignancies caused by deregulation of protein kinases. Note here that the poor solubility in water of betulin was reported in the literature: betulin was shown to be soluble to only 0.08 µg/mL (0.18 µM) [43,44] and had a high predicted Octanol-Water Partition Coefficient (LogP) of 9.01 (data from ChemSpider database, Royal Society of Chemistry). Consequently, in this study, betulin was solubilized in DMSO at 10 mM final concentration just before use, avoiding storage at −20 • C. For cell-based assays, dilutions were performed in culture media to achieve 0.5-1% DMSO final concentration. For kinase assays, dilutions were prepared in water to reach 1% DMSO final concentration. Cell Lines and Culture K562 (ATCC ® , CCL-243, described here as K562S to indicate sensitivity to doxorubicin), a human chronic myelogenous leukemia cell line, was obtained from American Type Culture Collection (Manassas, VA, USA). A doxorubicin-resistant cell line (K562R, also known as K562/Adr) was kindly provided by the IRSET institute (Research Institute for Environmental and Occupational Health, INSERM, University of Rennes 1, France). The cells were maintained at 37 • C and 5% CO 2 in Gibco™ 1640 Roswell Park Memorial Institute (RPMI-1640) medium containing 10% fetal bovine serum (FBS) (Life Technologies TM , Thermo Fisher Scientific, Waltham, MA, USA). Purification of Natural Products from Plant Material Plant material (consisting of bark) was collected in Samaru-Zaria, Nigeria in September, 2018, and was identified by U.S Gallah, the plant taxonomist of Biological Sciences Department, Kaduna State University, where a voucher specimen (number 1292) was deposited in the herbarium. All protocols involving the collection and use of plant material adhered to relevant ethical guidelines. Air-dried, pulverized bark was extracted with 70 % ethanol at room temperature for 7 days. The combined ethanol extract was concentrated using a rotary evaporator to give a semi-solid mass (45 g). Thirty-two grams of the crude extract was suspended in 100 mL of water and partitioned with 5 × 300 mL of ethyl acetate, and 5 × 300 mL of n-butanol was used to yield 3.6 g and 2.3 g of ethyl acetate-and nbutanol-soluble fractions respectively. A portion of the ethyl acetate-soluble fraction (2.4 g) was packed into a column of silica gel G (200-400 mesh, Silicycle, 5 cm × 50 cm) and eluted first with 100% dichloromethane and then with a stepwise gradient of dichloromethane and methanol mixtures, as follows: 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 80:20, 60:40, 50:50, 30:70, 10:90, and 100% methanol. The progress of elution was monitored by thin layer chromatography (carried out on pre-coated silica gel TLC plates aluminum backed (Silicycle) using the solvent system ethyl acetate:dichloromethane:methanol:water (15:8:4:1 and 6:4:4:1, respectively). Fractions eluted with 2% methanol in dichloromethane were further purified using Sephadex LH-20 (Pharmacia), eluted with methanol to give natural product betulin, yielding a white solid. The identification of betulin was performed by NMR spectroscopy, carried out on a Bruker Avance NMR spectrophotometer (500 MH Z 1 H, and 125 MH Z 13 C). Protein Kinase Assays Kinase enzymatic activities were assayed in 384-well plates using the ADP-Glo TM assay kit, following the recommendations of the manufacturer (Promega, Madison, WI). Controls were performed in appropriate dilutions of dimethyl sulfoxide (DMSO). Kinase activities, measured in the presence of 10 µM ATP, are expressed as percentage of maximal activity, i.e., measured in the absence of inhibitor. In order to determine the half-maximal inhibitory concentration (IC 50 ), the assays were performed in duplicate in the absence or presence of increasing doses of the tested compounds. Data were analyzed using GraphPad PRISM (GraphPad Software, San Diego, CA, USA) software to fit a sigmoïdal curve that allowed determination of the IC 50 values. The experimental conditions used for measuring kinase activities are comprehensively described in Ibrahim et al. [45]. Human recombinant ABL1 mutant (T315I) was purchased from Promega (Catalog # V5320) and tested following the recommendations of the manufacturer (Promega, Madison, WI, USA). Molecular Docking Molecular docking simulations were carried out with Discovery Studio 3.1 (Accelrys) and AutoDock Vina software [27]. The crystal structure of ABL1 tyrosine kinase complexed with imatinib was obtained from the Brookhaven protein data bank (PDB code: 1IEP) [28]. The protein was prepared and protonated for docking in Discovery Studio with the 'prepare protein' function. The pKa values and protonation states of the ionizable amino acids were subsequently calculated at pH 7.4, and the protein model was typed with the Momany and Rone CHARMm forcefield. A fixed atom constraint was applied to the backbone and the protein was energy minimized with the Smart Minimiser algorithm (50,000 steps maximum) using the implicit generalised Born solvation model with molecular volume. Discovery studio was used to construct structures for betulin and imatinib, which were submitted to the 'prepare ligands' protocol. The co-crystallised ligand and water molecules were removed from the protein model, and AutoDock Vina was used for the docking. The highest-ranked solution of each ligand was further refined with the Smart Minimizer algorithm. Illustrations were prepared with the PyMOL molecular graphics system [46].	v3-fos-license
2018-04-03T02:42:05.675Z	2016-07-14T00:00:00.000	491003	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.nature.com/articles/srep29415.pdf", "pdf_hash": "259b38a66e0ba209afebbac92a55ce2cee732e5b", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2161", "s2fieldsofstudy": [ "Medicine" ], "sha1": "259b38a66e0ba209afebbac92a55ce2cee732e5b", "year": 2016 }	pes2o/s2orc	Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. RCC tissues, DNA-PKcs expression in RCC tissues was about 4-times higher than that in normal renal tissues (Fig. 1B). Real-time PCR assay results showed that DNA-PKcs mRNA level was also increased in RCC tissues (Fig. 1C). Expression of DNA-PKcs in human RCC cells was also analyzed. As shown in Fig. 1D,E, DNA-PKcs protein expression was significantly higher in established (A498 and 786-0 lines) 20 and primary human RCC cells than that in non-cancerous proximal tubule epithelial HK-2 cells 20,21 . In addition, DNA-PKcs mRNA level was over-expressed in above HCC cells (Fig. 1F). Thus, these results show that DNA-PKcs is over-expressed in human RCC tissues and RCC cells. DNA-PKcs inhibitors induce proliferation inhibition and apoptosis in RCC cells. Above results demonstrate that DNA-PKcs is over-expressed in human RCC cells and RCC tissues. Next, we studied the potential effect of DNA-PKcs in RCC cell proliferation. Three different DNA-PKcs inhibitors, including NU-7026 22 , NU-7441 23 and LY-294002 24 were applied. Simply through the viable cell (trypan blue exclusive) counting assay, our results showed that the DNA-PKcs inhibitors remarkably inhibited 786-0 RCC cell proliferation ( Fig. 2A). Meanwhile, the results of MTT viability assay (Fig. 2B) and clonogenicity assay (Fig. 2C) further confirmed the anti-proliferative activity by these DNA-PKcs inhibitors. We also noticed significant apoptosis activation in 786-0 cells after treatment of DNA-PKcs inhibitors, which was shown by ssDNA apoptosis ELISA assay (Fig. 2D) and caspase-3 activity assay (Fig. 2E). These DNA-PKcs inhibitors were also anti-proliferative in A498 RCC cells (Fig. 2F), and in primary human RCC cells 20 (Fig. 2G). Apoptosis induction, evidenced by ssDNA ELISA OD increase (Fig. 2H), was also observed in the primary cancer cells after the DNA-PKcs inhibitor treatment. On the other hand, the proliferation of non-cancerous HK-2 cells (low DNA-PKcs expression, Fig. 1) 21 were not affected by the same DNA-PKcs inhibitor treatment (Fig. 2I). Note that expression of DNA-PKcs was not affected by these inhibitors in above cells (Data not shown). Together, these results demonstrate that DNA-PKcs inhibitors exert anti-proliferative and pro-apoptotic activities to cultured RCC cells. For the primary human RCC cells, siRNA method was applied to transiently knockdown DNA-PKcs, and Western blot assay showed DNA-PKcs silence by the two targeted siRNAs (Fig. 3H, upper). Primary human RCC cell proliferation, tested by MTT assay, was also inhibited by the non-overlapping DNA-PKcs siRNAs (Fig. 3H, lower). The proliferation of proximal tubule epithelial HK-2 cells was not affected by DNA-PKcs siRNAs (Fig. 3I,J). Thus, these results show that DNA-PKcs knockdown inhibits RCC cell proliferation in vitro. DNA-PKcs is in the complex of mTOR and SIN1, required for mTORC2 activation and HIF-2α expression. Recent studies have demonstrated a potential role of DNA-PKcs in mTORC2 activation 16,25,26 . It has been shown that DNA-PKcs could form a complex with SIN1, a key component of mTORC2 27,28 , thus regulating AKT Ser-473 phosphorylation 16,25 . Thus, we examined the potential role of DNA-PKcs in AKT-mTOR for applied time, cell proliferation was analyzed by viable cell counting assay (A, for 786-0 cells), MTT assay (B,F,G,I) or clonogenicity assay (C, for 786-0 cells); Cell apoptosis was tested by the ssDNA ELISA assay (D,H) or the caspase-3 activity assay (E, for 786-0 cells). Experiments in this figure were repeated three times, and similar results were obtained. For each assay, n = 5. * p < 0.05 vs. "DMSO" group. signaling activation in RCC tissues. First, using Co-IP assay, we noticed a physical interaction between DNA-PKcs, SIN1, Rictor and mTOR in multiple human RCC tissues, and in 786-0 RCC cells (Fig. 4A). Raptor, the mTORC1 component 29 , was not in the complex (Fig. 4A). Same IP method failed to detect a significant SIN1-DNA-PKcs association in the above normal renal tissues (Data not shown), possibly due to low expression of both proteins (Fig. 1). As shown in Fig. 4B, DNA-PKcs-shRNA knockdown significantly inhibited AKT Ser-473 phosphorylation in 786-0 cells. Yet AKT Thr-308 phosphorylation was almost unaffected. Meanwhile, expression of HIF-2α , a mTORC2-regulated gene 30 , was also downregulated with DNA-PKcs knockdown (Fig. 4B). The HIF-1α expression, which was mainly regulated by mTORC1 30 , was not changed (Fig. 4B). In addition, DNA-PKcs inhibitors, including NU-7026, NU-7441 and LY-294002, dramatically inhibited AKT Ser-473 phosphorylation and HIF-2α expression in 786-0 cells (Fig. 4C). Since LY-294002 was also a PI3K-AKT-mTOR pan inhibitor 31 , it thus blocked AKT Thr-308 phosphorylation and downregulated HIF-1α expression in 786-0 cells (Fig. 4C). As expected, SIN1-shRNA expressing cells showed similar results as DNA-PKcs-shRNA cells, showing decreased AKT Ser-473 phosphorylation and HIF-2α expression (Fig. 4D). AKT Thr-308 phosphorylation and HIF-1α expression were again not affected by SIN1 shRNA knockdown (Fig. 4D). Note that above experiments were also repeated in A498 cells and primary human HCC cells, and similar results were obtained (Data not shown). Together, these results indicate that DNA-PKcs is in the complex of mTORC2, regulating AKT Ser-473 phosphorylation and HIF-2α expression in RCC cells. Results demonstrated that oral administration of a single dose of the DNA-PKcs inhibitor NU-7441 (10 mg/kg, daily for three weeks) resulted in a significant inhibition of 786-0 xenograft growth in nude mice (Fig. 5A). Meanwhile, the in vivo growth of stable 786-0 cells with DNA-PKcs shRNA was also slower than the cells expressing scramble control shRNA (Fig. 5A). The daily xenograft growth volume was significantly lower with NU-7441 administration or DNA-PKcs silencing (Fig. 5B). Note that mice body weights were not affected by NU-7441 treatment nor by DNA-PKcs silencing (Fig. 5C). We also did not notice any signs of apparent toxicities, such as diarrhea, fever, severe piloerection or a sudden weight loss (> 10%), in the tested animals (Data not shown). The signaling changes in the above xenografted tumors were also analyzed. In line with the in vitro findings, Western blot analysis of 786-0 xenografts (week-2 and week-4 after initial treatment) showed that AKT Ser-473 phosphorylation and HIF-2α expression were both inhibited by NU-7441 administration or DNA-PKcs shRNA knockdown in vivo (Fig. 5D). DNA-PKcs band confirmed its silence in 786-0 xenografts expressing DNA-PKcs-shRNA-1 (last for at least 2 to 4 weeks, Fig. 5D). Immunohistochemistry (IHC) results further confirmed that NU-7441 administration or DNA-PKcs shRNA suppressed AKT Ser-473 phosphorylation in 786-0 xenografts (two weeks after initial treatment, Fig. 5E). Collectively, these results show that DNA-PKcs inhibition or silencing suppresses AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 xenograft growth in vivo. miR-101 downregulation correlates with DNA-PKcs overexpression in RCC. Above results have shown that DNA-PKcs overexpression regulates mTORC2 activation, HIF-2α expression and RCC cell proliferation. Next, we studied the underlying mechanisms of DNA-PKcs overexpression by focusing on miRNA regulation. A recent study by Yan et al. showed that miR-101 targeted 3′ UTR of DNA-PKcs mRNA, leading to DNA-PKcs mRNA degradation 19 . We thus analyzed level of miR-101 in human RCC tissues and RCC cells. Real-time PCR assay results in (Fig. 6A) showed that miR-101 was significantly decreased in RCC tissues ("Tumor") than that in surrounding normal renal tissues ("Normal") (n = 10). In addition, miR-101 level was also lower in established (A498 and 786-0 lines) and primary human RCC cells, as compared to HK-2 cells (Fig. 6B). Discussions In the present study, our results indicate that DNA-PKcs might be a novel oncogene for the RCC. First, we demonstrate that DNA-PKcs is over-expressed in multiple human RCC tissues. Second, inhibition of DNA-PKcs, through pharmacological inhibitors or siRNA/shRNA knockdown, significantly reduced RCC cell proliferation in vitro and in vivo. Third, DNA-PKcs was found in the complex of mTORC2, and was required for AKT activation (Ser-473 phosphorylation) and HIF-2α expression in RCC cells. Thus, DNA-PKcs might be a valuable target for RCC intervention. Overactivity of AKT is observed in many RCCs, which plays a vital role in cell survival, proliferation, migration, apoptosis-resistances and other cancerous behaviors 33,34 . Complete activation of AKT requires both Ser-473 and Thr-308 phosphorylations 33,34 . Studies have indicated a potential role of DNA-PKcs in regulating AKT activation. For example, Feng et al. showed that DNA-PKcs directly associates and activates AKT in the plasma membrane, causing a 10-fold enhancement of AKT activity 35 16 . Similarly, Xu and co-authors found that, upon low-dose X-ray irradiation (LDI), DNA-PKcs associates with mTORC2 to mediate AKT Ser 473 phosphorylation 25 . In the current study, we showed that DNA-PKcs formed a complex with mTOR and SIN1 in both human RCC tissues and RCC cells, and was required for mTORC2 activation (AKT Ser-473 phosphorylation) and HIF-2α expression. Inhibition or silencing of DNA-PKcs in RCC cells reduced AKT Ser-473 phosphorylation and HIF-2α expression. Thus, DNA-PKcs may regulate RCC cell proliferation through regulating mTORC2 signaling. Scientific RepoRts \| 6:29415 \| DOI: 10.1038/srep29415 production and tumor angiogenesis 38 . Studies have shown that 50% or more sporadic RCCs have somatic mutations in pVHL 38 . Although the role HIF-1α in tumor progression and angiogenesis has been extensively studied, existing evidences indicated that HIF-2α is far more important than HIF-1α in the pathogenesis of RCC 20,39,40 . As a matter of fact, HIF-2α silencing was shown to inhibit the ability of pVHL-knockout RCC cells to form tumors in vivo 39 . Kondo and colleagues showed that pVHL-mediated tumor suppression is abolished with overexpression of HIF-2α , but not HIF-1α 40 . In the current study, we showed that HIF-2α expression was inhibited with DNA-PKcs silencing or blockage. These results were not surprising, since translation of HIF-2α is solely dependent upon the activity of mTORC2 30 , and we showed that DNA-PKcs was required for mTORC2 activation in RCC cells. As a matter of fact, SIN1 shRNA knockdown similarly decreased HIF-2α expression in 786-0 cells. Notably, HIF-1α expression was not affected by DNA-PKcs or SIN1 knockdown. One reason could be that HIF-1α translation is controlled mainly by mTORC1 30 . Collectively, we suggest that DNA-PKcs is in the complex of mTORC2, regulating AKT Ser-473 phosphorylation and HIF-2α expression in RCC cells. miRNA-mediated gene regulation plays a fundamental role in controlling gene expression at the post-transcriptional level 41 . These miRNAs are vital in modifying many key biologic processes of human cells, possibly via regulating expression of signaling molecules including growth factors, cytokines, transcription factors and other proteins (i.e. DNA-PKcs 19 ) 41,42 . In addition, recent studies have shown that at least half of the miRNAs are linked to human cancers, these miRNAs are either upregulated or downregulated in human cancer cells. Specifically, many oncogenes and tumor suppressor genes are virtually regulated by miRNAs 42 . DNA-PKcs is shown to be negatively regulated by miRNA-101 19 . In the current study, we showed that miR-101 level was significantly lower in human RCC tissues, and in established or primary RCC cells, which might be a reason for DNA-PKcs over-expression. Introduction of miR-101 in RCC cells downregulated DNA-PKcs expression, and inhibited AKT activation, HIF-2α expression and cell proliferation. Reversely, over-expression of antagomiR-101 downregulated miR-101, and further enhanced DNA-PKcs expression and RCC cell proliferation. These results indicate that miR-101 downregulation might be at least one key reason for DNA-PKcs overexpression in RCC cells. In summary, our results demonstrate that DNA-PKcs over-expression in RCC cells regulates mTORC2-AKT activation, HIF-2α expression and RCC cell progression. DNA-PKcs might be a valuable target for RCC treatment. 20 . For all the cell lines, DNA fingerprinting and profiling were performed every 6 months to confirm the origin of the cell line, and to distinguish the cell line from cross-contamination. All cell lines were subjected to mycoplasma and microbial contamination examination. Population doubling time, colony forming efficiency, and morphology under phase contrast were also measured every 6 months under defined conditions to confirm the phenotype of cell line. Methods Human RCC tissues. Tissue specimens were obtained from ten RCC patients with total nephroureterectomy. All patients were administrated in the Second Affiliated Hospital of Nantong University. Each patient received no irradiation or chemotherapy prior to surgery. In each fresh-isolated specimen, tumor tissue and the surrounding normal renal tissue were separated and paired. Tissues were thoroughly washed in PBS with antibiotics and DTT (2.5 mM, Sigma), and then minced into small pieces, which were then maintained in DMEM plus 10% FBS and necessary antibiotics. Tissues were lysed and analyzed by Western blots and real-time PCR. All patients enrolled provided individual written-informed consent. Using human specimens in this study was approved by the Nantong University's Scientific Ethical Committee (Approve ID: 2013-002). The methods were carried out in accordance with the principles set out in the Declaration of Helsinki and the NIH Belmont Report. Primary culture of human RCC cells. Part of the minced RCC tumor tissues were also subjected to collagenase I (Sigma, 0.05% w/v) digestion for 30 min. Afterwards, individual cells were pelleted and rinsed twice with DMEM, and then cultured in DMEM, supplied with 10% FBS, 2 mM glutamine, 1 mM pyruvate, 10 mM HEPES, 100 units/mL penicillin/streptomycin, 0.1 mg/mL gentamicin, and 2 g/liter fungizone. Primary RCC cells of passage 3-6 were utilized for experiments. Single-stranded DNA analysis of apoptosis. The single-stranded DNA (ssDNA) Apoptosis ELISA Kit (Chemicon International, Temecula, CA) was utilized to quantify cell apoptosis. This assay was based on selective DNA denaturation in apoptotic cells by formamide, and detection of the denatured DNA by monoclonal antibody to single-stranded DNA. The detailed procedure was described in other studies 45,46 . Caspase-3 activity assay. Caspase-3 activity assay was described in our previous study 20 . Briefly, ten micrograms of cytosolic extracts per treatment were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) and the caspase-3 substrate (Calbiochem, Darmstadt, Germany). The release of 7-amido-4-(trifluoromethyl)-coumarin (AFC) was quantified via a Fluoroskan system set to an excitation value of 355 nm and emission value of 525 nm. Western blots. As previously reported 20 , cells or minced tissues were lysed by the lysis buffer containing 10 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 10 μ g/mL leupeptin, 10 μ g/mL aprotinin, 100 mM NaF and 200 μ M sodium orthovanadate. Aliquots of 30 μ g of protein samples were separated by electrophoresis in SDS-PAGE, transferred to the PVDF membrane and detected with the specific antibody. The immunoreactive proteins after incubation with appropriately labeled secondary antibody were detected with an enhanced chemiluminescence (ECL) detection kit (Amersham, Buckinghamshire, UK). Band intensity was quantified by ImageJ software (NIH) after normalization to the loading control. Co-Immunoprecipitation (Co-IP). As previously reported 20 , aliquots of 1000 μ g of protein samples in 1 mL of lysis buffer from each treatment were pre-cleared by incubation with 30 μ L of protein A/G Sepharose (Sigma) for 2 hours at 4 °C rotation. The pre-cleared samples were incubated with the specific anti-SIN1 antibody (1 μ g/mL) overnight at 4 °C rotation. 20-30 μ L of protein A/G Sepharose were added to the samples 2 hours at 4 °C rotation. The beads were washed and boiled, followed by Western blot assay. MTT assay of cell proliferation. Cells were seeded onto 96-well plates (3,000 per well) and allowed to attach overnight. After treatment of cells, cell viability/proliferation was tested using MTT [3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Sigma) according to the manufacturer's instructions. Absorbance was measured at 490 nm through a Microplate Reader. The OD value of treatment group was always normalized to that of the control group. Clonogenic assay. 786-0 cells were plated onto 60 mm plates with 2000 cells per plate. After treatment of cells, surviving colonies were fixed, stained with coomassie blue, and manually counted. shRNA and stable cell selection. For shRNA experiments, lentiviral particles were produced by constructing a lentiviral GV248 expression vector (Genechem, Shanghai, China) containing a puromycin resistance gene and either scramble control shRNA (5′ -AATTCTCCGAACGTGTCACGT) 47 , shRNAs to DNA-PKcs (5′ -GAACACTTGTACCAGTGTT, DNA-PKcs shRNA-1) 47 and (5′ -GATCGCACCTTACTCTGTT, DNA-PKcs shRNA-2) 48 . SIN1 shRNA lentiviral particles were purchased from Santa Cruz Biotech (Santa Cruz, CA). For infection, RCC cells were grown in 6-well culture plates in the presence of 2.0 μ g/mL polybrene (Sigma) to 60% confluence, lentiviral-shRNAs were added to the cells. Virus-containing medium was replaced with fresh medium after 12 hours. Stable clones were selected by puromycin (0.5 μ g/mL) for 10 days, expression of targeted protein in the resistant colonies was tested by Western blots or real-time PCR. antagomiR-101 expression vector as well as miR-control ("miR-C") and pSuper-puro-GFP vector were gifts from Dr. Lu's Lab at Nanjing Medical University 32 . Cells were seeded onto 6-well plates at 50% confluence with 2.0 μ g/mL polybrene (Sigma). After 24 hours, cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, USA). Twelve hours later, transfection medium was replaced with 2 mL of complete medium. Puromycin (2.5 μ g/mL, Sigma) was then added to select stable cells (8-10 days). Cells were always tested for miR-101. Xenograft model. As previously reported 20 , eight-week-old female, nude/beige mice were purchased from Nantong University Animal Laboratories. Approximately 5 × 10 6 786-0 cells were injected into mice right flanks, and tumors were allowed to reach 10 mm in maximal diameter. Mice were divided into four groups (n = 10 of each group): stable 786-0 cells with scramble control shRNA, stable 786-0 cells with DNA-PKcs shRNA (-1), NU-7441 oral administration or vehicle administration. NU-7441 was initially solubilized as a stock solution of 10 mg/mL in ethanol. Prior to gavage, NU-7441 was brought up to volume (0.2 mL) in PBS with 0.5% TWEEN 80 Scientific RepoRts \| 6:29415 \| DOI: 10.1038/srep29415 and 2.5% N,N-dimethylacetamide. Mice body weight and bi-dimensional tumor measurements were taken every 7 days. Tumor volume was estimated using the standard formula: (length × width 2 )/2. Mice (1 mice per group) were sacrificed 7 day or 14 days after initial treatment, and the primary tumors were excised for Western blot and IHC staining analysis. Tumor xenografts were stored in liquid nitrogen. All experimental protocols were approved by the Nantong University's Institutional Animal Care and Use Committee (IACUC, Approve ID: 2013-015) and Nantong University's Scientific Ethical Committee (Approve ID: 2013-002). The methods were carried out in accordance with Nantong University's IACUC regulations. Animal surgery and euthanasia using decapitation were performed under Hypnorm/Midazolam anesthesia, and all efforts were made to minimize suffering. Immunohistochemistry (IHC) staining. The IHC staining was performed on cryostat sections (4 μ m/section) of xenograft tumors according to the described methods 32 . The slides were incubated with the primary antibody (anti-AKT Ser-473, 1:50), and subsequently stained with horseradish peroxidase (HRP)-coupled secondary antibody (Santa Cruz). The slides were then visualized via peroxidase activity using 3-amino-9-ethyl-carbazol (AEC) method (Merck, Shanghai, China). Statistical analyses. All experiments were repeated at least three times, and similar results were obtained. Data were expressed as mean ± standard deviation (SD). Statistical analyses were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons were performed using Tukey's honestly significant difference procedure. A p value of < 0.05 was considered statistically significant.	v3-fos-license
2016-05-17T13:32:17.378Z	2013-07-31T00:00:00.000	6809088	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fnbeh.2013.00093/pdf", "pdf_hash": "9d3a33ca6717153336b7a3cc505b08d90fef785a", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2176", "s2fieldsofstudy": [ "Biology" ], "sha1": "9d3a33ca6717153336b7a3cc505b08d90fef785a", "year": 2013 }	pes2o/s2orc	Involvement of the endogenous opioid system in the psychopharmacological actions of ethanol: the role of acetaldehyde Significant evidence implicates the endogenous opioid system (EOS) (opioid peptides and receptors) in the mechanisms underlying the psychopharmacological effects of ethanol. Ethanol modulates opioidergic signaling and function at different levels, including biosynthesis, release, and degradation of opioid peptides, as well as binding of endogenous ligands to opioid receptors. The role of β-endorphin and µ-opioid receptors (OR) have been suggested to be of particular importance in mediating some of the behavioral effects of ethanol, including psychomotor stimulation and sensitization, consumption and conditioned place preference (CPP). Ethanol increases the release of β-endorphin from the hypothalamic arcuate nucleus (NArc), which can modulate activity of other neurotransmitter systems such as mesolimbic dopamine (DA). The precise mechanism by which ethanol induces a release of β-endorphin, thereby inducing behavioral responses, remains to be elucidated. The present review summarizes accumulative data suggesting that the first metabolite of ethanol, the psychoactive compound acetaldehyde, could participate in such mechanism. Two lines of research involving acetaldehyde are reviewed: (1) implications of the formation of acetaldehyde in brain areas such as the NArc, with high expression of ethanol metabolizing enzymes and presence of cell bodies of endorphinic neurons and (2) the formation of condensation products between DA and acetaldehyde such as salsolinol, which exerts its actions via OR. Long-term exposure to ethanol primarily induces a decrease in POMC expression (Boyadjieva and Sarkar, 1997;Rasmussen et al., 2002;Oswald and Wand, 2004) and in hypothalamic β-endorphin release and levels (Boyadjieva and Sarkar, 1994;Oswald and Wand, 2004). A limited number of studies reported an increase in biosynthesis of POMC and POMC mRNA expression (Seizinger et al., 1984;Gianoulakis et al., 1988) as well as an initial increase followed by a gradual return to normal levels (Wand, 1990). Also, some authors found an increase or no effect on β-endorphin release (Boyadjieva and Sarkar, 1994;Oswald and Wand, 2004). Discrepancies might be attributable to the method of ethanol administration, ethanol dose, time course of drug exposure, administration route and differences in the development of tolerance. Also, it has been observed that alcohol-induced changes depend on the brain region investigated as well as the species and strain of animals used (Gianoulakis, 2001;Méndez and Morales-Mulia, 2008). EVIDENCE OF BEHAVIORAL EFFECTS OF ETHANOL MEDIATED BY THE ENDOGENOUS OPIOID SYSTEM Given that β-endorphin, and also enkephalin, activate μ-OR, extensive research has investigated the role of μ-OR in the behavioral effects of ethanol (Gianoulakis, 1993;Herz, 1997;Sanchis-Segura et al., 2000;Thorsell, 2013). Here we will focus on the involvement of these components of the EOS in several behavioral effects of ethanol, including psychomotor stimulation and sensitization, consumption, and associative learning (with a special focus on conditioned place preference (CPP)). Psychomotor stimulation and sensitization Increased psychomotor stimulation induced by ethanol in mice can be blocked with non-selective opioid receptor antagonists such as naloxone or naltrexone (Kiianmaa et al., 1983;Camarini et al., 2000;Sanchis-Segura et al., 2004;Pastor et al., 2005;Pastor and Aragon, 2006). Some pharmacological strategies have suggested the existence of three so-called subtypes of μ-OR; μ 1 , μ 2 , and, μ 3 (Pasternak, 2001a,b;Cadet et al., 2003) and several studies have shown that μand specifically the μ 1/2and μ 3 -OR subtypes, but not δor κ-OR, are involved in the motor stimulant effects of ethanol in adult mice , and also in rats during early development (Arias et al., 2010;Pautassi et al., 2012). Other studies conducted in mice have suggested that this involvement of μ-OR in ethanol stimulation is debatable (Cunningham et al., 1998;Gevaerd et al., 1999;Holstein et al., 2005). Consistent with the EOS involvement, however, a lesion of the NArc produces a decrease in ethanol-induced stimulation in mice (Sanchis-Segura et al., 2000), and knockout mice deficient in β-endorphin showed attenuated ethanolinduced stimulation (Dempsey and Grisel, 2012). Also, in rats, naltrexone prevents activation produced by ethanol when locally administered in the NArc (Pastor and Aragon, 2008) and intra-VTA blockade of the μ-OR using either naltrexone or the irreversible and selective μ-OR antagonist β-funaltrexamine reduces ethanol-induced locomotor stimulation . Additionally, chronic naltrexone, which upregulates μ-OR (Unterwald et al., 1998;Lesscher et al., 2003), enhances the stimulant effects of ethanol in mice . A critical role of the EOS in the motor sensitizing effects of ethanol has also been proposed (Camarini et al., 2000;Miquel et al., 2003;Pastor and Aragon, 2006). Unspecific OR antagonism prevents development (Camarini et al., 2000) but not expression (Abrahao et al., 2008) of ethanol-induced locomotor sensitization. μ-OR are particularly involved in ethanol sensitization (Camarini et al., 2000), without a clear role of any of the μ-OR subtypes in mediating this process; μ 1/2 -OR antagonism slowed down, but did not block development of sensitization (Pastor and Aragon, 2006). Facilitation of ethanol-induced sensitization found after a period of voluntary alcohol consumption in mice was also seen to be absent in μ-OR deficient CXBK mice (Tarragón et al., 2012). The involvement of μ-OR in ethanol sensitization might be related to ethanol-induced increases in β-endorphin release as a recent study demonstrated that βendorphin-deficient mice do not show locomotor sensitization to ethanol (Dempsey and Grisel, 2012). Also, animals with selective lesions of the NArc show prevented sensitization to ethanol (Miquel et al., 2003;Pastor et al., 2011). Altogether these data suggest that opioids and specifically β-endorphins, via μ-OR, might be critical mediators of ethanol-induced neuroplasticity underlying psychomotor sensitization. Ethanol consumption Numerous studies conducted during the last few decades showed that systemic, as well as local administration of opioid antagonists decrease ethanol consumption under a variety of schedules in different animal species (for reviews see Herz, 1997;Gianoulakis, 2001;Oswald and Wand, 2004;Modesto-Lowe and Fritz, 2005). These conclusions have also been supported by the use of OR knockout mouse models (Roberts et al., 2000;Méndez and Morales-Mulia, 2008). This strong pre-clinical basis has lead to the use of opioid antagonists in alcoholism pharmacotherapy (O'Malley et al., 1992). In rodents, the use of non-selective, as well as selective μ-OR antagonists proved to be effective at reducing ethanol consumption (Méndez and Morales-Mulia, 2008). However, the effects of these manipulations have been seen to be, in some cases, non-specific; fat, saccharin, sucrose and water intake were also reduced by these manipulations (Krishnan-Sarin et al., 1995;Nielsen et al., 2008;Rao et al., 2008;Simms et al., 2008;Corwin and Wojnicki, 2009;Wong et al., 2009). These data are compatible with the interpre-Frontiers in Behavioral Neuroscience www.frontiersin.org July 2013 \| Volume 7 \| Article 93 \| 2 tation that OR, and especially μ-OR might be a key mediator of the processing of positive reinforcement, both at emotional and motivational levels (Herz, 1997;Peciña and Berridge, 2005). In general, data obtained with κ-OR or δ-OR manipulations are less conclusive. A recent review of the literature indicates that κ-OR stimulation generally antagonizes the reinforcing effects of alcohol whereas κ-OR blockade has no consistent effect (Wee and Koob, 2010). Dynorphin/κ-OR system appears to be involved in the negative reinforcing effects of ethanol by producing an aversive effect rather than by directly modulating the rewarding mechanism of ethanol (Wee and Koob, 2010;Walker et al., 2012). However, under an alcohol dependent-state, antagonism of κ-OR results effective in decreasing ethanol voluntary consumption (Wee and Koob, 2010;Walker et al., 2012). It has been reported that blockade of δ-OR either attenuates (Lê et al., 1993;Froehlich, 1995;Krishnan-Sarin et al., 1995;June et al., 1999;Hyytiä and Kiianmaa, 2001;Ciccocioppo et al., 2002), increases (Margolis et al., 2008) or has no effect on ethanol intake (Ingman et al., 2003). These discrepancies may be related to dynamic changes in δ-OR efficacy during ethanol exposure (Margolis et al., 2008). All these data support the participation of the POMC and PENK systems in maintaining alcohol consumption (Froehlich et al., 1991;Vengeliene et al., 2008). Associative learning and conditioned place preference It has been suggested that the EOS participates in the underlying mechanisms mediating conditioned effects induced by abused drugs, including ethanol. This implication is supported by two groups of experiments. On one hand, evidence indicates that OR antagonists attenuate cue-induced reinstatement of previously extinguished responding for ethanol self-administration (Lê et al., 1999;Ciccocioppo et al., 2002Ciccocioppo et al., , 2003Liu and Weiss, 2002;Burattini et al., 2006;Dayas et al., 2007;Marinelli et al., 2009), which suggests a role of EOS in cue-induced incentive motivational effects influencing ethanol-seeking behavior. This interpretation is consistent with clinical data showing that opioid antagonists increase abstinence duration periods in alcohol abusers (O'Malley et al., 1992), probably by reducing cueinduced seeking behavior. On the other hand, pretreatment with opioid receptor antagonism, while not influencing the acquisition of ethanol-induced CPP, reduces the expression and facilitates the extinction of this drug-free conditioned response (Bormann and Cunningham, 1997;Middaugh and Bandy, 2000;Kuzmin et al., 2003;Pastor et al., 2011). Mice lacking μ-OR also showed attenuated ethanol CPP (Hall et al., 2001). Further studies have suggested that expression of ethanol-induced CPP depends on OR located in the VTA, CeA, as well as anterior cingulated cortex (Bechtholt and Cunningham, 2005;Bie et al., 2009;Gremel et al., 2011). Additionally, a neurotoxic lesion of the β-endorphin neurons of the NArc, showed a facilitated extinction of ethanol-induced CPP (Pastor et al., 2011). β-endorphin and μ-OR appear to be therefore critically involved in the mechanisms underlying ethanol CPP. As Cunningham and collaborators have suggested, it is possible that altered opioid signaling might in turn alter conditioned motivation that normally maintains cueinduced seeking behavior during CPP testing (Cunningham et al., 1998). It is interesting to mention that pharmacological blockade of δ-OR with naltrindole in the CeA reduces expression of CPP induced by ethanol in rats (Bie et al., 2009). Activation of κ-OR has been shown to blunt acquisition of ethanol CPP (Logrip et al., 2009). Supporting these results, κ-OR knockout mice also showed enhanced ethanol CPP (Femenía and Manzanares, 2012). ACETALDEHYDE: A PSYCHOACTIVE METABOLITE The specific mechanism by which ethanol modulates the activity of the EOS remains to be understood. Evidence indicates that one possible mechanism might involve the role of acetaldehyde, the first metabolite of ethanol (Miquel et al., 2003;Sanchis-Segura et al., 2005b;Pastor and Aragon, 2008). Acetaldehyde is a psychoactive compound that produces behavioral and neurochemical effects suggested to mediate at least some of the effects of ethanol. Acetaldehyde is self-administered orally (Peana et al., , 2012Cacace et al., 2012) and directly into the brain (Brown et al., 1979;McBride et al., 2002;Rodd-Henricks et al., 2002;Peana et al., 2011). Its administration induces CPP (Smith et al., 1984;Quertemont and De Witte, 2001;Peana et al., 2009;Spina et al., 2010) as well as behavioral stimulation and sensitization when centrally administered (Arizzi et al., 2003;Correa et al., 2003aCorrea et al., ,b, 2009Rodd et al., 2005;Arizzi-LaFrance et al., 2006;Sánchez-Catalán et al., 2009). The oxidation of ethanol to acetaldehyde in the brain is essentially mediated by the catalase-H 2 O 2 system (Aragon et al., 1992a;Gill et al., 1992). Reduced brain catalase activity, which have been seen to decrease ethanol-derived central acetaldehyde formation in brain tissue preparations (Hamby-Mason et al., 1997) and in the brain of free-moving rats (Jamal et al., 2007), decreases ethanol consumption (Aragon and Amit, 1992; Koechling and Amit, 1994;Correa et al., 2004;Karahanian et al., 2011), ethanol-induced locomotor stimulation (Aragon et al., 1992b;Correa et al., 1999bCorrea et al., , 2004Sanchis-Segura et al., 1999a,b,c;Pastor et al., 2002;Pastor and Aragon, 2008), the anxiolityc effects of alcohol (Correa et al., 2008) and modulates ethanol-induced CPP . Strategies aimed at increasing the production of brain acetaldehyde via an enhancement in activity of the enzymatic catalase system have also been used. These manipulations produced an increase in the motor stimulant properties of ethanol in mice Pastor et al., 2002). Other ethanol-induced effects such as taste aversion (Aragon et al., 1985) and social memory recognition have also been seen to be modulated by changes in brain catalase (Manrique et al., 2005). ACETALDEHYDE-INDUCED CHANGES IN THE OPIOIDERGIC NEUROTRANSMISSION The NArc, the main site of β-endorphin synthesis in the brain, is one of areas with the highest levels of catalase expression (Moreno et al., 1995;Zimatkin and Lindros, 1996) and lower levels of the acetaldehyde-degrading enzyme aldehyde dehydroge-Frontiers in Behavioral Neuroscience www.frontiersin.org July 2013 \| Volume 7 \| Article 93 \| 3 nase (Zimatkin et al., 1992). Therefore, it has been thus suggested that catalase-dependent formation of acetaldehyde into the NArc might mediate ethanol-induced increases in the release of βendorphin from the NArc in turn activating OR at the level of the VTA/NAcb to stimulate behavioral and neurophysiological actions (Sanchis-Segura et al., 2005a;Pastor and Aragon, 2008). Supporting this hypothesis, several authors (Reddy and Sarkar, 1993;Pastorcic et al., 1994;Reddy et al., 1995) have demonstrated that ethanol-induced increases in hypothalamic β-endorphin release are, indeed, mediated by acetaldehyde (Reddy and Sarkar, 1993;Pastorcic et al., 1994;Reddy et al., 1995). Hypothalamic cell cultures exposed to ethanol (12.5-100 μM) led to the formation of acetaldehyde (8-24 μM) and similar concentrations of acetaldehyde (12.5-50 μM) were able to stimulate β-endorphin release when tested in the absence of ethanol (Reddy and Sarkar, 1993;Pastorcic et al., 1994). Moreover, pretreatment of hypothalamic cell cultures with catalase inhibitors caused dose-dependent decreases in ethanol-stimulated βendorphin secretion (Reddy et al., 1995). Another line of research linking the EOS and acetaldehyde is the investigation of the actions of salsolinol (for a review see Hipólito et al., 2012), the condensation product of DA and acetaldehyde. Salsolinol has been shown to alter enkephalinreceptor site binding (Lucchi et al., 1982) and other OR an effect that is blocked by naloxone (Fertel et al., 1980). Interestingly, intra-NAcb administration of salsolinol increases DA levels when microinjected in the core and decreases DA levels if the administration is in the NAcb shell ) in a similar way to μand δ-OR agonists (Hipólito et al., 2008). It has been demonstrated that μ 1 -OR receptors exert a tonic modulatory control over activity of the DA system (Di Chiara and North, 1992;Devine et al., 1993). Thus, one possible mechanism by which salsolinol exerts its effects on the OR could be disinhibiting DA neurons in the VTA. Upholding this hypothesis, intra-posterior VTA administration of salsolinol induces a μ-OR dependent increase in DA levels in the NAcb shell (Hipólito et al., 2011). Accordingly, it has been recently shown that salsolinol excites DA neurons of the VTA, by activating μ-OR on local GABA interneurons (Xie et al., 2012). EVIDENCE OF BEHAVIORAL EFFECTS OF ACETALDEHYDE MEDIATED BY THE ENDOGENOUS OPIOID SYSTEM Whereas accumulating evidence indicates that the EOS participates in the behavioral effects of ethanol, only few studies have studied the involvement of this system in acetaldehyde effects. Self-administration of acetaldehyde appears to be mediated by the EOS; high doses of naloxone reduced intravenous acetaldehyde self-administration in rats, and naltrexone reduced the maintenance, the deprivation effect, and operant break points of acetaldehyde voluntary consumption (Myers et al., 1984;Peana et al., 2011). Treatment with naloxonazine, a specific μ 1 -OR antagonist reduces maintenance of acetaldehyde oral selfadministration . Blockade of μ-OR using either naltrexone or the irreversible and selective μ-OR antagonist β-funaltrexamine suppress the locomotor stimulation effect of acetaldehyde when microinjected into the rat posterior VTA . Additionally, Hipólito et al. (2010) have provided data supporting the hypothesis that acetaldehyde may mediate the actions of ethanol through a mechanism dependent on μ-OR activation. These authors showed that intraposterior VTA injections of salsolinol induced locomotor stimulation and sensitization in rats; stimulation (but not sensitization) was prevented by μ-OR antagonism. Finally, Sanchis-Segura et al. (2005b) demonstrated that administration of a catalase inhibitor directly into the NArc is sufficient to prevent the effects of ethanol on rat locomotion. Conversely, locomotor stimulation induced by ethanol injected directly into the NArc, was prevented by catalase inhibition or naltrexone, indicating a link between the behavioral effects of a reduction in acetaldehyde formation and the antagonism of μ-OR (Pastor and Aragon, 2008). The NArc, therefore, may represent a critical site to link two independent but related hypotheses: (1) the hypothesis proposing that acetaldehyde may mediate some of the psychopharmacological actions attributed to ethanol (Aragon et al., 1992a;Smith et al., 1997;Quertemont et al., 2005;Correa et al., 2012) and (2) the hypothesis that suggests that the β-endorphin/μ-OR system participate in the reinforcing and psychomotor effects of ethanol (Stinus et al., 1980;Herz, 1997;Gianoulakis, 2001;Sanchis-Segura et al., 2005b;Pastor and Aragon, 2008). Early findings also suggested a role of the opioidergic system in mediating CPP induced by salsolinol in rats (Matsuzawa et al., 2000). Antagonism of μ-OR attenuated CPP induced by salsolinol when achieved under fear stress (Matsuzawa et al., 2000). Moreover, intra-posterior VTA administration of salsolinol, that produced CPP in rats, also produced an increase in DA in the NAcb that was suppressed by β-funaltrexamine administration (Hipólito et al., 2011). SUMMARY AND PERSPECTIVES In the present review we have summarized consistent results indicating that the EOS, and particularly β-endorphin and μ-OR, are critically involved in the psychopharmacological effects of ethanol. Additionally, we have reviewed a large body of data that indicates that the first metabolite of ethanol, acetaldehyde, might be responsible for the activation of β-endorphin release and μ-OR signaling after ethanol administration. There are two main lines of research suggesting a link between acetaldehyde and the EOS: (1) formation of acetaldehyde in brain areas such as the NArc, with high expression of ethanol metabolizing enzymes and presence of cell bodies of endorphinic neurons and (2) the formation of condensation products between DA and acetaldehyde such as salsolinol, which exerts its actions via μ-OR. To a certain degree both lines of research show important incompatibility. The fact that the lesions of the NArc are sufficient to block ethanol-induced behaviors challenge the putative role of salsolinol formed in other non-hypothalamic areas. Future studies will need to explore how to reconcile those two sets of data, and to clarify what is sufficient and/or necessary for acetaldehyde to induce behavioral responses mediated by the EOS. Finally, it is interesting to mention that most of the data suggesting a role of the EOS in acetaldehyde-induced behavioral effects have been linked to acetaldehyde-induced changes in the opioid system that are suggested to impact behavior via modulation of the DA system . Ethanol as well Frontiers in Behavioral Neuroscience www.frontiersin.org July 2013 \| Volume 7 \| Article 93 \| 4 as acetaldehyde activate firing of dopaminergic neurons in the VTA (Foddai et al., 2004;Diana et al., 2008) and stimulate DA transmission in the NAcb (Melis et al., 2007;Enrico et al., 2009;Sirca et al., 2011), effects that are prevented by D-penicillamine, a sequestering agent of acetaldehyde . A recent study demonstrates that in rats, ethanol and acetaldehyde induce via DA D 1 receptors, ERK phosphorylation in the NAcb and extended amygdala . This effect is blocked by D-penicillamine and by naltrexone, suggesting that the opiodergic modulation of the reinforcing properties of acetaldehyde could be mediated by the dopaminergic system Peana et al., 2011). There are other effects such as ethanol-induced CPP, ethanol drinking in some nonoperant conditions and even ethanol-induced sensitization that appear to have a less straightforward involvement of DA signaling (Risinger et al., 1992;Broadbent et al., 1995;Spina et al., 2010;Young et al., 2013). Future research will need to investigate DA-dependent and independent mechanisms by which acetaldehyde might induce behavioral responses via its modulation of the EOS.	v3-fos-license
2018-04-03T02:01:03.762Z	2015-05-08T00:00:00.000	18605180	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "BRONZE", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/anie.201502157", "pdf_hash": "9b98db4d14b0919734113525e1beee4149db9752", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2199", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "f65e5427aebf72b4ae901d4c73037e4cd07eebc7", "year": 2015 }	pes2o/s2orc	Two-Photon Voltmeter for Measuring a Molecular Electric Field** We present a new approach for determining the strength of the dipolar solute-induced reaction field, along with the ground- and excited-state electrostatic dipole moments and polarizability of a solvated chromophore, using exclusively one-photon and two-photon absorption measurements. We verify the approach on two benchmark chromophores N,N-dimethyl-6-propionyl-2-naphthylamine (prodan) and coumarin 153 (C153) in a series of toluene/dimethyl sulfoxide (DMSO) mixtures and find that the experimental values show good quantitative agreement with literature and our quantum-chemical calculations. Our results indicate that the reaction field varies in a surprisingly broad range, 0–107 V cm−1, and that at close proximity, on the order of the chromophore radius, the effective dielectric constant of the solute–solvent system displays a unique functional dependence on the bulk dielectric constant, offering new insight into the close-range molecular interaction. Materials and 1-Photon Absorption Measurements Toluene (> 99.9%), DMSO (> 99.9%), C153 (99%) and PRODAN (98%) were purchased from Sigma-Aldrich and used as received. Linear spectroscopic measurements were performed on a Perkin Elmer Lambda 950 spectrophotometer in 1 cm spectroscopic grade quartz cuvettes for all mixtures of Toluene and DMSO in ratios given in Table S1. Extinction coefficients were measured by the weight method. Table S1: Solvent mixtures used for each chromophore (e.g. 7:1 indicates 7 parts Toluene to 1 part DMSO by volume). X indicates that a 1PA and 2PA spectroscopic measurements were performed in that solvent mixture. 2-Photon Absorption Measurements Spectral shapes were obtained using the two-photon excited fluorescence method by employing a tunable optical parametric oscillator (OPO, Insight DeepSee, Spectra Physics) with a step size of 1 nm over the ranges of 680-900 nm (PRODAN) and 700-1025 nm (C153). Fluorescence was collected at a right angle to the incident beam with a PMT. Power dependence was measured at each wavelength with a stepper motor-controlled variable neutral density filter wheel (Thorlabs) that measured the fluorescence intensity for 20 steps over the full range of the wheel (OD 2). The intercept corresponding to the linear fit to the loglog plot of the fluorescence vs. incident power data was used to determine the relative value of the 2PA cross section at each wavelength. Only those points were used where the slope of the power dependence was verified to be in the range 2.00 ± 0.05. Correction for the beam shape was determined by imaging the beam at each wavelength with a CCD camera (Stingray, Basler). Pulse durations were measured using a home-built second harmonic generation autocorrelator using BBO as the doubling material, and were in the range 90-120 fs. The pulse repetition rate of the laser is 80 Mhz, and was chopped down to 70 Hz using a mechanical chopper wheel to alleviate thermal effects in the sample. Excitation power values were adjusted for the solvent absorption based on the linear spectroscopic measurements of each respective mixture. Absolute cross section values were measured for PRODAN (C153) at 750 nm relative to BDPAS (Fluorescein) using the reference method as described in detail earlier [1] . 2-Level Model Description Degenerate two-photon absorption is formally described via second order perturbation theory with the sumover-states expression [2] ( where σ2 is the 2PA cross section, ν/2 is the two-photon excitation energy, νim is the transition energy between states i and m, h is Planck's constant, c is the speed of light in vacuum, n is the refractive index of the medium, e is the polarization unit vector of the optical electric field, im mj , µ µ are the dipole moments between states i and m and between states m and j, respectively (note that if i = m or if m = j, these are permanent dipole moments), ( ) j g ν is the transition line shape of the final state, f is the local field factor, taken here to be the Lorentz factor, 2 2 3 n + , and Ω represents isotropic averaging over all orientations of the absorbing molecules relative to the direction of e . For the 2-level system approximation, i = 0, j = 1, and only the indices m = 0,1 are included in the summation, so that where 10 µ ∆ is the permanent dipole moment difference between the ground (0) and first excited (1) states, and β is the angle between the 10 µ ∆ and 10 µ vectors. Note here that the averaging over Ω assumes an isotropic distribution of molecular orientations in solution. Finally, using the relationship between the transition moment and molar extinction coefficient, ( ) M ε ν [3] , where NA is Avogadro's number, and assuming that 0 β ≈ , the 2-level 2PA cross section is given as Rearranging for 10 µ ∆ and converting into the units of Eqn. (1) in the main paper, . MATLAB Optimization Routine Description The model line shape is calculated based on Eq. (10) from the main text, where the peak transition energy and width are extracted from the fit to Eq. (7) for approximately 250 million combinations of the 5 unknown parameters over a broad input value interval (see below). The program then subtracts these model results from the experimental data to calculate the residual, and stores the product of the absolute value of the residuals (peak and width) in an array corresponding to the parameter values used for that particular calculation. The parameter interval ranges are then iteratively reduced until convergence of the results is achieved within the expected experimental uncertainty (±10%). The coordinates of the minimum value in this final 5-D array then correspond to the parameter values that best reproduce the experimental spectra, according to our model. Note that the experimental data are sufficient to solve simultaneously for 5 parameters, given that for convergence, a global fit must be obtained for the values of the peak transition energy and its first 2 derivatives vs. ε, as well as the values for the width and its first derivative vs. ε (see Figures S4, S5). The initial parameter variation intervals for both chromophores were the same and are given as follows: S S a : Flow chart outlining MATLAB parameter optimization procedure. Computational Details Ground state conformational analysis of Prodan and Coumarin C153 compounds in a gas phase has been carried out at BP86/TZVPP level using the Turbomole [4] software package. The resulting structures were re-optimized at B3LYP/6-311G(d,p) level in Gaussian09 [5] and the absence of imaginary frequencies was confirmed. Vertical excited state properties of the lowest singlet states were obtained from Gaussian09 TDDFT calculations using the Coulomb Attenuated Method CAM-B3LYP functional and the 6-311++G(d,p) basis set for the ground state geometries of Prodan and Coumarin 153. For Prodan, adiabatic properties of the first excited singlet state were also explored by optimizing the structure of the excited state at TD/CAM-B3LYP/6-311++G(d,p) level. The effects of solvation on excited state properties of Prodan were studied in toluene and dimethyl sulfoxide (DMSO) using linear response (LR) equilibrium IEF-PCM solvation (toluene and DMSO), as well as non-equilibrium LR and state-specific IEF-PCM solvation (DMSO) combined with the TDDFT approach. Excited-state dipole moments were obtained from relaxed excited-state electron densities computed by Z-vector method as implemented in Gaussian09. Excited-state polarizability was computed as the numerical derivative of the dipole moment. The isotropic average of the polarizability in the ground and excited states is used for comparison in Table 1. Unless specified otherwise, the results correspond to the lowest energy conformers of Prodan and Coumarin 153 (ground state gasphase reference). Figure S1 shows the measured 1PA (solid line) and 2PA (black markers) spectra for PRODAN (left) and C153 (right), in all measured solvent mixtures. C153 was measured in fewer mixtures for the sake of expediency, once it was determined that this was sufficient for reasonable determination of the parameters. Figures S4 and S5 show the dependence of the 1PA peak energy (Gaussian fit for the lowest energy transition) on the dielectric constant for PRODAN (left) and C153 (right). The symbols are experimental data and the red curves show the fits to the data. Figure S6 shows the dependence of the 1PA lowest energy band width (Gaussian fit for the lowest energy transition) on the dielectric constant for PRODAN (left) and C153 (right). The symbols are experimental data and the red curves show the fits to the data. Figure S7 shows the final predicted 1PA spectra (black markers) and fits to the experimental spectra (red lines). The model predictions almost exactly reproduce the experimental data. This is to be expected, as we optimized the parameters in order to reproduce the 1PA spectral data in the MATLAB routine. Discussion of Cavity Model: The empirical formula for the power dependence of the interior dielectric constant on that of the bulk solvent was chosen in order to account for close-range intramolecular interactions while still maintaining the conditions defining the reaction field. These conditions maintain that: 1) in ε ε ≤ for all dielectric constants so that the reaction field points in the direction of 0 µ and therefore lowers, instead of increasing, the energy of the system, and; 2) = , so that in the vacuum limit the reaction field goes to zero. It is commonly assumed that the interior of the cavity is vacuum, but this does not account for the effect of close-range interactions which can sometimes play a dominant role. We take the approach of assuming that the "dielectric" response of the solute molecule is related to the dielectric properties of the solvent. How the value for p is related to the type and magnitude of close-range interaction remains to be determined. However, an interesting comparison can be made between our power dependence and the Lippert-Mataga orientational polarizability function ∆f, given in Eq. (S6), where n is the solvent refractive index, taken here to be the average value of 1.488 [6] . Figure S8 compares the ε dependence of f from Eq. It can be observed that the ∆f function provides a reasonable estimate to the behavior, but saturates more rapidly than our function f as a consequence of the constant (vacuum) values for the interior dielectric constant and refractive index. Changing the value of p slightly from 0.65 to 0.8 yields a lower scaling for the reaction field, perhaps indicative of a different electrostatic response to the solvent environment. Uncertainty Estimation: 1) Experimentally determined parameters (νvac, ∆νvac, ∆µvac, ∆α): The error in νvac ( Figure S5) is expected to be primarily determined by the accuracy of the Gaussian fitting. This is because: 1) f(p) was optimized in order to minimize the standard error between the linear fits to the peak transition energy dependence (8 cm -1 and 15 cm -1 for PRODAN and C153, respectively); and 2) The lack of resolution of the peaks in the absorption band prevented locating the peaks with an accuracy less than these standard errors. The uncertainty in νvac is therefore estimated at ~5% of the width of the total 1PA band, or ±200 cm -1 . The main contribution to the uncertainty in the accuracy of our optimized parameter values is due to that associated with the reference standards used for the determination of 2PA cross section values. This uncertainty is estimated at ±10% of σ2. This corresponds to approximately ±5% of ∆µ, and is a systematic error expected to propagate to all solvent mixtures. Therefore, the result of this error is a systematic discrepancy in ∆µvac of ±5%.	v3-fos-license
2018-04-03T04:36:47.148Z	2017-02-13T00:00:00.000	4490251	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/292/13/5253.full.pdf", "pdf_hash": "67608ccd3d74df87a1379b088f59f5094b24be27", "pdf_src": "Highwire", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2210", "s2fieldsofstudy": [ "Biology", "Medicine", "Chemistry" ], "sha1": "3d00c80d1f2f0702ecf264cabfb7adfd2da1b121", "year": 2017 }	pes2o/s2orc	Metformin Promotes AMP-activated Protein Kinase-independent Suppression of ΔNp63α Protein Expression and Inhibits Cancer Cell Viability* The blood glucose modifier metformin is used to treat type II diabetes and has also been shown to possess anticancer activities. Recent studies indicate that glucose deprivation can greatly enhance metformin-mediated inhibition of cell viability, but the molecular mechanism involved in this inhibition is unclear. In this study, we report that, under glucose deprivation, metformin inhibited expression of ΔNp63α, a p53 family member involved in cell adhesion pathways, resulting in disruption of cell matrix adhesion and subsequent apoptosis in human squamous carcinoma cells. We further show that metformin promoted ΔNp63α protein instability independent of AMP-activated protein kinase and that WWP1, an E3 ligase of ΔNp63α, was involved in metformin-mediated down-regulation of ΔNp63α levels. In addition, we demonstrate that a combination of metformin and the glycolysis inhibitor 2-deoxy-d-glucose significantly inhibited ΔNp63α expression and also suppressed xenographic tumor growth in vivo. In summary, this study reveals a new mechanism for metformin-mediated anticancer activity and suggests a new strategy for treating human squamous cell carcinoma. Metformin is widely used to treat type II diabetes. Metformin can decrease blood glucose via enhancing cell sensitivity to insulin (1), inhibition of the mitochondrial respiratory chain (complex I), activation of AMP-activated protein kinase (AMPK), 3 and inhibition of glucagon-induced elevation of cAMP. Metformin can also suppress hepatic glucose production and enhances peripheral glucose uptake (2)(3)(4). In recent years, accumulating clinical evidence indicates that metformin may have anticancer activities. It has been reported that tumor incidence is reduced in cancer patients with diabe-tes treated with metformin (5). Metformin can inhibit tumor cell growth and survival, induce cellular senescence (6,7), and enhance cancer cell chemosensitivity (8). At the molecular level, metformin has been shown to inhibit mammalian target of rapamycin (mTOR) signaling (9) and suppress expression of cyclin D1 and ErbB2 (Her2) (10 -12). p63 is a p53 family member involved in multiple facets of biological processes, including embryonic development, cell proliferation, differentiation, survival, apoptosis, senescence, and aging (21). The p63 gene encodes multiple protein isoforms that are derived from two different promoters at the N terminus and alternative splicing at the C terminus. ⌬Np63␣ is the predominant p63 protein isoform expressed in epithelial cells and is essential for epithelial development (22). ⌬Np63␣ is frequently overexpressed in squamous cell carcinoma and has been reported to promote cancer cell proliferation and survival (23). On the other hand, it has been shown that ⌬Np63␣ is a master transcriptional regulator of cell adhesion program through regulation of several adhesion molecules, including integrin ␤4, integrin ␣6, fibronectin1, and E-cadherin (24). 2-Deoxy-D-glucose (2-DG) is a glucose analog in which the 2-hydroxyl group is replaced by hydrogen. It suppresses glycolysis by competitively inhibiting the production of glucose-6-P from glucose (25). Because 2-DG hampers cell growth, it was developed as a potential tumor-therapeutic drug, and it is currently in clinical trials (26). However, it is not completely clear how 2-DG inhibits cell growth. In this study, we show that metformin inhibited ⌬Np63␣ expression, resulting in disruption of cell matrix adhesion and subsequent apoptosis in human squamous carcinoma FaDu cells. Glucose deprivation dramatically facilitated the action of metformin. Combination of metformin and 2-DG significantly inhibited ⌬Np63␣ expression and xenographic tumor growth in vivo. Together, this study reveals a new mechanism for metformin-mediated anticancer activity and suggests a new strategy for treating human squamous cell carcinoma. Metformin Reduces Cancer Cell Viability under Glucose Deprivation-It has been reported that metformin possesses anticancer activities, but the molecular mechanism(s) is/are not entirely clear. To examine the effects of metformin on human squamous cancer cells, we first treated human head and neck squamous cell carcinoma FaDu cells in the absence or presence of metformin. As shown in Fig. 1, A and B, Metformin treat-ment of FaDu cells (grown under 4.5 mg/ml glucose) for 36 h had little effect on viability. Treatment with metformin for a longer time (48 h) led to significantly reduced cell viability, examined by MTS and trypan blue exclusion assay. By contrast, cell viability was comparable in the absence of metformin. Because culturing cells consumed glucose, we speculated that glucose concentration may be an important factor affecting metformin efficacy. We therefore examined the influence of glucose deprivation (1.0 mg/ml) on the effects of metformin. As shown in Fig. 1, C-F, under 1.0 mg/ml glucose, treatment of metformin for 24 h dramatically reduced cell viability. In addition, data from Western blotting analyses for cleaved PARP1, a marker of apoptosis, and data from FACS analyses indicated a significant increase in apoptosis. 12,24,36,48, or 60 h) in DMEM containing 4.5 mg/ml glucose (Glu). Cell viability was determined using an MTS assay (A) or trypan blue exclusion assay (B). OD, optical density. C-F, FaDu cells were treated with metformin (20 mM) for the indicated time (0, 12, 24, or 36 h) in DMEM containing 1.0 mg/ml glucose. MTS (C) and trypan blue exclusion (D) assays for cell viability, Western blotting analyses (E), and an apoptotic assay using Annexin V/PI staining (F) were performed. G-J, FaDu cells were treated with or without 20 mM metformin in the presence or absence of 20 mM 2-DG in DMEM containing 4.5 mg/ml glucose for 24 h. MTS (G) and trypan blue exclusion (H) assays for cell viability, Western blotting analyses (I), and an apoptotic assay using Annexin V/PI staining (J) were performed. Con, control. K and L, H596 and H292 cells were treated with metformin (20 mM) for 24 h in DMEM containing either 1.0 mg/ml glucose (K) or 4.5 mg/ml glucose in the presence or absence of 20 mM 2-DG (L). Cell viability was determined using MTS and trypan blue exclusion assays. Data are presented as means Ϯ S.E. from three independent experiments in triplicates. *, p Ͻ 0.001; , p Ͻ 0.01. 2-DG Is an Inhibitor of Glycolysis. Therefore, we examined whether 2-DG can affect the effect of metformin on cancer cell viability. As shown in Fig. 1, G-J, treatment with metformin or 2-DG, alone had little effect on the viability of FaDu cells under 4.5 mg/ml glucose. By sharp contrast, combined treatment of metformin and 2-DG significantly reduced FaDu cell viability, as evidenced by MTS and trypan blue exclusion assays, Western blotting for cleaved PARP1, and FACS analyses. Furthermore, similar to FaDu cells, metformin significantly reduced the cell viability of human lung adenosquamous carcinoma H596 cells and human mucoepidermoid pulmonary carcinoma H292 cells under glucose deprivation or treatment with 2-DG ( Fig. 1, K and L). Metformin Disrupts Cell Matrix Adhesion Concomitant with Reduced ⌬Np63␣ Expression-The abovementioned results indicated that metformin can dramatically reduce cancer cell viability under glucose deprivation. Notably, metformin treat-ment resulted in dramatic cell detachment under either low glucose (1.0 mg/ml) or with 2-DG (Fig. 2, A and B). We therefore examined the effects of metformin on the expression of adhesion molecules involved in cell matrix interaction, including integrin ␤1, integrin ␤4, integrin ␣6, and fibronectin 1. As shown in Fig. 2C, metformin significantly inhibits these adhesion molecules, except for integrin ␤1. Because ⌬Np63␣ is known to regulate the cell adhesion program in epithelial cells (24), we investigated whether metformin can impact the expression of ⌬Np63␣. Treatment of metformin for 24 h did not affect ⌬Np63␣ expression at 4.5 mg/ml glucose whereas it led to a dramatic decrease in ⌬Np63␣ expression at 1.0 mg/ml glucose (Fig. 2D) in a dose-and time-dependent manner (Fig. 2, E and F). In addition, 2-DG alone had little effect on ⌬Np63␣ expression. However, 2-DG significantly enhanced metformininduced down-regulation of ⌬Np63␣ under 4.5 mg/ml glucose (Fig. 2G). Moreover, metformin also dramatically decreased . Microscopic images of the cells were taken using a phase-contrast microscope, and typical images are shown. Culture plates were gently washed with cold PBS, and attached cells were trypsinized and counted with a hemocytometer. Con, control. C, FaDu cells were treated with metformin (10 mM) for 0, 12, or 24 h under 1.0 mg/ml glucose and subjected to Western blotting analyses for the indicated protein as shown. D, FaDu cells treated with metformin (10 mM) for 24 h in DMEM containing either 4.5 mg/ml or 1.0 mg/ml glucose. Whole-cell lysates were subjected to Western blotting analyses as indicated. E and F, FaDu cells grown in DMEM containing 1.0 mg/ml glucose were treated with metformin (0, 5, 10, 15, or 20 mM) for 24 h (E) or with 10 mM metformin for 0, 6, 12, 18, or 24 h (F). Cell lysates were subjected to Western blotting as indicated. G, FaDu cells were treated with metformin (10 mM) in the presence or absence of 2-DG at the indicated dose for 24 h. Whole-cell lysates were subjected to Western blotting analyses as indicated. H, H596 and H292 cells were treated with metformin (10 mM) under 1.0 mg/ml glucose for 24 h. Cell lysates were subjected to Western blotting as indicated. Data are presented as means Ϯ S.E. from three independent experiments in triplicates. Scale bars ϭ 50 m. *, p Ͻ 0.001. ⌬Np63␣ expression in H596 and H292 cells under 1.0 mg/ml glucose (Fig. 2H). Together, these findings indicate that metformin can significantly down-regulate ⌬Np63␣ expression under glucose deprivation concomitant with cell matrix disruption. Metformin Induces ⌬Np63␣ protein Instability Independent of AMPK-We next investigated the molecular mechanism with which metformin down-regulates ⌬Np63␣ protein expression. We examined the effects of metformin on ⌬Np63␣ protein half-life by cycloheximide (CHX) treatment. As shown in Fig. 3A, metformin dramatically reduced ⌬Np63␣ protein half-life. In addition, metformin-induced down-regulation of ⌬Np63␣ protein was significantly, but not completely, blocked by MG132, suggesting that the proteasome is largely involved in metformin-mediated ⌬Np63␣ protein instability (Fig. 3B). Because we have shown previously that WWP1, an ubiquitin E3 ligase, regulates ⌬Np63␣ protein stability (27), we examined the effect of metformin on WWP1 expression. As shown in Fig. 3C, metformin evidently increased WWP1 protein levels but not ITCH, which is documented as another ⌬Np63␣ E3 ligase (28). Silencing of WWP1 led to a clear increase in ⌬Np63␣ protein levels and restored, in part, expression of ⌬Np63␣ upon metformin treatment (Fig. 3D). These data indicate that WWP1 is important in metformin-mediated destabilization of ⌬Np63␣. It has been reported that metformin can inhibit mTOR signaling to impact cell proliferation and survival (29). We therefore examined the effect of mTOR on the expression of ⌬Np63␣. Our data showed that metformin significantly inhibited phosphorylation of both mTOR and S6K concomitant with down-regulation of ⌬Np63␣. Knockdown of mTOR1 or rapamycin treatment led to a marked reduction in ⌬Np63␣ protein FIGURE 3. WWP1 is important for metformin-induced ⌬Np63␣ protein instability. A, FaDu cells were treated with or without metformin (Met, 10 mM) for 12 h prior to treatment with 50 g/ml CHX for the indicated time intervals under 1.0 mg/ml glucose (Glu). Whole-cell lysates were subjected to Western blotting analyses as indicated. Western blotting images were analyzed using Image Lab analysis, and protein half-life was plotted as shown. Con, control. B, FaDu cells were treated with metformin (10 mM) for 12 h prior to treatment of MG132 (5 and 10 M) for 12 h under 1.0 mg/ml glucose. Whole-cell lysates were subjected to Western blotting analyses as indicated. C, FaDu cells were treated with metformin (10 mM) for 0, 6, 12, or 18 h under 1.0 mg/ml glucose. Cell lysates were subjected to Western blotting as indicated. D, FaDu cells stably expressing shWWP1-1, shWWP1-2, or shGFP were treated with metformin (10 mM) for 24 h under 1.0 mg/ml glucose. Whole-cell lysates were subjected to Western blotting analyses as indicated. E, FaDu cells were treated with metformin (10 mM) for 0, 12, or 24 h under 1.0 mg/ml glucose. Cell lysates were subjected to Western blotting as indicated. F, whole-cell lysates derived from FaDu cells stably expressing shmTOR or shGFP were subjected to Western blotting analyses as indicated. G, FaDu cells were treated with rapamycin (0, 0.1, 1, 10, and 100 M) for 24 h. Cell lysates were subjected to Western blotting as indicated. H, FaDu cells were left untreated or treated with 10 mM metformin for 24 h. Steady-state ⌬Np63␣ mRNA levels were determined by Q-PCR analysis. I, FaDu cells were treated with metformin (10 mM) in DMEM containing 1.0 mg/ml glucose for the indicated time intervals (0, 1, 2, 4, or 6 h). Whole-cell lysates were subjected to Western blotting analyses as indicated. J, whole-cell lysates derived from FaDu cells stably expressing shAMPK␣ or shGFP were subjected to Western blotting analyses as indicated. K, FaDu cells stably expressing shAMPK␣ were treated with metformin (10 mM) for 0, 18, or 24 h under 1.0 mg/ml glucose. Cell lysates were subjected to Western blotting analyses as indicated. Data are presented as means Ϯ S.E. from three independent experiments. , p Ͻ 0.001. levels (Fig. 3, E-G). These data suggest that mTOR signaling is involved in metformin-mediated down-regulation of ⌬Np63␣. Furthermore, Q-PCR analyses showed that metformin significantly decreased steady-state ⌬Np63␣ mRNA levels (Fig. 3H). It is well documented that metformin can activate AMPK (30,31) and inhibit mTOR signaling (9). When examined, metformin reduced phosphorylation of mTOR and S6K, as expected. However, metformin had little effect on AMPK phosphorylation in FaDu cells (Figs. 2 and 3I), suggesting that metformin failed to activate AMPK in this experimental setting. To further examine the role of AMPK in metformin-mediated regulation of ⌬Np63␣, we performed shRNA experiments. As shown in Fig. 3J, silencing of AMPK␣1 had no significant effect on ⌬Np63␣ expression. Furthermore, metformin could still dramatically decrease ⌬Np63␣ expression upon silencing of AMPK␣1 (Fig. 3K). Together, these data indicate that metformin inhibits ⌬Np63␣ expression independently of AMPK. ⌬Np63␣ Is Critical in Metformin-induced Cell Matrix Adhesion and Cell Death-Because metformin disrupts cell matrix adhesion and induces subsequent apoptosis concomitant with down-regulation of ⌬Np63␣ expression, we first demonstrated that suspension of FaDu cells led to a significant decrease in cell viability (Fig. 4A). We also found that silencing of p63 led to significant down-regulation of several adhesion molecules, including fibronectin and integrin ␤4 (Fig. 4B). We therefore investigated whether reduced ⌬Np63␣ expression accounts for metformin-mediated cell detachment and apoptosis. As shown in Fig. 4, C-E, ectopic expression of ⌬Np63␣ failed to reduce cell detachment and/or apoptosis, suggesting that other factors, in addition to ⌬Np63␣, are critical to overcome the effects of metformin. Ectopic expression of ⌬Np63␣ increased expres- , and cell viability was measured using an MTS assay (E). F, FaDu cells stably expressing HA/FLAG-⌬Np63␣ was treated with or without metformin (10 mM) for 24 h under 1.0 mg/ml glucose and subjected to Western blotting analyses as indicated. G and H, FaDu cells were seeded on tissue culture plates coated with either 50 g/ml fibronectin, collagen 1, or BSA. Cells were then cultured for 36 h in the presence or absence of metformin (5 or 10 mM) under 1.0 mg/ml glucose. Quantitation of attached cells is presented (G); cell viability was determined using an MTS assay (H). I and J, FaDu cells stably expressing ⌬Np63␣ or vector control were seeded on tissue culture plates coated with or without fibronectin. Cells were then cultured in the presence or absence of 10 mM metformin in DMEM containing 1.0 mg/ml glucose for 36 h. Microscopic images of the cells and attached cells are presented (I), and cell viability was determined using an MTS assay (J). Data are presented as means Ϯ S.E. from three independent experiments in triplicates. , p Ͻ 0.001; , p Ͻ 0.01; *, p Ͻ 0.05; NS, not significant. sion of fibronectin 1, consistent with a previous report (24). However, expression of ⌬Np63␣ could only partially restore expression of fibronectin in the presence of metformin (Fig. 4F). To demonstrate that down-regulation of fibronectin plays an important role in metformin-induced disruption of cell matrix adhesion, we applied exogenous fibronectin, collagen 1, or BSA to the surface of culture plates prior to growing FaDu cells in the presence or absence of metformin. As shown in Fig. 4, G and H, although collagen 1 failed to restore cell attachment and cell viability, fibronectin could, in part, restore cell attachment and cell viability in the presence of metformin. Combination of ectopic ⌬Np63␣ expression and exogenous fibronectin led to complete restoration of cell attachment and cell viability (Fig. 4, I and J). Together, these results indicate that downregulation of ⌬Np63␣ contributes to metformin-induced cell detachment and death. Combination of Metformin and 2-DG Inhibits ⌬Np63␣ Expression and Suppresses Tumor Growth in Vivo-Because our aforementioned results indicate that glucose deprivation or 2-DG significantly enhances metformin-induced cell apoptosis, we examined the effects of metformin and 2-DG on tumor cell growth in vivo. Results from tumor xenograph experiments showed that metformin and 2-DG together significantly inhibited tumor growth, as evidenced by measurement of tumor volume (reduced about 50%) (Fig. 5A), weight (reduced about 40%) (Fig. 5B), and tumor size (Fig. 5C). Notably, metformin and 2-DG together dramatically down-regulated p63 expression, as evidenced by immunohistochemical staining of p63 in tumor sections (Fig. 5D). These results indicate that inhibition of glycolysis is critical in metformin-mediated suppression of tumor growth in vivo. Discussion Metformin is frequently used to treat type II diabetes. Emerging evidence indicates that metformin also has antitumor activities. In this study, we show that metformin induces cell death upon glucose deprivation or inhibition of glycolysis. We further demonstratethatmetforminpromotes⌬Np63␣instabilityindependent of AMPK, leading to disrupted cell matrix adhesion and subsequent cell apoptosis. In addition, we show that WWP1 is critical in metformin-mediated inhibition of ⌬Np63␣ stability. Moreover, we demonstrate that combination of metformin and 2-DG significantly inhibits xenographic tumor growth in vivo. It has been well documented that metformin can decrease blood glucose levels via several means, including enhancing cell sensitivity to insulin (1), suppressing hepatic glucose production, and enhancing peripheral glucose uptake (2)(3)(4). Notably, it has been reported that glucose deprivation significantly impacts metformin anticancer activity (32,33), and most recently, Sabatini and co-workers (34) showed that glucose limitation is important for cancer sensitivity toward phenoformin, a member of the biguanide class like metformin; however, the mechanisms remain unclear. Our data clearly demonstrate that glucose deprivation or inhibition of glycolysis by 2-DG is essential for metformin effects on cell viability, and down-regulation of ⌬Np63␣ is critically important in this process. Notably, glucose deprivation has a more profound effect on metformin-induced apoptosis than that of 2-DG, suggesting that use of 2-DG (20 mM) under normal glucose (4.5 mg/ml) cannot mimic glucose deprivation (1.0 mg/ml) with regard to down-regulation of ⌬Np63␣. Our results show that metformin effectively induces cell detachment from the matrix and subsequent apoptosis under glucose deprivation. Work by us and other groups has demonstrated that ⌬Np63␣ functions as a master transcription regulator for a set of genes involved in cell adhesion, including integrins and fibronectin (24) as well as CD82 (35). Our results show that metformin inhibits ⌬Np63␣ expression upon glucose deprivation, resulting in decreased expression of fibronectin, integrin ␣6, and integrin ␤4, which leads to cell detachment from the matrix and subsequent apoptosis, a process termed anoikis (36). Notably, ectopic expression of ⌬Np63␣ can completely reverse metformin-induced cell detachment and apoptosis in the presence of exogenous fibronectin. In our experimental system, exogenous fibronectin is required likely because of inefficient restoration of fibronectin expression in the presence of metformin. How does metformin inhibit ⌬Np63␣ expression? We show that metformin promotes proteasome-dependent ⌬Np63␣ protein degradation. ITCH and WWP1 are two major ubiquitin E3 ligases of ⌬Np63␣ (27,28). Notably, treatment of metformin leads to up-regulation of WWP1 expression. Silencing of WWP1 leads to partial recovery of ⌬Np63␣ expression in the presence of metformin, suggesting that WWP1 and other factors as well play important roles in metformin-mediated inhibition of ⌬Np63␣. In addition, it has been reported that metformin can inhibit mTOR signaling. In this study, we also found that metformin inhibits mTOR signaling and that silencing of mTOR or inhibition of mTOR by rapamycin can also decrease ⌬Np63␣ expression. Furthermore, metformin can also suppress ⌬Np63␣ mRNA expression. Together, our data indicate that metformin impacts ⌬Np63␣ expression at multiple levels. AMPK plays a critical role in maintaining cell energy homeostasis and is important in the regulation of various biological processes, including cell proliferation, survival, senescence, and autophagy (16,17,37). It has been reported that metformin inhibits cancer cell growth via activation of AMPK, leading to activation of p53 (38) and inhibition of the mTOR pathway (29). In this study, we show that metformin inhibits of ⌬Np63␣ expression in an AMPK-independent manner. First, metformin fails to activate AMPK, whereas it inhibits ⌬Np63␣ expression in FaDu cells. Second, knockdown of PRKAA1 (AMPK␣1) does not affect ⌬Np63␣ expression. Most importantly, silencing of AMPK␣1 does not block metformin-mediated down-regulation of ⌬Np63␣ expression. Notably, metformin effects on cancer cell survival independent of AMPK have been reported previously (39,40). This study reveals a new mechanism by which metformin induces anoikis under glucose deprivation (Fig. 6). It is of interest that combination of metformin and 2-DG significantly inhibits ⌬Np63␣ expression and xenographic tumor growth in a mouse model, suggesting a new strategy for treating squamous cell carcinoma. Experimental Procedures Cell Culture and Drug Treatments-Human head and neck squamous cell carcinoma (HNSCC) FaDu and HEK 293T cells were cultured in DMEM supplemented with 10% FBS (Hyclone). Human lung adenosquamous carcinoma H596 cells and human mucoepidermoid pulmonary carcinoma H292 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. All Cells were grown in media supplemented with 100 units/ml penicillin and 100 g/ml streptomycin. Cells were maintained in a humidified 37°C incubator under a 5% CO 2 atmosphere. Cells grown to 75-85% confluence in high-glucose DMEM (4.5 mg/ml glucose) or low-glucose DMEM (1.0 mg/ml glucose) were treated with chemical compound(s), as indicated. Metformin (PHR1084), 2-DG (D8375), cycloheximide (R750107), and MG132 (M8699) were purchased from Sigma. FACS, Cell Viability, and Protein Stability Assays-For the Annexin V and PI double staining assay (C1062, Beyotime), both floating and adherent cells were collected and subjected to FACS analyses according to the instructions of the manufacturer. The cell viability assay (MTS) was performed using the CellTiter 96 kit (Promega) as described in the instructions from the manufacturer. The trypan blue exclusion assay (C0011, Beyotime) was performed according to the instructions of the manufacturer. To determine the protein half-life of ⌬Np63␣, FaDu cells were grown in DMEM containing 1.0 mg/ml glucose in the presence or absence of 10 mM metformin for 12 h, followed by addition of CHX (50 g/ml). Cells were then collected at the indicated time intervals (0, 3, 6, 9, and 12 h). Cell lysates were subjected to Western blotting analyses. In Vivo Tumor Formation Assay-All animal care and animal experiments in this study were carried out according to the principles outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Female BALB/c nude mice at an age of 5 weeks were used to establish xenografts. Cells (2 ϫ 10 6 /0.1 ml) were subcutaneously inoculated into the right scruff of each nude mouse, 8 mice/group. On day 16 (when tumor volumes reached ϳ250 mm 3 ), nude mice were intraperitoneally injected with metformin (400 mg/kg) and 2-DG (400 mg/kg) every other day. The volumes of xenograft tumors were measured 16, 20, 24, 28, and 32 days after inoculation. The tumor volumes were calculated according to the following formula: volume ϭ length ϫ width 2 / 2. The xenografts were dissected, and weights were measured 32 days after inoculation. Statistical Analysis-Quantitative data were analyzed statistically using Student's t test to assess significance. Data are presented as means Ϯ S.E. Author Contributions-Z. X. X., Y. Y., and Y. Z. conceived and designed the experiments. Y. Y., D. C., J. A., S. S., M. W., and X. L. performed the experiments. Z. X. X., Y. Y., and J. B. analyzed the data. Z. X. X. and Y. Y. wrote the paper.	v3-fos-license
2020-08-19T14:52:38.398Z	2020-08-19T00:00:00.000	221167762	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://ann-clinmicrob.biomedcentral.com/track/pdf/10.1186/s12941-020-00381-z", "pdf_hash": "72c6427a09c497e553cdc3577638efa1147e1eea", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2257", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "sha1": "72c6427a09c497e553cdc3577638efa1147e1eea", "year": 2020 }	pes2o/s2orc	Safety and bactericidal efficacy of cold atmospheric plasma generated by a flexible surface Dielectric Barrier Discharge device against Pseudomonas aeruginosa in vitro and in vivo Background Cold atmospheric plasma (CAP), which is ionized gas produced at atmospheric pressure, could be a novel and potent antimicrobial therapy for the treatment of infected wounds. Previously we have shown that CAP generated with a flexible surface Dielectric Barrier Discharge (sDBD) is highly effective against bacteria in vitro and in ex vivo burn wound models. In the current paper, we determined the in vitro and in vivo safety and efficacy of CAP generated by this sDBD device. Methods The effect of CAP on DNA mutations of V79 fibroblasts was measured using a hypoxanthine–guanine-phosphoribosyltransferase (HPRT) assay. Furthermore, effects on cell proliferation, apoptosis and DNA damage in ex vivo burn wound models (BWMs) were assessed using immunohistochemistry. Next, 105 colony forming units (CFU) P. aeruginosa strain PAO1 were exposed to CAP in a 3D collagen-elastin matrix environment to determine the number of surviving bacteria in vitro. Finally, rat excision wounds were inoculated with 107 CFU PAO1 for 24 h. The wounds received a single CAP treatment, repeated treatments on 4 consecutive days with CAP, 100 µL of 1% (wt/wt) silver sulfadiazine or no treatment. Wound swabs and punch biopsies were taken to determine the number of surviving bacteria. Results Exposure of V79 fibroblasts to CAP did not increase the numbers of mutated colonies. Additionally, the number of proliferative, apoptotic and DNA damaged cells in the BWMs was comparable to that of the unexposed control. Exposure of PAO1 to CAP for 2 min resulted in the complete elimination of bacteria in vitro. Contrarily, CAP treatment for 6 min of rat wounds colonized with PAO1 did not effectively reduce the in vivo bacterial count. Conclusions CAP treatment was safe but showed limited efficacy against PAO1 in our rat wound infection model. Page 2 of 10 Dijksteel et al. Ann Clin Microbiol Antimicrob (2020) 19:37 wound areas and a compromised host defense system [1]. Treatment of infected burns remains a challenge due to the emergence of antibiotic-resistant and persistent bacteria [2]. Additionally, current topical treatments for colonized and infected burns display sub-optimal bactericidal efficacy and may impair wound healing [3][4][5]. Therefore, novel antimicrobial therapies are needed for the treatment of wound colonization and infection. A potential antimicrobial therapy to limit bacterial colonization is ionized gas, known as plasma. Plasma is the fourth state of matter in physics and consists of a mix of ions, electrons, highly reactive molecules, excited species, electric fields and ultraviolet radiation [6]. It can be artificially generated by subjecting a neutral gas to an extremely high temperature or a strong electromagnetic field. Often, plasma is accompanied by the production of heat due to the collision of electrons, and the subsequent excitation, ionization and dissociation processes of the gas particles [6]. In the medical field, plasma has been shown to be effective for sterilization, skin resurfacing and coagulation purposes [7][8][9]. However, these plasmas are generated in vacuum or are extremely hot, making them unsuitable for the treatment of (infected) burns. An alternative approach to treat colonized or infected tissue is cold atmospheric plasma (CAP) [10,11]. CAP devices generally consist of a powered electrode and a ground electrode of stainless-steel wire mesh. They operate under atmospheric pressure and preferably do not raise the temperature above 40 °C. Temperature rise can be further limited by applying CAP in a pulsed mode [6]. In the current study, we investigated the safety and efficacy of a CAP source called the flexible surface Dielectric Barrier Discharge (sDBD). This plasma source consists of a dielectric plate that separates the powered electrode from the ground electrode, resulting in the formation of gas plasma on the ground electrode ( Fig. 1). An advantage of this plasma source is that its use is not limited to small or flat surfaces. Previously, we have shown that CAP generated with this device has excellent bactericidal properties and has no effect on the re-epithelialization process of ex vivo human burn wound models (BWMs) [12]. The current study describes additional efficacy and safety tests. We investigated potential deoxyribonucleic acid (DNA) damage and mutagenesis upon exposure to CAP in vitro. Thereafter, in vivo experiments using rat excision wounds were performed to determine the efficacy of CAP against P. aeruginosa in these circumstances. Plasma source The flexible sDBD consists of a polyimide (100 μm thick) dielectric barrier strip [12]. The strip has a diameter of 2.5 cm and is integrated into a holder for research purposes. It was operated at 7 kHz, 850-900 mA, 0.032 V rms for up to 6 min at atmospheric pressure in air. The surface between the strip and the treated sample was set at 4 mm and closed from the surroundings to achieve an optimal CAP effluent. The temperature of the samples was measured using a thermal imaging camera FLIR One (FLIR Systems, Inc., Wilsonville, OR, USA) attached to an iPad mini (Apple Inc., Cupertino, CA, USA). To mimic the in vivo environment, collagen-elastin matrices (Matriderm ® ; MedSkin Solutions Dr. Suwelack AG, Billerberck, Germany) with a diameter of 15 mm and a thickness of 1 mm were used. Matriderm ® scaffolds were soaked in FBM, the FBM medium was removed and V79 fibroblasts were seeded (2300 cells/ mm 2 ) onto these scaffolds. After 2 h incubation at 37 °C and 5% CO 2 , 2 mL of FBM was added to the scaffolds. The scaffolds were incubated overnight. Prior to exposure, V79 fibroblasts in Matriderm ® were washed twice using sterile saline. Cell viability To determine the activity of V79 fibroblasts in Matriderm ® , 2 mL of resazurin (Merck KGaA, Darmstadt, Germany) with a final concentration of 75 µM in FBM was added to the scaffolds. After 3 h incubation at 37 °C and 5% CO 2 , the fluorescence of the medium (100 µL) was measured using the SpectraMax M2 (Molecular Devices, California, USA) at an excitation and emission wavelength of 540 nm and 595 nm, respectively. To estimate the number of cells, we determined the amount of double-stranded DNA (dsDNA) of the same samples. Resazurin was discarded and 2 mL of 0.05% (v/v) Triton X-100 (Merck KGaA) was added to the cells. Three freeze and thaw cycles were performed at − 80 and 37 °C to lyse DNA mutation To determine possible mutations in the DNA of V79 fibroblasts, a modified hypoxanthine-guanine-phosphoribosyltransferase (HPRT) protocol of Davies et al. [14] was used. After exposure of V79 fibroblasts in Matriderm ® to CAP, the scaffolds were incubated in 2 mL of FBM overnight at 37 °C and 5% CO 2 . Cells were isolated as follows: scaffolds were incubated with 300 µL of 0.25% (wt/v) collagenase and dispase (Gibco) for 10-15 min at 37 °C and 5% CO 2 . To neutralize collagenase and dispase, 5 mL of 1 mM ethylenediaminetetraacetic acid (EDTA; Gibco-BRL Life Technologies, N.Y., USA) in sterile phosphate-buffered saline (PBS; Gibco) was added to the suspension. The suspension was filtered using a 70 µm cell strainer. After centrifugation at 180g for 10 min, V79 fibroblasts were sub-cultured in FBM at 10 cells/cm 2 (equivalent to 100 cells) to assess the plating efficiency and at 1300 cells/ cm 2 (equivalent to 10 5 cells) to estimate the mutation frequency. After 5 days, the FBM of the cell cultures for the estimation of mutation frequency was supplemented with 20 µL of 5 µg/mL 6-thioguanine (6-TG; Merck KGaA). This was added daily during a subsequent 10 days culture. Colonies were stained using crystal violet (Klinipath, Duiven, the Netherlands) and counted microscopically using NIS Elements (Nikon Instruments Europe B.V., Amstelveen, the Netherlands). As positive controls, V79 fibroblasts in Matriderm ® were exposed to the mutagenic compound ethyl methanesulfonate (EMS) at a concentration of 0.8 µL of 0.3 mg/mL for 3 h. Negative controls were prepared by washing V79 fibroblasts in Matriderm ® with sterile saline. Negative controls were performed in absence of the first antibody. All sections stained for BrdU and Caspase-3 were counterstained using hematoxylin and were dehydrated and mounted using Entellan (Merck KGaA). γH2AX stained sections were aqueously mounted. NIS Elements (Nikon Instruments Europe B.V) was used to microscopically measure the newly formed epidermis (outgrowth) and the number of positively stained cells in this area. Bacterial culture A mid-log growth culture of a P. aeruginosa strain, PAO1 (ATCC BAA47), was prepared in Luria Bertani (LB) medium at 37 °C, which was shaken at 200 rpm for approximately 3 h. After centrifugation of the bacterial suspension at 3600×g for 5 min, the pellet was resuspended in sterile saline to the required concentration, based on the optical density of the bacterial culture at 600 nm. In vitro efficacy test To determine the efficacy of gas plasma in a biologically relevant environment, Matriderm ® scaffolds were soaked in sterile saline, inoculated with 10 µL of 1 × 10 7 colony forming units (CFU)/mL PAO1 for 30 min at room temperature and then exposed to CAP. Thereafter, the scaffolds were transferred to polypropylene vials containing a metal bead and 1 mL of PBS. After homogenizing the samples using a TissueLyser (Qiagen, Venlo, the Netherlands) set at 50 Hz for 4 min, tenfold serial dilutions of the homogenates were prepared. Dilutions were cultured on LB agar plates to quantify the number of viable bacteria after an overnight incubation at 37 °C and 5% CO 2 . In vivo efficacy of CAP in a rat model The experimental protocol for the study of CAP was approved by the Central Authority for Scientific Procedures on Animals (protocol AVD114002016601), according to governmental and international guidelines for animal experimentation. Twelve male and 12 female rats (Wistar) of 8 to 10 weeks old and a minimum weight of 160 g were purchased from Envigo (Horst, the Netherlands). The animals were acclimatized for 2 weeks prior to wounding. The animals were kept under specific pathogen-free conditions and were housed in individually ventilated cages with tap water and an irradiation-sterilized pelleted diet ad libitum. Wood-shavings were used as bedding material and long paper strips were used as enrichment. The sample size calculation and detailed experimental procedure for antimicrobial efficacy tests using a rat excision wound model were previously described [17]. To minimize the number of experimental animals, two partial thickness excision wounds of approximately 1 cm 2 large and 2 cm apart were prepared on the back of the rats using a dermatome set at 0.7 mm. The wounds were equally divided into four treatment groups. Each group had 12 wounds, i.e. one wound on 6 male and 6 female rats. The wounds were inoculated with 100 µL of 10 8 CFU/ mL PAO1 at t = 0. Twenty-four h after inoculation, the wounds received no treatment (group 1) or a single CAP treatment (group 2) on day 1. Wounds in group 3 and group 4 received repeated treatments on 4 consecutive days with CAP or 100 µL of 1% (wt/wt) silver sulfadiazine in cetomacrogol cream (group 4; SSD; Pharmacy of the Medical Center Alkmaar, Alkmaar, the Netherlands), respectively. To determine the bacterial load, wound swabs were taken before and after CAP treatment. The untreated wounds were swabbed twice on day 1 to assess the effect of swabbing on the bacterial load of the wound. Wound swabs were taken only before SSD treatment to prevent the removal of this topical. Six male and 6 female rats were euthanized on day 3 and on day 7 using saturated CO 2 /O 2 followed by CO 2 only. Four mm punch biopsies were taken from the wounds of the euthanized rats to determine the bacterial load within the tissue. Samples were homogenized in 1 mL of PBS (Gibco) using a TissueLyser set at 50 Hz for 4 min. Ten-fold serial dilutions of the homogenates were plated on LB agar and Pseudomonas isolation agar supplemented with cetrimide (50 mg/L) and sodium nalidixate (3.8 mg/L) (Oxoid ltd, Basingstoke, UK) to selectively identify P. aeruginosa from commensal bacteria. After overnight incubation of the plates at 37 °C and 5% CO 2 , the number of viable bacteria was determined. Statistical analysis Statistically significant differences were determined using SPSS version 24. For differences between groups the Kruskal-Wallis test followed by the Mann-Whitney-U test were used. To compare two related groups, the Wilcoxon singed rank sum test was used. In vitro efficacy of CAP against P. aeruginosa To determine the optimal exposure time to CAP for an effective bactericidal elimination under the same conditions as in the in vitro safety tests, 10 5 CFU of PAO1 in Matriderm ® were exposed to CAP for 1-4 min. After exposure to CAP for 1 min, 6.2 CFU/mL of PAO1 survived on average (Fig. 2). Exposure to CAP for 2 min or longer resulted in no surviving bacteria. Effect of CAP on the viability of V79 fibroblasts CAP might induce membrane changes, such as loss of membrane symmetry or integrity, which ultimately results in loss of cell viability. To assess this, we exposed V79 fibroblasts cultured in Matriderm ® scaffolds to CAP for 1-4 min and determined the metabolic activity per V79 fibroblast as a measurement for viable cells. Compared to the unexposed control samples, exposure to CAP up to 3 min did not affect the viability of V79 fibroblasts. However, exposure to CAP for 4 min reduced the cell viability to 76% (Fig. 3). Effect of reactive species on cell viability and DNA mutations The generation of CAP is accompanied by some heat. In addition, CAP commonly generates highly reactive molecules such as H 2 O 2 , O 3 and NO 2 − that could decrease pH of non-buffered solutions and induce oxidative and DNA damage. Therefore, we assessed these factors in relation to cell viability and DNA mutations. We exposed V79 fibroblasts in Matriderm ® to heat from 50 to 70 °C water at a distance of 4 mm between sample and source and determined the cell viability. Additionally, we determined the effect of pH of the medium ranging from 6 to 3 and of H 2 O 2 concentration in PBS ranging from 0 to 0.15% (v/v) on cell viability. Exposure to temperatures up to 70 °C or pH as low as 3 for 4 min did not affect the viability of V79 fibroblasts (data not shown). However, 0.15% (v/v) H 2 O 2 reduced the cell viability to 69% (Fig. 4). Furthermore, we exposed V79 fibroblasts in Matriderm ® to CAP for 1-4 min and determined the mutation frequency by measuring the colony forming ability of the cells in the presence of cytotoxic 6-TG. Exposure of V79 fibroblasts in Matriderm ® to CAP resulted in 1-2 mutated colonies/10 5 cells, independent of the exposure period (Fig. 5). This was comparable to the number of mutated colonies for the unexposed samples. Unlike CAP, EMS induced sevenfold higher numbers of mutations in V79 fibroblasts. Effect of repeated CAP exposure on wound healing in an ex vivo wound model During 2 weeks of culture, ex vivo BWMs were exposed four times to CAP for 4 or 6 min to assess the effect on re-epithelialization, proliferation, apoptosis and DNA damage. Compared to the unexposed samples, exposure to CAP did not affect the re-epithelialization of BWMs. The re-epithelialization varied between 600 and 700 µm [12]. The additional safety assessments revealed that the number of proliferative, apoptotic and DNA damaged cells after exposure to CAP was not significantly different from those of the unexposed-control samples (Fig. 6). In vivo efficacy of CAP in a rat wound model Twenty-four hours after inoculation, just prior to treatment, the wound swabs showed a bacterial count of approximately 10 5 CFU, which increased to 10 6 CFU on day 7. Swabbing the same wounds twice resulted in approximately 0.5 log-reduction of the bacterial count on day 1 (Fig. 7a). A single CAP treatment on day 1 did not reduce the bacterial count significantly (data not shown). Repeated CAP treatment on 4 consecutive days resulted in a tenfold lower bacterial count of 1.7 × 10 5 CFU on day 4, which increased to approximately 10 6 CFU on day 7. Compared to the untreated wounds, CAP treatment increased the wound temperature with 3 °C on day 1 and with 5.9 °C on day 4. The discrepancy in temperature rise is most likely due to healing of the wounds. Furthermore, repeated treatment with SSD gradually and significantly Effect of CAP on mutations in V79 fibroblasts. V79 fibroblasts in Matriderm ® were exposed to CAP for 1-4 min or EMS (positive control) and the number of mutated V79 fibroblasts was determined using a HPRT assay. As negative control the samples were washed using sterile saline. Results are expressed as the number of mutated colonies/10 5 cells. Data represent the means of five independent experiments performed in duplicate. Statistical differences compared to the unexposed control samples are indicated: p < 0.05; p < 0.01; **p < 0.001 (MWU) Fig. 6 Effect of repeated exposure to CAP on ex vivo wound healing. During 2 weeks culture, BWMs were exposed four times (twice weekly) to CAP for 4 or 6 min or not exposed (negative control). Subsequently, the number of proliferative (a), apoptotic (b) and DNA damaged (c) cells per µm of newly formed epidermis (outgrowth) was determined using immunohistochemistry. The arrows indicate the positively-stained cells in the outgrowth of the unexposed BWMs (scale bars: 50 µm). This is also shown at a smaller magnification in the inset (scale bars: 100 µm). Data represent the means of five independent experiments performed in duplicate. No statistically significant differences were measured (Wilcoxon S-R; p > 0.05) reduced the bacterial count to 25 CFU PAO1 at day 7 but with high variation. Notably, SSD treatment was more than a 100-fold less effective against PAO1 which had penetrated the tissue (in biopsies) than against superficially located PAO1 (in swabs, Fig. 7a versus 7b). Discussion CAP displays antimicrobial activity against a wide range of micro-organisms, such as bacteria [18]. It is efficacious regardless of the kind/species of bacteria and the antibiotic resistance level [19,20]. This makes CAP an interesting therapy for the treatment of burn wound infections. CAP's rapid mode of action against bacteria involves among others membrane lipid peroxidation, oxidative DNA damage and acidification [18], which might be harmful for human skin cells as well. Therefore, we assessed several safety aspects of CAP generated by the flexible sDBD in vitro. Our findings show that CAP exposure for 4-6 min did not induce mutations, apoptosis and DNA damage or affect the wound healing process, i.e. reepithelialization and proliferation. The ability of CAP to induce mutations, apoptosis and DNA damage has been shown in a number of studies [21][22][23][24][25]. In fact, CAP could be a more potent mutagenesis tool compared to conventional mutagenesis systems [26]. Similar to our findings, several studies show that a relatively short treatment time with CAP has no mutagenic potential and does not induce apoptosis or DNA damage [27][28][29]. Additionally, Maisch et al. reports that CAP has no or a negligible effect on the viability of skin cells [29]. These findings indicate that CAP treatment can be used at specific settings for therapeutically safe applications. Several studies show that CAP increases the temperature and decreases the pH of the exposed solution [30][31][32][33], which might be harmful for human skin (cells). Dobrynin et al. reports that toxic effects of CAP are related to the increase of the skin temperature, which is highly dependent on several factors such as the frequency of the discharge and the treatment time [34]. We found that 4 min exposure to heat or low pH alone did not affect the viability of the V79 fibroblasts. In contrast, the viability of V79 fibroblast was significantly reduced by 0.15% (v/v; equivalent to 49 mM) H 2 O 2 . However, H 2 O 2 concentrations generated by CAP typically range from 0.3 to 1 mM [35] and these concentrations did not reduce the viability of V79 fibroblasts in our experiments. Next to H 2 O 2, also other reactive species such as O 3 , NO 3 − and NO 2 − are formed in the exposed liquid [36,37]. The synergic interactions between the different reactive species could be responsible for the reduced viability of V79 fibroblasts. Such synergic interactions are also required to effectively eradicate bacteria [38][39][40]. Low pH or H 2 O 2 alone were previously found to be insufficient to kill bacteria [35,41]. Relatively high H 2 O 2 concentrations of 490 mM or temperatures of 60 °C for a duration of 30 min were required to eradicate P. aeruginosa [41,42]. Furthermore, we have shown that CAP generated by the flexible sDBD completely eradicated P. aeruginosa in vitro after a relatively short exposure period of 2 min. Additionally, CAP was efficacious against P. aeruginosa in ex vivo human skin models, whereby bacteria were effectively eliminated after 3 min exposure to CAP or after 6 min in BWM [12]. In view of these results, we anticipated that an exposure period of 6 min would result in an effective bacterial elimination in a rat wound infection model. However, CAP displayed a limited bactericidal efficacy against P. aeruginosa in this in vivo model. This suggests that wound environmental factors such as biofilm formation and wound exudate could have played a role in the limited efficacy of CAP in vivo. It was shown that bacteria in biofilms can be more tolerant against CAP [43][44][45]. Additionally, bacterial colonization and wound exudate could increase pH and/or introduce buffering effects to the wounds [46]. As a consequence, the bactericidal effect of CAP may be impeded because several reactive species are not generated at alkaline and buffered conditions [35,47,48]. For example, the concentration of free hydroxyl radicals from the decomposition of HNO 3 is pH dependent, resulting in low radical concentrations at high pH [49]. Recently, Assadian et al. reported that plasma is safe but less effective in reducing the wound size or bacterial count as compared to current antimicrobial agents [50]. This is in agreement with our findings. Yet, several in vivo studies demonstrate an effective (but limited) elimination of bacteria using gas plasma treatments [51][52][53]. The successful elimination of bacteria using CAP is dependent on a number of factors such as the design of the device, treatment time, gas flow and composition, plasma power and frequency, the distance to the sample and environmental factors, such as the wound type, extracellular matrix or wound debris and exudate [54][55][56]. Possibly, it is more complex to achieve an effective bacterial elimination using CAP in the micro-environment of in vivo systems such as our rat wound infection model. We suggest to study CAP generated by this flexible sDBD device in combination with other antimicrobial or antibiofilm agents to combat bacteria. Previously, combination therapy of CAP and chlorhexidine for the disinfection of root canals resulted in a more effective elimination of bacteria than chlorhexidine or CAP alone [57]. Hence, combination therapy rather than monotherapy using CAP could potentially eliminate pathogenic bacteria more effectively in vivo. Conclusions CAP did not induce mutations, apoptosis and DNA damage or affect the wound healing process in our in vitro and ex vivo (wound) models. Therefore, CAP can be considered a safe treatment option. CAP demonstrated a fast bactericidal effect in vitro, however, in our rat wound infection model CAP displayed a limited efficacy against PAO1.	v3-fos-license
2020-10-22T18:55:02.444Z	2020-10-01T00:00:00.000	224826601	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1422-0067/21/20/7699/pdf", "pdf_hash": "f435f07c34fc064598d7dde87da8fc919da0cb58", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2284", "s2fieldsofstudy": [ "Materials Science", "Chemistry" ], "sha1": "9f61b5e6cef0ebaf2a58470d11d76ac1193f60a9", "year": 2020 }	pes2o/s2orc	Constructing 3-Dimensional Atomic-Resolution Models of Nonsulfated Glycosaminoglycans with Arbitrary Lengths Using Conformations from Molecular Dynamics Glycosaminoglycans (GAGs) are the linear carbohydrate components of proteoglycans (PGs) and are key mediators in the bioactivity of PGs in animal tissue. GAGs are heterogeneous, conformationally complex, and polydisperse, containing up to 200 monosaccharide units. These complexities make studying GAG conformation a challenge for existing experimental and computational methods. We previously described an algorithm we developed that applies conformational parameters (i.e., all bond lengths, bond angles, and dihedral angles) from molecular dynamics (MD) simulations of nonsulfated chondroitin GAG 20-mers to construct 3-D atomic-resolution models of nonsulfated chondroitin GAGs of arbitrary length. In the current study, we applied our algorithm to other GAGs, including hyaluronan and nonsulfated forms of dermatan, keratan, and heparan and expanded our database of MD-generated GAG conformations. Here, we show that individual glycosidic linkages and monosaccharide rings in 10- and 20-mers of hyaluronan and nonsulfated dermatan, keratan, and heparan behave randomly and independently in MD simulation and, therefore, using a database of MD-generated 20-mer conformations, that our algorithm can construct conformational ensembles of 10- and 20-mers of various GAG types that accurately represent the backbone flexibility seen in MD simulations. Furthermore, our algorithm efficiently constructs conformational ensembles of GAG 200-mers that we would reasonably expect from MD simulations. Introduction Proteoglycans (PGs) are a diverse group of proteins in the extracellular matrix (ECM), as well as on and within cells in animal tissue. PGs play key roles in signal transduction [1,2], tissue morphogenesis [3][4][5][6][7], and matrix assembly [7][8][9][10] by binding growth factors [3][4][5][6][7][11][12][13][14][15][16][17], enzymes [7,17], membrane receptors [17], and ECM molecules [2,7,17]. Many of these functions are mediated by glycosaminoglycans (GAGs), which are linear, highly negatively-charged, and structurally diverse carbohydrate chains covalently-linked to PGs. Specifically, GAGs can form covalent and noncovalent complexes with proteins or inhibit complex formation with other biomolecules. All of these functions allow GAGs to modulate disease. For example, hyaluronan can predict disease outcome and is used as Monosaccharide ring geometries are quantified by all bond lengths, bond angles, and dihedral angles within the ring and in exocyclic functional groups that are not part of a glycosidic linkage. Cremer-Pople (C-P) ring puckering parameters (ϕ, θ, Q) [91] of each monosaccharide ring in the MDgenerated 20-mer ensembles were computed to characterize potential ring puckering patterns. Conformations of each monosaccharide and each linkage of the 20-mers were extracted separately from each snapshot of the MD trajectories. First, to determine if conformational data in different runs matched, and if there was interdependency in the individual linkage and ring conformations, data were separated by run and monosaccharide/linkage number and aggregated across all snapshots in each MD simulation run. Next, all individual conformations were aggregated across all snapshots in all runs (e.g., 10,000 snapshots * 4 runs * 10 GlcA monosaccharides = 400,000 samples of GlcA monosaccharide conformations in hyaluronan 20-mer) to generate a single dataset for each of the two monosaccharide types and two linkage types in each GAG (e.g., GlcA monosaccharide, GlcNAc Monosaccharide ring geometries are quantified by all bond lengths, bond angles, and dihedral angles within the ring and in exocyclic functional groups that are not part of a glycosidic linkage. Cremer-Pople (C-P) ring puckering parameters (φ, θ, Q) [91] of each monosaccharide ring in the MD-generated 20-mer ensembles were computed to characterize potential ring puckering patterns. Conformations of each monosaccharide and each linkage of the 20-mers were extracted separately from each snapshot of the MD trajectories. First, to determine if conformational data in different runs matched, and if there was interdependency in the individual linkage and ring conformations, data were separated by run and monosaccharide/linkage number and aggregated across all snapshots in each MD simulation run. Next, all individual conformations were aggregated across all snapshots in all runs (e.g., 10,000 snapshots * 4 runs * 10 GlcA monosaccharides = 400,000 samples of GlcA monosaccharide conformations in hyaluronan 20-mer) to generate a single dataset for each of the two monosaccharide types and two linkage types in each GAG (e.g., GlcA monosaccharide, GlcNAc monosaccharide, β1-3 glycosidic linkage, and β1-4 glycosidic linkage conformations in hyaluronan 20-mer). Construction Algorithm to Generate GAG Conformational Ensembles The MD-generated conformational datasets described above made up the database used for the algorithm we developed to generate GAG polymer conformational ensembles of user-specified length and with a user-specified number of conformations (previously described) [33]. Essentially, our algorithm (1) incorporates all bond, bond angle, and dihedral angle conformational parameters from MD simulation of GAG 20-mers, (2) treats monosaccharide rings and glycosidic linkages independently, (3) performs a restrained energy minimization on each constructed conformation to relieve steric overlap while maintaining polymer conformation, and (4) applies a bond potential energy cutoff to exclude conformations that remain nonphysical after minimization. A nonphysical conformation results from either overlapping bonds or a bond that pierces the center of another monosaccharide ring in the initial constructed conformation ( Figure S1). As dihedral angles are restrained, minimization addresses these issues by stretching the overlapping or ring-piercing bonds to nonphysical lengths, which increases the post-minimization bond potential energy by more than 132 kcal/mol [33]. The energy cutoff is the sum of a 100 kcal/mol buffer and a polymer-length specific cutoff, which is equal to the post-minimization bond potential energy of the fully-extended conformation. This conformation was constructed by assigning energetically-favorable glycosidic linkage dihedrals from the corresponding MD simulations of each GAG type that give a fully-extended conformation (i.e., with the maximum end-to-end distance observed in simulation). We used the algorithm to construct 20-mer conformational ensembles of each nonsulfated GAG. For internal validation of the algorithm, glycosidic linkage dihedral free energies ∆G(φ,ψ), monosaccharide ring C-P parameters, end-to-end distance distributions, and radii of gyration from MD-generated ensembles and constructed ensembles were compared. Additionally, post-minimization bond potential energy distributions from constructed ensembles were analyzed to validate the calculated bond potential energy cutoff and verify that the ensemble had expected energy distributions for the given polymer length. To evaluate application of MD-generated 20-mer conformations to construct GAG polymers of variable length, we constructed 10-mer ensembles using the algorithm and compared them to 10-mer ensembles generated using the same protocol as 20-mer MD simulations. To assess the efficacy and efficiency of our algorithm to construct conformational ensembles of GAG polymers with biologically-relevant chain lengths, we also constructed 200-mer ensembles. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility To examine the backbone flexibility of hyaluronan 20-mers, end-to-end distances and radii of gyration were analyzed in each of the four MD simulation runs. One of the four runs produced more compact conformations, i.e., with lower end-to-end distances and radii of gyration than the other three (Figures 2 and S2a and Table 1). The system potential energy distributions were identical for all four runs, suggesting that this outlying run was energetically stable ( Figure S3). To uncover the conformational factors that contributed to end-to-end distance and explain the differences in backbone flexibility in the different MD runs, the monosaccharide ring and glycosidic linkage conformations were examined. 1 Probabilities were calculated for end-to-end distances sorted into 0.5 Å bins for the 20-mer ensembles and 0.25 Å bins for the 10-mer ensembles. 2 All = end-to-end distance distribution aggregated across all four runs. Figure 2. End-to-end distance probability distribution of MD-generated hyaluronan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for end-to-end distances sorted into 0.5 Å bins. Analysis of C-P parameters of GlcNAc monosaccharide rings revealed mostly 4 C 1 chair conformations, as found in crystal structures [92][93][94][95][96][97][98][99] and NMR and force field studies [67,[100][101][102], with a minority of samples in boat and skew-boat conformations, namely B 3,O , 1 S 3 , 1,4 B, and 1 S 5 (Figures 3a and 4a). While non-4 C 1 conformations of GlcNAc are rare, they have been observed in crystal structures of protein co-complexes [95,[103][104][105][106]. For example, one GlcNAc ring in the crystal structure of a hyaluronan tetramer in complex with hyaluronidase was found in the 1,4 B boat conformation [95]. Additionally, GlcNAc boat/skew-boat conformations have been sampled in biased MD [107], 1-µs unbiased MD [108], and 10-µs unbiased MD simulations of GlcNAc monosaccharides [100]. One NMR and force field study suggested that these conformations were important for GAG-protein interactions [100]. Analysis of individual GlcNAc rings in each run revealed that these non-4 C 1 conformations were not specific to any particular region of the 20-mer and did not occur simultaneously in multiple rings in the same snapshot ( Figure S4). Furthermore, GlcNAc rings that sampled non-4 C 1 puckers returned to 4 C 1 chair conformations relatively quickly, i.e., in under 25 ns ( Figure S4). -mer; geometries from the four sets of each ensemble are represented by red, green, blue, and magenta dots, respectively and the force-field geometry is represented by a single large black dot; Cremer-Pople parameters (ϕ, θ) for all rings in every tenth snapshot from each ensemble were plotted (i.e., 10 rings × 1,000 snapshots per run × 4 runs = 40,000 parameter sets for the 20-mer and 20,000 parameter sets for the 10-mer). -mer; geometries from the four sets of each ensemble are represented by red, green, blue, and magenta dots, respectively and the force-field geometry is represented by a single large black dot; Cremer-Pople parameters (φ, θ) for all rings in every tenth snapshot from each ensemble were plotted (i.e., 10 rings × 1000 snapshots per run × 4 runs = 40,000 parameter sets for the 20-mer and 20,000 parameter sets for the 10-mer). C-P analysis of GlcA monosaccharide rings in the hyaluronan 20-mer showed that GlcA is found mostly in 4 C 1 chair conformations (Figure 3b), as seen in crystal structures [92][93][94][95] and NMR and force field studies [51,67,101,102,107,109,110], with some boat and skew-boat conformations, including 3 S 1 , B 1,4 , 5 S 1 , 2,5 B, 2 S O , 1 S 3 , 1,4 B, and 1 S 5 (Figures 3b and 4b,c), which were also observed in GlcA rings in unbiased MD simulations of nonsulfated chondroitin 20-mers [33]. Some of these conformations were also sampled rarely in unbiased MD simulations of GlcA monosaccharides [51,107]. The slight differences between boat/skew-boat conformations in our simulations and monosaccharide simulations may have arisen from intramolecular interactions in the hyaluronan 20-mer. As with GlcNAc rings, these conformations did not occur consistently in any one region of the 20-mer, did not occur simultaneously in multiple rings in the same snapshot, and returned to 4 C 1 chair conformations quickly, i.e., within 40 ns ( Figure S5). Except for 2 S O , each of these GlcA and GlcNAc boat and skew-boat conformations brought the linker oxygen atoms closer together, causing a kink in the polymer chain ( Figure 4), which may have contributed to more compact polymer conformations. To investigate this, end-to-end distances and radii of gyration of 20-mer conformations with non-4 C 1 ring puckers were analyzed ( Figure S6) and both compact and extended conformations were observed. This can be explained by the flexibility in the glycosidic linkages. Of note, MD-generated atomic-resolution snapshots before and after a monosaccharide ring underwent a conformational change between 4 C 1 and non-4 C 1 were visualized in VMD to check for ionic interactions (e.g., between Na + and carboxylate O − atoms and/or partially-charged hydroxyl O atoms in GlcA, between Cl − and hydroxyl H atoms in GlcA, between Na + and partially-charged N-acetyl N or O atoms and/or hydroxyl O atoms in GlcNAc, or between Cl − and H atoms of methyl and/or hydroxyl groups in GlcNAc). Specifically, we looked for ions within 5 Å of the exocyclic atoms of the monosaccharide of interest and found that ionic interactions were most often absent during, and thus, did not significantly contribute to, ring puckering in the MD simulation of hyaluronan 20-mer. 20-mer with a 5 S1 GlcA conformer (ring atoms in cyan; linkage atoms in red) and 3 S1, B1,4, 5 S1, 2,5 B, 1 S3, 1,4 B, and 1 S5 GlcA monosaccharide conformations (endocyclic ring atoms and linker oxygen atoms only); (c) 20-mer with a 2 SO GlcA conformer (ring atoms in cyan; linkage atoms in red) and 4 C1 and 2 SO GlcA monosaccharide conformations (endocyclic ring atoms and linker oxygen atoms only); in each 20-mer snapshot, all gray rings are in 4 C1 conformations. C-P analysis of GlcA monosaccharide rings in the hyaluronan 20-mer showed that GlcA is found mostly in 4 C1 chair conformations (Figure 3b), as seen in crystal structures [92][93][94][95] and NMR and force field studies [51,67,101,102,107,109,110], with some boat and skew-boat conformations, including 3 S1, B1,4, 5 S1, 2,5 B, 2 SO, 1 S3, 1,4 B, and 1 S5 (Figures 3b and 4b,c), which were also observed in GlcA rings in unbiased MD simulations of nonsulfated chondroitin 20-mers [33]. Some of these conformations were also sampled rarely in unbiased MD simulations of GlcA monosaccharides [51,107]. The slight To analyze glycosidic linkage conformations, free energies of glycosidic linkage dihedrals ∆G(φ, ψ) were calculated (Figure 5a,b and Table 2). If we assume O 5 -C 1 -O-C n = H 1 -C 1 -O-C n -120 • for φ values and C 1 -O-C 3 -C 2 = C 1 -O-C 3 -H 3 -120 • and C1-O-C 4 -C 3 = C 1 -O-C 4 -H 4 + 120 • for ψ values, our data match well with X-ray diffraction and force field data from hyaluronan tetrasaccharides [49], as well as NMR and molecular mechanics data from hyaluronan tetrasaccharides [111], hexasaccharides [112], and octasaccharides [113]. If we assume C 1 -O-C 3 -C 2 = C 1 -O-C 3 -C 4 + 120 • and C 1 -O-C 4 -C 3 = C 1 -O-C 4 -C 5 -120 • , our data are also in close agreement with X-ray diffraction and quantum molecular modeling data [65,[114][115][116], as well as NMR and MD data [102]. This agreement is with conformations sampled about the global minimum in each linkage in our MD simulations. All of these conformations were helical, so it stands to reason that 2-, 3-, and 8-fold helices were observed in hyaluronan oligosaccharides. This does not necessarily mean that adjacent glycosidic linkage conformations were interdependent. In fact, variation in the types of helices observed in the force field and experimental studies in only short oligosaccharide units suggests a lack of consistent and uniform helical structures in long hyaluronan polymers. Our data also show that a secondary basin was sampled in each linkage type in our MD simulations. In GlcAβ1-3GlcNAc linkages, conformations in the upper left quadrant (−ϕ, +ψ) were sampled. A molecular modeling study that reportedly sampled the full allowable range of glycosidic linkage conformations in agreement with experimental data for several disaccharide units also Our data also show that a secondary basin was sampled in each linkage type in our MD simulations. In GlcAβ1-3GlcNAc linkages, conformations in the upper left quadrant (−φ, +ψ) were sampled. A molecular modeling study that reportedly sampled the full allowable range of glycosidic linkage conformations in agreement with experimental data for several disaccharide units also revealed hyaluronan GlcAβ1-3GlcNAc linkage conformations in a secondary basin similar to ours [117]. Furthermore, crystal structures of hyaluronan oligosaccharides in complexes with proteins [92][93][94][95] were examined and we found that most glycosidic linkage conformations were similar to the most energetically-favorable conformations observed in MD simulation, with the exception of one GlcAβ1-3GlcNAc linkage conformation with −φ, +ψ dihedrals, which was seen in a hyaluronan hexasaccharide in complex with hyaluronidase [94]. This suggests that, although rare, this is a physical conformation. Analysis of individual glycosidic linkages in individual runs ( Figure S7) showed that this basin was sampled mostly in two particular GlcAβ1-3GlcNAc linkages in the MD run with the lowest end-to-end distance distribution. Therefore, 20-mer MD snapshots with these glycosidic linkage conformations were analyzed; we found that these linkage conformations were associated with lower end-to-end distances ( Figure S8 and Table S1). Visual analysis of these snapshots showed that GlcAβ1-3GlcNAc glycosidic linkages with −φ, +ψ dihedrals caused a kink in the polymer, which explains the more compact polymer conformations in comparison to those containing only linkage conformations at the global free energy minimum ( Figure S8). To find out if there was a connection between these glycosidic linkage conformations and non-4 C 1 puckers of adjacent monosaccharide rings, glycosidic linkages flanking non-4 C 1 ring puckers were plotted, and they were all centered about the global minimum, i.e., −φ, −ψ ( Figure S9). In fact, there did not appear to be any correlation between GlcAβ1-3GlcNAc linkage −φ, +ψ conformations and a non-4 C 1 ring pucker in any region of the polymer. GlcNAcβ1-4 GlcA Analysis of GlcNAcβ1-4GlcA glycosidic linkage conformations ( Figure 5b and Table 2) revealed secondary minima in the lower left quadrant (−φ, −ψ), which was also seen in a molecular modeling study of GAG disaccharides with results validated by experimental data [117]. In hyaluronan 20-mer MD simulations, these conformations were sampled more in the GlcNAcβ1-4GlcA linkages neighboring the GlcAβ1-3GlcNAc linkages with −φ, +ψ dihedrals in the MD run with the lowest end-to-end distances. The same analyses to those performed on 20-mer conformations with GlcAβ1-3GlcNAc linkages with −φ, +ψ dihedrals were performed on MD-generated 20-mer snapshots with GlcNAcβ1-4GlcA linkages with −φ, −ψ dihedrals. Similarly, these glycosidic linkage conformations caused a kink in the polymer chain and were associated with lower end-to-end distances ( Figure S10 and Table S1). To determine if there was a correlation between GlcAβ1-3GlcNAc −φ, +ψ linkage dihedrals and GlcNAcβ1-4GlcA −φ, −ψ linkage dihedrals, snapshots with both of these linkage conformations flanking the same monosaccharide ring were examined. It was found that there were very few snapshots in which this was the case. GlcNAcβ1-4GlcA linkage conformations flanking non-4 C 1 monosaccharide rings were also analyzed, and these conformations were centered about the global minimum, i.e., −φ, +ψ ( Figure S9). Additionally, ionic interactions (e.g., between Na + , O − in GlcA carboxylate group, and O in C=O of adjacent GlcNAc N-acetyl group, between Cl − , H in GlcNAc methyl group, and H in GlcA hydroxyl groups, or between Na + and O atoms or Cl − and H atoms of hydroxyls in adjacent monosaccharides) did not appear to play a role in the transition of glycosidic linkage conformations between their primary and secondary φ, ψ basins. Hyaluronan 10-mers were also simulated and showed the same conformations as the 20-mer in MD simulation except for GlcNAc monosaccharide rings, which sampled only 4 C 1 chair conformations in the 10-mer (Figures 3c,d and 5c,d and Table 2). This was expected, as these conformations occurred very few times in 20-mer MD simulations, and there were half as many samples (i.e., monosaccharide rings) in the 10-mer. Construction Algorithm Our construction algorithm was used to construct four sets of hyaluronan 20-mer ensembles with 10,000 conformations each (40,000 conformations total). A comparison of end-to-end distance distributions ( Figure 6a and Table 1) and radii of gyration ( Figure S2a,b) showed that our construction algorithm produces ensembles that mimic backbone flexibility observed in MD simulations. Furthermore, the C-P parameters ( Figure S11) and glycosidic linkage dihedral free energies ∆G(φ, ψ) ( Figure S12) of constructed conformations post-minimization matched those of MD-generated ensembles (i.e., construction algorithm input data; Figures 3a,b and 5a,b and Table 2), indicating that dihedral angles were properly restrained and that minimization did not change the overall ring and linkage conformation. A hyaluronan 10-mer ensemble with 40,000 conformations was also constructed using the algorithm; similarly, the end-to-end distances and radii of gyration of the constructed 10-mer ensemble matched those of the MD-generated 10-mer ensemble (Figures 6b and S2c,d and Table 1). Both ensembles contained mostly extended conformations, as expected for short oligosaccharides with fewer opportunities for kinks or curves. Finally, a hyaluronan 200-mer ensemble containing four sets of 10,000 conformations was A hyaluronan 10-mer ensemble with 40,000 conformations was also constructed using the algorithm; similarly, the end-to-end distances and radii of gyration of the constructed 10-mer ensemble matched those of the MD-generated 10-mer ensemble (Figures 6b and S2c,d and Table 1). Both ensembles contained mostly extended conformations, as expected for short oligosaccharides with fewer opportunities for kinks or curves. Finally, a hyaluronan 200-mer ensemble containing four sets of 10,000 conformations was constructed using the algorithm. The construction procedure produced end-to-end distances, radii of gyration, and bond potential energies of all conformations, as well as PDB files of conformations with most probable end-to-end distances, bond potential energies above that of the fully-extended conformation, and all excluded conformations (for a total of about 1000 PDBs for each of the four sets) for visualization and validation of the bond potential energy cutoff. As seen in MD-generated hyaluronan 10-and 20-mer ensembles, we expected the end-to-end distance distribution curve skewness to shift toward the right (i.e., lower end-to-end distances indicating more compact conformations) with increasing polymer length, as there were more opportunities for kinks and curves resulting from ring puckering and the flexibility of the glycosidic linkages. This pattern was seen in the end-to-end distance distributions of the hyaluronan 200-mer constructed ensembles ( Figure 7) and in those of nonsulfated chondroitin 100-and 200-mer constructed ensembles [33]. The relationship between end-to-end distance and radius of gyration was decreasingly linear with increasing polymer length. In other words, conformations with the same end-to-end distance have a wider range of radii of gyration in longer polymers. This was evidenced by comparison of R 2 values of the end-to-end distance vs. radius of gyration regression lines of hyaluronan 20-and 10-mer ensembles, both MD-generated and constructed ( Figure S2a-d). As expected, the end-to-end distance and radius of gyration relationship was decreasingly linear with increasing polymer length, as shown by comparison of hyaluronan 10-, 20-, and 200-mer constructed ensembles ( Figure S2b,d,e). This suggests that our algorithm constructs hyaluronan 200-mer ensembles with the backbone conformations that we would expect to see in MD simulations. hyaluronan 10-and 20-mer ensembles, we expected the end-to-end distance distribution curve skewness to shift toward the right (i.e., lower end-to-end distances indicating more compact conformations) with increasing polymer length, as there were more opportunities for kinks and curves resulting from ring puckering and the flexibility of the glycosidic linkages. This pattern was seen in the end-to-end distance distributions of the hyaluronan 200-mer constructed ensembles ( Figure 7) and in those of nonsulfated chondroitin 100-and 200-mer constructed ensembles [33]. The relationship between end-to-end distance and radius of gyration was decreasingly linear with increasing polymer length. In other words, conformations with the same end-to-end distance have a wider range of radii of gyration in longer polymers. This was evidenced by comparison of R 2 values of the end-to-end distance vs. radius of gyration regression lines of hyaluronan 20-and 10-mer ensembles, both MD-generated and constructed ( Figure S2a-d). As expected, the end-to-end distance and radius of gyration relationship was decreasingly linear with increasing polymer length, as shown by comparison of hyaluronan 10-, 20-, and 200-mer constructed ensembles ( Figure S2b,d,e). This suggests that our algorithm constructs hyaluronan 200-mer ensembles with the backbone conformations that we would expect to see in MD simulations. Figure 7. End-to-end distance probability distribution of constructed ensemble of hyaluronan 200mer; most probable end-to-end distance across all four sets is 235 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility MD simulations of nonsulfated dermatan 20-mer revealed relatively rigid and linear backbone conformations, as evidenced by the narrow and highly left-skewed end-to-end distance distributions, which match in all four MD runs ( Figure 8 and Table 3), and the linear relationship between end-toend distance and radius of gyration ( Figure S13a). To understand the factors contributing to this rigid behavior of the dermatan 20-mer in simulation, conformations of monosaccharide rings and glycosidic linkages were analyzed. . End-to-end distance probability distribution of constructed ensemble of hyaluronan 200-mer; most probable end-to-end distance across all four sets is 235 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility MD simulations of nonsulfated dermatan 20-mer revealed relatively rigid and linear backbone conformations, as evidenced by the narrow and highly left-skewed end-to-end distance distributions, which match in all four MD runs ( Figure 8 and Table 3), and the linear relationship between end-to-end distance and radius of gyration ( Figure S13a). To understand the factors contributing to this rigid behavior of the dermatan 20-mer in simulation, conformations of monosaccharide rings and glycosidic linkages were analyzed. Figure 8. End-to-end distance probability distribution of MD-generated nonsulfated dermatan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for endto-end distances sorted into 0.5 Å bins. 1 Probabilities were calculated for end-to-end distances sorted into 0.5 Å bins for the 20-mer ensembles and 0.25 Å bins for the 10-mer ensembles. 2 All = end-to-end distance distribution aggregated across all four runs. GalNAc monosaccharide ring C-P parameters ( Figure 9a) show mostly 4 C1 conformations, as well-established by X-ray diffraction [49,106,118], NMR [47,119], and force field [33,47,49,107] data. Biased MD simulations of β-GalNAc monosaccharides showed that nonsulfated β-GalNAc sampled boat and skew-boat conformations, namely B3,O, 1 S3, and 1,4 B, with relatively high free energies [107]. In line with this study, our simulations showed very few boat and skew-boat puckers, namely 1 S3, 1,4 B, 1 S5 (Figure 4a; -3GalNAcβ1-endocyclic ring and linker oxygen atoms were identical to those of -3GlcNAcβ1-), and B2,5, on the microsecond timescale. These puckers were sampled by different rings of the 20-mer in different MD runs and returned to 4 C1 within 10 ns ( Figure S14), suggesting that this behavior was random, and confirming that non-4 C1 GalNAc puckers are not energetically favorable. While these boat and skew-boat conformations caused a kink in the polymer chain, there were so few occurrences of this that they did not impact the overall end-to-end distance distribution. Figure 8. End-to-end distance probability distribution of MD-generated nonsulfated dermatan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for end-to-end distances sorted into 0.5 Å bins. Table 3. Most Probable End-to-End Distances (d) in MD-Generated and Constructed Nonsulfated Dermatan Ensembles 1 . 1 Probabilities were calculated for end-to-end distances sorted into 0.5 Å bins for the 20-mer ensembles and 0.25 Å bins for the 10-mer ensembles. 2 All = end-to-end distance distribution aggregated across all four runs. 20-mer Ensembles 10-mer Ensembles GalNAc monosaccharide ring C-P parameters ( Figure 9a) show mostly 4 C 1 conformations, as well-established by X-ray diffraction [49,106,118], NMR [47,119], and force field [33,47,49,107] data. Biased MD simulations of β-GalNAc monosaccharides showed that nonsulfated β-GalNAc sampled boat and skew-boat conformations, namely B 3,O , 1 S 3 , and 1,4 B, with relatively high free energies [107]. In line with this study, our simulations showed very few boat and skew-boat puckers, namely 1 S 3 , 1,4 B, 1 S 5 (Figure 4a; -3GalNAcβ1-endocyclic ring and linker oxygen atoms were identical to those of -3GlcNAcβ1-), and B 2,5 , on the microsecond timescale. These puckers were sampled by different rings of the 20-mer in different MD runs and returned to 4 C 1 within 10 ns ( Figure S14), suggesting that this behavior was random, and confirming that non-4 C 1 GalNAc puckers are not energetically favorable. While these boat and skew-boat conformations caused a kink in the polymer chain, there were so few occurrences of this that they did not impact the overall end-to-end distance distribution. IdoA in the 20-mer and (c) GalNAc and (d) IdoA in the 10-mer; geometries from the four sets of each ensemble are represented by red, green, blue, and magenta dots, respectively and the force-field geometry is represented by a single large black dot; Cremer-Pople parameters (ϕ, θ) for all rings in every tenth snapshot from each ensemble were plotted (i.e., 10 rings × 1,000 snapshots per run × 4 runs = 40,000 parameter sets for the 20-mer and 20,000 parameter sets for the 10-mer). IdoA monosaccharide rings in 20-mer MD sampled a majority of 1 C4 and, to a lesser degree, 2 SO and 4 C1 puckers ( Figure 9b and Table S2), as observed in NMR and force field studies [29,45,47,51,107,[120][121][122][123][124][125]. Literature reports of the relative proportions of these three ring puckers in nonsulfated IdoA vary, which may be explained by differences in the structure of neighboring residues (or lack thereof) [45,120,123,124] and differences in ion concentrations [123]. Furthermore, existing studies were conducted on IdoA monosaccharides and short IdoA-containing GAG oligosaccharides (i.e., <20 monosaccharides). NMR data have shown that, when at the nonreducing terminal in GAG oligosaccharides, nonsulfated IdoA is in equilibrium with 1 C4, 2 SO, and 4 C1 puckers, and when flanked by nonsulfated N-acetylated sugar derivatives (i.e., GalNAc or GlcNAc), nonsulfated IdoA is primarily in 1 C4 and 2 SO conformations [123], with fewer than 10% of samples in 4 C1 [45,124]. Indeed, we found this to be the case in our dermatan 20-mer MD simulations (Table S2). Importantly, none of the 1 C4, 2 SO, or 4 C1 puckers introduced a kink in the polymer chain, so we would not expect variations in the relative proportions of these ring puckers to alter overall polymer backbone conformations. IdoA also sampled other boat and skew-boat conformations in 20-mer MD simulations, specifically B1,4, 5 S1, 2,5 B, B3,O, 1 S3, 1,4 B, and 1 S5 (Figure 9b and Table S2), which is in agreement with results from MD simulations of IdoA monosaccharides [107,126]. Another force field study showed that 2,5 B and B3,O IdoA conformers are also feasible interpretations of existing NMR data [127]. Furthermore, a force field study that mapped X-ray diffraction and NMR data for dermatan sulfate [122] and a molecular modeling study of IdoA monosaccharides [126] showed that interconversion between different boat/skew-boat forms of IdoA is much more common than that between boat/skew-boat and chair forms, as it is less energetically costly, which helps explain the sampling of multiple different boat/skew-boat IdoA conformations. As each of these boat/skew-boat puckers (except for 2 SO) introduces a kink in the polymer chain (Figures 10 and 4b,c; -4IdoAα1endocyclic ring and linker oxygen atoms are identical to those of -4GlcAcβ1-), end-to-end distances of 20-mer conformations with these puckers were analyzed to determine if they were associated with more compact conformations. The end-to-end distance distribution of conformations with boat/skew- IdoA monosaccharide rings in 20-mer MD sampled a majority of 1 C 4 and, to a lesser degree, 2 S O and 4 C 1 puckers (Figure 9b and Table S2), as observed in NMR and force field studies [29,45,47,51,107,[120][121][122][123][124][125]. Literature reports of the relative proportions of these three ring puckers in nonsulfated IdoA vary, which may be explained by differences in the structure of neighboring residues (or lack thereof) [45,120,123,124] and differences in ion concentrations [123]. Furthermore, existing studies were conducted on IdoA monosaccharides and short IdoA-containing GAG oligosaccharides (i.e., <20 monosaccharides). NMR data have shown that, when at the nonreducing terminal in GAG oligosaccharides, nonsulfated IdoA is in equilibrium with 1 C 4 , 2 S O , and 4 C 1 puckers, and when flanked by nonsulfated N-acetylated sugar derivatives (i.e., GalNAc or GlcNAc), nonsulfated IdoA is primarily in 1 C 4 and 2 S O conformations [123], with fewer than 10% of samples in 4 C 1 [45,124]. Indeed, we found this to be the case in our dermatan 20-mer MD simulations (Table S2). Importantly, none of the 1 C 4 , 2 S O , or 4 C 1 puckers introduced a kink in the polymer chain, so we would not expect variations in the relative proportions of these ring puckers to alter overall polymer backbone conformations. IdoA also sampled other boat and skew-boat conformations in 20-mer MD simulations, specifically B 1,4 , 5 S 1 , 2,5 B, B 3,O , 1 S 3 , 1,4 B, and 1 S 5 (Figure 9b and Table S2), which is in agreement with results from MD simulations of IdoA monosaccharides [107,126]. Another force field study showed that 2,5 B and B 3,O IdoA conformers are also feasible interpretations of existing NMR data [127]. Furthermore, a force field study that mapped X-ray diffraction and NMR data for dermatan sulfate [122] and a molecular modeling study of IdoA monosaccharides [126] showed that interconversion between different boat/skew-boat forms of IdoA is much more common than that between boat/skew-boat and chair forms, as it is less energetically costly, which helps explain the sampling of multiple different boat/skew-boat IdoA conformations. As each of these boat/skew-boat puckers (except for 2 S O ) introduces a kink in the polymer chain (Figures 10 and 4b,c; -4IdoAα1endocyclic ring and linker oxygen atoms are identical to those of -4GlcAcβ1-), end-to-end distances of 20-mer conformations with these puckers were analyzed to determine if they were associated with more compact conformations. The end-to-end distance distribution of conformations with boat/skew-boat ring puckers (other than 2 S O ) matched that of the average of the four runs from the full MD-generated 20-mer ensemble ( Figure S15), indicating that these ring puckers do not necessarily give compact polymer conformations. Additionally, the end-to-end distance distribution of 20-mer conformations with 2 S O IdoA conformations was compared to that of the full MD ensemble ( Figure S15). These distributions were similar, further suggesting that 2 S O conformations are not associated with more or less compact 20-mer conformations. To determine if IdoA ring puckering in nonsulfated dermatan 20-mer MD was random, an analysis was performed of C-P parameters of individual IdoA monosaccharides in each of the four MD runs ( Figure S16). This revealed that (1) not all IdoA rings overcame the energy barrier to sample a 4 C 1 pucker, (2) no single IdoA ring sampled 4 C 1 in all four MD runs, (3) each IdoA ring overcame the energy barrier to sample boat/skew-boat puckers (i.e., C-P θ 90 • ) in at least one MD run, and (4) after sampling boat/skew-boat puckers, some IdoA rings returned to 1 C 4 in as little as 165 ns, while others remained in boat/skew-boat conformations for the remainder of the 1-µs trajectory. These observations support the idea that monosaccharide rings in a nonsulfated dermatan 20-mer behave randomly and independently in unbiased MD simulation. boat ring puckers (other than 2 SO) matched that of the average of the four runs from the full MDgenerated 20-mer ensemble ( Figure S15), indicating that these ring puckers do not necessarily give compact polymer conformations. Additionally, the end-to-end distance distribution of 20-mer conformations with 2 SO IdoA conformations was compared to that of the full MD ensemble ( Figure S15). These distributions were similar, further suggesting that 2 SO conformations are not associated with more or less compact 20-mer conformations. To determine if IdoA ring puckering in nonsulfated dermatan 20-mer MD was random, an analysis was performed of C-P parameters of individual IdoA monosaccharides in each of the four MD runs ( Figure S16). This revealed that (1) not all IdoA rings overcame the energy barrier to sample a 4 C1 pucker, (2) no single IdoA ring sampled 4 C1 in all four MD runs, (3) each IdoA ring overcame the energy barrier to sample boat/skew-boat puckers (i.e., C-P θ ~ 90°) in at least one MD run, and (4) after sampling boat/skew-boat puckers, some IdoA rings returned to 1 C4 in as little as 165 ns, while others remained in boat/skew-boat conformations for the remainder of the 1-µs trajectory. These observations support the idea that monosaccharide rings in a nonsulfated dermatan 20-mer behave randomly and independently in unbiased MD simulation. Analysis of dihedral free energies revealed a single ΔG(ϕ, ψ) minimum for each of IdoAα1-3GalNAc and GalNAcβ1-4IdoA glycosidic linkages (Figure 11a,b and Table 4). This is consistent across all linkages in all runs for each linkage type. The lack of secondary basins in ϕ, ψ dihedral samples may explain the higher degree of rigidity and tendency toward extended backbone conformations of the nonsulfated dermatan 20-mer in comparison to hyaluronan and nonsulfated chondroitin [33] 20-mers in MD simulation. An X-ray diffraction study of sodium dermatan sulfate observed three different helical forms (i.e., right-handed, left-handed, and achiral) [118] and quantified their dihedral angles, which were defined by: ϕ = O5-C1-O-Cn and ψ = C1-O-Cn-C(n + 1). Assuming C1-O-C3-C2 = C1-O-C3-C4 + 120° and C1-O-C4-C3 = C1-O-C4-C5 -120°, we see that the righthanded and achiral helical forms corresponded to high ΔG(ϕ, ψ) values in our nonsulfated dermatan 20-mer MD simulations, and the left-handed helical form corresponded to glycosidic linkage conformations very close to the ΔG(ϕ, ψ) global minimum. In line with our results, force field studies that compared MD results to existing NMR and X-ray diffraction data of dermatan sulfate dismissed right-handed and achiral helical forms and confirmed the presence of left-handed helical structure Analysis of dihedral free energies revealed a single ∆G(φ, ψ) minimum for each of IdoAα1-3GalNAc and GalNAcβ1-4IdoA glycosidic linkages (Figure 11a,b and Table 4). This is consistent across all linkages in all runs for each linkage type. The lack of secondary basins in φ, ψ dihedral samples may explain the higher degree of rigidity and tendency toward extended backbone conformations of the nonsulfated dermatan 20-mer in comparison to hyaluronan and nonsulfated chondroitin [33] 20-mers in MD simulation. An X-ray diffraction study of sodium dermatan sulfate observed three different helical forms (i.e., right-handed, left-handed, and achiral) [118] and quantified their dihedral angles, which were defined by: φ = O 5 -C 1 -O-C n and ψ = C 1 -O-C n -C (n + 1) . Assuming C 1 -O-C 3 -C 2 = C 1 -O-C 3 -C 4 + 120 • and C1-O-C 4 -C 3 = C 1 -O-C 4 -C 5 -120 • , we see that the right-handed and achiral helical forms corresponded to high ∆G(φ, ψ) values in our nonsulfated dermatan 20-mer MD simulations, and the left-handed helical form corresponded to glycosidic linkage conformations very close to the ∆G(φ, ψ) global minimum. In line with our results, force field studies that compared MD results to existing NMR and X-ray diffraction data of dermatan sulfate dismissed right-handed and achiral helical forms and confirmed the presence of left-handed helical structure [49,122]. A quantitative comparison of our MD-generated glycosidic linkage data to their findings and other NMR data for dermatan tetrasaccharides [47] showed close agreement if we assume O 5 [49,122]. A quantitative comparison of our MD-generated glycosidic linkage data to their findings and other NMR data for dermatan tetrasaccharides [47] showed close agreement if we assume O5- MD simulations of a nonsulfated dermatan 10-mer produced mostly linear backbone conformations ( Figure S13c) and similar monosaccharide ring ( Figure 9) and glycosidic linkage ( Figure 11 and Table 4) geometries to the nonsulfated dermatan 20-mer. The major difference was that in 10-mer MD simulations, 9% of all IdoA rings in all runs were found in 4 C1 form; this was almost exclusively in the nonreducing terminal IdoA (i.e., ring #10; Figure S17 and Table S2), which is mostly in line with the aforementioned NMR study of IdoA ring conformations [45]. However, based on the findings from the NMR study and from our dermatan 20-mer MD simulations, we would still expect the nonterminal IdoA rings in a GAG polymer to sample some 4 C1 conformations. This supports our position that GAG 20-mers are better-suited for predictions of long-chain backbone conformations than short GAG oligosaccharides. GalNAcβ1-4 IdoA Min MD simulations of a nonsulfated dermatan 10-mer produced mostly linear backbone conformations ( Figure S13c) and similar monosaccharide ring ( Figure 9) and glycosidic linkage ( Figure 11 and Table 4) geometries to the nonsulfated dermatan 20-mer. The major difference was that in 10-mer MD simulations, 9% of all IdoA rings in all runs were found in 4 C 1 form; this was almost exclusively in the nonreducing terminal IdoA (i.e., ring #10; Figure S17 and Table S2), which is mostly in line with the aforementioned NMR study of IdoA ring conformations [45]. However, based on the findings from the NMR study and from our dermatan 20-mer MD simulations, we would still expect the nonterminal IdoA rings in a GAG polymer to sample some 4 C 1 conformations. This supports our position that GAG 20-mers are better-suited for predictions of long-chain backbone conformations than short GAG oligosaccharides. These findings suggest that (1) monosaccharide rings and glycosidic linkages in nonsulfated dermatan GAG 20-mers behave randomly and independently in MD simulation, (2) nonsulfated dermatan polymers take on rigid left-handed helical structure with a tendency toward linear backbone conformations in unbiased MD simulations, and (3) nonsulfated dermatan 20-mer conformations in MD simulation provide a realistic representation of longer nonsulfated dermatan polymer conformations. Construction Algorithm Four sets of 10,000 conformations of a nonsulfated dermatan 20-mer were constructed using our algorithm and compared to conformations seen in nonsulfated dermatan 20-mer MD. As expected, the C-P parameter plots ( Figure S18) and glycosidic linkage free energies ( Figure S19) in the constructed 20-mer ensemble matched those in the MD-generated 20-mer ensemble (Figures 9a,b and 11a,b and Table 4). Furthermore, the end-to-end distances (Figure 12a) and radii of gyration ( Figure S13a,b) in the constructed ensemble matched those in the MD-generated ensemble, with only a 2.42% difference in most probable end-to-end distance ( Table 3). This demonstrates that our algorithm produces nonsulfated dermatan 20-mer ensembles that mimic 20-mer backbone flexibility seen in MD simulations. Construction Algorithm Four sets of 10,000 conformations of a nonsulfated dermatan 20-mer were constructed using our algorithm and compared to conformations seen in nonsulfated dermatan 20-mer MD. As expected, the C-P parameter plots ( Figure S18) and glycosidic linkage free energies ( Figure S19) in the constructed 20-mer ensemble matched those in the MD-generated 20-mer ensemble (Figures 9a,b and 11a,b and Table 4). Furthermore, the end-to-end distances (Figure 12a) and radii of gyration ( Figure S13a,b) in the constructed ensemble matched those in the MD-generated ensemble, with only a 2.42% difference in most probable end-to-end distance ( Table 3). This demonstrates that our algorithm produces nonsulfated dermatan 20-mer ensembles that mimic 20-mer backbone flexibility seen in MD simulations. The nonsulfated dermatan 10-mer ensemble with 40,000 conformations constructed by the algorithm had very similar end-to-end distance and radius of gyration data compared to the MD- The nonsulfated dermatan 10-mer ensemble with 40,000 conformations constructed by the algorithm had very similar end-to-end distance and radius of gyration data compared to the MD-generated ensemble (Figures 12b and S13c,d and Table 3). The algorithm was also used to create a nonsulfated dermatan 200-mer ensemble. As expected, the 200-mer end-to-end distance distribution ( Figure 13) and radii of gyration ( Figure S13e) showed that conformations tended to be more compact as polymer length increased. The shift in skewness of the end-to-end distance distribution with increasing polymer length was more subtle in nonsulfated dermatan than in hyaluronan, as there were fewer linkage and ring conformations causing kinks in the polymer. Table 3). The algorithm was also used to create a nonsulfated dermatan 200-mer ensemble. As expected, the 200-mer end-to-end distance distribution ( Figure 13) and radii of gyration ( Figure S13e) showed that conformations tended to be more compact as polymer length increased. The shift in skewness of the end-to-end distance distribution with increasing polymer length was more subtle in nonsulfated dermatan than in hyaluronan, as there were fewer linkage and ring conformations causing kinks in the polymer. Figure 13. End-to-end distance probability distribution of constructed ensemble of nonsulfated dermatan200-mer; most probable end-to-end distance across all four sets is 365 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility In MD simulations, nonsulfated keratan 20-mers showed rigid backbone behavior and favored extended backbone conformations, as evidenced by end-to-end distance distributions ( Figure 14 and Table 5) which were identical in all four MD runs, and the high correlation of end-to-end distance to radius of gyration ( Figure S20a). To understand the causes of this rigid behavior, monosaccharide ring and glycosidic linkage conformations were analyzed. Figure 14. End-to-end distance probability distribution of MD-generated nonsulfated keratan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for endto-end distances sorted into 0.5 Å bins. Figure 13. End-to-end distance probability distribution of constructed ensemble of nonsulfated dermatan200-mer; most probable end-to-end distance across all four sets is 365 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility In MD simulations, nonsulfated keratan 20-mers showed rigid backbone behavior and favored extended backbone conformations, as evidenced by end-to-end distance distributions ( Figure 14 and Table 5) which were identical in all four MD runs, and the high correlation of end-to-end distance to radius of gyration ( Figure S20a). To understand the causes of this rigid behavior, monosaccharide ring and glycosidic linkage conformations were analyzed. In MD simulations, nonsulfated keratan 20-mers showed rigid backbone behavior and favored extended backbone conformations, as evidenced by end-to-end distance distributions ( Figure 14 and Table 5) which were identical in all four MD runs, and the high correlation of end-to-end distance to radius of gyration ( Figure S20a). To understand the causes of this rigid behavior, monosaccharide ring and glycosidic linkage conformations were analyzed. Figure 14. End-to-end distance probability distribution of MD-generated nonsulfated keratan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for endto-end distances sorted into 0.5 Å bins. Figure 14. End-to-end distance probability distribution of MD-generated nonsulfated keratan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for end-to-end distances sorted into 0.5 Å bins. C-P parameters of GlcNAc monosaccharides in keratan 20-mer MD (Figure 15a) showed similar conformations to GlcNAc in hyaluronan 20-mer. There were predominantly 4 C 1 chair conformations, which are also found in a keratan sulfate tetrasaccharide crystal structure [128], with very few transitions (i.e., no more than one per ring or two per run) to boat/skew-boat conformations (i.e., 2 S O , 1 S 3 , 1,4 B, and 1 S 5 ), which were sampled for no more than 20 ns before returning to 4 C 1 chair ( Figure S21). Analysis of end-to-end distance and radius of gyration of 20-mer conformations containing non-4 C 1 ring puckers showed that these ring puckers were not associated with compact 20-mer conformations ( Figure S22). C-P analysis of Gal monosaccharides showed that for the duration of the keratan 20-mer MD simulations, Gal remained in 4 C 1 chair (Figure 15b), which was the predominant conformation found in NMR [119,[129][130][131] and force field data [107] for Gal in general, and in a keratan sulfate tetrasaccharide crystal structure [128]. 4 C1 ring puckers showed that these ring puckers were not associated with compact 20-mer conformations ( Figure S22). C-P analysis of Gal monosaccharides showed that for the duration of the keratan 20-mer MD simulations, Gal remained in 4 C1 chair (Figure 15b), which was the predominant conformation found in NMR [119,[129][130][131] and force field data [107] for Gal in general, and in a keratan sulfate tetrasaccharide crystal structure [128]. Glycosidic linkage free energy ∆G(φ, ψ) analysis of Galβ1-4GlcNAc linkages revealed a global minimum in the basin with −φ, +ψ and a secondary minimum in the basin with −φ, −ψ ( Figure 16a and Table 6), similar to hyaluronan GlcNAcβ1-4GlcA linkages (Figure 5b,d and Table 2), but with additional rare conformations in +φ, +ψ. A molecular modeling study, which was shown to agree with experimental data, showed that β1-4 linkages in keratan sulfate disaccharides took on the same conformations as β1-4 linkages in hyaluronan disaccharides [117], confirming the primary and secondary basins sampled in our 20-mer MD simulations. Keratan 20-mer conformations with glycosidic linkage conformations in the tertiary basin (i.e., +φ, +ψ) were visualized and end-to-end distances were analyzed ( Figure S23). These conformations formed a slight bend in the polymer chain and were associated with more compact polymer backbone conformations. However, there did not appear to be any close contacts between atoms of adjacent monosaccharides in this conformation or in previous snapshots that could explain these linkage bond rotations. Furthermore, this conformation was not specific to any particular region of the polymer. Therefore, this behavior was likely random. As −φ, −ψ β1-4 linkage conformations are associated with compact hyaluronan polymer conformations, and nonsulfated keratan 20-mer is relatively rigid and favors extended conformations in contrast to hyaluronan, we sought to determine the effects of Galβ1-4GlcNAc linkage conformations in the secondary (i.e., −φ, −ψ) basin on keratan 20-mer backbone flexibility in MD simulations. As in hyaluronan, these conformations caused a kink in the keratan 20-mer chain and were associated with more compact conformations ( Figure S23 and Table S1). However, these conformations were slightly less stable in keratan 20-mer MD (Table 6) than in hyaluronan MD (Table 2), and were, therefore, less common. conformations in contrast to hyaluronan, we sought to determine the effects of Galβ1-4GlcNAc linkage conformations in the secondary (i.e., −ϕ, −ψ) basin on keratan 20-mer backbone flexibility in MD simulations. As in hyaluronan, these conformations caused a kink in the keratan 20-mer chain and were associated with more compact conformations ( Figure S23 and Table S1). However, these conformations were slightly less stable in keratan 20-mer MD ( Table 6) than in hyaluronan MD (Table 2), and were, therefore, less common. Analysis of GlcNAcβ1-3Gal glycosidic linkages showed a global free energy minimum in the −φ, −ψ basin ( Table 6) similar to that of IdoAβ1-3GalNAc linkages in nonsulfated dermatan MD (Figure 11a,c and Table 4), but with additional rare occurrences in a secondary basin in −φ, +ψ, confirmed by the aforementioned molecular modeling study [117], and a small tertiary basin in +φ, −ψ (Figure 16b). To determine the effects of GlcNAcβ1-3Gal linkage conformations in these secondary and tertiary basins on backbone conformation, we visualized 20-mer conformations containing these glycosidic linkage conformations and analyzed end-to-end distances ( Figure S24). Glycosidic linkage conformations in the tertiary basin caused a kink and were associated with more compact 20-mer conformations. In contrast to hyaluronan, nonsulfated keratan β1-3 glycosidic linkage conformations in the secondary basin (i.e., −φ, +ψ) caused only a slight bend in the polymer chain, but were still associated with compact 20-mer conformations (Table S1). Both secondary and tertiary GlcNAcβ1-3Gal linkage conformations were rare in keratan 20-mer MD, which helps explain why the keratan 20-mer chain was relatively rigid and favored extended conformations in MD simulation. GlcNAcβ1-3 Gal Backbone conformational analysis of nonsulfated keratan 10-mer MD revealed that the 10-mer chain was rigid and favored extended conformations ( Figure S20c and Table 5), which is similar behavior to that of nonsulfated keratan 20-mer in MD simulation. This stands to reason, as conformations of monosaccharide rings ( Figure 15) and glycosidic linkages ( Figure 16 and Table 6) in keratan 10-mer MD matched those in keratan 20-mer MD, with the only differences being that (1) Gal briefly (i.e., for <5 ns) sampled a 1 S 5 skew-boat conformation once in each of two of the 10-mer MD runs ( Figure S25), and (2) GlcNAcβ1-3Gal linkages in the 10-mer did not sample conformations in the tertiary basin (i.e., +φ, −ψ) sampled in the 20-mer. Boat/skew-boat conformations including 1 S 5 were shown to have a high secondary free energy minimum (i.e., > 4 kcal/mol) in biased MD simulations of Gal monosaccharides [107], indicating that while possible, these conformations are not very stable in Gal monosaccharides. In both biased and unbiased MD simulations of a tetrasaccharide with a central Gal-GlcNAc disaccharide unit, the free energy minimum of Gal boat/skew-boat conformations was much higher (i.e.,~7 kcal/mol) [108], suggesting that non-4 C 1 conformations are less stable in Gal linked to other monosaccharides. This could explain why Gal does not sample non-4 C 1 conformations in keratan 20-mer MD, and suggests that these conformations would not be seen in long polymers. Although +φ, −ψ conformations in GlcNAcβ1-3Gal linkages are rare, they are physical conformations [117] that minorly contribute to polymer backbone flexibility. This further supports our belief that conformational landscapes from 20-mer MD are better-suited to construct conformational ensembles of GAGs with biologically-relevant chain lengths than those of short GAG oligosaccharides. The higher degree of rigidity and probability of extended conformations in nonsulfated keratan MD compared to hyaluronan MD can be explained by the facts that: (1) for each glycosidic linkage type, the conformations that caused a kink in the polymer chain were less stable and, thus, rarer in nonsulfated keratan MD than in hyaluronan MD; and (2) GlcNAc and Gal are mostly rigid 4 C 1 chair conformers in nonsulfated keratan whereas GlcNAc and GlcA took on more boat/skew-boat conformations that caused kinks in hyaluronan. Importantly, nonsulfated keratan 10-and 20-mers behaved randomly, and glycosidic linkages and monosaccharide rings behaved independently in MD simulations. Construction Algorithm Nonsulfated keratan 20-mer conformational ensembles (four sets of 10,000 conformations) were constructed using our algorithm and compared to MD-generated keratan 20-mer ensembles. The monosaccharide ring ( Figure S26) and glycosidic linkage ( Figure S27) conformations in the constructed keratan 20-mer ensemble were identical to the input data (i.e., MD-generated keratan 20-mer conformations; Figures 15a,b and 16a,b and Table 6), as expected. The end-to-end distances ( Figure 17a and Table 5) and radii of gyration ( Figure S20a,b) were similar in constructed and MD-generated ensembles, demonstrating that nonsulfated keratan 20-mer conformational ensembles provide an accurate representation of backbone flexibility in nonsulfated keratan 20-mer MD simulations. Four sets of nonsulfated keratan 10-mer ensembles with 10,000 conformations each were constructed, and the results were compared to those of nonsulfated keratan 10-mer MD. The end-to-end distances (Figure 17b and Table 5) and radii of gyration ( Figure S20c,d) in constructed 10-mer ensembles matched those of MD-generated 10-mer ensembles, demonstrating that MD-generated 20-mer conformations can be applied to construct ensembles of keratan polymers of different lengths that mimic backbone flexibility seen in MD simulation. A nonsulfated keratan 200-mer ensemble was constructed and, as expected, the end-to-end distance distribution ( Figure 18) and radii of gyration ( Figure S20e) showed that as the polymer length increased, conformations tended to be more compact. Notably, this shift in the end-to-end distance distribution curve was more subtle in keratan than in hyaluronan, which stands to reason, as monosaccharide rings and glycosidic linkages are more rigid and tend to be more extended in keratan 20-mer MD than in hyaluronan 20-mer MD. constructed keratan 20-mer ensemble were identical to the input data (i.e., MD-generated keratan 20mer conformations; Figures 15a,b and 16a,b and Table 6), as expected. The end-to-end distances (Figure 17a and Table 5) and radii of gyration ( Figure S20a,b) were similar in constructed and MDgenerated ensembles, demonstrating that nonsulfated keratan 20-mer conformational ensembles provide an accurate representation of backbone flexibility in nonsulfated keratan 20-mer MD simulations. Four sets of nonsulfated keratan 10-mer ensembles with 10,000 conformations each were constructed, and the results were compared to those of nonsulfated keratan 10-mer MD. The end-toend distances (Figure 17b and Table 5) and radii of gyration ( Figure S20c,d) in constructed 10-mer ensembles matched those of MD-generated 10-mer ensembles, demonstrating that MD-generated 20mer conformations can be applied to construct ensembles of keratan polymers of different lengths that mimic backbone flexibility seen in MD simulation. A nonsulfated keratan 200-mer ensemble was constructed and, as expected, the end-to-end distance distribution ( Figure 18) and radii of gyration ( Figure S20e) showed that as the polymer length increased, conformations tended to be more compact. Notably, this shift in the end-to-end distance distribution curve was more subtle in keratan than in hyaluronan, which stands to reason, as monosaccharide rings and glycosidic linkages are more rigid and tend to be more extended in keratan 20-mer MD than in hyaluronan 20-mer MD. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility The backbone flexibility of nonsulfated heparan 20-mer, quantified by the end-to-end distances ( Figure 19 and Table 7) and radii of gyration ( Figure S28a) in MD simulations was analyzed. The wide end-to-end distance distribution curves and tendency toward lower end-to-end distances, relative to those of the extended 20-mer conformation, indicated that nonsulfated heparan 20-mer was highly flexible and tended toward compact conformations in MD simulations. To determine factors contributing to this conformational flexibility, monosaccharide ring and glycosidic linkage conformations were examined for patterns. Figure 18. End-to-end distance probability distribution of constructed ensemble of nonsulfated keratan 200-mer; most probable end-to-end distance across all four sets is 305 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Molecular Dynamics Simulations: Glycosidic Linkage and Monosaccharide Ring Geometry Effects on Polymer Backbone Flexibility The backbone flexibility of nonsulfated heparan 20-mer, quantified by the end-to-end distances ( Figure 19 and Table 7) and radii of gyration ( Figure S28a) in MD simulations was analyzed. The wide end-to-end distance distribution curves and tendency toward lower end-to-end distances, relative to those of the extended 20-mer conformation, indicated that nonsulfated heparan 20-mer was highly flexible and tended toward compact conformations in MD simulations. To determine factors contributing to this conformational flexibility, monosaccharide ring and glycosidic linkage conformations were examined for patterns. Figure 19. End-to-end distance probability distribution of MD-generated nonsulfated heparan 20-mer ensemble; each of the four runs includes 10,000 conformations; probabilities were calculated for end-to-end distances sorted into 0.5 Å bins. 1 Probabilities were calculated for end-to-end distances sorted into 0.5 Å bins for the 20-mer ensembles and 0.25 Å bins for the 10-mer ensembles. 2 All = end-to-end distance distribution aggregated across all four runs. C-P parameters of GlcNAc rings (Figure 20a) revealed all 4 C 1 chair conformations, in line with NMR and force field data for α-GlcNAc [119]. This stands to reason, as we would not expect much difference in ring conformation between αand β-GlcNAc, because the only structural difference is the orientation of the exocyclic oxygen atom on C 1 (i.e., the linker oxygen on nonterminal GlcNAc monosaccharides in GAG polysaccharides). This structural difference will impact glycosidic linkage conformation but is less likely to impact GlcNAc ring conformation. The IdoA monosaccharide C-P parameters (Figure 20b) from nonsulfated heparan 20-mer MD were similar to those of IdoA in nonsulfated dermatan 20-mer MD (Figure 9b): predominantly 1 C 4 , 2 S O , and some 4 C 1 , which is also in line with NMR and force field data for nonsulfated IdoA in heparan sulfate oligosaccharides [120,123] and nonsulfated heparan trisaccharides [126], and occasional boat and skew-boat conformations (Figure 4b,c; -4IdoAα1-endocyclic ring and linker oxygen atoms in heparan are identical to those of -4GlcAβ1-in hyaluronan). As with GalNAc in dermatan, the presence of neighboring GlcNAc monosaccharides substantially decreased sampling of 4 C 1 conformations [123], which is in line with our results. One NMR and force field study reported 19-49% 2 S O conformations of nonsulfated IdoA in heparan sulfate hexasaccharides, and found that as the degree of GlcNAc sulfation increased, the percentage of 2 S O conformations in adjacent nonsulfated IdoA decreased [120]. As we studied only nonsulfated heparan, we would expect to see a higher proportion of IdoA 2 S O conformations (i.e., close to 49%) if simulated under the same conditions. However, the aforementioned study performed MD in aqueous solution with only neutralizing Na + ions, whereas our systems contained an additional 140 mM NaCl. Another NMR and molecular modeling study showed that increasing NaCl salt concentrations caused a shift in equilibrium of 1 C 4 and 2 S O toward 1 C 4 conformations of 2-O-sulfated IdoA flanked by sulfated glucosamine [123]. This may explain the tendency toward 1 C 4 IdoA conformations, with~54% of IdoA rings across all four 20-mer MD runs in 1 C 4 ,~25% in 2 S O , and~4% in 4 C 1 (Table S3). Furthermore, very few IdoA rings sampled 4 C 1 chair conformations, and one run contained no IdoA 4 C 1 chair conformations ( Figure S29), which is in contrast to the more random distribution of IdoA 4 C 1 chair conformations in nonsulfated dermatan 20-mer MD ( Figure S16). This is likely the result of random kinetic trapping during nonsulfated heparan 20-mer MD simulations, which means IdoA only rarely overcame energy barriers between boat/skew-boat and 4 C 1 conformations. Importantly, it appears that all relevant IdoA conformations (i.e., those in line with the literature) were sampled in nonsulfated heparan 20-mer MD simulations. Therefore, we believe our database of MD-generated 20-mer conformations contains a full conformational landscape of IdoA rings in nonsulfated heparan. The primary goal of our algorithm is to predict backbone conformations of long GAG chains, which requires that (1) monosaccharide rings in GAG 20-mers behave independently in MD simulation and (2) contributions of ring puckers to backbone flexibility match those expected in nonsulfated heparan polymers in aqueous solution. There did not appear to be any interdependency The primary goal of our algorithm is to predict backbone conformations of long GAG chains, which requires that (1) monosaccharide rings in GAG 20-mers behave independently in MD simulation and (2) contributions of ring puckers to backbone flexibility match those expected in nonsulfated heparan polymers in aqueous solution. There did not appear to be any interdependency between adjacent rings, suggesting that monosaccharide rings behave independently in nonsulfated heparan 20-mer MD simulations. To determine the effects of IdoA ring puckering on polymer backbone conformations, end-to-end distances of 20-mer conformations containing monosaccharides in (1) boat and skew-boat conformations that caused a kink in the polymer chain and (2) 2 S O conformations, were analyzed ( Figure S30). The end-to-end distance distribution of 20-mer conformations with the ring puckers that caused a kink was similar to that of the average of the four MD runs and the most probable end-to-end distance was only 1 Å lower in conformations with ring puckers that caused a kink, suggesting that boat and skew-boat conformations that introduce a kink do not necessarily give less compact 20-mer conformations, and thus are not major factors contributing to backbone flexibility. The distribution curve of 20-mer conformations with 2 S O IdoA ring puckers and that of 20-mer conformations with non-2 S O boat/skew-boat puckers that caused a kink were qualitatively similar to that of the full 20-mer MD ensemble, but had additional peaks in probability of higher end-to-end distances. These findings suggest that IdoA boat/skew-boat conformations are associated with the full range of 20-mer backbone conformations in MD simulations with only a slight tendency toward extended 20-mer conformations. Therefore, the proportion of IdoA 2 S O conformations likely does not have a major effect on nonsulfated heparan 20-mer backbone flexibility. Next, glycosidic linkage dihedral free energies ∆G(φ, ψ) were examined ( Figure 21 and Table 8). IdoAα1-4GlcNAc glycosidic linkages sampled primarily −φ, +ψ with secondary basins in −φ, −ψ and GlcNAcα1-4IdoA glycosidic linkages sampled conformations in a single basin (+φ, +ψ). We compared our results to those of an NMR and force field study that performed two sets of MD on each of nonsulfated IdoAα1-4GlcNAc and GlcNAcα1-4IdoA disaccharides, one with IdoA restrained to 1 GlcNAcα1-4IdoA glycosidic linkage conformations differ from any other GAG glycosidic linkage conformation because of the orientation of the linker oxygen atom with respect to GlcNAc. Specifically, the oxygen atom on C1 of α-GlcNAc is in the opposite orientation as that on C1 of any other GAG monosaccharide (Figure 1). The GlcNAcα1-4IdoA linkages have a coiled conformation ( Figure S31), which helps explain the high tendency toward compact conformations in nonsulfated heparan compared to other GAGs. IdoAα1-4 GlcNAc As IdoAα1-4GlcNAc linkages have secondary conformations, we sought to determine if their behavior was random and independent. ΔG(ϕ, ψ) was plotted for each individual IdoAα1-4GlcNAc GlcNAcα1-4IdoA glycosidic linkage conformations differ from any other GAG glycosidic linkage conformation because of the orientation of the linker oxygen atom with respect to GlcNAc. Specifically, the oxygen atom on C 1 of α-GlcNAc is in the opposite orientation as that on C 1 of any other GAG monosaccharide (Figure 1). The GlcNAcα1-4IdoA linkages have a coiled conformation ( Figure S31), which helps explain the high tendency toward compact conformations in nonsulfated heparan compared to other GAGs. GlcNAcα1-4 IdoA As IdoAα1-4GlcNAc linkages have secondary conformations, we sought to determine if their behavior was random and independent. ∆G(φ, ψ) was plotted for each individual IdoAα1-4GlcNAc linkage in each MD run and there did not appear to be any connection between adjacent IdoAα1-4GlcNAc linkages or any patterns across different MD runs. To determine the effects of IdoAα1-4GlcNAc linkages with −φ, −ψ dihedrals on 20-mer backbone flexibility, end-to-end distances of 20-mer conformations with these glycosidic linkage conformations were analyzed. Although these linkage conformations caused a kink in the polymer chain (as with hyaluronan GlcNAcβ1-4GlcA linkages), they were not associated with more compact conformations ( Figure S32). In fact, the end-to-end distance distribution of heparan 20-mer conformations with IdoAα1-4GlcNAc linkage −φ, −ψ dihedrals was similar to that of the full MD-generated 20-mer ensemble. This was likely because these linkage conformations occurred infrequently in nonsulfated heparan 20-mer MD, meaning a single heparan 20-mer conformation was not likely to have many kinks resulting from these −φ, −ψ linkage dihedrals. These findings are in line with the literature, which suggests that IdoA ring conformational flexibility in heparin/heparan sulfate oligo-and polysaccharides does not affect glycosidic linkage conformation or overall backbone shape [132]. To determine if there was any interdependency between different IdoA ring puckers and flanking IdoAα1-4GlcNAc linkage geometries in 20-mer MD simulations, conformations of all IdoAα1-4GlcNAc linkages flanking each of 1 C 4 , 2 S O , 4 C 1 , and boat/skew-boat (non-2 S O ) conformations were analyzed separately ( Figure S33). There did not appear to be strong associations between any particular IdoA ring conformation and flanking IdoAα1-4GlcNAc linkage conformation, which is in line with IdoAα1-4GlcNAc disaccharide data from NMR and MD with restrained 1 C 4 and 2 S O IdoA ring conformations and a biasing potential on glycosidic linkage dihedrals [30]. This supports our hypothesis that glycosidic linkages and monosaccharide rings behave independently in MD simulation of nonsulfated heparan 20-mer. Backbone conformational analysis of nonsulfated heparan 10-mer in MD simulation revealed flexible, compact conformations ( Figure S28c and Table 7), as seen in nonsulfated heparan 20-mer MD. Monosaccharide ring and glycosidic linkage conformations in nonsulfated heparan 10-mer MD were similar to those of nonsulfated heparan 20-mer MD. These findings indicate that in MD simulations of nonsulfated heparan 10-and 20-mers, (1) IdoAα1-4GlcNAc glycosidic linkages with −φ, −ψ dihedrals cause a kink in the polymer chain but are rare and thus do not contribute to compact polymer conformations and (2) monosaccharide rings and glycosidic linkages behave randomly and independently. Construction Algorithm A nonsulfated heparan 20-mer ensemble with 40,000 conformations was constructed by the algorithm. Monosaccharide ring C-P parameters ( Figure S34) and glycosidic linkage conformations ( Figure S35) post-minimization in the constructed ensemble matched the input data (Figures 20a,b and 21a,b), as expected. Additionally, the end-to-end distance distribution and radii of gyration of the constructed ensemble closely resembled those of the MD-generated 20-mer ensemble (Figures 22a and S28a,b and Table 7), demonstrating that our algorithm generates nonsulfated heparan 20-mer ensembles with backbone conformations that mimic backbone flexibility seen in 20-mer MD. A nonsulfated heparan 10-mer ensemble with 40,000 conformations was also constructed by the algorithm and had end-to-end distances and radii of gyration that matched those of MD-generated nonsulfated heparan 10-mer ensembles (Figures 22b and S28c,d and Table 7). This demonstrates that nonsulfated heparan 20-mer conformations from MD can be used to construct 10-mer ensembles that mimic the backbone flexibility seen in nonsulfated heparan 10-mer MD. The constructed nonsulfated heparan 200-mer ensemble had reasonably expected end-to-end distance probability distributions (Figure 23), i.e., the skewness of the curves shifted toward the right, indicating more compact conformations with increasing polymer length. The heparan 200-mer endto-end distance distribution curve was qualitatively similar to that of hyaluronan 200-mer (i.e., the probability peak was of similar magnitude and most probable end-to-end distances were similar), which stands to reason, as ring and linkage conformations in nonsulfated heparan 20-mer MD cause more compact backbone conformations, as in hyaluronan 20-mer MD. A nonsulfated heparan 10-mer ensemble with 40,000 conformations was also constructed by the algorithm and had end-to-end distances and radii of gyration that matched those of MD-generated nonsulfated heparan 10-mer ensembles (Figures 22b and S28c,d and Table 7). This demonstrates that nonsulfated heparan 20-mer conformations from MD can be used to construct 10-mer ensembles that mimic the backbone flexibility seen in nonsulfated heparan 10-mer MD. The constructed nonsulfated heparan 200-mer ensemble had reasonably expected end-to-end distance probability distributions (Figure 23), i.e., the skewness of the curves shifted toward the right, indicating more compact conformations with increasing polymer length. The heparan 200-mer end-to-end distance distribution curve was qualitatively similar to that of hyaluronan 200-mer (i.e., the probability peak was of similar magnitude and most probable end-to-end distances were similar), which stands to reason, as ring and linkage conformations in nonsulfated heparan 20-mer MD cause more compact backbone conformations, as in hyaluronan 20-mer MD. Figure 23. End-to-end distance probability distribution of constructed ensemble of nonsulfated heparan 200-mer; most probable end-to-end distance across all four sets is 260 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Conclusions Collectively, our findings support our hypotheses that (1) glycosidic linkages and monosaccharide rings in hyaluronan and nonsulfated dermatan, keratan, and heparan GAG 20-mers behave randomly and independently in MD simulation and (2) using a database of conformations from corresponding GAG 20-mer MD simulations and treating glycosidic linkages and monosaccharide rings independently, our algorithm can efficiently construct conformational ensembles of hyaluronan and nonsulfated dermatan, keratan, and heparan GAG 10-and 20-mers that mimic backbone flexibility observed in corresponding MD simulations. Additionally, our algorithm constructed sets of 10,000 molecular conformations of nonsulfated GAG 200-mers with backbone conformations that we would reasonably expect to see in MD simulation and did so within 12 h. This suggests that the algorithm can generate conformational ensembles of nonsulfated heterogeneous GAG polymers of arbitrary length in under a day. For perspective, 1-µs MD simulations, each producing 10,000 snapshots (i.e., 3-D atomic coordinate sets), of GAG 10-and 20-mers were completed in about 1-2 weeks and 1-2 months, respectively, and using modern GPU-accelerated hardware and software. Furthermore, the algorithm's potential energy minimization and bond potential energy cutoff criterion exclude nonphysical conformations from constructed ensembles, leaving only conformations with reasonably expected bond energies ( Figures S36, S37, S38, and S39). A comparison of the different GAG 20-mer MD simulations provided further insights into the relationship between GAG structure and conformation. For example, no associations between adjacent monosaccharide conformations were seen in any of the GAG 20-mer MD simulations, but differences in conformations of the same monosaccharide ring type in different GAGs were observed. This indicates that the structure of adjacent monosaccharides contributes to ring conformation. Furthermore, IdoA monosaccharides were much more conformationally flexible than GlcA monosaccharides in GAG 10-and 20-mer MD simulations, even though the structural difference between these two monosaccharide types (specifically, the orientation of the carboxylate group on C5) is subtle. Interestingly, dermatan and heparan are the only GAGs with IdoA monosaccharides, yet hyaluronan and heparan showed the most conformational flexibility, and heparan showed the greatest tendency toward compact conformations compared to other GAGs. This was likely because of variability in glycosidic linkage conformation, which is independent of monosaccharide ring conformation. Independence of glycosidic linkages and monosaccharide rings was further evidenced by the fact that despite differences in flanking monosaccharide structure in different GAG types, there were similarities between all 1-3 linkage conformations and between all 1-4 linkage conformations, with the exception of GlcNAcα1-4IdoA linkages in heparan. Figure 23. End-to-end distance probability distribution of constructed ensemble of nonsulfated heparan 200-mer; most probable end-to-end distance across all four sets is 260 Å; probabilities were calculated for end-to-end distances sorted into 5 Å bins; the ensemble contains four sets of 10,000 conformations. Conclusions Collectively, our findings support our hypotheses that (1) glycosidic linkages and monosaccharide rings in hyaluronan and nonsulfated dermatan, keratan, and heparan GAG 20-mers behave randomly and independently in MD simulation and (2) using a database of conformations from corresponding GAG 20-mer MD simulations and treating glycosidic linkages and monosaccharide rings independently, our algorithm can efficiently construct conformational ensembles of hyaluronan and nonsulfated dermatan, keratan, and heparan GAG 10-and 20-mers that mimic backbone flexibility observed in corresponding MD simulations. Additionally, our algorithm constructed sets of 10,000 molecular conformations of nonsulfated GAG 200-mers with backbone conformations that we would reasonably expect to see in MD simulation and did so within 12 h. This suggests that the algorithm can generate conformational ensembles of nonsulfated heterogeneous GAG polymers of arbitrary length in under a day. For perspective, 1-µs MD simulations, each producing 10,000 snapshots (i.e., 3-D atomic coordinate sets), of GAG 10-and 20-mers were completed in about 1-2 weeks and 1-2 months, respectively, and using modern GPU-accelerated hardware and software. Furthermore, the algorithm's potential energy minimization and bond potential energy cutoff criterion exclude nonphysical conformations from constructed ensembles, leaving only conformations with reasonably expected bond energies ( Figures S36-S39). A comparison of the different GAG 20-mer MD simulations provided further insights into the relationship between GAG structure and conformation. For example, no associations between adjacent monosaccharide conformations were seen in any of the GAG 20-mer MD simulations, but differences in conformations of the same monosaccharide ring type in different GAGs were observed. This indicates that the structure of adjacent monosaccharides contributes to ring conformation. Furthermore, IdoA monosaccharides were much more conformationally flexible than GlcA monosaccharides in GAG 10and 20-mer MD simulations, even though the structural difference between these two monosaccharide types (specifically, the orientation of the carboxylate group on C 5 ) is subtle. Interestingly, dermatan and heparan are the only GAGs with IdoA monosaccharides, yet hyaluronan and heparan showed the most conformational flexibility, and heparan showed the greatest tendency toward compact conformations compared to other GAGs. This was likely because of variability in glycosidic linkage conformation, which is independent of monosaccharide ring conformation. Independence of glycosidic linkages and monosaccharide rings was further evidenced by the fact that despite differences in flanking monosaccharide structure in different GAG types, there were similarities between all 1-3 linkage conformations and between all 1-4 linkage conformations, with the exception of GlcNAcα1-4IdoA linkages in heparan. A comparison of MD-generated GAG 20-mer conformations to those of GAG 10-mers and to existing experimentally-determined conformations of monosaccharides and GAG oligosaccharides supported the use of 20-mer data for the construction of longer GAG polymers. For example, certain IdoA conformations (namely 4 C 1 chair) were found more in unbound monosaccharide rings and terminal rings of GAG oligosaccharides than in central rings. Similarly, galactose occasionally sampled non-4 C 1 conformations in monosaccharide rings and short oligosaccharides, including nonsulfated keratan 10-mer in MD simulation, but boat/skew-boat conformations of galactose were decreasingly common in central rings of polymers of increasing length. This is in line with our observation that only 4 C 1 conformations of galactose were sampled in nonsulfated keratan 20-mer MD. Additionally, some nonhelical glycosidic linkage conformations that caused a kink in the polymer chain, and thus contributed to compact GAG backbone conformations, were found more in GAG 20-mers than in short GAG oligosaccharides. Therefore, conformational landscapes from GAG 20-mer MD simulations likely provide a better representation of conformations of long GAG polymers than existing conformational landscapes of monosaccharides and GAG oligosaccharides. A comparison of backbone conformational analyses of 200-mers of different GAG types provided insights into structural features and conformational behaviors contributing to GAG polymer backbone flexibility. For example, 200-mer end-to-end distance distribution curves were qualitatively similar in nonsulfated dermatan ( Figure 13), keratan (Figure 18), and chondroitin [33], whereas hyaluronan and nonsulfated heparan polymer end-to-end distance distributions (Figures 7 and 23, respectively) showed an even higher tendency toward compact conformations with increasing polymer length than other nonsulfated GAGs. To find possible explanations for this, we compared observations from MD analyses of all GAG types. In hyaluronan 10-and 20-mer MD simulations, (1) GlcNAc conformations were similar to those in nonsulfated keratan and heparan MD, and (2) GlcA conformations were similar to those in nonsulfated chondroitin MD [33]. Furthermore, IdoA rings showed more flexibility in MD simulation of nonsulfated dermatan and heparan than GlcA rings in hyaluronan and chondroitin MD [33]. However, boat/skew-boat conformations (except for 2 S O ) in IdoA did not appear to cause more compact polymer backbone conformations than 1 C 4 , 2 S O , and 4 C 1 IdoA conformations, while boat/skew-boat GlcA ring conformations were more highly associated with more compact polymer backbone conformations in hyaluronan and chondroitin [33]. Additionally, in hyaluronan 10-and 20-mer MD simulations, (1) GlcNAcβ1-4GlcA linkages took on the same conformations as Galβ1-4GlcNAc linkages in nonsulfated keratan MD, IdoAα1-4GlcNAc linkages in nonsulfated heparan MD, and GalNAcβ1-4GlcA in nonsulfated chondroitin MD [33] (i.e., primarily −φ, +ψ with a secondary basin at −φ, −ψ) and (2) GlcAβ1-3GlcNAc linkages took on the same conformations as IdoAα1-3GalNAc in nonsulfated dermatan MD, GlcNAcβ1-3Gal in nonsulfated keratan MD, and GlcAβ1-3GalNAc linkages in nonsulfated chondroitin MD [33] (i.e., primarily −φ, −ψ), but with more conformations at −φ, +ψ. All secondary and tertiary conformations (i.e., −φ, −ψ in 1-4 linkages and −φ, +ψ in 1-3 linkages) were nonhelical and caused a kink or slight bend in the polymer chain, and were, therefore, associated with more compact conformations. The differences in energetic stability of secondary and tertiary linkage conformations between different GAG types can be explained by the adjacent monosaccharide structure. The major observation that is unique to hyaluronan is that both glycosidic linkage types take on more of these nonhelical secondary conformations in 10-and 20-mer MD simulations than any of nonsulfated dermatan, keratan, or chondroitin [33] in 10-and 20-mer MD simulations. Heparan is unique, in that it has all 1-4 linkages, while all other GAGs have alternating 1-3 and 1-4 linkages, and it contains α-GlcNAc, which gives unique conformations in GlcNAcα1-4IdoA linkages. These characteristics give nonsulfated heparan a tendency toward more coiled structures than other GAGs. Based on these observations and the fact that nonsulfated dermatan, keratan, and chondroitin showed more extended polymer backbone conformations than hyaluronan and nonsulfated heparan in MD simulations, it is likely that: (1) monosaccharide structure and conformational flexibility determine adjacent monosaccharide conformational flexibility; (2) although there is a much higher degree of ring flexibility in IdoA than in GlcA, the flexibility of GlcA rings is more highly associated with GAG polymer backbone flexibility than that of IdoA rings; and (3) nonhelical glycosidic linkage conformations, which cause kinks in GAG polymer chains, contribute to more compact GAG polymer backbone conformations and, thus, a higher degree of GAG polymer backbone flexibility. These are valuable insights that would be difficult to obtain from conformational analyses of only solid-state structures and MD-generated conformational ensembles of short GAG oligosaccharides. Other important insights that can be gained from 3-D atomic-resolution conformational ensembles of GAG polymers include potential binding properties, and thus, predictions of binding poses with other biomolecules of interest. Models of long-chain GAG polymers ( Figure 24 and [33]) can help characterize complete PGs and GAG-mediated complexes between multiple biomolecules, and consequently, improve our understanding of the bioactivity and function of GAG biopolymers in animal tissue. with GAG polymer backbone flexibility than that of IdoA rings; and (3) nonhelical glycosidic linkage conformations, which cause kinks in GAG polymer chains, contribute to more compact GAG polymer backbone conformations and, thus, a higher degree of GAG polymer backbone flexibility. These are valuable insights that would be difficult to obtain from conformational analyses of only solid-state structures and MD-generated conformational ensembles of short GAG oligosaccharides. Other important insights that can be gained from 3-D atomic-resolution conformational ensembles of GAG polymers include potential binding properties, and thus, predictions of binding poses with other biomolecules of interest. Models of long-chain GAG polymers ( Figure 24 and [33]) can help characterize complete PGs and GAG-mediated complexes between multiple biomolecules, and consequently, improve our understanding of the bioactivity and function of GAG biopolymers in animal tissue. Figure S1: Constructed hyaluronan 20-mer conformation with a pierced ring, Figure S2: Scatterplots of radius of gyration as a function of end-to-end distance in MD-generated and constructed hyaluronan ensembles, Figure S3: System potential energy probability distribution of the MD-generated hyaluronan 20-mer ensemble, Figure S4: C-P plots and C-P parameter θ timeseries for each GlcNAc monosaccharide ring in the MD-generated hyaluronan 20-mer ensemble, Figure S5: C-P plots and C-P parameter θ timeseries for each GlcA monosaccharide ring in the MD-generated hyaluronan 20-mer ensemble, Figure S6: Scatterplot of radius of gyration as a function of end-to-end distance in MD-generated hyaluronan 20-mer conformations with non-4 C 1 ring puckers, Figure S7: ∆G(φ,ψ) plots for each glycosidic linkage in the MD-generated hyaluronan 20-mer ensemble, Figure S8: MD snapshots of hyaluronan 20-mer GlcAβ1-3GlcNAc glycosidic linkages with dihedrals in different basins and end-to-end distance distributions of 20-mer conformations with linkages in secondary basin, Figure S9: ∆G(φ,ψ) plots for glycosidic linkages flanking non-4 C 1 ring puckers in the MD-generated hyaluronan 20-mer ensemble, Figure S10: MD snapshots of hyaluronan 20-mer GlcNAcβ1-4GlcA glycosidic linkages with dihedrals in different basins and end-to-end distance distributions of 20-mer conformations with linkages in secondary basin, Figure S11: C-P plots for GlcNAc and GlcA in the constructed hyaluronan 20-mer ensemble, Figure S12: ∆G(φ, ψ) for aggregated glycosidic linkage data from the constructed hyaluronan 20-mer ensemble, Figure S13: Scatterplots of radius of gyration as a function of end-to-end distance in MD-generated and constructed nonsulfated dermatan ensembles, Figure S14: C-P parameter θ timeseries for each GalNAc monosaccharide ring in the MD-generated nonsulfated dermatan 20-mer ensemble, Figure S15: End-to-end distance distributions of MD-generated nonsulfated dermatan 20-mer conformations with boat/skew-boat ring puckers that cause a kink in the polymer chain and 2 S O conformations, Figure S16: C-P plots and C-P parameter θ timeseries for each IdoA monosaccharide ring in the MD-generated nonsulfated dermatan 20-mer ensemble, Figure S17: C-P plots and C-P parameter θ timeseries for each IdoA monosaccharide ring in the MD-generated nonsulfated dermatan 10-mer ensemble, Figure S18: C-P plots for GalNAc and IdoA in the constructed nonsulfated dermatan 20-mer ensemble, Figure S19: ∆G(φ, ψ) for aggregated glycosidic linkage data from the constructed nonsulfated dermatan 20-mer ensemble, Figure S20: Scatterplots of radius of gyration as a function of end-to-end distance in MD-generated and constructed nonsulfated keratan ensembles, Figure S21: C-P plots and C-P parameter θ timeseries for each GlcNAc monosaccharide ring in the MD-generated nonsulfated keratan 20-mer ensemble, Figure S22: Scatterplot of radius of gyration as a function of end-to-end distance in MD-generated nonsulfated keratan 20-mer conformations with non-4 C 1 ring puckers, Figure S23: MD snapshots of nonsulfated keratan 20-mer Galβ1-4GlcNAc glycosidic linkages with dihedrals in different basins and end-to-end distance distributions of 20-mer conformations with linkages in secondary and tertiary basins, Figure S24: MD snapshots of nonsulfated keratan 20-mer GlcNAcβ1-3Gal glycosidic linkages with dihedrals in different basins and end-to-end distance distributions of 20-mer conformations with linkages in secondary and tertiary basins, Figure S25: C-P plots and C-P parameter θ timeseries for each Gal monosaccharide ring in the MD-generated nonsulfated keratan 10-mer ensemble, Figure S26: C-P plots for GlcNAc and Gal in the constructed nonsulfated keratan 20-mer ensemble, Figure S27: ∆G(φ, ψ) for aggregated glycosidic linkage data from the constructed nonsulfated keratan 20-mer ensemble, Figure S28: Scatterplots of radius of gyration as a function of end-to-end distance in MD-generated and constructed nonsulfated heparan ensembles, Figure S29: C-P parameter θ timeseries for each IdoA monosaccharide ring in the MD-generated nonsulfated heparan 20-mer ensemble, Figure S30: End-to-end distance distributions of MD-generated nonsulfated heparan 20-mer conformations with boat/skew-boat ring puckers that cause a kink in the polymer chain and 2 S O conformations, Figure S31: Snapshot of nonsulfated heparan 20-mer MD-generated ensemble highlighting GlcNAcα1-4IdoA linkages, Figure S32: End-to-end distance distributions of MD-generated nonsulfated heparan 20-mer conformations with linkage dihedrals in secondary basins, Figure S33: ∆G(φ,ψ) plots for IdoAα1-4GlcNAc linkages flanking different IdoA conformations in the MD-generated nonsulfated heparan 20-mer ensemble, Figure S34: C-P plots for GlcNAc and IdoA in the constructed nonsulfated heparan 20-mer ensemble, Figure S35: ∆G(φ, ψ) for aggregated glycosidic linkage data from the constructed nonsulfated heparan 20-mer ensemble, Figure S36: Bond potential energy probability distributions from constructed hyaluronan ensembles, Figure S37: Bond potential energy probability distributions from constructed nonsulfated dermatan ensembles, Figure S38: Bond potential energy probability distributions from constructed nonsulfated keratan ensembles, Figure S39: Bond potential energy probability distributions from constructed nonsulfated heparan ensembles.	v3-fos-license
2021-05-04T22:05:33.338Z	2021-04-03T00:00:00.000	233598436	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.cell.com/article/S2372770521000504/pdf", "pdf_hash": "daf08b9c0b00f13f50d023ade444972c442db41f", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2289", "s2fieldsofstudy": [ "Medicine", "Biology" ], "sha1": "8dbb4d02d28ed790eac963c979ce6a1236a972d9", "year": 2021 }	pes2o/s2orc	MicroRNA-499 serves as a sensitizer for lung cancer cells to radiotherapy by inhibition of CK2α-mediated phosphorylation of p65 The present study aimed to define the tumor-suppressive role of microRNA-499 (miR-499) in lung cancer cells and its underlying mechanism. First, qRT-PCR analysis revealed poor expression of miR-499 in clinical samples and cell lines of lung cancer. Next, we performed loss- and gain-of-function experiments for the expression of miR-499 in lung cancer cells exposed to irradiation (IR) to determine the effect of miR-499 expression on cell viability and apoptosis as well as tumor growth. Results showed that overexpression of miR-499 inhibited cell viability, enhanced the radiosensitivity of lung cancer cells, and promoted cell apoptosis under IR. Furthermore, CK2α was verified to be a target of miR-499, and miR-499 was identified to repress p65 phosphorylation by downregulating CK2α expression, which ultimately diminished the survival rate of lung cancer cells under IR. Collectively, the key findings of the study illustrate the tumor-inhibiting function of miR-499 and confirmed that miR-499-mediated CK2α inhibition and altered p65 phosphorylation enhances the sensitivity of lung cancer cells to IR. INTRODUCTION Lung cancer not only remains one of the most frequently diagnosed cancers but also ranks first for cancer-related mortality, accounting for nearly 2 million deaths worldwide on an annual basis. 1,2 However, approximately 20% to 25% of all lung cancer patients are diagnosed at an early disease stage (stage IA-IIIA), which is amenable to curative surgery. 3 Although various diagnostic procedures are currently available, tissue biopsy remains the mainstay of routine lung cancer diagnostics. 4 This being said, the advancement of prevention strategies and the development of novel treatment strategies are urgently required to improve the rather dismal prognosis of lung cancer. 5 The dysregulation of microRNAs (miRNAs) has been well documented to be associated with the development of lung cancer, 6,7 such as miR-1276, 8 thus highlighting this as a potential treatment target for lung cancer. In general, miRNAs reportedly play a crucial role in the development of lung cancer by regulating various target genes associated with this malignancy. 9 A previous study suggested that the sequence variation of mature miR-499 conferred an adverse prognosis on lung cancer patients. 10 In addition, serum miR-499 has been suggested to be a promising biomarker for early exploration and prognostic prediction of non-small cell lung cancer (NSCLC). 11 In this regard, we speculated that miR-499 might target CK2a and regulate the development of lung cancer. Interestingly, transcription factors and miRNAs have been reported to have synergistic effects, characterized by unique molecular mechanisms and evolutionary background, which correspond to the two main levels of gene regulatory networks. 12 Transcription factor CK2a has been reported to contribute to lung cancer metastasis by targeting BRMS1 nuclear output and degradation. 13 Additionally, lung cancer patients have been suggested to benefit from treatment with CK2a inhibitors via activation of the Notch1 signaling pathway. 14 Intriguingly, CK2a has been demonstrated to affect several cellular signaling pathways by phosphorylation mechanisms. 15 Additionally, inhibition of EZH2 by reducing transcription factor p65 presents a new mechanism to suppress human lung cancer cells. 16 Transcription factor p65 also plays a crucial role in the induction of cell anti-viral responses. 17 Based on the aforementioned literature, we designed this study to investigate whether miR-499 could regulate CK2a expression to affect p65 phosphorylation, thereby modulating the sensitivity of lung cancer to irradiation (IR), thus defining new alternative therapeutic strategies for the treatment of lung cancer. Downregulation of miR-499 is observed in lung cancer and associated with poor prognosis After differential analysis of the NSCLC-associated miRNA microarray GSE102286, we screened 94 differentially expressed miRNAs (Table S1). Next, we obtained 430 and 519 NSCLC-related miRNAs through the HMDD database (http://www.cuilab.cn/hmdd) and MNDR database (http://www.rna-society.org/mndr/), respectively, and then obtained five candidate miRNAs, namely hsa-miR-34c-5p, hsa-miR-499-5p, hsa-miR-140-3p, hsa-miR-193a-3p, and hsa-miR-34c-3p, after intersection of differentially expressed miRNAs and NSCLC-related miRNAs ( Figure 1A). The differential expression heatmap of candidate miRNAs in control and NSCLC samples was also plotted ( Figure 1B). It has been reported that miR-499-5p overexpression can inhibit the proliferation and metastasis of NSCLC by targeting VAV3, 18 and that miR-499 overexpression can impart poor prognosis by regulating tumor-related gene expression, enhancing tumorigenesis and chemoresistance. As such, miR-499 may be a relevant biomarker for predicting the prognosis of NSCLC patients. 10 In order to verify the correlation between miR-499 expression in lung cancer cells and to clarify its relationship with poor prognoses, we analyzed the expression of miR-499 in lung cancer tissues and adjacent normal tissues by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The qRT-PCR results demonstrated that the expression of miR-499 was diminished in the tumor tissues compared with the normal tissues ( Figure 1C). For further validation, we selected four human lung cancer cell lines (A549, Calu-3, NCI-H209, and NCI-H292) and one human lung normal cell line (MRC-5) and assessed the expression of miR-499 in these five cell lines by qRT-PCR detection. The results demonstrated that, compared with MRC-5 cells, the expression of miR-499 was reduced in all four cancer cells lines examined (Figure 1D), providing evidence indicating that miR-499 was downregulated in lung cancer cells. Furthermore, receiver operating characteristic (ROC) analysis revealed a cutoff value of 0.798 and indicated that miR-499 could be a diagnostic indicator for patients with lung cancer (Figure 1E). We conducted 2-year follow-up for 12 cases with miR-499 expression above the medium (miR-499-above median) and 75 cases with miR-499 expression below the median value (miR-499-low median). Finally, Kaplan-Meier survival analysis of the overall survival of patients was performed as a function of the tumor level of miR-499 expression. The results illustrated that lung cancer patients with high expression of miR-499 exhibited a higher overall survival rate ( Figure 1F). Overexpression of miR-499 enhances the sensitivity of lung cancer cells to IR exposure in vitro We further found that the expression of miR-499 was elevated in miR-499 mimic-IR-treated cells relative to miR-499 mimic-treated cells, in inhibitor negative control (NC)-IR-treated cells compared to inhibitor NC-treated cells, and in miR-499 inhibitor-IR-treated cells compared to miR-499 inhibitor-treated cells (Figure 2A). We subsequently overexpressed or knocked down miR-499 using miR-499 mimic or miR-499 inhibitor in NCI-H292 cells. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the cell viability in the miR-499 mimic group was significantly reduced compared with the mimic NC group and that cell viability in the miR-499 inhibitor group was remarkably increased compared with the inhibitor NC group. Furthermore, compared with the corresponding non-irradiated groups (mimic NC-IR versus mimic NC, miR-499 mimic-IR versus miR-499 mimic, inhibitor NC-IR versus inhibitor NC, miR-499 inhibitor-IR versus miR-499 inhibitor), the proliferation rate of cells with different treatments was significantly reduced following IR ( Figure 2B). Meanwhile, we evaluated the viability of A549 ( Figure 2C) and NCI-H292 ( Figure S1A) cells via the MTT assay after 1-10 Gy IR exposure. The cell viability after miR-499 mimic treatment was diminished with identical doses of IR, while cell viability was elevated after miR-499 inhibitor treatment under the same doses of IR. In addition, compared with the corresponding non-irradiated groups (mimic NC-IR versus mimic NC, miR-499 mimic-IR versus miR-499 mimic, inhibitor NC-IR versus inhibitor NC, miR-499 inhibitor-IR versus miR-499 inhibitor), the proliferation rate following IR of cells with different treatments was significantly reduced. Finally, colony formation abilities in A549 ( Figure 2D) and NCI-H292 (Figure S1B) cells displayed a reduced cell colony formation upon treatment of miR-499 mimic and elevated cell colony formation following treatment with miR-499 inhibitor after 10 Gy IR exposure; compared with the corresponding non-irradiated groups (mimic NC-IR versus mimic NC, miR-499mimic-IR versus miR-499 mimic, inhibitor NC-IR versus inhibitor NC, miR-499 inhibitor-IR versus miR-499 inhibitor), the colonies of cells with different treatments were significantly reduced following IR. Moreover, we found that the invasion and migration abilities of miR-499 mimic-treated cells were decreased miR-499 promotes IR-induced apoptosis of lung cancer cells in vitro Flow cytometry assay was employed to detect apoptosis in A549 (Figure 3A) and NCI-H292 ( Figure S1C) cell lines after varying doses of IR (0, 6, 10 Gy) in response to miR-499 mimic/inhibitor. Without IR exposure, apoptosis was augmented in miR-499 mimic-transfected cells, while it was reduced upon miR-499 inhibitor transfection. Apoptosis was enhanced in all the groups following IR exposure, but a higher rate of apoptosis was observed in the cells transfected with miR-499 mimic when compared to cells transfected with mimic NC. Relative to the inhibitor NC-treated A549 cells, the apoptosis was decreased in miR-499 inhibitor-treated A549 cells. Higher doses of IR led to a more pronounced difference in apoptosis between miR-499 mimic-and mimic NC-treated cells. Meanwhile, western blot analysis of the protein expression apoptosis-related factors in A549 ( Figure 3B) and NCI-H292 ( Figure S1D) cell lines further validated the aforementioned results. A549 cells treated with miR-499 mimic displayed no change in caspase-3 expression but elevated expression of cleaved PARP and cleaved caspase-3. In addition, higher IR doses resulted in greater increases in the expression of cleaved PARP and cleaved caspase-3. On the other hand, miR-499 inhibitor transfection exerted no effect on caspase-3 expression but resulted in reduced expression A B Figure 3. miR-499-enhanced IR-induced apoptosis of lung cancer A549 cells in vitro (A) The effect of overexpression or silencing of miR-499 on IR-induced apoptosis detected by flow cytometry assay. (B) The effect of overexpression or silencing of miR-499 on IR-induced caspase-3, cleaved PARP, and cleaved caspase-3 protein expression determined by western blot analysis. *p < 0.05 versus cells treated with mimic NC; # p < 0.05 versus cells treated with inhibitor NC. The measurement data were expressed as mean ± standard deviation. Data between the two groups were compared using independent sample t test. The experiments were repeated for three times, with the representative result presented. of cleaved PARP and cleaved caspase-3. Furthermore, higher IR doses attenuated the decreases in expression of cleaved PARP and cleaved caspase-3. Therefore, we conclude that overexpression of miR-499enhanced IR-induced apoptosis of lung cancer cells. miR-499 increases the sensitivity of lung cancer cells to IR exposure in vivo We subsequently subcutaneously injected A549 cell lines transfected with miR-499 mimic into the nude mice. The nude mice were subjected to 10 Gy IR on the ninth day after xenografting and photographed every 3 days to monitor the tumor growth of different groups ( Figures 4A-4C). The in vivo experiments demonstrated that, in comparison to the mice injected with mimic NC, there was a slight decrease in tumor volume and weight in mice injected with miR-499 mimic, and, furthermore, the tumor volume and weight of mice treated with mimic NC + IR were reduced. In comparison with mice treated with mimic NC + IR, the tumor volume and weight were notably diminished in mice treated with miR-499 mimic and IR. Thus, miR-499 overexpression augmented the sensitivity of lung cancer cells to IR exposure in vivo. The results of hematoxylin and eosin (H&E) staining assay of the resected tumors showed that, compared with the mimic NC group, the cytoplasmic content of some cells in the miR-499 mimic group was concentrated, along with absent nucleoli, and likewise in the mimic NC + IR group. Compared with the mimic NC + IR group, cells in the miR-499 mimic + IR group had the most significant shrinkage, absence of nucleoli, and cell necrosis ( Figure 4D). The results of immunohistochemistry (IHC) showed that the number of Ki67-, CK2a-, and p-p65-positive cells was significantly decreased in the miR-499 mimic group compared with the mimic NC group, and the number of Ki67-, CK2a-, and p-p65-positive cells was significantly reduced in the mimic NC + IR group. Compared with the mimic NC + IR group, the number of Ki67-, CK2a-, and p-p65-positive cells in the miR-499 mimic + IR group was significantly reduced ( Figure 4E). miR-499 upregulates the sensitivity of lung cancer cells to IR exposure via inhibition of CK2a to repress p65 phosphorylation We had first predicted 600, 1,461, and 1,576 downstream target genes of miR-499 by scrutiny of the bioinformatics databases TargetScan, mirDIP, and RNAInter, respectively, and also obtained 2,856 NSCLC-related genes by GeneCards database. Next, 58 candidate genes were flagged by intersection of target genes with NSCLC-related genes ( Figure 5A). The candidate gene interaction relationship The measurement data were expressed as mean ± standard deviation. Data between two groups were compared using independent sample t test. Comparisons among multiple groups were conducted by ANOVA, followed by Bonferroni's post hoc test. www.moleculartherapy.org network was obtained by the STRING tool, and the interaction relationship network of the genes was plotted. The results showed that five genes, namely FGF2, CDKN1A, GATA3, IKBKB, and CSNK2A2 (alias: CK2a), were in the core position (degree R 17) ( Figure 5B). Further, we found that CK2a knockout can significantly enhance the radiosensitivity of a variety of lung cancer cells. 19 Binding sites be-tween miR-499 and CK2a were predicted and subsequently verified via the bioinformatics website (http://www.targetscan.org/ mamm_31/) and confirmed by dual luciferase gene assay in HEK293T and A549 cells, which showed that CK2a could indeed augment the tolerance of lung cancer cells to radiotherapy ( Figures 5C and 5D). qRT-PCR results ( Figures 5E and 5F) demonstrated The measurement data were expressed as mean ± standard deviation. Data between two groups were compared using independent sample t test. that, in comparison to the cells treated with mimic NC, miR-499 expression was increased, while the expression of CK2a was diminished in the cells treated with miR-499 mimic. In comparison to the cells transfected with inhibitor NC, miR-499 expression was reduced, while the expression of CK2a was markedly elevated in the cells transfected with miR-499 inhibitor. In comparison to oe-NC treated cells, there was no significant difference detected in the expression of miR-499, whereas the expression of CK2a was elevated in oe-CK2a-treated cells. Relative to the si-NC-transfected cells, CK2a expression was notably diminished in the si-CK2a-transfected cells. In the miR-499 mimic and oe-CK2a-treated cells, there was a significant increase in CK2a expression relative to treatment with miR-499 mimic and oe-NC. In the cells treated with miR-499 inhibitor and si-CK2a, there was a considerable reduction in CK2a expression observed in comparison to cells treated with miR-499 inhibitor and si-NC. miRNAs exist in the cytoplasm as components of the RNA-induced silencing complex (RISC), of which Ago2 is a key component that is required for miRNA-mediated gene silencing. 20 The results of qPCR and western blot analyses showed no significant difference in the expression levels of Ago2 between cancer tissues and adjacent normal tissues, as well as between lung cancer cells and immortalized human bronchial epithelial cell line cells ( Figure S2). In this study, an RNA binding protein immunoprecipitation (RIP) experiment was performed in lung-cancer-resistant cell extracts using an antibody against Ago2 to determine whether CK2a and miR-499 belong to the same RISC. RIP results showed that miR-499 mimic-treated cells had Ago2-rich miRNAs compared with control immunoglobulin G (IgG) immunoprecipitation. High CK2a expression was detected in the same precipitate ( Figure 5G). p65 expression and phosphorylation of p65 content was analyzed in A549 cells by western blot after overexpression or silencing of CK2a. The results revealed no notable change in the expression of p65, while the relative phosphorylation of p65 content was elevated in cells transfected with oe-CK2a compared with cells transfected with oe-NC ( Figure 5H). In comparison to the treatment with si-NC, there was no significant change of p65 expression, while the relative phosphorylation of p65 content was remarkably reduced in the cells with si-CK2a treatment ( Figure 5H). Next, to verify further whether miR-499 could increase the sensitivity to radiotherapy by targeting CK2a to regulate p65 phosphorylation, we treated lung cancer A549 cells with a CK2a inhibitor (CX-4945) following the inhibition of miR-499. Cell survival rate was determined using colony-formation assay after IR exposure at different doses (0, 2, 4, 6, 8, and 10 Gy), and the expressions of the related proteins were analyzed by western blot analysis. After 10 Gy IR exposure, the higher doses of IR resulted in lower cell survival rate. Compared with inhibitor NC treatment, the cell survival rate increased in the presence of miR-499 inhibitor but decreased in cells treated with CX-4945. In addition, cell survival rate was increased in response to miR-499 inhibitor and CX-4945 treatment compared to CX-4945 alone ( Figure 5I). Cell survival in different groups was detected by colony-formation assay. The results showed that the colony formation was significantly reduced with increasing IR dose; compared with the inhibitor NC group, the cell colonies in the miR-499 inhibitor group were significantly increased, whereas the cell colonies in the inhibitor NC + CX-4945 group were significantly reduced. Compared with the inhibitor NC + CX-4945 group, the miR-499 inhibitor + CX-4945 group had significantly increased colonies ( Figure 5J). Following miR-499 inhibitor treatment, there was no significant difference in p65 expression; the relative phosphorylation of p65 content was elevated, while the expression of cleaved PARP, caspase-3, and cleaved caspase-3 was diminished in comparison to inhibitor NC treatment. Furthermore, while inhibitor NC + CX-4945 treatment exerted no effect on p65 expression, it decreased phosphorylation of p65 content and elevated the expression of cleaved PARP, caspase-3, and cleaved caspase-3. Relative to miR-499 inhibitor treatment, miR-499 inhibitor and CX-4945 treatment exerted no effect on p65 expression but resulted in reduced phosphorylation of p65, as well as elevated expression of cleaved PARP, caspase-3, and cleaved caspase-3. Together, these results provided evidence demonstrating that miR-499 could target CK2a to inhibit p65 phosphorylation and repress apoptosis (Figure 5K). The same results were also obtained in NCI-H292 cells (Figures S3A-S3D). Altogether, the aforementioned results indicated that targeted regulation of miR-499 could potentially be used as a treatment to prevent the spread of lung cancer and the onset of radioresistance. DISCUSSION A diverse group of miRNAs continues to be implicated in the occurrence and development of lung cancer. 21 Thus, the overexpression of miR-499 has been previously reported to repress the proliferation and metastasis of NSCLC. 22 The present study further evidenced that miR-499 overexpression could inhibit the development of lung cancer and enhance the sensitivity of lung cancer cells to IR exposure. The results obtained demonstrated low levels of miR-499 expression in lung cancer tissues and cells compared to non-cancer samples. In addition, the forced overexpression of miR-499 resulted in elevated radiotherapy sensitivity of lung cancer cells in vitro. Overexpression of miR-499 was found to repress proliferation while elevating the apoptosis of lung cancer cells. Existing literature has suggested that the knockdown of miR-499 could augment apoptosis at the later stages of cell differentiation. 23 Previous research has indicated that miR-499a may enhance the apoptosis of glioma cells by inhibiting the MAPK signaling pathway. 24 The aforementioned findings were partially consistent with our earlier observations that miR-499 contributed to the apoptosis of lung cancer cells. Furthermore, the overexpression of miR-499 enhanced the sensitivity of lung cancer cells to IR, decreased cell viability, and promoted cell apoptosis under different doses of IR, as well as increasing the levels of the apoptosis-related proteins cleaved PARP and cleaved caspase-3. Additionally, apoptosis was reflected by the regulation of markers such as the elevated level of cleaved-caspase 3 and cleaved-PARP proteins. 25 Interestingly, miR-499-5p has been reported to trigger an increase in the expression levels of Bcl-2 and downregulate the expression of caspase-3. 26 www.moleculartherapy.org In the subsequent experiments, bioinformatics website analysis and dual-luciferase gene assay were performed to predict and validate the relationship between miR-499 and CK2a. We then uncovered that miR-499 targeted and inhibited CK2a, thus contributing to elevated sensitivity of lung cancer cells to IR exposure. The upregulation of miR-760 and miR-186 has been suggested to induce replicative senescence in human lung fibroblast cells by CK2a. 27 Additionally, a previous study also demonstrated that the inhibition of miR-125b could target CK2a to ameliorate cerebral ischemia/reperfusion injury. 28 Transcription factors are DNA-binding proteins that bind to transcription co-regulators, ultimately leading to histone modifications capable of altering chromatin structure to regulate gene transcription. 29 CK2a has been speculated to be a promising target for enhancing the radio-sensitivity in NSCLC. 19 Our investigation demonstrated that CK2a could augment lung cancer inhibition or could aggravate the sensitivity of lung cancer cells to IR exposure. Numerous previous studies have evidenced that miR-499 could inhibit p65 phosphorylation by targeting CK2a to heighten radiosensitivity of cell lines. Phosphorylation is the prototypical posttranslational modification of key proteins, which is capable of influencing diverse physiological functions of cells. 30 Accordingly, we have considered the possibility that the survival of CK2 in prostate cancer may be mediated by the maintenance and promotion of androgen receptor and NF-kB p65 expression. 31 Moreover, the ectopic expression of miR-506 has also been demonstrated to repress the expression of NF-kB p65 and to subsequently activate p53, resulting in the inhibition of lung cancer cell survival, 32 which seems partially consistent with the present findings. Furthermore, diminished levels of p65 expression may enhance the role of solamargine in human lung cancer cells. 33 Taken together, the key findings of the present study highlight that miR-499 may target and downregulate CK2a expression to repress p65 phosphorylation, ultimately contributing to the enhancement of lung cancer cell sensitivity to IR exposure ( Figure 6). Our findings Figure 6. The molecular mechanism of the regulatory network and function of miR-499 Overexpression of miR-499 increases the sensitivity of lung cancer cells to IR exposure by targeting CK2a and suppressing p65 phosphorylation. emphasize the potential of miR-499 as a promising therapeutic target for enhancing radiotherapy in lung cancer treatment. However, this study has certain limitations, which call for further clinical and biological verifications of the therapeutic action of miR-499 in lung cancer. Ethics statement The study protocols were performed with the approval of the Ethics Committee of Shanghai Tenth People's Hospital, Tongji University School of Medicine and conducted in strict accordance with the Helsinki Declaration. All the participants signed informed written consent documentation. All animal experiments were performed in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Bioinformatics analysis Through the GEO database (https://www.ncbi.nlm.nih.gov/geo/), the NSCLC-associated miRNA microarray GEO: GSE102286 was obtained, which included 88 normal samples and 91 NSCLC samples. The R language "limma" package (http://www.bioconductor.org/ packages/release/bioc/html/limma.html) was used for differential analysis of microarray miRNA expression profiles with \|log2FC\| > 1 and p < 0.05 as screening criteria of differentially expressed miRNAs. Using the R language "pheatmap" package (https://cran.r-project.org/ web/packages/pheatmap/index.html), the differential expression heatmap of candidate miRNAs was plotted. Through the HMDD database (http://www.cuilab.cn/hmdd), the MNDR database (http:// www.rna-society.org/mndr/), and a literature search, NSCLC-associated miRNAs were obtained. By using the jvenn tool (http://jvenn. toulouse.inra.fr/app/example.html), the overlapping parts of differentially expressed miRNAs and NSCLC-related miRNAs were obtained. Through the bioinformatics database TargetScan (http://www. targetscan.org/vert_72/), mirDIP (http://ophid.utoronto.ca/mirDIP/ , minimum score = high), and RNAInter (http://www.rna-society. org/rnainter/, score > 0.17), the downstream target genes of miRNAs were predicted. NSCLC-related genes were retrieved through the GeneCards database (https://www.genecards.org/, score > 7), and the jvenn online tool (http://jvenn.toulouse.inra.fr/app/example. html) was employed to search for the overlapping parts of predicted miRNA target genes with NSCLC-related genes. By the STRING tool (https://string-db.org/), the interaction between candidate targets was analyzed, the interaction relationship network of genes was visualized using Cytoscape 3.5.1 software, and the node and edge of the network were analyzed using the network analyzer built-in tool to obtain the degree of the gene (degree). To further predict the downstream regulators of target genes, we used the BioGRID database (https:// thebiogrid.org/, evidence R 2) to search for the interaction factors of genes and intersect them with NSCLC-related genes in the Gene-Cards database to screen potential targets. Finally, the Phenolyzer tool (http://phenolyzer.wglab.org/) was used to obtain the correlation ranking and score of candidate genes with NSCLC. Study subjects Lung cancer tissues (n = 87) and adjacent normal tissues (n = 87) were collected from 87 lung cancer patients who had undergone lung cancer resection at the Shanghai Tenth People's Hospital, Tongji University School of Medicine from June 2013 to April 2016. Lung cancer tissues and adjacent normal tissues were fixed with formalin and embedded in paraffin for histopathological diagnosis. Adjacent normal tissues were confirmed by tissue biopsy (R5 cm away from cancer tissues). All the patients were followed up for a period of at least 3 years, with follow-up ending in April 2019. qRT-PCR Total RNA was extracted from the tissues or cells using a TRIzol kit (15596-018; Beijing Solarbio Science & Technology, Beijing, China) in accordance with the manufacturer's instructions, with RNA concentration determined accordingly. The primers applied in this study were synthesized by Dalian Takara (Dalian, China) (Table S2). Reverse transcription was subsequently performed using a complementary DNA (cDNA) Reverse Transcription Kit (K1622, Beijing Yaanda Biotechnology, Beijing, China) in accordance with the manufacturer's instructions. The reversely transcribed cDNA was diluted to 50 ng/mL for fluorescent qPCR in a real-time PCR instrument (ViiA 7, Daan Gene of Sun Yat-sen University, Guangzhou, China). U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were regarded as internal references. The relative transcription level of the target gene was calculated by relative quantification (2 À OO CT method). MTT assay A single-cell suspension was prepared using culture medium containing 10% fetal bovine serum (FBS) and seeded into a 96-well plate at a volume of 200 mL/well and cultured for 3-5 days. Next, 20 mL of MTT solution was added to the cells for incubation for an additional 4 h. The culture was terminated with the supernatant in the well discarded. Next, 150 mL of dimethyl sulfoxide (DMSO) was added to each well followed by shaking for 10 min. The absorbance of each well was subsequently determined at 490 nm on the enzyme-linked immunosorbent monitor, and the results were recorded. The cell growth curve was then plotted with time as the abscissa and absorbance as the ordinate. Colony-formation assay When the cells reached approximately 70% confluence, a cell-colonyformation assay test was performed. RPMI 1640 medium containing 10% FBS was mixed with 10% agarose gum solution in a ratio of 1:10. More specifically, 5 mL of the mixture was placed in a sterile Petri dish and allowed to solidify and subsequently preserved in a 37 C, 5% CO 2 incubator for later use. The cells were initially digested with 0.25% trypsin into single cells, centrifuged, dispersed with 10% FBS and RPMI 1640, counted and diluted to a cell density of 500 cells/mL. Next, 2% agarose gum solution was mixed with cell suspension at a ratio of 10:1, followed by the addition of 2 mL of the mixture to the prepared agarose, and placed in a 37 C, 5% CO 2 incubator for 7 days. The formation of the cell colonies was then observed and the colony-formation assay rate (clone formation rate = number of formed clones/number of cells inoculated) was calculated. Flow cytometry assay Following a 24 h period of transfection, apoptosis of the lung cancer cells was detected by Annexin V fluorescein isothiocyanate (FITC)/ propidium iodide (PI) double-staining kit (556547, Shanghai Solja Technology, Shanghai, China). In brief, 10Â binding buffer was diluted into 1Â binding buffer with deionized water. Cells were digested and dispersed into single cells, followed by centrifugation at 2,000 rpm for 5 min at ambient temperature for cell collection. The cells were then resuspended with pre-cooled 1Â PBS, centrifuged at 2,000 rpm for 5-10 min, and suspended by 300 mL of 1Â binding buffer. The cells were then mixed with 5 mL of Annexin V-FITC and incubated for 15 min at ambient temperature in the dark. The cells were added with 5 mL of PI and protected from light for 5 min in an ice bath 5 min prior to analysis with a flow cytometer (Cube 6, Partec, Munster, Germany). The excitation wavelength was set at 419 nm, with FITC detected at 519 nm and PI detected at a wavelength greater than 575 nm. www.moleculartherapy.org Wound-healing assay Cells from each group in the logarithmic phase were collected and seeded in 6-well plates for culture at 10 6 cells/well. A marker pen was used to evenly draw lines on the back of the 6-well plates, which were used as the positioning for photographic recording. After the cells were completely distributed on the surface of the 6-well plates, the original culture medium was replaced with cell culture medium containing 1% FBS, and the cells were starved for 12 h. With the help of a ruler, a 200 mL pipette tip was used to scratch vertical lines on the 6-well plate. The cells were washed three times with 2 mL of PBS, and the scratched cells were washed off and treated as described above followed by photographing at 0 and 24 h. Cell migration rate (%) was calculated as (1 À scratch width/initial scratch width) Â 100%. SPSS software was used for statistical analysis of data at each time point in each group. Transwell assay Tubes and pipette tips were pre-cooled at À20 C before testing. Basement membranes of the Transwell chambers were coated with Matrigel glue (356234, Corning, Corning, NY, USA) diluted with serum-free cold DMEM cell medium. A total of 100 mL of the diluted gel was added to the upper chamber of a 24-well Transwell, which was incubated at 37 C for at least 4-5 h. The basement membrane was hydrated, and the gel was gently washed with serum-free medium. The cells were then detached and washed three times with culture medium. Cells were resuspended in a density of 5 Â 10 5 cells/mL with 1% FBS. Next, a total of 200 mL of cell suspension was added to the upper chamber, and 600 mL of cell culture medium was added to the lower chamber. After incubation at 37 C for 20-24 h, a cotton swab was used to wipe off the non-invasive cells on the upper chamber. Transwell chambers were removed from the incubator and washed twice with PBS for 2 min each. The cells were then fixed with methanol and acetic acid at a ratio of 3:1 for 15-30 min and dried. Next, 500 mL solution containing 1% crystal violet was added to a 24-well plate, and the chamber membranes were immersed in the culture medium. The membrane was removed after 30 min at 37 C and washed with PBS. Four visual fields were photographed, and the number and diameter of migrated cells were counted. Tumor formation in nude mice Forty-eight BALB/c nu/nu mice (aged 4-6 weeks; weighing 12-19 g; 24 male mice and 24 female mice; Shanghai Lingchang Systems, Shanghai, China) were raised in the specific-pathogen-free (SPF) environment in the Animal Experimental Center (Experimental Animal Qualification certificate no. 159) of Shanghai Tenth People's Hospital, Tongji University School of Medicine prior to the commencement of experimental procedures. The mice were acclimatized for 7 days and provided free access to food and drinking water in a 12 h light-dark cycle in aseptic conditions. The mice were then subcutaneously injected through the dorsal area with commercially purchased lung cancer A549 and NCI-H292 cell lines, which were transfected with mimic NC, miR-499 mimic, and/or subjected to IR exposure, with 12 mice in each group. After inoculation, the mice were placed in a laminar flow hood in the SPF animal room. Tumor growth was observed every 3 days following inoculation and recorded for 30 days. The tumors were then removed and weighed. H&E staining After animal dissection, the tumors were placed in PBS at pH 7.4 for 2 h. Next, the tumors were dehydrated in a graded alcohol system, embedded in paraffin, and cut to a thickness of 5 mm before staining with H&E. RIP The binding of miR-499 with CK2a was detected using the RIP kit (Millipore, Burlington, MA, USA). Cells from each group were washed with pre-chilled PBS and the supernatant discarded. Washed cells were lysed with an equal volume of RIPA lysate (P0013B, Beyotime) in an ice bath for 5 min and centrifuged at 14,000 rpm and 4 C for 10 min. A portion of the cell extract was removed as input, and the remainder was incubated with the antibody for co-precipitation. RNA was extracted from the samples and input after detachment with proteinase K for subsequent qRT-PCR detection of miR-499 and CK2a expression. The antibody used for RIP (rabbit anti-Ago2 (1:100, ab32381, Abcam, UK) was mixed for 30 min at room temperature, and rabbit anti-human IgG (1:100, ab109489, Abcam, UK) was used as a negative control. Each experiment was repeated three times. Dual-luciferase reporter gene assay As per the binding sequence between CK2a mRNA 3 0 untranslated region (UTR) and miR-499, the target sequence and the mutant sequence were designed and synthesized. The artificially synthesized gene fragments of CK2a 3 0 UTR and the mutant were introduced into the pGL3 vector (Promega, Madison, WI, USA) for the construction of mutant-type (MUT) plasmids. After transfection for 48 h, the luciferase reporter plasmids wild type (WT) or MUT were co-transfected with miR-499 mimic or NC mimic into HEK293T cells, which were then collected and lysed. Luciferase activity was measured using a Luminometer TD-20/20 detector (E5311, Promega, Madison, WI, USA) and Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA). Statistical analysis Statistical analysis was performed using SPSS 21.0 software (IBM, Armonk, NY, USA). Following the application of a homogeneity test of normal distribution and variance, data conforming to normal distribution were expressed as the mean ± standard deviation. Data between the two groups were compared using an independent sample t test. Comparisons between multiple groups were conducted by one-way analysis of variance (ANOVA), followed by Tukey multiple comparison test. Statistical analysis in relation to time-based measurements within each group was analyzed using repeated-measures ANOVA, followed by a Bonferroni's post hoc test for multiple comparisons. Statistical analysis in relation to different concentrations within each group was analyzed using two-way ANOVA, followed by Bonferroni's post hoc test. ROC was applied to identify the optimal cutoff value for miR-499 expression, whereas the Kaplan-Meier method was employed to analyze the relationship between high and low expression of miR-499 in the lung cancer tissues and total survival (log-rank test). A p value < 0.05 was considered to be indicative of statistically significant difference. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.	v3-fos-license
2016-03-22T00:56:01.885Z	2012-08-01T00:00:00.000	15292813	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://www.mdpi.com/1420-3049/17/8/9421/pdf", "pdf_hash": "ddbe1e9ffe35408ba380d01fd474995c66e2a68f", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2312", "s2fieldsofstudy": [ "Chemistry", "Agricultural And Food Sciences" ], "sha1": "ddbe1e9ffe35408ba380d01fd474995c66e2a68f", "year": 2012 }	pes2o/s2orc	A New Natural Lactone from Dimocarpuslongan Lour. Seeds A new natural product named longanlactone was isolated from Dimocarpuslongan Lour. seeds. Its structure was determined as 3-(2-acetyl-1H-pyrrol-1-yl)-5-(prop-2-yn-1-yl)dihydrofuran-2(3H)-one by spectroscopic methods and HRESIMS. Introduction Longan [Dimocarpus longan Lour. (syn. Euphoria longana Lam.)] is an evergreen tree of the Sapindaceae family, which is widely cultivated in Southern China, India, and Southeast Asia [1]. Longan fruit is one of the most favoured tropical fruits in China [2]. Longan seeds have long been used as a folk medicine in China for treatment of acariasis, hernia, wound hemorrhages, eczema and scrofula [3], which have recently also been proven to possess free radical-scavenging activity [4], antifatigue properties [5], growth inhibition of colorectal carcinoma cells [6], and hypoglycemic effects [7]. Longan seeds have been found to be a rich source of antioxidant phenolic compounds which are promising as functional food ingredients or natural preservatives. Soong and Barlow reported that longan seeds contained thirteen polyphenols, such as gallic acid, corilagin and ellagic acid [8]; recently Sudjaroen et al., identified eleven polyphenolic compounds from longan seed, including chebulagic acid, ellagic acid 4-O-α-L-arabinofuranoside, isomallotinic acid and geraniin, etc. [9]. We have investigated the chemical constituents of longan seeds and succeeded in the isolation of eight polyphenols and twelve other compounds [10,11]. This paper deals with the isolation and structure elucidation of a new natural lactone product named longanlactone from longan seeds. Results and Discussion The new compound (Figure 1) was obtained from the chloroform extracts of longan seeds as white acicular crystals with the melting point of 198-200 °C. The HR-ESI-MS spectrum revealed a quasi-molecular ion peak at 232.0971 [M+H] + (calculated 232.0973) corresponding to the molecular formula of C 13 H 14 NO 3 . The 1 H-1 H COSY and 1 H-13 C COSY spectra revealed the partial structures shown by bold lines in Figure 2. Further connectivities were deduced from the HMBC and NOESY spectra ( Table 1). The HMBC correlations from H-7' to C-2', C-3', C-6' indicated the acetyl group was attached on C-2'. The HMBC correlations from H-3 to C-1 and C-2, indicated connectivity of C-2 to C-1 and C-3. The correlations from H-7 to C-5 and C-4, from H-4 to C-6, indicated connectivity of the propargyl to the lactone ring on C-4. Key HMBC correlations (arrows) of this compound are shown in Figure 2. Figure 2. 1 H-1 H COSY (bold line) and key HMBC correlation (arrow) in longanlactone. In the NOESY spectrum (Table 1 and Figure 3), the presence of mutual NOE correlations between H-5' and H-2 indicated the pyrrole ring and the lactone ring are connected through C-2 and the N atom. The mutual NOE correlations between H-2, H-3a and H-4 indicated that H-2 and H-4 are located on the same side of the lactone ring, so the relative configuration of this compound was established as depicted in Figure 3. Plant Material Longan seeds were collected from a commercial longan orchard located in Maoming, Guangdong, China, in September 2005. The seeds were sun dried, and ground to powder. Extraction and Isolation The longan seed powder (10.5 kg), after defatting with petroleum ether, was extracted with 95% EtOH (150 L) three times at room temperature (24 h per time). The EtOH solutions were combined and concentrated under vacuum. The residue was suspended in H 2 O and partitioned successively with petroleum ether and CHCl 3 (10 L, room temperature, 6 h) to obtain CHCl 3 -soluble extracts (39.0 g). The CHCl 3 -soluble extract was subjected to silica gel column chromatography (CC) and eluted with CHCl 3 -MeOH mixtures with increasing polarities (10:0-4:1) to obtain sixteen fractions A-P. Fraction D (31 mg), obtained on elution with 99:1 CHCl 3 -MeOH was further subjected to silica gel CC, eluted with petroleum ether-acetone mixtures with increasing polarities (99:1-9:1), to afford five subfractions D1-D5. Subfraction D4 was separated by CC on Sephadex LH-20 with MeOH as eluant to yield the title compound (3 mg).	v3-fos-license
2020-10-28T19:12:58.574Z	2020-10-07T00:00:00.000	225169690	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/fsn3.1913", "pdf_hash": "9d51bdd2161334ce6885835909cd942e0f361bb9", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2326", "s2fieldsofstudy": [ "Environmental Science" ], "sha1": "aeb70b9d95651d8b5a5d3a44fd6c0190ba42202e", "year": 2020 }	pes2o/s2orc	Effect of storage time on biochemical characteristics and antioxidant activity of hawk tea (Litsea coreana) processed by boiling water fixation Abstract This study investigated the effect of storage time on biochemical characteristics of hawk tea (Litsea coreana) and explored the correlation between the content of flavonoids and polyphenols and antioxidant activity. The antioxidant activity and the content of inclusions, amino acid, and mineral elements in hawk tea processed by boiling water fixation and packed in airtight polypropylene bags and stored in 0°C refrigerator under different storage time (one year, three years, and six years) were analyzed. Results indicated that the biochemical characteristics of hawk tea changed less within 12 months. The total content and types of amino acids in hawk tea reached the maximum in the third year, as well as the content of total trace elements. The water extracts, tea polyphenol, caffeine, lysine, valine, isoleucine, glycine, proline, Ca, and Zn decreased continuously in the storage period of 6 years, but the total flavonoids, Mg, and Ni changed just the opposite. Total polyphenol is the main antioxidant material in hawk tea. Results of the present study provided useful information for people to systematically understand the changes of tea in the storage process and to reasonably develop hawk tea product. Hawk tea tree is a woody plant of Lauraceae, belonging to the tropical species of East Asia, mainly distributed in China, and may also be distributed in Korea or Japan, but it is rarely reported. As an undergrowth crop in forests, hawk tea tree is widely cultivated in southwestern China, particularly in Guizhou Province where it is a major agroforestry crop . According to the processed material (buds or leaves) of L. coreana var. lanuginose, hawk tea is divided into bud hawk tea (buds) and hawk tea (tender and mature leaves). Hawk tea is traditionally processed by picking the buds and new leaves in middle and late April and drying them slowly after fixing in an iron pot. But in Xifeng county and Chishui city, Guizhou province, the newly mature leaves are picked in early May and then dried slowly after fixing in boiling water. This is used to feed the larvae of Aglossa dimidiate or Hydrillodes morosa, and the feces of the larvae are commonly known as insect hawk tea or sandy tea (Jia et al., 2017). Hawk tea is abundant in proteins, amino acids, sugars, polyphenols, and flavonoids, which are closely related to its physiological activity, and varies with the processing methods (Huang et al., 2016;Park, 2017;Xu, et al. 2017;Xu, et al., 2016). It had been devoted more attention to the extraction and health benefits of flavonoids, polysaccharides, and essential oils from new hawk tea in recent years (Jia et al., 2014(Jia et al., , 2017Qin et al., 2018). The contents of total flavonoids and total polyphenol in hawk tea were varied with the seasons (Xiao et al., 2015), and the contents of trace elements (Pb, Cd, Mn, Fe, Zn, and Ca) in hawk tea from different areas were varied (Gu and Peng, 2013). However, less attention has been paid to the research on the change of chemical constituents in hawk tea with storage time. This paper intended to explore the effect of storage time on biochemical characteristics of hawk tea processed by boiling water fixation. The variation of biochemical characteristics such as the inclusions, amino acids, and mineral elements of the hawk tea with storage for 0, 1, 3, and 6 year was analyzed systematically for the first time. The correlation between the content of flavonoids and polyphenols and antioxidant activity was explored. These results would provide scientific basis for commercial utilization of hawk tea. \| Materials Tender leaves (one bud and five to six leaves) from wild L. coreana var. lanuginose plants growing in Xifeng County, Guizhou Province, China, were collected in April 2014. They were processed by the Guizhou Institute of Tea Science on the same day according to the method of tea fixation by boiling water. The teas were packed in airtight polypropylene bags and stored in 0°C refrigerator until further use. \| Tea fixation by boiling water The process flow chart of preparing hawk tea fixation by boiling water was shown in Figure 1. Go through 9 steps to obtain hawk tea processed by boiling water fixation. \| Biochemical quality test Determination of water extracts in the hawk tea was according to the National Standard of the People's Republic of China GB/T 8305-2013 "Tea determination of water extracts content." Two grams of dry and ground sample was weighed and placed into a 500-ml conical flask, to which 300 ml of boiling distilled water was added, following which the mixture was immediately transferred to a boiling water bath for 45 min (with shaking every 10 min). Following the extraction, the tea residue was filtered immediately under hot decompression and washed several times with about 150 ml of boiling distilled water. The tea residue and a known quality of filter paper were then placed into a baking dish and transferred to a constant temperature drying oven at 120°C ± 2°C for 1 hr. Following this, the tea was cooled for 1 hr, baked for 1 hr, immediately transferred to a dryer in order to cool to room temperature, and then weighed. The content of water extract in the hawk tea was calculated according to formula (1): where m 0 is the mass of the dry sample, g; and m 1 is the mass of the dried tea residue, g. Determination of soluble sugar in the hawk tea was according to the National Standard of the People's Republic of China GB/T 8305-2013 "Method for analysis of forestry biomass-Determination of soluble saccharides content." a) The glucose standard curve was prepared to evaluate the soluble sugar in terms of glucose equivalents. Ten milligrams of glucose and deionized water was mixed and diluted to concentrations of 5, 10, 15, 20, 25, or was weighed into a 10-ml centrifuge tube, to which 5 ml of 70% methanol solution (preheated at 70°C) was added, following which the mixture was stirred and wet evenly and immediately transferred to a 70°C water bath and extracted for 10 min (with stirring every 5 min). The mixture was then cooled to room temperature and centrifuged at 3,500 r/min for 10 min, and then, the supernatant was transferred to a 10-ml volumetric flask. The residue was extracted twice with 5 ml of 70% methanol solution, and the above operation was repeated. The extraction solution was combined to make 15 ml, shaken well, and passed through a 0.45-µm membrane. Two milliliters was transferred to a 10-ml volumetric flask with a pipette. 3) For the determination, a pipette was used to transfer 1 ml of glucose working fluid, water, and measurement solution, respectively, to a 10-ml graduated tube, to which 3 ml of anthrone reagent was added. The mixture was kept at 90°C for 15 min and then cooled, following which the absorbance value was measured at 620 nm using a UV/Vis spectrophotometer (Schimadzu, Japan) to calculate the total soluble sugar content using the glucose standard curve. The protein content in the hawk tea was determined by spectrophotometry according to the National Food Safety Standard GB/T 36056-2018 "Determination of protein in foods" using the ammonia nitrogen standard curve. 1) An ammonia nitrogen standard curve was prepared to evaluate the protein in terms of the standard series and the conversion coefficient. Ammonium sulfate (0.4720 g) was dissolved in water and made up to 100 ml in a volumetric flask. Ten milliliters was pipetted into a 100-mL volumetric flask with water added to make up the volume and then mixed well. Each mL of this solution is equivalent to 0.1 mg nitrogen. We then pipetted 0.00 ml, 0.05 ml, 0.10 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, and 1.00 ml ammonia nitrogen standard solution (equivalent to 0.00 μg, 5.00 μg, 2) The test solution was prepared by transferring 0.1 g-0.5 g tea sample into a dry 100-ml Kjeldahl flask, to which 0.1 g copper sulfate, 1 g potassium sulfate, and 5 ml sulfuric acid were added. The mixture was slowly heated until the contents were carbonized and foaming, and the temperature was increased to keep the liquid in the bottle boiling slightly until the liquid become blue-green and transparent, following which it was heated for 0.5 hr. After cooling, 20 ml of water was slowly added and the mixture was transferred into a 50-ml volumetric flask. The Kjeldahl flask was mixed with a small amount of water, following which water was added to scale and mixed well. Two to five milliliters was pipetted into a 50-ml volumetric flask, to which 1-2 drops of p-nitrophenol indicator solution was added. The mixture was shaken well, and sodium hydroxide solution was added to neutralize it to yellow, following which acetic acid solution was added to make the solution colorless. The mixture was then diluted to scale with water and mixed well. 3) For determination, 0.5-2 ml (about equal to nitrogen < 100 μg) of sample solution and the same amount of reagent blank solution were, respectively, pipetted into 10-ml colorimetric tubes. The absorbance at 400 nm was measured in the same way as the standard curve above, and the protein content was measured in the hawk tea by using the ammonia nitrogen standard curve. The fat content of the hawk tea was determined by using the Two to three grams of sample was used for those with ash contents greater than 10 g/100 g, while 3 g to 10 g was weighed for samples with ash contents less than 10 g/100 g. 3) For determination, the sample was first heated on an electric heating plate with a small fire to fully carbonize it until there was no smoke, following which it was placed in a muffle furnace and burned for 4 hr at 550 ± 25°C. It was then cooled to about 200°C, removed, and placed into a desiccator for 30 min. If carbon particles were present in the burned residue before weighing, a little water was dropped into the sample to moisten it, the water was evaporated, and the sample was burned until there were no visible carbon particles. The burning process was repeated until the weight difference was less than 0.5 mg. The following formula was used (3): where m 1 is the mass of the crucible and ash, g; m 2 is the mass of the crucible, g; and m 3 is the mass of crucible and sample, g. Determination of total flavonoids in the hawk tea used the sodium nitrite-aluminum nitrate-sodium hydroxide color system as described by Xiao et al. (2015) with some modifications. A rutin standard curve was prepared to evaluate the total flavonoids in terms of rutin equivalents. Six milligrams of rutin and 70% methanol solution were mixed and diluted in concentrations of 1.2, 2.4, 3.6, 4.8, 6.0, or 7.2 μg/ml. 2) The test solution was prepared using the same step as the above soluble sugar. 3) For determination, 1 ml of rutin working fluid, water, and measurement solution were, respectively, pipetted into a 10-ml graduated tube to which 1 ml of sodium nitrite (5%) was added, shaken well, and allowed to stand for 6 min. One milliliter of 10% aluminum nitrate was added, shaken well, and allowed to stand for 6 min. Four milliliters of 4% sodium hydroxide was added. The total volume of the mixture was made to 10 ml by adding 70% methanol, and the tubes were vigorously stirred. The resulting solution was pink, and its absorbance was determined using a spectrophotometer at 510 nm against a 70% methanol. The content of total flavonoids in the hawk tea was calculated using the rutin standard curve. Determination of total polyphenols in the hawk tea using the Folin-Ciocalteu reagent method as described by Xiao et al. (2015) and the National Standard of the People's Republic of China GB/ T8313-2008 "Determination of total polyphenols and catechins content in tea." a) The gallic acid standard curve was prepared to evaluate the total phenolics in terms of gallic acid equivalents. Ten milligrams of gallic acid and deionized water was mixed and diluted in concentrations of 5, 10, 15, 20, 25, or 30 μg/ml. b) The test solution was prepared using the same step as the above soluble sugar. c) For determination, a pipette was used to transfer 1 ml of gallic acid working fluid, water, and measurement solution, respectively, to a 10-ml graduated tube, to which 5 ml of Folin-Ciocalteu reagent (10%) was added and shaken well. After reacting for 3-8 min, 4 ml of 7.5% Na 2 CO 3 solution was added, topped up with water to 10 ml, and shaken well. The mixture was allowed to stand at room temperature for 60 min, following which the absorbance was measured at 765 nm using distilled water as a blank. The content of total polyphenols in the hawk tea was calculated using the gallic acid standard curve. The caffeine content was determined by ultraviolet spectrophotometry according to the National Standard of the People's Republic of China GB/T 8312-2013 "Tea determination of caffeine content." a) A caffeine standard curve was prepared to evaluate the caffeine in terms of caffeine equivalents. Ten milligrams of caffeine and deionized water was mixed and diluted to concentrations of 5, 10, 15, 20, 25, or 30 μg/ml. One milliliter of caffeine working fluid was transferred to a 25-ml volumetric flask, to which 1.0 ml of 0.01 mol/L hydrochloric acid was added and diluted to scale with distilled water and mixed well. Distilled water was used as a blank reference, the absorbance was measured at 274 nm, and a curve between the absorbance and the corresponding caffeine concentration was generated. b) The test solution was prepared using the same step as the above water extract. c) For determination, a pipette was used to transfer 10 ml of measurement solution into a 100-ml volumetric flask, to which 4 ml of 0.01 mol/L hydrochloric acid and 1 ml of basic lead acetate (add 100 ml of water to 50 g basic lead acetate, allow it to stand still overnight, and filter the supernatant) were added. The solution was diluted to scale with distilled water, mixed well, allowed to stand still overnight, and the supernatant filtered. Twenty-five milliliters of filtrate was transferred into a 50-ml volumetric flask, to which 0.1 ml 4.5 mol/L sulfuric acid solution was added. The mixture was diluted with water to scale, mixed well, allowed to stand still overnight, and the supernatant filtered. Using distilled water as a blank reference, the absorbance at 274 nm was measured. The content of caffeine in the hawk tea was calculated using the caffeine standard curve. The content of amino acids was determined by an amino acid au- in a hydrolysis tube, to which 10-15 ml of 6 mol/L hydrochloric acid solution was added, followed by 3-4 drops of phenol. The hydrolysis tube was placed into a refrigerant (commercially available salt and ice cubes mixed in a ratio of 1:3 by mass) for 3-5 min, vacuumed and filled with nitrogen three times, sealed, and placed into a hydrolysis furnace at 110 ± 1°C and hydrolyzed for 22 hr. It was then removed, cooled to room temperature, and filtered into a 50-ml volumetric flask. The hydrolysis tube was washed with a small amount of water several times, following which water was added to scale. One milliliter of filtrate was accurately pipetted into a 15-ml test tube, dried at 40-50°C by a tube concentrator, and the residue dissolved with 1-2 ml water and then dried under reduced pressure. One to two milliliters of pH 2.2 sodium citrate buffer solution was added for dissolution, shaken and mixed well, and passed through a 0.22-μm filter membrane for determination. For the determination of tryptophan, the solution was prepared as follows: A certain amount of crushed dry sample (accurate to 0.0001 g) was accurately weighed in a polytetrafluoroethylene liner, to which 4 mol/L 1.50 ml hydrogen lithium oxide solution was added. The liner was inserted into the hydrolysis glass tube, vacuum-packed to 7 Pa, sealed, and placed in a hydrolysis furnace at 110 ± 1°C and hydrolyzed for 22 hr. It was then removed, cooled to room temperature, transferred to a 50-ml volumetric flask with a small amount of sodium citrate buffer solution at pH 4.3 [c(Na + ) 0.2 mol/L], and neutralized with 6 mol/L hydrochloric acid solution. The volume was made up with the above buffer solution and passed through a 0.22 μm filter membrane for determination. For the determination of cystine, the solution was prepared as follows: The dried tea sample was crushed and sieved through a 40-mesh sieve. A sample not exceeding 75 mg (accurate to 0.0001 g) was placed in a 20-ml concentration bottle and cooled in an ice-water bath for 30 min, to which 2 ml of cool performic acid solution [30% hydrogen peroxide and 88% formic acid mixed at 1:9 (V/V), placed at room temperature for 1 hr, placed in an ice-water bath, and cooled for 30 min] was added, ensuring that the sample was completely moist. The sample was then sealed and placed in a 0°C refrigerator with an ice bath and reacted for 16 hr. We then added 0.5 ml of biased sodium sulfite solution (33.6 g of biased sodium sulfite, water added to 100 ml). The mixture was shaken well, and 17.5 ml of 6.8 mol/L hydrochloric acid solution was added and the sample hydrolyzed at 110 ± 3°C for 22-24 hr. The hydrolysis tube was removed and cooled, and the contents were transferred to a 50-ml volumetric flask with water and neutralized with 7.5 mol/L sodium hydroxide solution to pH 2.2. Sodium citrate buffer (pH 2.2, 0.2 mol/L Na) was then added to a constant volume, centrifuged, and the supernatant removed for instrument measurement. 3) For determination, a mixed amino acid standard working solution and sample measurement solution were injected into the amino acid analyzer in the same volume, and the concentration of amino acids in the sample was calculated by calculating the peak area with the external standard method. The minimum detection limit of the various amino acids was 10 pmol. The content of mineral elements was determined by inductively coupled plasma-mass spectrometry (ICP-MS) with microwave-assisted digestion pretreatment as described by Xin et al. (2010) with some modifications, and As and Hg were determined using an AFS-230E atomic fluorescence spectrophotometer as described by Gao et al. (2018) with some modifications. Crushed dry tea (0.5 g) was placed into the electrothermal digestion tube, to which 8 ml nitric acid was added and the mixture stirred at room temperature for 2 hr. The mixture was then heated to 130°C for 2 hr and then 145°C for 2 hr until it became clear and transparent. It was then cooled to room temperature and transferred into a 50-ml volumetric flask and shaken well. Five parallel samples were made for each sample, and the average value was used. The blank solution was tested at the same time. The linear ranges for Ca and Mg were from 0 to 100 μg/L; those for Fe, Zn, and Mn were from 0 to 10 μg/L; those for Cu and Pb were from 0 to 5 μg/L; and those for Se, Ni, and Cr were from 0 to 0.5 μg/L. As and Hg were determined using an AFS-230E atomic fluorescence spectrophotometer as described by Gao et al. (2018) with some modifications. For these, 0.5 g crushed dry tea was placed into an electrothermal digestion tube, to which 9 ml nitric acid and 1 ml perchloric acid were added, stirred at room temperature for 2 hr, and then heated up to 130°C for 2 hr and 145°C for 2 hr until the solution became transparent. The mixture was then cooled to room temperature, 10 ml of water was added, and 10% sodium hydroxide solution was added to neutralize until phenolphthalein discoloration. Two milliliters hydrochloric acid, 2.5 ml 5% thiourea, and 5% ascorbic acid mixed solution were added, and the mixture was transferred into a 25-ml volumetric flask and shaken well. Five parallel samples were measured for each sample, and the average value was used. The blank solution was measured at the same time. The linear ranges for As and Hg were from 0 to 0.5 μg/L. DPPH• scavenging activity The DPPH• scavenging activity was determined as described previously (Wan et al., 2013). An acid methanolic solution of DPPH (1,1-diphenyl-2-picrylhydrazyl) was used to assess the antioxidant activity of the hawk tea sample. Trolox was used as the positive control. A 2-ml sample was added to a methanolic solution of DPPH (2.0 × 10 −4 mol/L, 2.0 ml) and 5 ml of methanol. The mixture was then vigorously shaken for 10 s and left to stand at room temperature for 30 min. The scavenging activity of the hawk tea on DPPH• was determined by the absorbance at 517 nm, and the percentage of scavenging activity was calculated according to the following equation: where Ab scontrol is the absorbance of the control (without the test sample), and Abs sample is the absorbance of the sample (with the test sample). The results were expressed as the EC 50 values (µg/ml) for the 50% DPPH• scavenging effect concentration. Ferric reducing activity power assay Ferric reducing activity power (FRAP) was evaluated according to the previous report using FeSO 4 ⋅7H 2 O as the standard (Frank et al., 2012). The fresh FRAP reagent was prepared before using, which contains 25 ml acetate buffer (300 mmol/L, pH 3.6), 2.5 ml TPTZ solutions (10 mmol/L in 40 mmol/L HCl), and 2.5 ml of FeCl 3 ⋅6H 2 O solution (20 mmol/L). The reagent was warmed to 37°C, and then, 500 µl was placed in a cuvette and the initiate absorbance was read. 20 µl of the sample solutions was added to the cuvette, and the absorption was determined at 593 nm using the spectrophotometer (UV-1750, Shimadzu). Values were calculated using the FeSO 4 ⋅7H 2 O standard curve. \| Statistical analysis All of the analyses were carried out in triplicate, and the results were reported as means ± SD (standard deviation). ANOVA and Duncan's tests with α = 0.05 were used to determine the differences between the assays. Differences at p < .05 were considered to be significant. All of the statistical analyses were performed with SPSS 18.0 (SPSS Inc., Chicago, US). \| Inclusions The inclusions including water extracts, protein, tea polyphenols, total ash, crude fat, soluble sugar, total flavonoids, and caffeine were analyzed. As can be seen from the results in Figure 2, the contents of water extracts and tea polyphenols gradually decreased with increasing storage time (Figure 2a). The water extracts content of tea reflects the amount of soluble substances in tea. A greater water extract value implies a more concentrated tea soup. The soluble extract of tea has traditionally been regarded as an important international Scavenging activity (%) = (Abs control − Abs sample )∕Abs control × 100, standard for tea quality, as it can be directly absorbed and utilized by human body (Harbowy et al., 1997). It is generally required to be more than 20% in the high-quality green tea. According to the results from Figure 2a, the contents of water extract detected in hawk tea prepared by boiling water fixation method were all higher than 20% during 6 years storage time, which exceeded the content standard of high-quality green tea. With the prolongation of storage time, the content of water extracts in hawk tea gradually decreased. When Similarly, tea polyphenols, one of the important chemical components that determine the quality of tea, exhibited a similar variation pattern to that of water extracts. The content of tea polyphenol was 96.17%, 53.62%, and 52.96% of the new tea (CK) at 1, 3, and 6 years of storage. This was because on the one hand, tea polyphenols would polymerize and oxidize into macromolecular substances, and on the other hand, tea polyphenols would undergo hydrolysis during storage. Protein is an important material foundation in human life activity organization. Proper intake of protein can not only ensure people's normal life, but also have an important impact on human health. It has been reported that proteins have biological activities such as antitumor, antibacterial, and immunoenhancement (Zhou et al., 2019). In the hawk tea, the protein content is higher (22.53%) and fluctuated up and down with a constant decrease in 3 years storage and then an increase to the same level as that of new tea in the second 3 year storage (Figure 2a). It might be caused by the different rates of protein hydrolysis and decomposition into amino acids, and amino acids conversion and polymerization into proteins. During the first three years of storage, the rate of protein hydrolysis and decomposition was fast, but in the next three years of storage, the rate of amino acid conversion and polymerization was fast. Total ash is one of the legal chemical inspection items for tea export and is negatively correlated with the quality of tea. The total ash contents of the hawk tea prepared by boiling water fixation fluctuated up and down with a continuous increase from 3.97% to 6.33% for the first 3 year storage (3Y) and then a decrease to 5.45% for the second 3 year storage (6Y). And the storage time had a significant effect (p < .05) on the total ash content of hawk tea. The change trend of crude fat content was just opposite to that of total ash content. Its content decrease from 3.95% to 1.75% for the first 3 year storage (3Y) and then an increase to 3.27% for the second 3 year storage (6Y). Soluble sugar is an important component of tea taste and leads to a unique sweet aftertaste. Generally, dried tea leaves contain approximately 4%-6% soluble sugars (Harbowy et al., 1997). The content of soluble sugar in the hawk tea ranged from 0.29% to 1.83% in 6 years storage time and reached the highest in the third year of storage, but was the lowest in the sixth year of storage. In the first three years of storage, the content of soluble sugars showed an upward trend, which might be attributed to the transformation of insoluble sugar. However, in the next three years of storage, the change was just the opposite, and the decrease was obvious (p < .05), which indicated that the insoluble sugar had been completely or little transformed during this period, but the degradation and conversion of soluble sugars were serious. Soluble sugars could be oxidized and fermented, could be transformed into various acidic substances, and could produce maillard reaction with amino acids. The content of total flavonoids increased gradually. When stored for 1 year (1Y), 3 years (3Y), and 6 years (6Y), the content of total flavonoids was 1.05, 2.36, and 3.32 times of the new tea (CK), respectively. Caffeine is a powerful central nervous system and cardiac muscle stimulant that contributes to the unique quality of tea. However, high levels of caffeine can irrigate the gastrointestinal tract and lead to other adverse effects (Gramza-Michalowska, 2014). Therefore, it is advisable that the caffeine content of tea of reduced. The change of caffeine content detected in hawk tea made by boiling water fixation was decreased significantly from 1.62% to 0%. And in the sixth year of storage, caffeine was not detected in hawk tea. The above results indicated that the contents of inclusions in hawk tea would change during the storage. Due to the influence of the external environment, hawk tea could occur hydrolysis, oxidation, and degradation, especially the effect of temperature and humidity. And the contents of inclusions in hawk tea changed less within 12 months. \| Amino acids Amino acids are the main substance of tea and are closely related to the aromatic properties of the tea. They also have an obvious influence on the taste and color of the tea soup, and the content of free amino acids in tea is positively related to tea quality (Harbowy et al., 1997). Eighteen types of amino acids were detected in the hawk tea prepared by boiling water fixation, and their contents are shown in Table 1. Overall, the total contents of 8 types of essential amino acids and 18 types of amino acids in hawk tea presented a trend of descending first, then increasing, and then decreasing with storage time and had significant differences (p < .05). The contents for lysine, valine, isoleucine, glycine, and proline decreased continuously in the storage period of 6 years. Especially for glycine, its content dropped to 0 in the sixth year. The contents for glutamic acid kept growing in the first 3 year storage (3Y), from 829.00 mg/kg to 2,942.50 mg/kg, and then drastically decreased to 0 in the next 3 years storage (6Y). But for tryptophan, its content did not detect in the first 3 years storage and then increased to 23.50 mg/kg in the next 3 years storage. As one of essential amino acids of animal, tryptophan has many biological functions, such as regulation of immune function, digestive function, protein synthesis, and animal stress . For arginine, its content increased from 0.00 mg/kg to 332.50 mg/kg in the first 3 year storage and then decreased to 110.00 mg/kg in the next 3 year storage. Amino acids, due to the physical and chemical properties of the amino acids themselves, can be oxidized, degraded, and transformed under certain temperature and humidity conditions. For example, amino acids can undergo decarboxylation and oxidative deamination under certain conditions to produce corresponding aldehyde compounds, such as glycine to formaldehyde, alanine to acetaldehyde, and phenylalanine to phenylacetaldehyde. In addition, amino acids, sugars, and catechins can undergo maillard reaction under suitable conditions. This is consistent with the decreasing trend of tea poly- \| 3 Mineral elements Tea contains a variety of mineral elements. These mineral elements play an important role in human metabolism, normal life activities and maintenance of acid-base balance. The content of mineral elements in tea is an important quality indicator that determines the quality and grade of tea to a certain extent (Meng et al., 2020). The mineral elements in hawk tea were determined. As shown in Table 2, none of the samples had Hg and all of the heavy metals were well below the National Limit Standard (NY 659-2003, GB 2762-2012 \| 4 Antioxidant activity A large number of studies have shown that polyphenols and flavonoids have strong antioxidant properties Ji et al., 2020). It was found that the polysaccharide and flavonoid of hawk tea had good antioxidant activity (Chen et al., 2019;Tan et al., 2016;Xiao, et al. 2017). However, it is unclear which kind of substance has a significant correlation with the antioxidant activity of hawk tea. This study explored the correlation between the content of flavonoids and polyphenols and antioxidant activity. As shown in Figure 3a, the DPPH radical scavenging activity for all of the hawk tea samples was higher than that of Trolox. The different storage time significantly affected the DPPH antioxidant activity (p < .05). Here, a larger EC 50 implies a lower scavenging activity. With the increase of storage time, the value of EC 50 increased. The DPPH antioxidant activity was CK > 1Y> 3Y > 6Y. Figure 3b showed that the reducing power of hawk tea samples was negatively correlated with the storage time (p < .05). The FRAP values of CK were highest than those of all tested samples, and with the increase of storage time, the FRAP value decreased. The Pearson correlation among total flavonoids, total polyphenol and DPPH radical scavenging activity, FRAP value were calculated using SPSS software. According to Pearson's significance test, there was a significant negative correlation between the DPPH radical scavenging activity and FRAP value, and the Pearson correlation coefficient was −0.835 and −0.948. However, there was no significant correlation between antioxidant capacity and total flavonoids content of hawk tea stored at different time (p > .05). This result indicated that total polyphenols are the main active antioxidant in the hawk tea and have powerful capacity to reduce Fe 3+ to Fe 2+ , which is consistent with a previous study (Xiao et al., 2015). \| CON CLUS ION This study reported the effect of storage time on inclusions, amino acids, and mineral compositions of hawk tea (Litsea coreana) produced by boiling water fixation. With the prolongation of storage time, the biochemical characteristics of hawk tea changed less within 12 months. The total content and types of amino acids in hawk tea reached the maximum in the third year, as well as the content of total trace elements. The contents of water extracts, tea polyphenol, caffeine, lysine, valine, isoleucine, glycine, proline, Ca, and Zn decreased continuously in the storage period of 6 years. And the contents of total flavonoids, Mg, and Ni were just the opposite, showing the trend of increasing year by year. Tryptophan was detected in the sixed year. During the storage period of 6 years, the content of other inclusions, amino acids, and mineral compositions in hawk tea fluctuated up and down. The antioxidant activity of the new hawk Tea (EC 50 6.56 μg/ml for DPPH radical scavenging activity and FRAP value 2,432 Trolox μmol/g) was best, and the activity decreased with the extension of storage time. Total polyphenol is the main antioxidant material in the hawk Tea. All in all, results from this paper showed that the components of hawk tea have different changes with the extension of storage time. In content, some of the components increased and some of the components decreased. This study provides a theoretical basis for clarifying the relationship between the storage time and the change of the content. At the same time, this study further proves that total polyphenol is the main antioxidant material in the hawk tea. ACK N OWLED G M ENT The authors are grateful for the financial support of Guizhou CO N FLI C T S O F I NTE R E S T The authors declare no conflict of interest.	v3-fos-license
2017-08-27T09:02:36.387Z	2015-07-15T00:00:00.000	29158677	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.17145/jab.15.016", "pdf_hash": "842168218bab732b3cde93fde86847182e92d4b9", "pdf_src": "ScienceParseMerged", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2334", "s2fieldsofstudy": [ "Biology" ], "sha1": "842168218bab732b3cde93fde86847182e92d4b9", "year": 2015 }	pes2o/s2orc	A simple and selective liquid chromatography-tandem mass spectrometry method for determination of ε-aminocaproic acid in human plasma Intraoperative hemorrhage during pediatric craniofacial reconstruction and other surgical procedures can be significant and may exceed the circulating blood volume [1] mandating substantial perioperative transfusion. The intraoperative administration of antifibrinolytic agents is increasingly used to minimize blood loss and the need for transfusion [2]. Fibrin binding by tissue-type plasminogen activator and plasminogen is the key to the initiation of fibrinolysis. Plasmin is generated and cleaves fibrin, producing new C-terminal lysines, which serve to mediate positive feedback in the fibrinolytic cascade [3]. Downregulation of fibrinolysis in vivo occur through the action of carboxypeptidases, which remove C-terminal lysines [4]. The antifibrinolytic epsilon-aminocaproic acid (EACA) is a synthetic lysine analogue that blocks the lysine-binding sites on plasminogen, resulting in antifibrinolytic activity through inhibition of plasmin formation [5, 6] and ultimately clot stabiliziation. The objective of this investigation was to develop and validate a simple and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of EACA in human plasma to support clinical pharmacokinetic studies of EACA in pediatric patients. There were several HPLC methods reported in the literature for analysis of EACA in biological samples. EACA was dansylated and measured by fluorescence detection [7] with an assay range of 50 to 1000 μg/mL in human serum. A similar HPLC-fluorimeter assay with a range of 62.5 to 1000 μg/mL was reported by Lacroix C et.al. [8]. An assay for EACA in plasJOURNAL OF APPLIED BIOANALYSIS, July 2015, p. 99-107. http://dx.doi.org/10.17145/jab.15.016 (ISSN 2405-710X) Vol. 1, No. 3 Introduction Intraoperative hemorrhage during pediatric craniofacial reconstruction and other surgical procedures can be significant and may exceed the circulating blood volume [1] mandating substantial perioperative transfusion.The intraoperative administration of antifibrinolytic agents is increasingly used to minimize blood loss and the need for transfusion [2].Fibrin binding by tissue-type plasminogen activator and plasminogen is the key to the initiation of fibrinolysis.Plasmin is generated and cleaves fibrin, producing new C-terminal lysines, which serve to mediate positive feedback in the fibrinolytic cascade [3].Downregulation of fibrinolysis in vivo occur through the action of carboxypeptidases, which remove C-terminal lysines [4].The antifibrinolytic epsilon-aminocaproic acid (EACA) is a synthetic lysine analogue that blocks the lysine-binding sites on plasminogen, resulting in antifibrinolytic activity through inhibition of plasmin formation [5,6] and ultimately clot stabiliziation.The objective of this investigation was to develop and validate a simple and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of EACA in human plasma to support clinical pharmacokinetic studies of EACA in pediatric patients.There were several HPLC methods reported in the literature for analysis of EACA in biological samples.EACA was dansylated and measured by fluorescence detection [7] with an assay range of 50 to 1000 µg/mL in human serum.A similar HPLC-fluorimeter assay with a range of 62.5 to 1000 µg/mL was reported by Lacroix C et.al.[8].An assay for EACA in plas-ma and urine after derivatization with o-phthalaldehyde and fluorescence detection was reported with an assay range of 50 to 250 µg/mL [9].One of the common challenges encountered with HPLC methods were separation of EACA from other naturally occurring amino acids in biological samples.Recently, a method was reported for analysis of EACA in ophthalmic formulation that utilized isocratic tandem-mode HPLC with reverse phase and strong cation exchange columns and an assay range of 500-1500 µg/mL [10].Previously, a LC-MS/ MS assay was reported for analysis of caprolactam and EACA in human urine [11].Assay range for EACA was 62.5-1000 ng/mL, when a 20 μL aliquot of urine was injected directly into (LC-MS-MS) system.Recently, two LC-MS/MS assays were reported for analysis of structurally related compound, tranexamic acid in human plasma with an assay range of 0.8-200 µg/mL [12] and 1-200 µg/mL [13].In the present study, we report a simple and selective LC-MS/MS assay for quantitation of EACA in human plasma in concentration range of 1-250 µg/mL.This assay has been successfully employed for analysis of EACA in pediatric plasma samples [14,15]. Reagents and chemicals EACA (epsilon-aminocaproic acid) and 7-aminoheptanoic acid were purchased from Sigma-Aldrich (Cambridge, MA, USA) (Fig. 1).The different lots of drug-free (blank) human plasma (prepared with citrate-phosphate-dextrose, or citrate-phosphate-dextrose-dextrose or citrate-phosphate-dextrose-adenine) were obtained from the blood bank at The Children's Hospital of Philadelphia.HPLC grade methanol and acetonitrile were purchased from Fisher-Scientific (Pittsburgh, PA, USA) and reagent-grade formic acid (~96%) was purchased from Sigma-Aldrich Co. (St.Louis, MO, USA).Deionized water was prepared using a Barnstead Nanopure™ water purifying system from Thermo Fisher Scientific (Marietta, OH, USA). Liquid chromatography The Shimadzu HPLC system consisted of two LC-20AD delivery pumps, a DGU-20A5 Shimadzu vacuum degasser, a SIL-20AC Shimadzu autosampler and a CBM-20A system controller (Shimadzu Scientific Instruments; Columbia, MD, USA).HPLC separations were performed on a Thermo Scientific Nucleosil C18 analytical column (3 x 100 mm, 3.5 µm 120 A).For chromatographic separation, water with 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The linear gradient was as follows: 0.00-0.50minutes mobile phase A 80%, mobile phase B 20%; 0.51-3.00minutes mobile phase A 20%, mobile phase B 80% with divert valve off; 3.00-3.51minutes mobile phase A 80%, mobile phase B 20%; and initial gradient maintained until 6 minutes.The flow rate was 0.50 mL/min and 5 µL of the sample was injected for each analysis.The column and autosampler were maintained at room temperature and 10°C, respectively.An electronic valve actuator with a Rheodyne selector valve was used to divert LC flow to waste, at the first 0.5 minute and the last 3 minutes, when no data acquisition was taking place. Mass spectrometry analysis Samples were analyzed with an ABI/Sciex 4000 triple quadropole mass spectrometer equipped with Turbo Ionspray.Software for controlling this equipment, acquiring and processing data was Analyst version 1.6.2software (MDS Sciex; Toronto, Canada).The positive ion mode for MS/MS analysis was selected.Nitrogen (purity >99.999 %) was used as the nebulizer, auxillary, collision and curtain gases.Analytes were detected by tandem mass spectrometry using multiple reaction monitoring (MRM) with a dwell time of 100 ms.For the determination of the mass of the precursor and product ions a solution of 500 µg/mL EACA or internal standard in mobile phase (1:1 methanol: water (v/v) with 0.05% formic acid) was infused directly into the ion sources with a Harvard Apparatus syringe pump at a flow rate of 10 µL/min.The most intense precursor-to-fragment transitions using positive turbo spray were: EACA m/z 132.20→79.10;7-aminoheptanoic acid (internal standard) m/z 146.20→55.10.We have used additional MRMs of 132 to 114, 132 to 69 and 132 to 55 to confirm EACA.Product ion spectrum of EACA and 7-aminoheptanoic acid is shown in Fig. 2. The conditions for ionization of EACA and internal standard were optimized using individual standard solutions, each at 500 ng/mL in 1:1 methanol: water (v/v) with 0.05% formic acid at 10 µL/min.EACA and internal standard were infused by a syringe pump alone or through a Tee device at a flow rate of 10 µL/min into the stream of mobile phase (1:1 methanol: water (v/v) with 0.05% formic acid at 0.5 mL/min).eluting from the LC column through a mixing Tee and then into the turbo spray source, to mimic the LC-MS/MS conditions.The main working parameters of the mass spectrometer were: collision activate dissociation (CAD) gas, 8; curtain gas 40; Gas 1 (nebulizer gas) 80; Gas 2 (heater gas) 50; source temperature 600°C.The optimized declustering potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CXP) were set at 46, 10, 23, 6 for EACA; 46, 10, 50, 10 for 7-aminoheptanoic acid (IS). Preparation of standards and quality control (QC) samples Two independent EACA stock solutions at 25 mg/mL in water were prepared: standard solutions were prepared from one stock solution and QC samples were prepared from the other.The stock solutions were stored at -20 °C and were stable at least for 1 year.The primary stock solution was diluted in human plasma to prepare intermediate stock solutions of 500 µg/mL.Working solutions of EACA were freshly prepared by appropriately diluting the respective stock solutions with plasma.Eight standards containing EACA concentrations of 1, 2.5, 5, 10, 25, 50, 100 and 250 µg/mL were prepared by adding the appropriate volumes of working solution into 1.5 mL eppendorf tubes containing plasma.Four QC concentrations were prepared in the same manner by adding appropriate volumes of working solution to obtain concentrations of 1, 4, 40 and 200 µg/mL, representing LLOQ, low, medium and high QCs.The internal standard stock solution was prepared by dissolving 4 mg of 7-aminoheptanoic acid in water to a final concentration of 1 mg/ mL.Internal standard working solution was prepared by diluting the internal standard stock solution with water into a single working solution with a final concentration of 2 µg/mL.Amber glass vials for storing stock solution were used.Representative pharmacokinetic plasma samples used in this study were collected and processed as described previously [15]. Sample preparation Blood samples were collected in Microtainer tubes (BD, Franklin Lakes, NJ, USA) containing lithium heparin and stored at 4°C for up to 12 h prior to centrifugation and separation of plasma [14].To 50 µL of each standard, QC, blank, and unknown plasma samples from clinical studies in 1.5 mL Eppendorf tube, 1 mL of 10% acetic acid in water was added (1:20 dilution).Samples were vortex mixed and 50 µL of the aliquot was transferred into a disposable culture tube.To each tube, internal standard solution (50 µL) and 5 mL of water was added and vortex mixed (1:102 dilution).The sample preparation involves net dilution of 1:2040.An aliquot (300 µL) of the final solution was transferred into the appropriate position of a 96 well MicroLiter® plate for analysis.Five µL of the supernatant was injected into the LC-MS/MS system for analysis. Method validation Method validation and documentation were performed according to guidelines set by the United States Food and Drug Administration (FDA) for bioanalytical method validation [16].This method was validated in terms of linearity, specificity, lower limit of quantitation (LLOQ), recovery, intra-and inter-day accuracy and precision, and stability of the analyte during sample storage and processing procedures [17].Each analytical run included a double blank sample (without internal standard), a blank sample (with internal standard), eight standard concentrations for calibration, and six sets of QC samples: LLOQ QC at 1 µg/mL, low QC at 4 µg/mL, medium QC at 40 µg/mL, and high QC at 200 µg/mL. Linearity and sensitivity For the evaluation of the linearity of the standard calibration curve, the analyses of EACA in plasma samples were performed on three independent days using fresh preparations.The calibrations curves were prepared over a linear range of 1-250 µg/mL at eight concentrations of 1, 2.5, 5, 10, 50, 25, 50, 100, and 250 µg/mL.Each calibration curves consisted of a double blank sample, a blank sample, a plasma blank sample and eight calibrator concentrations.Another double blank sample was analyzed immediately following the highest concentration standard in each run to monitor for carryover of EACA or the internal standard.The calibration curve was developed using the following criteria: (1) the mean value should be within ±15% of the theoretical value, except at the LLOQ, where it should not deviate by more than ±20%; (2) the precision around the mean value should not exceed a 15% coefficient of variation (CV), except for LLOQ, where it should not exceed a 20% CV. (3) at least 75% of the eight non-zero standards of each nominal concentration should meet the above criteria; (4) the linear correlation coefficient (r, Pearson product moment correlation coefficient) should be greater than or equal to 0.98.Each calibration curve was constructed by plotting the EACA to internal standard peak area ratio (y) against the EACA concentrations (x).The calibration curves were fitted using a least-square linear regression model y=ax+b, weighted by 1/x 2 using the Analyst ® software.The resulting a, and b parameters were used to determine back-calculated concentrations, which were then statistically evaluated. Specificity The specificity was defined as non-interference at retention times of EACA from the endogenous plasma components and no cross-interference between EACA and internal standard using the proposed extraction procedure and LC-MS/MS conditions.Six different lots of blank (EACA free plasma, prepared with lithium heparin as a anticoagulant) were evaluated with and without internal standard at low QC concentration (4 µg/mL) to assess the specificity of the method. Accuracy and precision The intra-and inter-assay precisions were determined using the CV (%), and the intra-and inter-assay accuracies were expressed as the percent difference between the measured concentration and the nominal concentration.The % accuracy of the method was expressed by the formula: %accuracy=(measured concentration)/(nominal concentration) x 100%.Intra-assay precision and accuracy were calculated using replicate (n=6) determinations for each concentration of the spiked plasma sample during a single analytical run.Inter-assay precision and accuracy were calculated using replicate (n=6) determination of each concentration made on three separate days. Recovery (extraction efficiency) and matrix effect The extraction efficiency of EACA was determined by analyzing three replicates of EACA plasma samples at three QC concentrations of 4, 40 and 200 µg/mL.Recovery was calculated by comparing the peak areas of EACA added into blank plasma and extracted using the protein precipitation procedure with those obtained from EACA spiked directly into post-protein precipitation solvent at the four QC concentrations.The matrix effect was measured by comparing the peak response of the post-extracted spiked sample with those of the pure standards containing equivalent amounts of the EACA prepared in mobile phases.In addition, post-column infusion method described by Bongfiglio et al. [18] was employed to test matrix effect in six lots of blank plasma. Stability study The stability of EACA in human plasma was assessed by analyzing replicates QC samples at concentrations of 4, 40 and 200 µg/mL (n=6), during the sample and storage procedure (in 4 mL amber vials).For all stability studies, freshly prepared and stability testing QC samples were evaluated by using a freshly prepared standard curve for the measurement.The short term stability was assessed after exposure of the plasma samples to room temperature for 24 hours (n=6).The long term stability was assessed after storage of the plasma samples -80°C for up to one year (n=6).The freeze/thaw stability was determined after three freeze/thaw cycles (room temperature to -80°C, n=6).The concentrations obtained from all stability studies were compared to freshly prepared QC samples (n=6), and the percentage concentration deviation was calculated.The analyte was considered stable in human plasma when the concentration difference was less than 15% between the freshly prepared samples and the stability testing samples. Pharmacokinetics of EACA in children This assay was implemented for analysis of PK samples from healthy infants aged 2-24 months undergoing craniofacial surgery without history of renal impairment or a history of a coagulation disorder [15].Subjects were sequentially enrolled in one of the three cohorts.Each subject received an intravenous (IV) loading dose (25 or 50 or 100 mg/kg) of EACA followed by a continuous IV infusion (CIVI) of EACA (10 or 20 or 40 mg/kg/h) as described previously [15].PK samples, consisting of 1 ml of blood, were drawn (in lithium heparin tubes) immedi-from 96.9-102% (Table 1).Validation results for the replicates of quality control samples are depicted in Table 2.The intraday precision (n=6) ranged from 4.71-10.4% with the accuracy ranging from 92.3-106%.The inter-day (n=18, 3 days) precision ranged from 4.68-9.79%with the accuracy ranging from 95.4-103%.These data con-ately before and after the loading dose, after initiation of CIVI (0.5, 2, 4-6 h), at the end of the CIVI, and after the end of the CIVI (0.5, 3, 6, 9, 12, and 15 h), for a maximum total of 12 PK samples [15].Plasma was separated by centrifugation and stored at -80ºC until analysis. Linearity, sensitivity and specificity The method was validated at the above criteria and found to be linear for the concentration range of 1 -250 µg/ mL.A representative calibration curve for EACA is shown in Fig. 3.The linear correlation coefficient (r) from inter-day analysis was found to be greater than 0.98 in all cases.The LLOQ was 1 µg/mL, demonstrating a %CV less than 10% (precision) and accuracy greater than 92%, with a signal-to-noise (S/N) ratio of greater than 10.LLOQ was determined as the concentration at which all acceptable criteria are met and the measured concentration was within ±20% of the nominal concentration.The limit of detection (LOD) was 0.25 µg/mL, which was determined by the lowest concentration with acceptable chromatography, the presence of precursor and product ions with a relative retention time within ±2% of average retention time of EACA, an accuracy of within ±20% and a S/N ratio of at least 3 [11].A representative chromatogram of double blank, LLOQ, internal standard and a clinical plasma sample are shown in Fig. 4. Analysis of six different blank plasma samples and the corresponding spiked low QC (4 µg/mL) showed no significant interference from endogenous compounds (data not shown).No carryover of peaks was observed at the retention time or ion channels of EACA or IS. Precision and Accuracy At the eight calibration standards, the intra-day (n=6) precision ranged from 1.1-7.1% and the accuracy ranged measured concentrations of EACA (mean ± SD, n=6) in the post-extract spiked samples were 4.11 ± 0.16, 39.5 ± 1.69 and 180 ± 2.23 µg/mL, respectively.To further evaluate matrix effect, blank extracts from 6 lots of human plasma were analyzed by post-column infusion method described by Bongfiglio et.al. [18].No significant ion suppression was seen at the retention time of EACA in the infusion chromatograms (Fig. 5), confirming that there was minimal or no matrix effect observed for EACA after 1: 2040 plasma dilution and the HPLC separation. EACA stability The stability of EACA was investigated to cover expected conditions during sample preparation and storage for all samples, which include data from freeze/thaw, shortterm and long-term stability studies (Table 3).The precision for freeze/thaw samples (n=6) ranged from 1.4-10.9%and the accuracy ranged from 92.1-105%.The results indicated that the analyte was stable in plasma for three cycles when stored at -80 °C and thawed to room temperature.The precision for 24 hour bench-top stability (n=6) ranged from 5 -11.3% and the accuracy ranged from 86.4-111%.After 1 year storage at -80°C (n=6), the precision for the quality control samples ranged from 2.6 -8.44% and the accuracy ranged from 93.3-99.7%.These results indicated reliable stability under the experimental conditions of the analytical runs and storage conditions. firm that the present method has an acceptable accuracy, precision and reproducibility for the quantification of EACA throughout the desired concentration range. Assay specificity and ionization suppression (matrix effect) Matrix effect can affect the reproducibility from the analyte or the internal standard of the assay.The matrix effect, or intensity of ion suppression or enhancement is caused by co-eluting matrix components.The matrix effect of EACA and IS were calculated using the following formula: % matrix effect = (A/B) x 100%.A represents the corresponding peak areas of the analytes in spiked plasma post-precipitation and B represents peak responses of the pure standards prepared in mobile phases.A value of >100% indicated ionization enhancement, and a value of <100% indicated ionization suppression.The matrix effect was tested on the three QC concentrations (n=6) and six individual lots of blank plasma were evaluated.At 4, 40 and 200 µg/mL QC concentrations the MOORTHY GS J. APPL.BIOANAL Pharmacokinetics of EACA in children The representative PK profiles of EACA in 3 subjects are shown in Fig. 6.Subject 1 received an IV loading dose of 25 mg/kg followed by a CIVI of 10 mg/kg/h of EACA (cohort 1), subject 2 received an IV loading dose of 50 mg/kg followed by a CIVI of 20 mg/kg/h of EACA (cohort 2) and subject 3 received an IV loading dose of 100 mg/kg followed by a CIVI of 40 mg/ kg/h of EACA (cohort 3) as described previously [15].The results show that the assay range was appropriate for analysis of EACA in pediatric plasma samples from the clinical trials [14,15].Time (min) EACA Concentration (µg/mL) Figure 6.Semi-logarithmic concentration-time plots of EACA for three subjects.Subject 1: intravenous loading dose of 25 mg/kg followed by a CIVI of 10 mg/kg/ h.Subject 2: loading dose of 50 mg/ kg followed by a CIVI of 20 mg/kg/ h.Subject 3: loading dose of 100 mg/kg followed by a CIVI of 40 mg/kg/ h. Discussion EACA is a potent antifibrinolytic agent that inhibits plasmin-plasminogen system [5].EACA is a natural product that is present in wine lees [19] and cyclized product of EACA, caprolactam is used as an intermediate in synthesis of nylon 6 [20,21].We developed an EACA assay that has a rapid sample preparation (1:2040 dilution min-plasminogen system [5].EACA is a natural product that is present in wine lees [19] and cyclized product of EACA, caprolactam is used as an intermediate in synthesis of nylon 6 [20,21].We developed an EACA assay that has a rapid sample preparation (1:2040 dilutionof plasma samples) and a selective LC-MS/MS method capable of analyzing small volumes of plasma samples, which is ideal for use in pediatric clinical trials [14,15].The LC-MS/MS assay reported here accurately and precisely quantitates EACA concentration in 50 µL plasma specimens with a limit of quantification of 1 µg/mL.The intra-day and inter-day coefficients of variation were less than 10.4% at all concentrations tested.Assay range of 1-250 µg/mL is well suited for expected concentration ranges in pediatric pharmacokinetic studies.EACA had good recovery and stability under assay conditions.Our stability data is consistent with the reported results that EACA was stable at 20ºC for 3 months [22].At higher temperature (50 -80ºC), EACA degrades to form small amounts of dimer, trimer and caprolactam.However, in urine samples EACA was reported to be stable only for 3 days at 4ºC [11].There was no significant carryover or matrix effect observed for EACA with six different lots of blank plasma in post-column infusion studies.However, there was a significant matrix effect observed with urine samples for EACA analysis and the issue was resolved by diluting and analyzing urine samples [11].This is consistent with our finding that there was no significant matrix effect from 1:2040 diluted plasma samples.We were able to take advantage of the selectivity and sensitivity of LC-MS/ MS and high concentrations of EACA expected in clinical samples and develop a simple and reliable assay for the determination of EACA in human plasma.The minimum effective concentration of EACA to control systemic fibrinolytic activity was determined to be 130 µg/mL [23][24][25].Various dosing strategies based on PK studies in adults [26,27] and children [28] have been targeted to maintain plasma EACA levels at or above this therapeutic concentration.Previous pharmacokinetic studies of EACA have shown that renal excretion was the major route of elimination, whether administered orally or IV [29][30][31][32][33]. Approximately 65-75% of the dose is eliminated in the urine as unchanged drug while ~11% of the dose excreted as adipic acid [29,30,32,33].EACA pharmacokinetics is influenced by weight, age, and perioperative conditions.Weight-based dosing was suggested based on the modelling, a loading dose of 100 mg/kg followed by a CIVI of 40 mg/kg/h is appropriate to maintain target plasma EACA concentrations in patients 6-24 months of age [15].EACA was well tolerated and no adverse events were attributed to its administration.This assay has been successfully utilized for analysis of EACA in clinical studies [14,15]. Figure 5 . Figure 5. Infusion chromatograms (3.2 min) of EACA (at 1 µg/mL at 10 µL/min) employing post-column infusion with EACA assay LC conditions (flow rate of 0.5 mL/min): plasma free extraction solvent (A and H) and six lots of blank human plasma extracts (B to G). Table 1 . Accuracy and precision of EACA calibration standards in human plasma (n =6). Table 3 . Summary of stability outcomes for EACA in human plasma. Table 2 . Summary of validation outcomes for EACA in human plasma.	v3-fos-license
2021-09-27T19:48:00.089Z	2021-08-10T00:00:00.000	238682332	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://www.researchsquare.com/article/rs-591255/latest.pdf", "pdf_hash": "bf78856d96a15c5390d4033686e77c1a2357ac2f", "pdf_src": "SpringerNature", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2338", "s2fieldsofstudy": [ "Biology" ], "sha1": "f73d55bb8aa719693f68c41a6c44a7b4c308005c", "year": 2021 }	pes2o/s2orc	Streptomyces blattellae, a novel actinomycete isolated from the in vivo of a Blattella germanica During a screening for novel and useful actinobacteria in desert animal, a new actinomycete was isolated and designated strain TRM63209T. The strain was isolated from in vivo of a Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China. The strain was found to exhibit an inhibitory effect on biofilm formation by Candida albicans ATCC 18,804. The strain was observed to form abundant aerial mycelium, occasionally twisted and which differentiated into spiral spore chains. Spores of TRM63209T were observed to be oval-shaped, with a smooth surface. Strain TRM63209T was found to grow optimally at 28 °C, pH 8 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM63209T were rhamnose ribose, xylose, mannose, galactose and glucose, and the principal polarlipids were found to be diphosphatidylglycerol, phos-phatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, phosphatidylinositol and an unknown phospholipid(L). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid. The predominant menaquinone was found to be MK-9(H6) (14.64%), MK-9(H2) (19.65%), MK-9(H8) (22.34%), MK-10(H2) (25.37%). The major cellular fatty acids were identified as iso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0 and Sum in Feature 3. Analysis of the 16S rRNA sequence showed that strain TRM63209T exhibits high sequence similarity to Streptomyces bungoensis strain DSM 41781T 98.20%. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and phylogenomic analysis also illustrated that strain TRM63209T should be assigned to the genus Streptomyces. The DNA G + C content of the strain was determined to be 70.2 mol%. Average nucleotide identity (ANI) between strain TRM63209T and S. bungoensis DSM 41781T, Streptomyces phyllanthi PA1-07T, Streptomyces longwoodensis DSM 41677T and Streptomyces caeruleatus NRRL B-24802T were 82.76%, 82.54%, 82.65%, 84.02%, respectively. Digtal DNA-DNA (dDDH) hybridization were 26.30%, 25.10%, 26.20%, 29.50%, respectively. Therefore, it is concluded that strain TRM63209T represents a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. The type strain is TRM63209T (CCTCC AA 2018093T = LMG 31,403 = TRM63209T). Introduction The genus Streptomyces was initially described in 1943 (Waksman and Henrici 1943) and more than 850 species with valid names of Streptomyces have been published (https://www.bacterio.net/streptomyces. html) (Zhang et al. 2017). Streptomyces are aerobic Gram-positive bacteria with well-developed mycelia, which can produce a large number of conidia for reproduction. The members of the genus are with high DNA G ? C content, and species are widely distributed in aquatic and terrestrial ecosystems. Many Streptomyces undergo morphological differentiation and also have a mycelial phase. Streptomyces are highly versatile and produce an abundant array of bioactive secondary metabolites (Genilloud 2017) that have been used as antibiotic, anti-carcinogenic, antihelminthic and antifungal compounds. Consequently, they are very important for biotechnology, medicine and agriculture (Barka et al. 2015). Strain TRM63209 T isolated from in vivo of an Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China(40°55 0 N,81°29 0 E) This strain inhibits the formation biofilm of Candida albicans ATCC 18,804. On the basis of phylogenetic, phenotypic and genetic data, TRM63209 T considered to a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. Isolation of Streptomyces strain and culture conditions As part of a program to unravel the diversity of symbiotic actinomycetes in insect-microbe and to discover novel actinomycetes and novel natural products, strain TRM63209 T was isolated from the in vivo of a Blattella germanica, the Tarim University, Alar, Xinjiang Province, north-west China. Blattella germanica is washed with sterile distilled water to remove surface impurities. The surface was sterilized in 70% ethanol for 60 s and then washed three times in sterile distilled water. Grind it to a powder and suspend in sterile distilled water incubated on a rotary shaker at 180 rpm 37°C for 30 min (Liu et al. 2017), ultrasound 3 min, and the suspension was appropriately diluted before being spread onto Czapek's agar (Wiese et al. 2008) supplemented with nystatin (100 mg/ml) and nalidixic acid (50 mg/ml) (Arocha-Garza et al. 2017). After 21 days of incubation at 28°C, the isolate was transferred and purified on International Streptomyces Project (ISP) 4 medium (Shirling and Gottlieb 1966) and the spore and mycelia maintained as glycerol suspensions (20%, v/v) at -80°C. Chemotaxonomy Isomers of diaminopimelic acid were analysed following the method of Hasegawa et al. (1983). The whole cell sugar composition was analysed following the method of Staneck and Roberts (1974). Polar lipids in cells of strain TRM63209 T were extracted and examined by two-dimensional TLC and identified following the methods of Minnikin et al. (1984). Menaquinones were extracted using the method of Collins (1985) and subjected to HPLC analysis (Groth et al. 1997). The cellular fatty acid composition was determined using the Microbial Identification System (MIDI Sherlock version 6.0) (Sasser 1990). Phylogenetic analyses Genomic DNA extraction and PCR amplification of the 16S rRNA gene from strain TRM63209 T were performed following Chun and Goodfellow (1995). The purified PCR product was cloned into the vector pMD19-T (Takara) and sent to Sangon for gene sequencing. Multiple alignments with sequences from closely related Streptomyces species and calculations of sequence similarity were performed using the EzTaxon-e server (Kim et al. 2012). Phylogenetic analyses were performed using MEGA version 7.0 (Kumar et al. 2016) selecting the neighbour-joining (Saitou and Nei 1987), Maximum-Evolution (Rzhetsky and Nei 1993) and maximum-likelihood (Felsenstein 1981) algorithms. Topologies of the resultant trees were evaluated using the Felsenstein's (1985) resampling method with 1000 replications. AtpD, gyrB, rpoB, recA and trpB genes were obtained using primers and amplification conditions as previously described (Guo et al. 2008;Hatano et al. 2003). Phylogenetic relationships were reconstructed using the Neighbour-Joining algorithm as described above. Phylogenomic analysis was performed online by Type (strain) Genome Server (TYGS) (Meier-Kolthoff et al. 2019). The whole genome of TRM63209 T was sequenced by Oxford Nanopore technologies. The DNA G ? C content of strain TRM63209 T was obtained by whole genome sequencing. The Average nucleotide identity (ANI) was determined as described by Lee et al. (2015). DNA-DNA relatedness values were determined online according to the method of Meier- Kolthoff et al. (2013). DNA-DNA hybridization (dDDH) values were calculated at the Genome-to Genome Distance Calculator (GGDC) website using formula 2, as originally described by Auch et al. (2010) and updated by Meier-Kolthoff et al. (2013). Anti-SMASH was used to predict the biosynthetic gene clusters of strain TRM63209 T (Blin et al. 2013). Antifungal and antibacterial activity C. albicans ATCC 18,804 was obtained from China Center for Type Culture Collection, and was cultured with Sabouraud dextrose agar/broth (SDA/SDB). Unless specified otherwise, ISP 3 was used to culture TRM63209 T strain. A 4% (v/v) inoculum of well-growing strain TRM63209 T was used to culture strain TRM63209 T in ISP 3 liquid culture medium (20 g oatmeal and 1 ml trace salt per L distilled water) and incubated at 28°C with shaking at 180 rpm for 10 days. Cells were removed by centrifugation to leave the supernatant, which was kept at 4°C for further screening of biofilm inhibition. The effect of the strain TRM63209 T growth supernatants on static biofilm formation measured according to Balasubramanian et al. (2017). Briefly, test organism cells were diluted 1:100 with fresh SDB to bring the test cell suspension to a concentration of 1 9 10 8 cells per mL. 100 lL aliquots of cells were added to the wells of a 96 well plate and 100 lL of supernatants was added, then the plates inoculated with C. albicans were incubated at 37°C for 72 h. Wells without the supernatant (100 lL SDB) was used as blank control. After crystal violet staining, the absorbance was measured at 490 nm by an enzymelinked immunosorbent assay reader (Bio-Rad). Relative activity of biofilm formation was indicated as Relative Biofilm Formation % (RBF %) calculated the following formula: RBF % = Treated OD 490 /Untreated OD 490 9 100%. Results and discussion Strain TRM63209 T was observed to grow optimally on ISP 3 and ISP 2, and showed moderate growth on ISP 1, ISP 4, ISP 5, nutrient agar and Gause's synthetic agar no. 1, with slow growth on ISP 6, ISP 1 and Czapek's medium. Light yellow soluble pigment was produced in ISP 5 and greenish White soluble pigment was produced in ISP 6, the colour of other the aerial mycelium is white, other no diffusible pigment was produced on the media test, the color of ISP 2 substrate mycelium is light yellow ( Table 1). The growth and cultural characteristics of strain TRM63209 T related type strains are listed in the species description and in Table S1. Morphological characteristics of strain TRM63209 T were observed using SEM (Fig. 1). The strain was observed to form an abundant white aerial mycelium, occasionally twisted, which differentiates into spiral spore chains. Each spore was observed to be oval-shaped with a smooth surface (Fig. 1). Strain TRM63209 T was found to grow only at 5-55°C, pH 4.0-12.0 and 0-20% (w/v) NaCl, with optimal growth at 28°C, pH 8.0 and with 1% (w/v) NaCl. Other physiological characteristics of strain TRM63209 T are listed in the species description and in Table 1. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain TRM63209 T belongs to the genus Streptomyces, with high sequence similarity to Streptomyces bungoensis DSM 41781 T (GenBank accession no. KQ948892; 98.20%), Streptomyces phyllanthi PA1-07 T (GenBank accession no. LC125632; 98.14%), Streptomyces longwoodensis DSM41677 T (GenBank accession no. KQ948572; Rong and Huang (2012), in multi-locus sequence analysis (MLSA) it is considered that pairs with evolutionary distances greater than 0.007 belong to different species. A MLSA of five house-keeping genes (atpD, gyrB, recA, rpoB, and trpB) indicated that the MLSA distances between strain TRM63209 T and similar species were greater than the 0.007 threshold (Fig. S7). Result of phylogenomic analysis also supported that strain TRM63209 T belonged to genus Streptomyces (supplementary Fig. S8). The DNA G ? C content in the draft genome sequence of strain TRM63209 T was determined to be 70.2 mol %. The complete genome of strain TRM63209 T has a size of 8.49 Mb, did not distribute among chromosomes and plasmids. In its genome, 7804 genes were annotated, of which 7732 are putative protein-coding genes. The number of hypothetical proteins is 2367, corresponding to 31% of the total number of putatively annotated proteins. 60 tRNAs and seven copies of the 16S rRNA gene were identified. The genomic characteristics of the compared strains are quite heterogeneous (Table. S4). The ANI relatedness between strain TRM63209 T and the phylogenetically related strain Streptomyces caeruleatus NRRL B-24802 T , Streptomyces bungoensis DSM 41781 T , Streptomyces longwoodensis DSM 41677 T and Streptomyces phyllanthi PA1-07 T were respectively determined to be 84.02%, 82.76%, 82.65%,82.54%. This value is significantly lower than the widely accepted threshold for describing prokaryote species (95-96%; Kim et al. 2014). The dDDH value between strain TRM63209 T and the phylogenetically related strain Streptomyces bungoensis DSM 41781 T , Streptomyces phyllanthi PA1-07 T , Streptomyces longwoodensis DSM 41677 T and Streptomyces caeruleatus NRRL B-24802 was respectively determined to be 26.30%, 25.10%, 26.20%, 29.50%. significantly lower than the 70% threshold value for delineation of prokaryotic genomic species (Wayne et al. 1987). It is thus proposed that strain TRM63209 T can be differentiated from closely related Streptomyces species and represents a novel species. The supernatant of strain TRM63209 T inhibited biofilm formation by both C. albicans, with inhibition ratios over 40% (Table S3). The anti-SMASH biosynthetic gene cluster prediction tool was used to investigate the draft genome sequence of strain TRM63209 T and found one type I, two type III polyketide biosynthetic gene clusters, five nonribosomal peptide synthetase biosynthetic gene clusters and one NRPS-like fragment. In addition, five terpene, three siderophore, three class i lanthipeptide clusters like nisin, one nonalpha poly-amino acids like e-Polysin (NAPAA), one ectoine, one arylpolyene, one other unspecified ribosomally synthesised and post-translationally modified peptide product (RiPP), one redox-cofactors such as Table 2 Differential characteristics between strain TRM63209 T and phylogenetically related species of the genus Streptomyces Table. S4. A product of one of these clusters may be involved in the antibiofilm activity observed. Through anti-SMASH analysis, the 7-prenylisatin antibiotic biosynthesis gene cluster can be found, which can effectively inhibit the growth of fungi and the similarity to 60%, whereby the strain TRM63209 T inhibits the formation of BF may be associated with this gene cluster (Liang D). In summary, the sequencing of the genome of strain TRM63209 T further clarified the evolutionary relationship between strains and will guide the screening for active secondary metabolites. The type strain, TRM63209 T (CCTCC AA 2018093 T = LMG 31,403), was isolated from the in vivo of a Blattella germanica in Tarim University, Alar City, Xinjiang Province, The GenBank/EMBL/ DDBJ accession numbers for the genome and 16S rRNA gene sequence of strain TRM63209 T are WJBG00000000 and MK795724, respectively.	v3-fos-license
2020-10-25T13:05:17.698Z	2020-10-01T00:00:00.000	225057838	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1422-0067/21/20/7774/pdf", "pdf_hash": "f372b8baa39ada0d03b4618f78a9388126bd3f05", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2342", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "deb86d7f719675940d5c3e3db3238ac6a6ffdbe6", "year": 2020 }	pes2o/s2orc	A Critical Review of the Abilities, Determinants, and Possible Molecular Mechanisms of Seaweed Polysaccharides Antioxidants Oxidative stress induces various cardiovascular, neurodegenerative, and cancer diseases, caused by excess reactive oxygen species (ROS). It is attributed to the lack of sufficient antioxidant defense capacity to eliminate unnecessary ROS. Seaweeds are largely cultivated for their edible and commercial purposes. Excessive proliferation of some seaweeds has occurred in coastal areas, causing environmental and economic disasters, and even threating human health. Removing and disposing of the excess seaweeds are costly and labor-intensive with few rewards. Therefore, improving the value of seaweeds utilizes this resource, but also deals with the accumulated biomass in the environment. Seaweed has been demonstrated to be a great source of polysaccharides antioxidants, which are effective in enhancing the antioxidant system in humans and animals. They have been reported to be a healthful method to prevent and/or reduce oxidative damage. Current studies indicate that they have a good potential for treating various diseases. Polysaccharides, the main components in seaweeds, are commonly used as industrial feedstock. They are readily extracted by aqueous and acetone solutions. This study attempts to review the current researches related to seaweed polysaccharides as an antioxidant. We discuss the main categories, their antioxidant abilities, their determinants, and their possible molecular mechanisms of action. This review proposes possible high-value ways to utilize seaweed resources. Introduction Reactive oxygen species (ROS) are byproducts of aerobic metabolism, mainly produced in the mitochondria. They contain free and non-free radical oxygen, such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 − ), singlet oxygen (1/2 O 2 ), and hydroxyl radicals (·OH). At high levels, ROS are toxic to cells as they impair the redox balance with high reactivity, resulting in damage to intracellular proteins, lipids, and nucleic acids. However, cells have evolved mechanisms to deal with ROS toxicity so that at low levels, they play an integral role in various cell signaling pathways as regulators, such as cytokine, transcription, neuro-modulation, immune-modulation, and apoptosis [1]. The balance between the formation of ROS and the ability to remove them is vital in biological systems, and a shift in the balance to ROS formation is termed "oxidative stress" [2], disordered redox signaling, and control [3]. Excess levels of ROS or the abnormal functioning of the antioxidant system, have been identified in ions with non-ionizing electromagnetic radiation of frequencies between 300 MHz and 300 GHz [40], and enzyme-assisted extraction (EAE) degrades cell walls [41]. However, no method is perfect, and yields may be low. In addition, the methods are not optimized and require adjustments to be boosted to an industrial scale [38]. The structural complexity of polysaccharides and their "unconventional" and heterogeneous sugar composition, sulfation, and other modifications limits their broader applications in the industry [38]. Polysaccharides have various brown, red, and green characteristics. Some important seaweed polysaccharides that have commercial value are fucoidan, alginates, and laminarin from brown algae; carrageenan and agar from red, and ulvan from green seaweeds [36,[42][43][44][45][46][47]. Brown Seaweed Polysaccharides Fucoidan, also called "fucan", "fucosan", or "sulfated fucan", is composed of high percentages of L-fucose and sulfated ester groups. It is found in brown seaweeds and some marine invertebrates (such as sea urchins and sea cucumbers) [48,49]. Fucose, the preponderant constituent, is combined with other monomers, such as galactose, mannose, xylose, and residues of glucuronic acid [50,51]. Based on the backbone structure, fucoidans are divided into two subgroups: one is made up of α-1,3-L-fucopyranose and the other alternating 1,3-and 1,4-linked α-L-fucopyranose [52]. Fucoidan molecular weights range from 40 kDa to 1600 kDa. The amount of fucoidans in seaweeds varies with the seasons (highest during autumn), the species, and the development stage, from 0.1% to 20% of dry weight [53]. Alginates are primarily extracted from Macrocystis pyrifera, Ascophyllum nodosum, Laminaria spp., Ecklonia maxima, Eisenia bicyclis, Lessonia nigricans, and Sargassum spp. [54]. They are usually distributed in the cell walls as calcium, magnesium, and sodium salts and enhance the strength and flexibility of seaweed tissue [55]. The extraction process usually converts the cationic salt from the insoluble form to the soluble one, followed by eliminating impurities by successive dissolutions and precipitations [54]. Alginate is widely used in medical and pharmaceutical technology, and the cosmetic, food, agricultural, textile, and paper industries [56]. They are linear anionic unbranched copolymers, composed of β-1,4-D-mannuronic acid (M) and α-1,4-L-guluronic acid (G) and are usually described by their M/G ratio and average molecular weight, because their functions, physical properties, mechanical strength, and biocompatibility are largely dependent on those parameters [57]. Laminarin, a major carbohydrate reserve, is mainly composed of β-1,3-D-glucopyranose, with the β-1,6-linked D-glucopyranose units as branch-points or interchain residues [58]. Its solubility in water depends on the branching structure, as affected by interchain β-1,6-linkages. The antioxidant activity of laminarin has been linked to its molecular structure, degree, and length of branching and monosaccharide constituents [59]. According to the difference in terminal reducing end of the polymer chain, laminarin is divided into the G-type, which only contains glucopyranose, and the M-type with 1-O-linked D-mannitol [58,60]. The ratio of M-type to G-type varies by species, as high as 3:1 in Laminaria digitate [61] and absent in Eisenia bicyclis. The biological activities of laminarins vary according to species and are characterized by content, type (branchpoints or interchain), and the spatial distribution of the β-1,6-linkages [38]. Red Seaweed Polysaccharides Carrageenan is a high molecular weight sulfated polysaccharide extracted from Chondrus, Gigartina, and various Eucheuma species from the red algal family, Rhodophyceae [62]. It is a major component of cell walls in red seaweeds and interacts with other bioactive compounds, such as proteins, lipids, and other polysaccharides [38]. It is composed of the base units, D-galactopyranosyl with one or two sulfate groups, linked via alternated (1→3)-βd -and (1→4)-βd -glucoside [36]. Depending on the number and position of the sulfate groups, carrageenans have been divided into 10 types, of which the kappa (κ), iota (ι), and lambda (λ) are of commercial significance. Carrageenan is edible and safe, largely used in food and pharmaceutical industries as a stabilizer, gelling agent, thickener, binder, and additive. However, there are reports that its consumption increases the risk of colitis [63,64]. In addition, in studies of carrageenan-induced paw edema and pleurisy and thrombosis in a tail thrombosis model, carrageenan is used to study the mechanisms involved in inflammation [65,66], antithrombosis, and thrombolysis [67,68] in laboratory animals, such as rats. Agar is a mixture of gel polysaccharides, including agarose and agaropectin, uniquely found in the cell walls of some red seaweeds, specifically species in the families Gracilariaceae, Gelidiaceae, Pterocladiaceae, and Gelidiellaceae [69]. Agar is composed of alternating monomers, d-and l-galactose, linked via glycosidic bonds. Agarose, the major fraction of agar, making up about 70% of the polysaccharides, is composed of repeating d-galactose and 3,6-anhydro-l-galactose, with high molecular weight [70,71]. Compared to agarose, agaropectin has a lower molecular weight and higher amount of sulfate ester groups. The backbone of agaropectin is the same as agarose, substituted with various amounts of sulfate esters and d-glucuronic and pyruvic acids [69]. Agar, a phycocolloid from species in the genera Gracilaria and Gelidium, is broadly used in the food, pharmaceuticals, cosmetics, medical, and biotechnology industries [72]. Green Seaweed Polysaccharides Ulvan is a uronic acid-rich sulfated polysaccharide, primarily composed of sulfated rhamnose, uronic acids (glucuronic acid and iduronic acid), and xylose, It is found in species mainly in the Ulvalean genera Ulva and Enteromorpha [73]. It is the major component of cell walls in green seaweed, occupying 9 to 36% of their dry weight [44]. The backbone of ulvan is mainly composed of repeating disaccharide units, αand β-(1,4)-linked monosaccharides (rhamnose, xylose, glucuronic acid and iduronic acid). Ulvan has been reported to be effective as an immune modulator [74,75], and with antiviral [76,77] and anticoagulant properties [78,79]. It is regarded to have potential in nutraceutical, pharmaceutical, and cosmetic applications. Radical Scavenging Capacity The radical scavenging capacity of seaweed polysaccharides have been evaluated by two categories of assays: hydrogen atom transfer (HAT) and electron transfer (ET) reaction-based assays, depending on particular reactions [12]. Based on calculations of the ability of antioxidants to donate hydrogen, HAT-based methods are usually quick and independent of solvent and pH conditions [12,80]. In contrast, ET-based methods require relatively longer times, are typically expressed as percent decrease in product rather than kinetics [80,81]. Because of the dominant role of hydrogen atom transfer in biological redox reactions, HAT-based methods are considered to be more relevant to biology [12]. ET-based methods usually employ the scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), the measurement of total phenolic content (TPC) by the Folin-Ciocalteu (FC) assay and the Ferric reducing antioxidant power (FRAP). The DPPH assay is simple and effective for evaluating the antioxidant capacity of extracts. It is based on the antioxidant potential to give a hydrogen atom to the synthetic nitrogen radical compound DPPH [82]. The Folin-Ciocalteu (FC) assay measures the reducing capacity of a sample by the Folin-Ciocalteu reagent (FCR), a mixture of phosphomolybdate and phosphotungstate [83]. The weight equivalents of standard antioxidants, such as gallic acid (GAE) [83], phloroglucinol (PGE) [84], and tannic acid equivalents (TAE), are used to measure the values of polyphenols in samples. Because of the similarity of the chemistry between FCR and an ET-based antioxidant capacity assay [12], good linear correlations have been frequently reported between the "total phenolic profiles" and "the antioxidant activity". However, the opposite results, i.e., a lack of correlation between TP content and antioxidant activity, have also been reported [85], suggesting that other components, such as chlorophyll and carotenoids, together with differences in the polyphenols profiles may affect the antioxidant activity [85]. The FRAP assay measures the power of the antioxidant to reduce Fe 3+ to Fe 2+ in acidic media (pH 3.6) maintained by 2,3,5-triphenyltetrazolium chloride (TPTZ). Due to the blue color of the ferrous (Fe 2+ ) complex, antioxidant ability is calculated by the absorbance at 593 nm [86]. The equivalent of Fe 2+ or reference standard antioxidants, such as Trolox, is expressed as the value of antioxidant ability. The assays of oxygen radical absorbance capacity (ORAC), 2,2'-azino-bis (3-ethylbenzothiazoline -6-sulphonic acid)(ABTS), superoxide anion (O 2 − ), and hydroxyl (·OH) radicals scavenging activity are typically the HAT-based methods applied to measure the antioxidant ability of seaweed polysaccharides [80,[87][88][89]. The ORAC assay measures the ability of the antioxidants to break radical chains by monitoring the inhibition of antioxidants to peroxyl radical induced oxidations [90]. Trolox is usually used as a standard antioxidant, and trolox equivalents (TE) are commensurately expressed as the ORAC values of the tested antioxidants [91]. Superoxide radical scavenging activity assay evaluates antioxidant capacities by supervising the inhibition in the photochemical reduction of nitro blue tetrazolium (NBT). The hydroxyl radical scavenging activity assay is based on the Fenton's reaction [92]. Gallic acid is usually used as a positive control in the two assays [92,93]. Generally, more than two assays are applied to measure the antioxidant ability (Table 1). For example, the ulvan extracted from U. pertusa by the method of microwave-assisted extraction exerted a high antioxidant ability as evaluated by the radical-scavenging activity of DPPH and ABTS, and reducing power [94]. However, the results from different assays do not always agree, which may be attributed to the diverse mechanisms of the antioxidants. According to the action mode, there are primary and secondary antioxidants also referred to as chain breaking and preventive antioxidants [95]. Primary antioxidants commonly accept free radicals, breaking the propagation chain of autoxidation by inhibiting the initial step or interfering with the propagation step [96]. The activities of primary antioxidants are determined by their ability to donate hydrogen atoms to free radicals [97]. Secondary antioxidants alleviate oxidative stress by decreasing the rate of oxidation reactions via various mechanisms [98], such as providing H to a primary antioxidant, scavenging reactive oxygen and decomposing hydroperoxide [95]. Koh et al. reported that the antioxidant activity of Undaria pinnatifida fucoidan had a significantly less free radical scavenging ability than both the synthetic antioxidants, ascorbic acid, and BHA, but it had a hydroxyl radical scavenging activity similar to BHA although still lower than ascorbic acid [99]. Similar results were found with the Sargassum binderi fucoidan [100], indicating that fucoidan is a better secondary antioxidant than a primary one. However, Costa et al. measured the antioxidants of sulfated polysaccharides from 11 species of tropical marine seaweed, and found that only four species (Caulerpa sertularioide, Dictyota cervicornis, Sargassum filipendula, and Dictyopteris delicatula) showed hydroxyl radical scavenging activities, far less than gallic acid. Meanwhile, all species showed antioxidant ability through the assay of a total capacity antioxidant [93]. Oliveira reported a similar result of a sulfated galactan prepared from the red seaweed, Gracilaria birdiae, that showed no hydroxyl radical scavenging ability, suggesting that the antioxidant mechanism of seaweed sulfated polysaccharides is likely different [92]. Endogenous Antioxidant Ability Besides their direct ROS scavenging ability, polysaccharides play a stronger role in the fight against oxidative stress by enhancing the endogenous antioxidant systems of humans and animals [44]. The endogenous antioxidant ability of polysaccharides is indirectly evaluated via the measurement of enzymatic (e.g., SOD, CAT, GPx) and oxidation products (such as malondialdehyde (MDA) and lipid peroxidation (LPO)) [44]. Tables 2 and 3 summarized the recently reported enhancement of the endogenous antioxidant ability of seaweed polysaccharides according to cell lines and animal models, as related to diabetes [104], nephropathy [105], immunity [106], Alzheimer's disease [42], pulmonary disease [107], and others. For examples, the neoagaro-oligosaccharides (NAOs), acid hydrolyzed from agar, have been reported to benefit the antioxidative system of type 2 diabetes mellitus (T2DM) mice by upregulating the activity of GPx and SOD while significantly reducing the concentration of MDA [108]. Ulvan alleviated the damage of RAW264.7 murine macrophage cell lines induced by H 2 O 2 through the upregulation of levels of SOD and CAT [94]. Chen et al. investigated the ROS scavenging ability of agaro-oligosaccharides in vitro through the DPPH assay, then further studied the attenuating effect of oligosaccharides on ROS production in human liver L-02 cells treated by the oxidative agents, H 2 O 2 and antimycin A [109]. However, agaro-oligosaccharides, such as agarobiose and agarotetraose, showed protection against oxidation in a concentration-dependent manner; they were also reported to induce oxidation at lower levels [109]. Additionally, pretreatment of polysaccharides before induction were applied in various studies with significantly protective effects, such as ABAP induced female Wistar rats [110], alcohol induced male Kunming mice [106], UVB radiation induced hairless Kun Ming mice [111], and H 2 O 2 -induced NT2 neurons [112]. Jin et al. compared four strategies of administering algal oligosaccharides (AOS) from Gracilaria lemaneiformis ( Table 2), suggesting that taking AOS 2 h orally before alcohol consumption is the best strategy to protect the liver from alcohol damage [106]. A similar result was reported by Liu et al.-pretreatment of laminarin shows more significant changes in SOD, MDA, GSH, and CAT [107]. CAT: Catalase; GPx: glutathione peroxidase; GSH: glutathione; MDA: malondialdehyde; SOD: superoxide dismutase; The arrows upward represent increase and the downward represent decrease. Determinants of Antioxidant Activity The influence of the molecular weight of polysaccharides on antioxidant activity has been mentioned in various papers. The lower molecular weight sulphated polysaccharides tend to have a higher antioxidant activity. Lim et al. reported that among the fucoidans from Fucus vesiculosus degraded by gamma rays, via the assay method of ferric-reducing antioxidant power (FRAP), the lesser the molecular weight, the higher the antioxidant activity [122]. The fucoidan fraction with molecular weight lower than 10 kDa from Undaria pinnatifida showed a significantly higher secondary antioxidant activity than the crude fucoidan, the fraction with molecular weight cut off (MWCO) of 300 kDa, and even synthetic antioxidant butylated hydroxyanisole BHA [99]. Chen et al. reported that the fucoidan fractions with molecular weight between 5-10 k Da promoted the highest DPPH radical scavenging activity (48.3%) among fractions with molecular weight below 5 kDa, between 5-10 kDa, 10-30 kDa, 30-50 kDa, and over 50 kDa [123]. The stronger antioxidant activity of lower molecular weight polysaccharides may be attributed to their non-compact structure, which potentially makes more hydroxyl and amine groups available to neutralize free radicals [124]. However, agar oligosaccharides from Gelidum amausii showed an inverse consequence in which the oligosaccharide with higher molecular weight (2000-3800 Da) showed a higher antioxidation activity [125]. Sulfated polysaccharides from Mastocarpus stellatus showed similar results in which the fraction with the highest and lowest molecular weight were the best antioxidants, compared to the other fractions [124]. Therefore, other factors should be considered when evaluating the antioxidative ability of polysaccharides. Sulfate content is also a vital factor affecting the antioxidant activity of fucoidan. There is a positive correlation between sulfate content and the scavenging superoxide radical ability in fucoidan from Laminaria japonica [126]. The ratio of sulfate content/fucose was proposed to be an effective indicator to evaluate the antioxidant activity of fucoidans [126]. Similar results were reported in the sulfated polysaccharides from the red seaweed Mastocarpus stellatus through the assay methods of FRAP-reducing power and ABTS-radical scavenging [124]. Other factors, such as the position of sulfate groups, and monosaccharide content and structure, influence the antioxidant activity of sulfated polysaccharides. However, Costa et al. analyzed the antioxidant ability of 11 seaweed polysaccharides, finding no correlation between sulfate content and superoxide anion scavenging ability [93]. Similar results were reported where concentration and structure were found to be the factors affecting the antioxidant ability of carrageenans rather than the degree of sulfation [47,102]. This indicates that not only sulfate content but also spatial patterns of sulfate groups determine the antioxidant activity of polysaccharides [47]. In Ulva intestinalis, alkaline-extracted sulfated polysaccharides exhibited no significantly higher DPPH radical scavenging ability, though they had higher sulfate content and lower molecular weight than water-extracted polysaccharides [127]. The study proposed that the antioxidant activity of the sulfated polysaccharides was more related to glucose content than sulfate content or molecular weight, which agreed with the conclusion by Lo et al. that the free-radical scavenging ability of polysaccharides were notably dependent on the monosaccharide composition [128]. Sokolova et al. measured the in vitro antioxidant properties of the various carrageenans (lambda-, iks-, kappa-, kappa/beta-and kappa/iota-) from Gigartinaceae and Tichocarpaceae, using the FRAP and PPM assays, and the radical scavenging of DPPH, superoxide anion, hydrogen peroxide, and nitric oxide [102]. Iks-carrageenan exhibited the most effective antioxidative ability, possibly because it has the highest content of sulfate groups and the 3,6-anhydrogalactose unit [102]. A similar result by Abad et al. reported that the antioxidant ability of κ-, ιand λ-carrageenans followed the order of λ < ι < κ, according to the hydroxyl radical scavenging, reducing power, and DPPH radicals scavenging capacity assays [47]. Extraction and degradation methods affect the sulfate content at the same level of molecular weight, which determines the antioxidant activity of polysaccharides. Peasura et al. found that the antioxidant activities of sulphated polysaccharides from Ulva intestinalis were influenced by the extraction solvent (distilled water, 0.1 NHCl, and 0.1 N NaOH) and time (1, 3, 6, 12, and 24 h). The acid extract exhibited higher antioxidants than distilled water or alkali extract [127]. In Fucus vesiculosus fucoidans, the radical degradation method acquired almost 0.8-fold more sulfate content than acidic heating [122]. The degree of polymerization, an important factor affecting the antioxidant ability of carrageenans, depends on the methods of depolymerization. Sun et al. used four degradation methods, free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, to degrade food-grade κ-carrageenan. Free radical depolymerization was proposed as an effective method to obtain hydrolysates with the highest antioxidant ability according to the structure analysis by ESI-MS and CID MS/MS and antioxidant activity assay [129]. It is attributed to the difference of the reducing sugar content, the degree of polymerization, and the carboxyl and sulfate groups affected by the methods of depolymerization [129]. Kang et al. explored the influence of the concentration of substrate and enzyme on the antioxidant activity of agaro-oligosaccharides degraded from agarose [103]. Using the assays of DPPH, ABTS, and FRAP, considerable antioxidant activities of agaro-oligosaccharides that depended on the degree of hydrolysis [103] were observed. Gamma irradiation also produced a low molecular weight laminarin through a random scission of chains, formatting more carbonyl groups and enhancing the antioxidation [59]. However, Rafiquzzaman et al. compared the antioxidant ability of carrageenans from Hypnea musciformis separately extracted by the conventional method and ultrasonic-assisted extraction (UAE). No significant difference was observed between the two methods [130]. Molecular Mechanism of Polysaccharide-Induced Antioxidant Ability A sequence of signaling cascades are available to eukaryotes to protect them from various harms and to maintain cellular redox homeostasis [131]. Besides immediately removing the generated ROS, antioxidants enhance the endogenous antioxidant system by upregulating the expression of genes encoding antioxidant enzymes and proteins to reduce the generation of noxious substances [2,10] ( Table 4 and Figure 1). They also play roles in repairing the damage caused by ROS [132] which, when induced by oxidative stress, inhibits cell proliferation and causes apoptosis that is also regulated by a complex network of signaling pathways [133]. Antioxidants protect against oxidative stress damage induced by the regulation of apoptotic-related signaling pathways [134] (Table 4 and Figure 2). ASK1: apoptosis signal-regulating kinase 1; Bax: Bcl-2 associated X protein; Bcl-2: B-cell lymphoma 2; ERK:extracellular regulated protein kinases; GSK-3β: Glycogen synthase kinase3β; HO-1: hemeoxygenase-1; JNK: jun N-terminal kinase; Keap1: Kelch ECH associating protein 1; Nrf2: nuclear factor erythroid 2-related factor 2; γ-GCS: Glutamylcysteine synthetase; The arrows upward represent increase and the downward represent decrease. Conclusions Polysaccharides are effective antioxidants both in vivo and in vitro. They are therapeutic for various diseases, such as diabetes, nephropathy, repressed immunity, Alzheimer's, and pulmonary disease. The effects are determined by many factors, including molecular weight, sulfate content, position of sulfate groups, and monosaccharide content and structure. However, the determinants are complex and need further study. The exogenous antioxidants, polysaccharides, have been demonstrated to promote endogenous antioxidants such as CAT, SOD and GPx via upregulation of the expression of genes encoding antioxidant enzymes and proteins. The Nrf2 pathway is crucial to the oxidative stress response. Seaweed polysaccharides effectively upregulate Nrf2 by increasing the levels of Nrf2 upstream molecules such as ERK and GSK-3β, thus promoting the production of corresponding downstream Phase II detoxifying enzymes and antioxidative proteins such as SOD-1, HO-1, and γ-GCS. Seaweed polysaccharides also protect against oxidative stress by regulating apoptotic-related signaling pathways, such as Caspase-9, Bax and JNKs, in order to repair the damage caused by oxidative stress. We hope this review might be conducive to boost their value as antioxidants and facilitate seaweed polysaccharides as therapy for various diseases, in order to utilize seaweed resources to the utmost. Endogenous Antioxidant System The activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-driven antioxidant response element (ARE) is a crucial pathway in the response to oxidative stress. It induces various cytoprotective phase II enzymes [131,135,136]. The regulation of Nrf2 dependent on Kelch ECH associating protein 1 (Keap1) is the most characterized mechanism of ARE activation [137]. Nrf2 is an inducible cap 'n' collar type of transcription factor, normally degraded by the promotion of its repressor, Keap1, through the ubiquitin proteasome pathway [9,136]. When exposed to oxidant stress, the Keap1-Cul3 complex normally linked to Nrf2 is dissociated because of the modifications of Keap1 [10]. Nrf2 bound to ARE accumulates, promoting the production of the corresponding downstream phase II detoxifying enzymes and antioxidative proteins (e.g., superoxide dismutase-1 (SOD-1), hemeoxygenase-1 (HO-1), Glutamylcysteine synthetase (γ-GCS)) [9,138,139]. Ryu and Chung investigated the molecular mechanisms of fucoidan in the protection of human keratinocytes (HaCaT cells) from mild oxidative stress, indicating that the expression levels of HO-1, SOD-1, and Nrf2 are positively time-dependent following fucoidan treatment, whereas Keap1 expression was the opposite [140]. A similar positive effect of fucoidan treatment was also observed in the translocation of elevated Nrf2 from the cytosol into the nucleus, through the nuclear localization of Nrf2 by immunocytochemistry [140]. Fucoidan was also reported to extend the lifespan of the Drosophila melanogaster under heat stress by enhancement of the endogenous antioxidant system of upregulation of Nrf2 [141]. Positive regulation of Nrf2 by alginate was also reported in the H 2 O 2 -induced NT2 neural cell line by an increase in the levels of HO-1 and γ-GCS from upregulation of the expression of Nrf2 [112]. Laminarin also regulated NRF2 signaling pathways, suppressed KEAP1, and promoted NQO1, GCLC, and HO1 [108]. Upstream molecules of the Nrf2 signaling pathway regulated by seaweed polysaccharides, such as extracellular regulated protein kinases (ERK) [140] and glycogen synthase kinase3β (GSK-3β) [142], have been reported. The ERK/Nrf2 pathway regulates cellular protection against oxidative stress in various types of cells [143,144]. It was reported that fucoidan increased the level of ERK by positively upregulating the expression of Nrf2 [140]. Fucoidan also activates the Nrf2 signaling pathway by increasing the levels of GSK-3β in lipopolysaccharide (LPS)-induced male BALB/c mice [145]. Apoptotic Pathway The Caspase and Bcl-2 families play important roles in apoptotic pathways [146]. Caspase-3 and Caspase-9, the former an effector and the latter an initiator, are crucial mediators of apoptosis [118,147]. Caspases are induced by an increase in intracellular ROS in response to oxidative damage [118]. Once Caspase-9 is activated, a cascade triggers the cleavage or activation of other downstream caspases such as caspase-3, causing cell death by apoptosis [132]. Fucoidans from Dictyota mertensii protect the pre-osteoblast-like cells (MC3T3-L1) from H 2 O 2 -induced apoptosis by decreasing intracellular ROS and depressing the activation of caspase-3 and caspase-9 [118]. Similar protections by polysaccharides, mainly fucoidans, depress the activation of caspase-3 and caspase-9 caused by inducers, such as FeSO 4 , CuSO 4 , ascorbate, and acetaminophen, and thereby ameliorate apoptosis [42,114,121,148]. Among ROS, H 2 O 2 is a well-known apoptosis-inducing factor, which acts by several pathways [149]. The expression of the Bcl-2 family is regulated by H 2 O 2 particularly by down-regulating anti-apoptotic Bcl-2 and increasing the expression of the pro-apoptotic Bcl-2-associated X protein (Bax). Bax plays a crucial role in mitochondria-dependent programed cell death. It induces necrotic cell death even without caspase activation in certain cases [150,151]. The ratio of Bax/Bcl has been demonstrated as a molecular control point in many apoptotic pathways and determines the progress of the cell death program [152]. Recent studies have shown that the decrease in activity of SOD, Gpx, and caspase-3, as well as the increased content of MDA and ratio of Bcl-2/Bax induced by Aβ-induced stress in Sprague-Dawley rats, were reversed by the fucoidan from Laminaria japonica [42]. Hong et al. also reported similar results in Sprague-Dawley rats, where fucoidan inhibited ROS accumulation and hepatic apoptosis was induced by acetaminophen with increasing activity of GSH, GPx, SOD, and expression of Bcl-2, as well as decreasing content of MDA and expression of Bax [114]. Polysaccharides from Sargassum fulvellum showed dose-dependent potential for scavenging intracellular ROS in 2,2-azobis (2-amidinopropane) hydrochloride (AAPH)-treated monkey kidney fibroblasts (Vero) cells. It inhibited apoptosis by downregulating Bax and caspase-3 and upregulating Bcl-xL and PARP [134]. Phosphorylation of c-Jun N-terminal protein kinases (JNKs) is another adverse consequence caused by oxidative stress, further aggravating oxidative stress in mitochondria, evoking dysfunction and DNA fragmentation, as well as cellular necrosis [113,153]. JNK activation upregulated the levels of Bax and promoted translocation to mitochondria in APAP hepatotoxicity [153]. Wang et al. showed that fucoidan markedly mitigated APPP-induced hepatotoxicity in the human normal hepatocyte HL-7702 cell line by alleviating mitochondria dysfunction, downregulation of a signal-regulating kinase 1(ASK1), followed by inhibition of JNK and Bax activation. Choi et al. also indicated that sulfated polysaccharide from Hizikia fusiformis effectively protected ethanol-induced cytotoxicity in IEC-6 cells by downregulation of JNK [119]. Conclusions Polysaccharides are effective antioxidants both in vivo and in vitro. They are therapeutic for various diseases, such as diabetes, nephropathy, repressed immunity, Alzheimer's, and pulmonary disease. The effects are determined by many factors, including molecular weight, sulfate content, position of sulfate groups, and monosaccharide content and structure. However, the determinants are complex and need further study. The exogenous antioxidants, polysaccharides, have been demonstrated to promote endogenous antioxidants such as CAT, SOD and GPx via upregulation of the expression of genes encoding antioxidant enzymes and proteins. The Nrf2 pathway is crucial to the oxidative stress response. Seaweed polysaccharides effectively upregulate Nrf2 by increasing the levels of Nrf2 upstream molecules such as ERK and GSK-3β, thus promoting the production of corresponding downstream Phase II detoxifying enzymes and antioxidative proteins such as SOD-1, HO-1, and γ-GCS. Seaweed polysaccharides also protect against oxidative stress by regulating apoptotic-related signaling pathways, such as Caspase-9, Bax and JNKs, in order to repair the damage caused by oxidative stress. We hope this review might be conducive to boost their value as antioxidants and facilitate seaweed polysaccharides as therapy for various diseases, in order to utilize seaweed resources to the utmost.	v3-fos-license
2020-03-18T13:04:37.768Z	2020-03-16T00:00:00.000	212739873	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/mbo3.1029", "pdf_hash": "4600c7425413616f55d754a62ead8443835574f6", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2350", "s2fieldsofstudy": [ "Chemistry", "Biology" ], "sha1": "48a7ea3a379a7d4a0c65ef0d058003ae1561fc3a", "year": 2020 }	pes2o/s2orc	A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation Abstract [NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator. and iron ions in the active site are coordinated to the protein backbone via four cysteine-stemming thiolates. Two cysteines ligate both metals and therefore serve as bridging ligands. Furthermore, two cyanides (CN) and one carbon monoxide (CO) belong to the ligand sphere of the iron and keep the metal in a low-spin Fe 2+ state. Maturation and insertion of the NiFe(CN) 2 (CO) cofactor into the apo-form of the large subunit require a sophisticated maturation machinery that consists of at least six auxiliary Hyp proteins (Böck, King, Blokesch, & Posewitz, 2006;Lacasse & Zamble, 2016). First, the Fe(CN) 2 (CO) moiety is assembled with the aid of the HypE and HypF proteins, which synthesize the cyanide ligands out of carbamoyl phosphate (Blokesch et al., 2004;Reissmann et al., 2003). The metabolic origin of CO under anaerobic conditions remains, however, unclear (Bürstel et al., 2011;Nutschan, Golbik, & Sawers, 2019), while under aerobic conditions, this diatomic ligand is derived from formyltetrahydrofolate (Bürstel et al., 2016;Schulz et al., 2020). Assembly takes place on a scaffold complex, consisting of the HypC and HypD proteins, from which the Fe(CN) 2 (CO) moiety is transferred to the apo-large subunit (Bürstel et al., 2012;Stripp et al., 2013). Nickel is subsequently delivered to the active site with the help of the metallochaperones HypA and HypB. HypB was shown to feed HypA with nickel, which is compatible with a model in which HypA donates nickel to the large subunit (Lacasse, Summers, Khorasani-Motlagh, George, & Zamble, 2019;Watanabe et al., 2015). The assumption that HypA delivers the active site nickel was recently supported by observations made for the apo-large subunit of the cytoplasmic [NiFe]-hydrogenase from Thermococcus kodakarensis that has been crystallized in a complex with the HypA protein (Kwon et al., 2018). Interestingly, the interaction of HypA with the flexible N-terminus of the large subunit brought the chaperone in close vicinity of the still vacant active site. This was in fact a surprising result, because so far only the C-terminal extension of the large subunit stood in the focus of [NiFe]-hydrogenase maturation. The apo-large subunit is usually synthesized with a C-terminal extension comprising 3-68 amino acids (Greening et al., 2015), which is cleaved off by a specific endopeptidase once the complete NiFe site has been incorporated (Böck et al., 2006;Fritsch, Lenz, & Friedrich, 2013;Theodoratou, Huber, & Böck, 2005). Modifications of this C-terminal extension, including amino acid exchanges, truncation (Theodoratou, Paschos, Mintz-Weber, & Böck, 2000), or even complete removal (Massanz, Fernandez, & Friedrich, 1997;Senger, Stripp, & Soboh, 2017;Thomas, Muhr, & Sawers, 2015), by genetic engineering generally lead to the formation of inactive hydrogenase. Interestingly, while exchanges and truncations revealed a premature large subunit that was unable to form a complex with the small subunit, genetic removal of the entire extension allowed the formation of hydrogenase with canonical subunit composition. This phenomenon has been observed for the soluble, NAD + -reducing [NiFe]hydrogenase of R. eutropha (Massanz et al., 1997) and, more recently, for membrane-bound [NiFe]-hydrogenase 2 (Hyd-2) from Escherichia coli (Thomas et al., 2015). In both cases, however, the large subunit was at least devoid of nickel (Massanz et al., 1997). Nickel-free Hyd-2 was also shown to lack the CN and CO ligands of the Fe(CN) 2 (CO) moiety, which are easily detectable by infrared spectroscopy (Senger et al., 2017). These observations suggest an essential role of the C-terminus in active site maturation. However, it has to be stressed at this point that numerous [NiFe]-hydrogenases are naturally devoid of a C-terminal extension, yet they seem to employ the canonical maturation machinery to acquire a NiFe active site. Prominent members are the H 2sensing hydrogenases (belonging to groups 2b and 2d according to (Greening et al., 2015)), CO dehydrogenase-associated hydrogenases (group 4c), Ech-type hydrogenases (group 4e), and certain so far uncharacterized hydrogenases (group 4g) (Greening et al., 2015). The parallel occurrence of [NiFe]-hydrogenase large subunits with and without C-terminal extension leaves the importance of the C-terminal extension in the maturation of [NiFe]hydrogenases ambiguous. To obtain further information, we investigated the role in maturation of the C-terminal extension of the large subunit of the (Lenz, Lauterbach, Frielingsdorf, & Friedrich, 2015). Three of them, the soluble cytoplasmic NAD + -reducing [NiFe]-hydrogenase, the membrane-bound [NiFe]-hydrogenase (MBH), and the actinobacterial-like [NiFe]-hydrogenase, harbor large subunits whose apo-forms carry C-terminal extensions. The large subunit of the H 2 -sensing regulatory hydrogenase (RH), by contrast, is devoid of a C-terminal extension (Kleihues, Lenz, Bernhard, Buhrke, & Friedrich, 2000), although being equipped with a canonical NiFe(CN) 2 (CO) center (Bernhard et al., 2001), which is incorporated by the Hyp machinery of R. eutropha ). The basic hydrogenase module of the MBH of R. eutropha consists of the large subunit HoxG carrying the NiFe(CN) 2 (CO) cofactor and the small subunit HoxK comprising three iron-sulfur (Fe-S) clusters . Upon incorporation of the NiFe(CN) 2 (CO) cofactor into HoxG by the Hyp machinery, the specific endopeptidase HoxM cleaves off the C-terminal extension. Deletion of the gene encoding the MBH-specific endopeptidase HoxM results in the accumulation of a HoxG preform still carrying the C-terminal extension (Bernhard, Schwartz, Rietdorf, & Friedrich, 1996;Hartmann et al., 2018). In this study, we deleted the C-terminal extension of HoxG by genetic engineering and show that the resulting truncated version of HoxG still receives a NiFe(CN) 2 (CO) cofactor and forms, together with the corresponding HoxK subunit, catalytically active MBH. \| Genetic constructions All bacterial strains and plasmids used in theis study are listed in Table 1. The sequence encoding the C-terminal extension of hoxG (amino acids 604-618 of HoxG, ) was eliminated by PCR amplification using the primers SFP43 5′-AAGAATGTATACGTGCCAGACGTG-3′ and SFP44 5′-ACTAAGCTTTTAGTGAGTCGAACACGCCAGAC-3′ using pJH5415 as template. The PCR product was digested with AccI/HindIII and ligated into AccI/HindIII-cut pJH5415 yielding pSF8.14. pSF8.14 was digested with SpeI/XbaI, and the resulting 3595-bp fragment was ligated into XbaI-cut pEDY309 yielding pSF10.8. This plasmid was transformed into E. coli S17-1 (AK2429) for subsequent conjugative transfer to R. eutropha strain HF1063 yielding strain HP9. The wild-type control strain was generated by digesting pJH5415 with SpeI/XbaI and transfer of the resulting fragment into pEDY309, yielding pJH5437. Plasmid pJH5437 was transferred by conjugation into strain HF1063, yielding strain HP3. \| Media composition and cell cultivation Ralstonia eutropha strains HF649, HF1063, HP3, and HP9 were cultivated in FGN mod medium as described elsewhere (Hartmann et al., 2018;Lenz et al., 2018). Cells were harvested by centrifugation (11,500 g, 4°C, 12 min), and the resulting cell pellet was frozen in liquid nitrogen and stored at − 80°C. Lithoautotrophic cultivation in liquid and on agar-solidified minimal medium devoid of organic carbon sources was carried out as described previously (Lenz et al., 2018) with the exception that a gas atmosphere of 10% (v/v) CO 2 , were inoculated with a preculture to an initial OD 436 nm of 0.1 and shaken at 120 rpm and 30°C for 16 days. Agar plates were incubated for 6 days at 30°C. \| Protein extract preparation, polyacrylamide gel electrophoresis, and immunological analysis To analyze the MBH subunit content in different cellular protein fractions, cell pellets were resuspended in 3 ml (per gram wet weight) of resuspension buffer (50 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.3, 150 mM NaCl) containing cOmplete EDTA-free protease inhibitor mixture (Roche Diagnostics) and DNase I (Roche Diagnostics). The resuspended cells were disrupted in a chilled French pressure cell at 124.11 MPa. This procedure resulted in whole-cell lysate ("lysate" sample). Unbroken cells and cell debris were subsequently sedimented by centrifugation (4,000 g, 4°C, 20 min), yielding an emulsion composed of membranes and soluble proteins. The emulsion was ultracentrifuged (100,000 g, 4°C, 1 hr), yielding a dark-brown membrane pellet and a brownish liquid supernatant ("soluble extract" sample). The membrane pellet was washed by homogenization with a Potter-Elvehjem homogenizer in 10 ml of resuspension buffer (per gram, wet weight, of the membrane) containing protease inhibitor cocktail. The suspension was then ultracentrifuged (100,000 g, 4°C, 35 min), yielding clean membranes as a pellet. Its resuspension in resuspension buffer yielded the sample "membrane". Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) was used for protein separation, which was followed by Western blot analysis (Towbin, Staehelin, & Gordon, 1979). Proteins in gels were either stained with Coomassie brilliant blue G-250 (Diezel, Kopperschläger, & Hofmann, 1972) or transferred to a nitrocellulose membrane (BioTrace, Pall Corp.) using a fast semidry transfer buffer. (Garić et al., 2013) Polyclonal antibodies raised against the MBH large subunit (anti-HoxG, (Bernhard et al., 1996)) in combination with a goat anti-rabbit secondary antibody (coupled with alkaline phosphatase, Dianova) were used for detection of HoxG. Protein concentrations were determined with the Pierce BCA Protein Assay Kit (Thermo Scientific), using bovine serum albumin as standard. \| MBH purification Purification of the MBH Strep variants derived from R. eutropha HP3, HP9, and HF649 was carried out as described previously (Goris et al., 2011;Lenz et al., 2018). The protein samples were frozen in liquid nitrogen and stored at − 80°C. \| H 2 oxidation activity assay H 2 -mediated reduction of methylene blue was determined spectrophotometrically as previously described (Lenz et al., 2018) using a Cary50 UV-Vis spectrophotometer (Varian, Agilent). The specific activity was given in Units (U) per mg of protein, where 1 U corresponds to the turnover of 1 µmol H 2 per minute. \| RE SULTS Previous studies revealed that the genetic removal of the C-terminal extension of the large subunit results in hydrogenase devoid of a functional NiFe active site (Massanz et al., 1997;Thomas et al., 2015). To test whether the absence of the C-terminal extension of the membrane-bound hydrogenase (MBH) of R. eutropha also leads to inactive protein, we deleted the sequence encoding the amino acid residues Val604-Arg618 of the HoxG subunit. The deletion resulted in a preprocessed HoxG subunit, henceforth designated as HoxG proc , terminating with residue His603 that also represents the very last residue upon cleavage of the native subunit with the protease HoxM . The corresponding hoxGproc gene was cloned together with hoxK Strep , which encodes the According to the current model, the C-terminus of the large subunit is cleaved off only if nickel had been inserted properly into the active site (Böck et al., 2006). In 2015, Sawers and coworkers have challenged this model. They observed that the genetically processed large subunit of E. coli Hyd2 lacks the native NiFe cofactor but forms a complex with the small subunit. The resulting inactive Hyd2 was even accepted by the Tat translocation apparatus and appropriately inserted into the cytoplasmic membrane (Thomas et al., 2015). Therefore, we analyzed the catalytic activity and the cofactor content of the MBH proc purified from the membrane fraction of R. eutropha HP9 and compared the results with those of R. eutropha HP3, synthesizing native MBH. The MBH yield from R. eutropha HP9 and HP3 was (115 ± 28) µg and (208 ± 11) µg, respectively, of protein per gram of cells (wet weight). Thus, membranes of strain HP9 had a ~45% lower MBH content than strain HP3, which is in line with the Western blot results (Figure 2b and c). Both MBH versions showed, however, identical specific activities for H 2 -mediated methylene blue reduction, with (87.8 ± 3.4) U/mg for native MBH and (88.5 ± 4.7) U/mg for MBH proc . Thus, despite the genetic removal of the C-terminal extension, the NiFe active site of MBH proc seemed to be correctly assembled. To investigate the integrity of the active site further, we performed infrared (IR) spectroscopy, which probes the C≡O and C≡N stretching vibrations associated with the CO and CNligands of the NiFe site. These vibrations are very sensitive to structural and redox modifications of the active site (Bagley, Duin, Roseboom, Albracht, & Woodruff, 1995). The resulting IR spectra of as-isolated, oxidized native MBH and MBH proc are shown in Figure 3. To obtain quantitative information on the loading of the proteins with the NiFe cofactor, the spectra were normalized based on the intensity of the amide II band, which is proportional to the protein concentration. Both spectra were almost identical to that of aerobically purified MBH (Goris et al., 2011) \| D ISCUSS I ON The MBH proc of R. eutropha is the first example of a [NiFe]hydrogenase that is equipped with a NiFe catalytic center, although the C-terminal extension of the large subunit was genetically removed. In fact, the purified MBH proc protein was indistinguishable from native MBH with respect to the active site architecture and catalytic activity. Thus, the C-terminal extension of the large subunit is not essential for Hyp protein-mediated insertion of the NiFe cofactor. Nevertheless, removal of the C-terminal extension led to significantly lowered MBH levels in the membrane. Thus, our results indicate that the C-terminal extension optimizes maturation efficiency. Notably, there was no indication for apo-MBH, that is, MBH without NiFe cofactor, in our membrane-derived protein preparation. This is in clear contrast to previous reports for E. coli Hyd-2, where catalytically inactive hydrogenase complexes were identified that contained genetically processed, but NiFe cofactor-free large subunits (Massanz et al., 1997;Senger et al., 2017;Thomas et al., 2015). It has been convincingly shown that the Fe-S cluster-containing hydrogenase small subunit, which is equipped with the Tat leader peptide, becomes transported through the cytoplasmic membrane only in complex with the large subunit (Rodrigue, Chanal, Beck, Müller, & Wu, 1999;Schubert et al., 2007). The results by Thomas et al. suggest that the mere attachment of the large subunit to the small subunit elicits the signal to initialize the Tat cofactor-free hydrogenase complexes may be subjected to proteolysis. The latter mechanism is rather unlikely, because the genetically processed large subunit of the soluble, NAD + -reducing [NiFe]-hydrogenase, SH, of R. eutropha forms a complex with the remaining hydrogenase subunit, although no nickel had been inserted into the active site (Massanz et al., 1997). Thus, the small subunits themselves might reject processed premature large subunits lacking a complete NiFe cofactor, and this capability seems not to be uniformly distributed among all hydrogenases. While the SH small subunit obviously cannot distinguish between processed mature and immature large subunits (Massanz et al., 1997), those of the MBH and the regulatory [NiFe]-hydrogenase (RH) obviously can. In fact, the RH belongs to the subclass of hydrogenases whose apo-large subunits natively lack the C-terminal extension, but are recognized by the canonical Hyp machinery that inserts the canonical NiFe cofactor . Purified RH protein consisted of the iron-sulfur cluster-containing small subunit and large subunit that was stoichiometrically loaded with nickel (Buhrke et al., 2005), indicating an intrinsic proofreading mechanism that prevents the complex formation of premature subunits. The same seems to be true for MBH proc . The fact that both HoxG proc and the RH large subunit properly receive the NiFe ( The protein backbones of the large subunits are depicted in blue with red N-termini and magenta C-termini (cartoon representation). The structural models with the PDB codes 4IUC (mature HoxG) (Frielingsdorf et al., 2014) and 5YXY (preform of apo-HyhL (Kwon et al., 2018)) were used. (a) The active site, including the coordinating cysteines and the C-terminal histidine of mature HoxG, are shown as stick models. The C-terminal extension is not visible, as it has been cleaved off. Note that the N-terminus (red) adapts to a β-sheet domain of the main protein, and one of the N-terminal β-strands is located at the position, which is occupied by a β-strand structure of the C-terminal domain in apo-HyhL (b). (b) The C-terminal extension (C ext) of apo-HyhL, which is cleaved off upon NiFe cofactor insertion, is shown in mint. The red N-terminus (the region between the two ends of the red line is structurally unresolved and depicted as a broken line) protrudes from the globular protein and forms a complex with HypA (gray). The four conserved cysteines that coordinate the NiFe(CN) 2 (CO) cofactor are represented as sticks. The terminal histidine residue (shown as sticks), which lies directly in front of the cleavage site of the C-terminal extension, was not resolved and therefore was modelled computationally into the structure using PyMol ( HyhL occupies parts of the position of the N-terminus in the mature structure, forcing the N-terminus in another direction (Kwon et al., 2018). As a consequence, the N-terminus acts like a "crane arm," which brings the HypA protein close to the active site cavity where it can deliver the nickel ion ( Figure 4). Nickel incorporation presumably leads to a dramatic conformational change in the C-terminal extension that upon cleavage moves close to the active site cavity, which unblocks the dedicated position of the N-terminus of the mature protein (Kwon et al., 2018). Therefore, a major role of the C-terminal extension might be providing indirectly the N-terminus with sufficient flexibility to interact with the Hyp machinery. A role of the C-terminal extension in enhancing structural flexibility to facilitate the interaction with Hyp proteins has also been proposed (Albareda, Pacios, & Palacios, 2019;Pinske et al., 2019). A function as a maturation facilitator would also explain why the removal of the C-terminal extension does not necessarily lead to immature hydrogenase. In case of R. eutropha MBH proc , the assembly process seemed to be just less efficient, resulting in a reduced amount of fully active MBH in the cytoplasmic membrane. It should be mentioned, however, that the genetic removal of the extension might result in reduced stability of the large subunit before it becomes equipped with NiFe(CN) 2 (CO) cofactor and oligomerizes with the small subunit. To clarify the overall necessity of the C-terminal extension, more large subunits need to be tested for their capacity to tolerate the absence of the C-terminal extension in the course of NiFe cofactor insertion. Ingo Zebger for providing access to the IR spectrometer. CO N FLI C T O F I NTE R E S T S None declared. E TH I C S S TATEM ENT None required. DATA AVA I L A B I L I T Y S TAT E M E N T All data are provided in full in the results section of this paper.	v3-fos-license
2018-06-07T13:19:41.702Z	2018-08-01T00:00:00.000	44138509	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://doi.org/10.1016/j.ejpb.2018.05.031", "pdf_hash": "5663da6afcecc197eb20485e4c76a8baa7adb430", "pdf_src": "MergedPDFExtraction", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2364", "s2fieldsofstudy": [ "Biology", "Materials Science" ], "sha1": "9d509ef62645dec12290820e65926999f3241eed", "year": 2018 }	pes2o/s2orc	Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery Graphical abstract Figure. No caption available. &NA; Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)‐based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co‐encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field‐flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 &mgr;g OVA, in NPs or as free antigen, by using near‐infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN‐delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP‐loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP‐loaded dissolving MNs. Introduction Skin is an attractive site for vaccination because of its rich network of immune cells and its accessibility [1]. However, the top layer of the skin, the stratum corneum, effectively prevents the penetration of vaccines into the skin. Conventional needles can be used for intradermal immunizations but this method is painful and technically challenging. Therefore, vaccination via the skin requires the development of a device that enables pain-free administration of vaccines into the epidermis and dermis, which both harbour a dense network of immune cells (e.g. Langerhans cells and dermal dendritic cells) [2]. Among several developed devices for intradermal vaccine administration, microneedles (MN) are currently one of the most promising technologies. They are needle-like structures with micron-scale lengths (< 1000 µm) typically assembled as arrays with the ability to pierce into the skin in a minimally invasive and pain-free manner [3,4]. In modern vaccine programmes, subunit antigens (e.g., proteins) are preferred since they can offer safer and better controlled alternatives to whole vaccines [5,6]. However, frequently the immunogenicity of plain subunit antigens is limited. Therefore, subunit vaccines typically contain adjuvants, such as an optimally designed delivery system and an immune modulator. Several types of nanocarriers have been examined for vaccine delivery and of those polymeric nanoparticles (NP) and liposomes are most commonly used [7][8][9]. Particle-based vaccines can improve the immune responses against the antigen due to their resemblance to pathogens, prolonged release of antigen and possibility to tailor particle properties depending on the required type of immune response [5,8,10]. In addition, particulate formulations allow the codelivery of antigen and adjuvant and enhanced uptake in DCs, which can further enhance the immunogenicity of the antigen against infectious diseases [11][12][13] or cancer [14][15][16]. However, the skin is still rarely used as non-invasive administration route for particulate vaccines. The major challenges are reproducible delivery of a sufficient dose of particulate vaccines into the skin and simultaneous the minimization of the fabrication wastage of the vaccine. Arrays of dissolving [17,18] and coated MNs [19] are dry-state MN technologies that have been used to deliver NP vaccines into the skin. In dissolving MNs, nanoparticles are localized in the MN matrix, and after the MN array is inserted into the skin, the MNs dissolve, thereby releasing the NPs. Beside dry-state MNs, single hollow MN can be used to inject NP suspensions into the skin without time-consuming development of dissolving or coated MN formulations. Therefore, it is an ideal tool to compare vaccine formulations for intradermal delivery [20]. The advantages of dissolving MNs are that the use of dry vaccine formulations can potentially increase the stability and eliminate the need for a cold chain, in contrast to hollow MNs which require a liquid formulation [21,22]. In addition, dissolving MNs are prepared from safe and biodegradable materials and do not create sharp waste in contrast to coated MNs [4]. Until now, a limited number of studies have been reported on NP-based intradermal vaccination using dissolving MNs [17,18]. Particularly, the formulation development of NP-loaded dissolving MNs has not been described in detail in the literature. Moreover, studies on the effect of the MN composition and the NP incorporation on the preparation and MN dissolution have not been reported yet. Finally, the quantification of the intradermally delivered antigen dose are only reported for coated MNs [23]. Challenging factors for developing dissolving MNs include the need for (1) reproducible MN penetration into the skin, (2) dissolution and subsequent release of NPs from the MNs into the skin, (3) delivery of a sufficient and reproducible antigen dose and (4) minimizing the loss of often expensive antigen and adjuvant. The aim of this study was to develop NP-loaded dissolving MNs to evoke humoral and cellular immune responses after intradermal vaccination. Poly(lactic-co-glycolic) acid (PLGA) NPs co-encapsulating model antigen ovalbumin (OVA) and TLR3 ligand poly(I:C) as adjuvant were selected. Dissolving MNs were prepared from hyaluronan (HA) and contained PLGA NPs that were loaded exclusively into the MN tips. Next, the most suitable formulation composition (i.e., the weight ratio of HA and NPs) and preparation conditions for MN dissolution was evaluated by using ex vivo human skin. Using the optimized MN composition, we quantified the delivered dose in mice. Finally, dissolving and hollow MN-mediated intradermal immunization with PLGA NPs were compared for the ability to evoke humoral and cellular immune responses in mice. To determine OVA and poly(I:C) loading of the NPs, 1 mg dried nanoparticles were dispersed in 0.15 ml DMSO and incubated for 1 h at 37°C. Next, 0.85 ml (determination of OVA) or 0.35 ml (determination of poly(I:C)) of 0.05 M NaOH with 0.5% (w/v) SDS was added to samples which were thereafter incubated for an additional 1 h at 37°C. The calibration curves were prepared in mixtures of DMSO and NaOH/ SDS. Micro BCA™ protein assay (Pierce Micro BCA™ protein Assay kit, ThermoFisher, Rockford, IL, USA) was used to measure the OVA concentration following the manufacturer's instructions and absorbance was determined with a Tecan infinite M1000 plate reader (Tecan Austria GmbH, Grödig, Austria). To analyse of poly(I:C) concentration, fluorescence of rhodamine-labelled poly(I:C) was measured by using the plate reader (λ ex 545 nm/λ em 576 nm). The encapsulation efficiency (EE) and loading capacity (LC) of OVA and poly(I:C) in the nanoparticles were calculated as below: where M loaded OVA/poly(I:C) represents the mass of loaded OVA or poly (I:C), M total OVA/poly(I:C) is the total amount of OVA or poly(I:C) added to the formulations and M NP+OVA+poly(I:C) is the total weight of NPs, OVA and poly(I:C). Dissolving microneedle preparation The preparation scheme of the dissolving PLGA NP-loaded MNs is presented in Fig. 1. PDMS molds were prepared from silicon MN arrays (16 MN tips in 5.4 × 5.4 mm array, length 300 µm) as described earlier [24]. First, HA was dissolved overnight in 10 mM phosphate buffer (5.8 mM Na 2 HPO 4 , 4.2 mM NaH 2 PO 4 , pH 7.0). On the next day, NPs were suspended in buffer and HA solution and NP suspension were mixed so that the final concentrations of NPs and HA were 10 mg/ml and 10 mg/ml (NP:HA weight ratio 1:1), 5 and 20 mg/ml (NP:HA weight ratio 1:4) and 5 mg/ml and 50 mg/ml (NP:HA weight ratio 1:10), respectively. When MNs were visualized with fluorescence microscopy 1% of total HA was replaced with FAM-HA that was prepared as described earlier [24]. Next, 30 µl NP:HA mixture was pipetted into each array of the PDMS mold that was placed in the vacuum for 10 min to remove any entrapped air from the mixture (Fig. 1). To deposit the nanoparticles into the MN tips, the mold was placed into a centrifuge (Beckman Coulter Allegra X-12R Indianapolis, IN, USA) for 15 min at 3270g. After the centrifugation, the excess mixture (20-25 µl) was removed from the mold, and the remaining mixture was dried for 30 min in a vacuum desiccator at room temperature to evaporate the water. First four steps were repeated once by adding again NP:HA mixture (20 µl) into each array of PDMS, followed by centrifugation, removal of the excess mixture and drying. Subsequently, 60 µl of 50 mg/mL HA solution was added to form an additional HA layer, followed by centrifugation (30 min, 930g). Finally, the PDMS mold was dried overnight in a vacuum desiccator at room temperature. After the complete drying, a vaccine-free backplate was prepared for all MNs. A mixture of vinylpolysiloxane base and catalyst was prepared and it was added to each array to form the backplate [25]. MNs were stored at room temperature in a desiccator until use. For in vivo studies, MNs containing only free OVA and poly(I:C) (free-OVA-PIC) were prepared, as follows: 60 µl solution containing HA (40 mg/ml), OVA (10 mg/ml) and poly(I:C) (10 mg/ml) was added to each array of the PDMS mold. This was placed in vacuum to remove any air and then centrifuged for 2 h at 3270g. After the centrifugation, another 60 µl of HA/OVA/poly(I:C) solution was added in arrays of the mold followed by the repetition of the centrifugation step. MNs containing 50% of the OVA and poly(I:C) dose encapsulated in NPs and 50% in free form (free-OVA-PIC/NP mixture) were prepared similarly to NP-loaded MNs but with some modifications. Free OVA (5 mg/ml) and poly(I:C) (5 mg/ml) were mixed to form the mixture for preparation of MN tips with HA (20 mg/ml) and subsequently NPs (2.5 mg/ml) were added to this mixture. The HA solution for backplate contained OVA (5 mg/ml) and poly(I:C) (5 mg/ml). Subsequently, the samples were dried overnight in a vacuum desiccator at room temperature. Finally, the vinylpolysiloxane backplate was prepared as described above. Microscopic analysis of microneedles The shape of MNs after the preparation and after the immunisation experiments were analysed by light microscopy (Zeiss, Sterni 2000 C, Carl Zeiss Microscopy GmbH, Göttingen, Germany). Scanning electron microscopy (SEM, NOVA nanoSEM, Eindhoven, the Netherlands) was used to examine additionally the shape, and external and internal surface morphology of the MNs. The arrays were coated with a platina/ palladium layer to increase the surface conductivity. Images were taken at magnifications of 31-15,000× by using a voltage of 15 kV between the electron gun and the sample surface. The distribution of the NPs containing OVA-AF647 in MNs with FAM-HA were analysed with fluorescence microscopy (Zeiss Imager D2, camera AxioCam MRm, light source HXP120V, Carl Zeiss Microscopy GmbH, Göttingen, Germany) before and after dissolution in the ex vivo human skin. ZEN Pro 2012 software (Carl Zeiss Microscopy GmbH) was used for imaging and image analysis in light and fluorescence microscopy. Reconstitution of PLGA nanoparticles after microneedle dissolution To examine whether the various steps of MN preparation influence the release of the NPs from the MNs upon the MN dissolution, asymmetrical flow field-flow fractionation (AF4) was utilized. The following samples were prepared: (1) NP suspension, (2) NP suspension with HA, (3) NP suspension with HA dried at +37°C in ambient pressure, (4) NP suspension with HA dried at room temperature in vacuum and (5) NPloaded MNs without silicone backplate. In all samples the NP:HA weight ratio was 1:4 and 10% of total HA was replaced with FAM-HA. After drying samples 3 and 4, and 5 on the flat PDMS surface or in PDMS mold, respectively, they were dispersed and dissolved in PBS (pH 7.4) overnight at +37°C. Before the analysis, all samples were diluted to a concentration of 1 mg/ml NPs and 4 mg/ml HA in PBS. The AF4 measurements were performed on an Agilent 1200 system (Agilent Technologies, Palo Alto, CA, USA) combined with Wyatt Eclipse detectors (Wyatt Technology Europe GmbH, Dernbach, German). For the separation of NPs and HA, a small channel equipped with a 350 µm spacer of medium width and a regenerated cellulose membrane with a cut-off of 5 kDa were used. Multiangle laser light scattering (MALLS) was used for the detection of the NPs and the fluorescence signal (λ ex 496 nm, λ em 520 nm) for the FAM-HA. The mobile phase was 200 mM NaNO 3 with 0.02% NaN 3 , filtered through a 0.1 µm filter. The injection volume was 20 µl per sample and the detector flow was 1 ml/min. The cross-flow was initially set to 0.3 ml/min which was gradually decreased to 0.1 (from 9 to 12 min), and set at 0.1 ml/min until 32 min thereafter to 0 ml/min as indicated in Fig. 4A. The fractions were collected at 16-21 min and the particle size of the NPs of the fractions were measured with DLS as described above. In vitro quantification of PLGA NPs and free OVA content of dissolving MNs For this experiment, MNs were prepared as described above except that NP-loaded MNs contained NPs encapsulating OVA-IR800 and for OVA MNs 5% of the total OVA was replaced with OVA-AF647. Each array (n = 9 and n = 6 for NP and OVA-loaded MNs, respectively) was dispersed in 1 ml of PBS (pH 7.4) for 2 h and the NP and OVA content was determined with a Tecan plate reader based on the fluorescence of OVA-IR800 (λ ex 779 nm, λ em 794 nm) and OVA-AF647 (λ ex 650 nm, λ em 668 nm), respectively. Dissolving MN penetration and dissolution in ex vivo human skin Ex vivo human skin was used to analyse the penetration and dissolution of dissolving MN in the skin. Within 24 h after the skin was obtained from the hospital, excess fat was removed and the skin was frozen and stored at −80°C until use. Upon use, the skin was thawed and fixed on the Styrofoam. The MNs were applied onto the skin by using an in-house developed impact-insertion applicator [26] by attaching MN array to the applicator head with double-sided tape before application to the skin. In the case of skin piercing studies, after inserting MNs into the skin for 15 s, 50 µl of 0.4% (w/v) trypan blue solution was applied onto the skin surface for 30 min [24]. After the removal of trypan blue solution, the skin was cleaned with MQ water to avoid visualization of trypan blue at the skin surface, and finally the stratum corneum was removed by tape stripping (∼10 strips) by using normal office tape. The skin piercing indicated by blue spots was visualised with a light microscope (Zeiss, Sterni 2000 C) and the penetration efficiency was calculated diving the number of the successful piercings with the total number of tips (i.e. 16) in each MN array. For dissolution studies, MNs were applied onto the skin for 20 min as described for penetration studies. After withdrawal, the MNs were analysed with a fluorescence microscope. To examine the deposition of NPs and HA into the skin, this was analysed after MN dissolution with confocal laser scanning microscopy as described previously [24]. Briefly, the experiment was carried out with a Nikon T-200-e inverted microscope supplied with a Nikon C1 confocal unit. For acquisition and analysis of scans, a Nikon Plan Apo 4 × (numerical aperture 4) objective and Nikon NIS Elements version 4.20.00 64-bit software were used. The xy resolution was 6.3 µm/pixel and xy scans were acquired every 10 µm, and rhodamine-labelled trimethyl chitosan (TMC) [27] was used to determine the skin surface during the analysis. NPs (i.e. AF647-OVA in NPs) and FAM-HA were visualised with a 637 nm (diode laser, intensity 75 and gain 100) and a 488 nm (Ar laser, intensity 75 and gain 100) laser, respectively. Animals Balb/c mice (haplotype H2 d , female, age 7-8 weeks) and C57BL/6 mice (CD45.2 + , H2 b , age 8 weeks) were purchased from Charles Rivers (Maastricht, The Netherlands). They were housed under standardised conditions in the animal facility of Leiden Academic Centre for Drug Research of Leiden University. Experiments were approved by the ethical committee on animal experiments of Leiden University (licence number 14241). Quantification of the delivered dose by dissolving microneedles in vivo Dissolving MNs containing NPs encapsulating IR800-OVA or free IR800-OVA were used to quantify amount of delivered OVA-NPs and OVA in the skin, respectively. First, balb/c mice were anaesthetised by intraperitoneal injection of ketamine (60 mg/kg) and xylanize (4 mg/ kg) and the insertion site was shaved. Next, MNs were applied on by using an impact-insertion applicator for 20 min, as described above (Section 2.8). The calibration standards were injected with a hollow MN applicator as described below (Section 2.11) in shaved ex vivo mouse skin by using either free IR800-OVA (63-1000 ng protein/injection) or IR800-OVA-NPs (0.625-20 µg NPs/injection). The variation of injected amount of OVA or NPs was achieved by varying the injection volume (0.31-10 µl). Mice were sacrificed before measuring the fluorescence intensity by using a Perkin-Elmer IVIS Lumina Series III in vivo imaging system (Waltham, MA, USA) with an excitation wavelength of 745 nm and an emission filter set at ICG. Other parameters were set as follows: acquisition time 8 s, F-stop 2, binning 4 and field of view of 12.5 cm. Perkin-Elmer Living Image software version 4.3.1.0 was used for image acquisition and analysis. Untreated sites of the skin were used to determine the background signal. Immunisation study for antibody responses Study groups (n = 8/group) were immunised either by intradermal injection with hollow MNs or 20 min insertion of dissolving MNs on the flank of the mice. Hollow MNs were fabricated by hydrofluoric acid etching from fused silica capillaries and injections were performed with an in-house developed applicator as described earlier [20,28,29]. The bore diameter of the hollow MNs was 50 µm, the injection depth 200 µm, the injection rate 12 µl/min and the injection volume 12 µl. Before the immunisation, mice were anaesthetised by intraperitoneal injection of ketamine (60 mg/kg) and xylanize (4 mg/kg) that was followed by the shaving of the immunisation site. In all study groups, the total dose of both OVA and poly(I:C) doses were ∼1 µg. Three different formulations were used and they were administered with both hollow and dissolving MNs: 1) free-OVA-PIC, 2) mixture of free-OVA-PIC (50% of dose) and OVA-PIC-NPs (50% of dose), and 3) OVA-PIC-NPs. The buffer with low ion concentration (10 mM phosphate buffer, pH 7.0) was used to dissolve OVA and poly(I:C), and to suspend NPs to prevent a premature release from NPs. Mice were immunised on the day 0 (prime), day 21 (1st boost) and day 42 (2nd boost), and before each immunisation venous blood sample was collected from the tail to measure the antibody responses. Mice were sacrificed on day 51 by first collecting a blood sample from abdominal/thoracic vein followed by cervical dislocation. ELISA analysis of OVA-specific IgG antibodies OVA-specific antibodies were analysed by a sandwich enzymelinked immunosorbent assay (ELISA) as described earlier [20]. The plates (Nunc-Immuno MaxiSorp 96, ThermoScientific, Roskilde, Denmark) were coated with 500 ng of OVA for 1.5 h at 37°C in 0.05 M carbonate/bicarbonate buffer (pH 9.6), followed by the blocking step with 1% (w/v) BSA in PBS (1 h at 37°C). Next, diluted sera of each mouse was further serially diluted three-fold with dilution buffer (PBS with 0.5% BSA and 0.05% Tween 80) and incubated for 1.5 h at 37°C. Subsequently, 1:5000 diluted horseradish peroxidase-conjugated goat antibodies against IgG, IgG1, IgG2a were added to the plates to be incubated for 1.5 h at 37°C. Finally, antibodies were detected by adding TMB and after 15 min, the reaction was stopped with 2 M H 2 SO 4 . The absorbance was measured at 450 nm and the antibody titre was determined as a log10 value of the mid-point dilution of S-shaped dilution-absorbance curve of the diluted serum level. Immunisation study for transgenic CD4 + and CD8 + T-cell responses One day before the immunisation, CD8 + T cells (OT-I, CD45.1 + ) and CD4 + T cells (OT-II, CD45.1 + ) were obtained from transgenic mice as described recently [20]. An equivalent 20 000 OT-I and 200 000 OT-II cells were adoptively transferred in the C57BL/6 mice (CD45.2 + , H2 b ) by i.v. injection in 200 µl PBS. On the following day, mice (n = 4/ group) were immunised with OVA-PIC-NPs (OVA and poly(I:C) doses ∼ 4 µg) either delivered by using four dissolving MN arrays per mouse or hollow MNs as described above. PBS was injected by hollow MNs for the negative control group (n = 3). At day 7, mice were sacrificed by cervical dislocation and spleens were collected to isolate splenocytes for flow cytometry analysis. Flow cytometry analysis of CD8 + and CD4 + T-cells Splenocytes were isolated from the collected spleens by using 70 µm Nylon cell strainers (Falcon, Corning, Corning, NY, USA) followed by 30 s lysis of erythrocytes by using Ammonium-Chloride-Potassium (ACK) lysis buffer. Thereafter, the splenocytes were washed with PBS and cell suspension (∼500 000 cells per well) was added to the 96-well plate. After the washing of the cells, the cell surfaces were stained with fluorescently labelled antibodies against CD45.1 (efluor450, 1:800 dilution), CD4 (APC, 1:800), CD8α (PE, 1:800) and Thy1.2 (PE-Cy7, 1:400) in 100 µl of FACS buffer. After 30 min incubation at +4°C, the excess antibodies were washed by FACS buffer before the incubation with Cytofix solution (BD Bioscience) for 10 min at 4°C. Before the flow cytometry analysis (FACS CantoII, BD Biosciences), the cells were washed once with FACS buffer. The data were analysed by using FlowJo software (version 10.0.7. TreeStar Inc). Statistical methods Statistical differences in immunisation studies were analysed by 1way ANOVA with Bonferroni's multiple comparison post-test (GraphPad Prism version 5.02). The data of DLS analysis of AF4 fractions was analysed by Kruskall-Wallis test followed by Dunn's multiple comparison test. The level of the significance was set at 0.05 in both cases. Development and characterisation of PLGA NP-loaded dissolving MNs PLGA NPs were loaded with OVA (OVA-NP) for MN development and characterisation studies, and with OVA and poly(I:C) (OVA-PIC-NP) for immunisation studies. The NPs had a size, determined by DLS and expressed as Z-average diameter, between 150 and 200 nm and their zeta potential was negative ( Table 1). The co-encapsulation of poly(I:C) did not significantly affect these values. The OVA loading capacity in OVA-NPs was 7.7% w/w and the encapsulation efficiency was 67%. When OVA and poly(I:C) were co-encapsulated, their loading capacities were 3.2% and 3.0% (w/w), respectively, and thus the weight ratio of their encapsulated doses was approximately 1:1. The difference in OVA loading capacity between OVA-NPs and OVA-PIC-NPs was probably due to adjustment of initial amount of OVA in order to obtain equal loading capacity of OVA and poly(I:C). The encapsulation efficiencies of OVA and poly(I:C) in OVA-PIC-NPs were 53% and 10%, respectively. HA-based dissolving MNs containing antigen and adjuvant loaded NPs were successfully developed. Preliminary studies indicated that sharp MNs with very high NP content could be produced without additional HA as matrix material in the MN tips (data not shown). However, these MNs did not have sufficient mechanical strength for reproducible skin penetrations. Consequently, it was decided to mix varying concentrations of HA solution with the NP suspension to study which compositions result in the formation of MNs with sufficient strength and sharpness for skin penetration. The examined weight ratios of NP:HA were 1:1, 1:4 and 1:10. During the preparation, the first four preparation steps was repeated once to increase the number of NPs deposited into the MN tips and consequently to increase the dose delivered into the skin (Fig. 1). All MNs (containing NPs, free-OVA-PIC/ NP mixture, or free-OVA-PIC) had equal dimensions (length 300 µm) and sharpness (Fig. 2). To minimize wastage of vaccine and increase the delivered dose, it is crucial that NPs are localized in the MN tips and minimally in the backplate. As can be inferred from SEM (Fig. 2) and fluorescence imaging (Fig. 3), the NPs were mainly concentrated in the tips of the MNs. SEM images showed that the upper parts of the MN tips had rough surfaces ( Fig. 2A-C) in contrast to smooth surfaces of MNs with free-OVA-PIC (Fig. 2E), suggesting the localization of NPs into the MN tips. Additionally, fluorescence microscope images showed that low HA concentrations (10 or 20 mg/ml, with a NP:HA weight ratio of 1:1 and 1:4, respectively) allowed the efficient deposition of NPs into the MNs tips. This is indicated by the yellow colour of the MN tips, resulting from overlay of OVA-AF647 loaded NPs (red label) and FAM-HA (green label) ( Fig. 3A and C). Furthermore, fluorescence microscope images suggested that the NP content of MN tips was relatively uniformly distributed between different MN tips with 1:4 and 1:1 NP:HA ratios ( Fig. 3A and C). In contrast, a high HA concentration (50 mg/ml) with 1:10 NP:HA ratio seems to prevent the homogenous NP distribution in the MN tips or backplate (Figs. 2C and 3E) due to the high viscosity caused by the elevated HA concentration. Finally, SEM images of cross-sections cut at the approximate half-height of MN tips were analysed. The comparison of the cross-sections of OVA-loaded and NP-loaded MNs indicated the presence of NPs in the HA matrix of NP-loaded MNs (Fig. 2G-H). Spherical shapes of NPs could be detected in the cross-sections of NP-loaded MNs, while OVAloaded MNs had smooth cross-section surface. After NP-loaded MNs have been applied into the skin, they should be able to release the NPs in non-aggregated form. Since it would be very difficult to study the particle size of NPs released in situ in skin, we decided to dissolve the MNs in PBS instead and analysed the size of the released NPs by AF4 and DLS. As the NP:HA 1:4 ratio had the most optimal characteristics concerning NP distribution and dissolution in the skin (see next Section 3.2), we selected this ratio for the experiments. AF4 was used to separate FAM-labelled HA and (unlabelled) NPs and analyse the samples with online fluorescence and MALLS detection, respectively. Based on the MALLS signal, it was selected which fractions were collected from AF4 analysis to measure NP size offline with DLS. In addition, the MALLS signal was used to compare the relative amounts of particulate material in AF4 elugrams between different samples prepared with same NP batches [30]. However, there are limitations in the use of MALLS signal since it is affected not only by particle concentration but also by the particle size. Alternatively, refractive index signal could be used but in this study the signal was too weak for meaningful analysis. First, the fluorescence signal of FAM-labelled HA was similar in all samples containing NPs, suggesting that different drying conditions did not affect HA dissolution (Fig. 4A). HA without NPs had the elution peak maximum at 9 min but in the presence of NPs the elution peak shifted to 12 min. However, the elution peaks of samples containing NPs had shoulder at 9 min. This indicates that in samples containing NPs, a part of HA is in original form while other part of HA was weakly interacting with NPs. As expected, the NP suspension showed a practically flat fluorescence baseline. According to the MALLS signal, the NPs started eluting at 12 min. Thus, it was not overlapping with the elution of HA, and additionally HA did not show a measurable MALLS signal (cf. Fig. 4A and B). The MALLS signal was high for non-dried NP control samples, i.e., NP suspensions with and without HA, but the presence of HA slightly delayed the elution of NPs (Fig. 4B), confirming the presence of interaction between the NPs and HA. NP:HA samples dried in a vacuum desiccator at room temperature had a high and similar MALLS signal to that of Table 1 Physicochemical properties and OVA and poly(I:C) loading characteristics of OVA-NPs (mean ± SD, n = 4) used for in vitro studies and OVA-PIC-NPs (n = 5) used in immunisation studies. Table 2). Compared to AF4-MALLS, DLS allows a more accurate quantification of the particle size. Mixing of HA solution and NPs did not affect the particle size of the NPs. Vacuum drying increased the particle size and PDI of the NPs only slightly (201 nm, 0.27), but after drying at ambient conditions (at 37°C), the light scattering signal was very low and no meaningful DLS results could be obtained. This latter observation indicates a low concentration of NPs in the samples. In dispersed NP-loaded MN samples, the NP size and PDI, and were higher (287 nm, 0.35) than that of vacuum dried samples (201 nm, 0.27), and additionally the variation between separate experiments was larger as indicated by larger SD. This suggests that NPs were more aggregated in MN samples than in vacuum dried samples. This could be due to the centrifugation step during the MN preparation. Centrifugation does force NPs into the microcavities of the PDMS mold and this may have caused the NP aggregation. Dissolving MN piercing and dissolution in ex vivo human skin Dissolving MNs need to have sufficient mechanical strength to penetrate into the skin and therefore their penetration into ex vivo human skin was examined by using a trypan blue assay. The skin penetration efficiency indicates the percentage of MN tips successfully penetrated into the skin and it was 91.9 ± 1.6% (n = 10), 100% (n = 6) and 85.4 ± 2.5% (n = 3) for MNs with NP:HA 1:1, 1:4 and 1:10 wt ratios, respectively (Fig. 5A). This experiment showed an excellent skin penetration efficiency for the different arrays, and especially for the MNs with a NP:HA weight ratio of 1:4. Dissolution of the MNs with NP:HA ratios of 1:1, 1:4 and 1:10 was examined by assessing MN tips with microscope after the application into the ex vivo human skin. The NP:HA ratio remarkably influenced the dissolution of MN tips. For MNs with a NP:HA ratio of 1:1, the dissolution rate was slow and no significant dissolution occurred within 20 min (Fig. 3B). Importantly, the dissolution rate was clearly improved for MNs with 1:4 and 1:10 NP:HA ratios (Fig. 3D and F) and thus, an increase of HA content facilitated the MN dissolution. Finally, the skin was analysed with confocal microscopy immediately after the MNs were inserted and left for 20 min in the skin. The results showed that both the HA and the NPs were deposited into the ex vivo human skin until an approximate depth of 200-300 µm ( Fig. 5B and C). Based on the results regarding the preparation of MNs and their dissolution, MNs with a NP:HA ratio of 1:4 were selected for in vivo studies. Dissolution rate and delivered antigen dose of dissolving MNs in mice The MN dissolution rate and antigen dose delivered into the skin were characterised in vivo in mice after administering dissolving MNs containing NPs (NP:HA ratio 1:4) loaded with OVA-IR800. The MNs were applied into the skin of mice (n = 4) and left in the skin for 20 min samples. Both signals were recorded simultaneously but for sake of clarity the data is presented in two panels. In panel A, the crossflow profile is indicated by the dashed line (right y-axis) and in panel B, the period during which the NP fraction was collected for DLS analysis is indicated. All NP:HA samples had a weight ratio of 1:4 and samples are described in detail in Section 2.7. One representative set of elugrams from three independent experiments is presented. Legend in panel B as presented in panel A. by using an impact-insertion applicator, similarly to the experiments with ex vivo human skin. The delivered dose of antigen was quantified by near-IR imaging [23]. Hollow MN injections of OVA-IR800 loaded NPs were used for calibration, since the encapsulated OVA-IR800 had a significantly lower fluorescence signal than OVA-IR800 solution. NPloaded MNs delivered 31.0 ± 14.3 µg (n = 7, mean ± SD) NPs into the mice, which corresponds to 0.99 ± 0.46 µg OVA and 0.93 ± 0.43 µg poly(I:C) based on loading capacities of OVA-PIC-NPs used for immunisation studies (Table 1). When the delivered dose of NPs was compared to NP content of total MN array (130 ± 25 µg, n = 9, mean ± SD) measured in vitro, it was found that 24% of the dose contained in NP-loaded MN arrays was actually delivered into the skin. In the case of MNs containing free-OVA, injections of free OVA-IR800 were used for calibration. These MNs delivered 1.02 ± 0.52 µg (n = 5, mean ± SD) OVA into the mice. The delivered poly(I:C) dose was not measured, but is expected to be approximately 1 µg based on the OVA:poly(I:C) ratio of 1:1 in the MNs. OVA-loaded MN arrays contained totally 652 ± 57 µg (n = 6, mean ± SD) OVA based on in vitro dissolution experiment. Consequently, only 0.16% of the total OVA in a MN array was delivered into the skin. Therefore, it can be concluded that NP-loaded MNs had much higher delivery efficiency into the skin than free-OVA loaded MN arrays, and that the wastage of the payload was moderate in NP-loaded MNs. Nevertheless, it must be noted that a part of the antigen is lost during the NP fabrication since the encapsulation efficiency is not 100%. For MNs containing a mixture of free OVA and NPs, it was assumed that MNs would have a similar delivery efficiency of NPs and free-OVA as MNs containing solely NPs or free-OVA. Immunisation studies Mice were immunised with OVA-PIC-NPs, a solution of OVA and poly(I:C) (free-OVA-PIC), or a 1:1 mixture of OVA-PIC-NPs and free-OVA-PIC administered either by dissolving MNs or a hollow MN. The major difference between these two immunisation methods is that a hollow MN delivers a known volume of either vaccine solution or suspension [20], whereas dissolving MNs are solid formulations requiring dissolution and release of vaccine. All dissolving MN arrays were characterised by light microscopy after the immunisations studies (Fig. 6). These analyses confirmed that > 95% of applied arrays could dissolve sufficiently to deposit the MN tip containing NPs or free-OVA-PIC into the mice. Furthermore, the visual analysis of MNs after the immunization did not reveal any substantial differences between the dissolution of MNs containing (1) OVA-PIC-NPs, (2) free-OVA-PIC or (3) a mixture of these two. In the case of immunization with soluble free-OVA-PIC, delivery with dissolving and hollow MNs resulted in comparable IgG1 and IgG2 responses (Fig. 7). When 50% of the OVA dose was in soluble form and the other 50% of the OVA loaded in NPs, hollow MN mediated delivery tended to evoke higher OVA specific IgG1 and IgG2a responses than dissolving MN mediated delivery, although no statistical differences were found. When the whole OVA dose was encapsulated in NPs, hollow MN mediated delivery resulted in statistically stronger IgG1 responses than NP-loaded dissolving MNs at all time points (Fig. 7). In addition, strong IgG2a response was detected after hollow MN-mediated delivery of NPs but not after the dissolving MN-mediated delivery. Moreover, in line with our previous results [20], encapsulation of OVA and poly(I:C) in NPs did not improve the IgG1 response, but was required for eliciting a robust IgG2a response after hollow MN mediated immunisation ( Fig. 7D-F). An increased IgG2a response after NP vaccination suggests triggering of cellular immune responses. Therefore, CD8 + and CD4 + T-cell responses were analysed by using adoptive transfer of transgenic OT-I and OT-II cells before the delivery of the OVA-PIC-NPs by dissolving and hollow MNs. Hollow MN mediated delivery of NPs resulted in robust CD8 + and CD4 + T-cell responses (Fig. 8). In contrast, dissolving MN mediated delivery resulted in absence of a CD8 + response and only a minimal CD4 + response. Discussion The aim of the study was to develop a dry-state nanoparticulate vaccine formulation for minimally invasive intradermal immunisation by using HA-based MNs. The developed MNs should be able to penetrate into the skin followed by fast dissolution, and subsequently deliver a sufficient antigen dose in a reproducible manner. Until now, only a small number of studies have described the use of nano- [17,18,31] or microparticle [32] -loaded dissolving MNs for intradermal vaccine delivery. However, these studies were mainly focused on the analysis of immunological effects, whereas the understanding of optimal MN formulation parameters is still limited. Therefore, in this study, important technical aspects of the pharmaceutical development of such a complex formulation combining NPs and dissolving MNs were explored. The clinical translation of dissolving MNs for vaccination has been recently reviewed [4] and it is not further discussed here. The ratio of HA as matrix material and NPs was found to be important for successful skin penetration and dissolution of MNs. The optimal NP:HA ratio obtained in this study was 1:4 and this was based on two observations. First, with a NP:HA weight ratio of 1:1 or 1:4, the viscosity of HA did not negatively affect MN preparation. In contrast, a high HA content of the MNs with NP:HA weight ratio 1:10 increased the viscosity of NP/HA suspension such that the suspension became difficult to handle in the preparation phase. In addition, the higher viscosity limited the deposition of NPs into the MN tips, as was reported also recently for PVP/PVA-based MNs [18]. Importantly, this can decrease the delivered vaccine dose. Secondly, MN dissolution in ex vivo human skin was strongly influenced by the NP:HA ratio. Whereas MNs with a NP:HA weight ratio of 1:4 or 1:10 dissolved within 20 min in the skin, those with a NP:HA ratio of 1:1 hardly dissolved and were therefore considered unsuitable for effective immunisation. Interestingly, MN dissolution in mouse skin was more complete than in ex vivo human skin, probably due to the higher humidity and temperature of the tissue of the mice. Based on the results of these studies, the dissolving MNs with a NP:HA weight ratio of 1:4 were selected for in vivo studies. The centrifugation and drying conditions were found to be critical for successful MN preparation. Without the optimization of these conditions, it would not be possible to deliver sufficient vaccine into the skin and to ensure the ideal re-dispersion of NPs from dry-state to a single particle suspension in the skin, both of which are required for an effective immunisation. First, the centrifugation step was crucial to increase the delivered antigen dose by at least 100-fold, by depositing the NPs into the MN tips. This was estimated by comparing the percentages of the delivered dose of the total vaccine content of a MN arrays between free OVA and NP-loaded MNs (0.16% vs. 24%) since free OVA as solute is not deposited into MN tips by the centrifugation as occurs for NPs. However, centrifugation bears the risk of potential aggregation of NPs, as indicated in this study by a slight increase of the particle size and the PDI of the NPs after in vitro dispersion of MN arrays. To study this, it was necessary to develop a novel analysis method of NP-loaded MNs combining AF4 and DLS, since the analysis with more straightforward methods (e.g., nanoparticle tracking analysis or DLS only) were confounded by the presence of HA in the dispersion medium (data not shown). The application of this developed method is not limited only for NP-loaded MNs but it is suitable also for other formulations embedding NPs in matrices. Secondly, besides the centrifugation, the importance of optimal drying conditions for good NP dispersion after MN dissolution was highlighted in this study. Vacuumdrying of the MNs during the preparation prevented practically completely NP aggregation, while drying of the MN arrays at ambient conditions, that were found to suitable for HA-based protein-loaded dissolving MNs in our previous study [24], led to severe NP aggregation. The superiority of vacuum drying can be attributed a lower temperature and vacuum at low relatively humidity, which can decrease interactions between PLGA NPs and consequently susceptibility to the aggregation. However, as some NP aggregation was still detected after the in vitro dispersion of NP-loaded MNs, there is still room for further optimization such as by screening excipients or alternative drying methods. For example, freeze drying is generally used for drying of nanoparticles as dry powder [33] or when embedded in matrices [34]. Although, in the case of HA, freeze drying produces sponge-like cakes without sufficient mechanical strength for the penetration into the skin, it could be potentially suitable method for other matrix materials. Finally, it is highlighted that the optimization of preparation conditions should be performed for each NP and matrix combination. Finally, the immunogenicity of dissolving MNs (dry state NPs) was compared to that of hollow MNs (NP suspension) using identical doses. The intradermal injection of OVA-PIC-NP suspension elicited strong cellular and humoral responses, which confirms our recent results showing the potency of this vaccine formulation [35]. Unfortunately, the NP-loaded dissolving MNs elicited inferior humoral responses and lacked cellular responses in comparison to hollow MN mediated immunization. However, immunisation with free-OVA-PIC by dissolving or hollow MN resulted in comparable immune responses, which suggests that the HA-based MNs are able to successfully deliver soluble antigens and adjuvants intradermally. Administration of a mixture of free and encapsulated OVA and poly(I:C) in dissolving MNs resulted in IgG1 responses between those of dissolving MNs containing either free antigen and adjuvant or antigen and adjuvant encapsulated in NPs. This supports the finding that OVA-PIC-NPs in dissolving MNs evoked inferior humoral responses compared to free-OVA-PIC. It is generally accepted that the nanoparticulate nature of the vaccine is required for strong T-cell and Th1-type responses since the NP uptake by dendritic cells is enhanced by small size (< 200 nm) [8,36] leading to co-delivery of antigen and adjuvant. Therefore, the absence of cellular or IgG2a immune responses in dissolving MN-mediated immunisation may be due to the absence or a low number of single NPs in the skin. As the MNs almost completely dissolved in the skin, our results suggest that NP-loaded dissolving MNs were unable to release NPs in non-aggregated form in the skin. This is different from the in vitro release analysed with AF4 and DLS. The discrepancy can be related to the differences between conditions in vitro and in vivo such as the limited amount of tissue fluid and low diffusivity in the skin as compared to PBS. Therefore, NP dispersion and distribution in the skin after the immunisation may be potentially hindered with dry vaccine formulation, such as dissolving MNs, leading to poor vaccine uptake by antigen presenting cells in comparison to administration of NP suspension. For future studies, the formulation of dissolving MNs should be improved to maintain NP integrity upon release into the skin and to obtain T-cell responses, for instance, by examining suitable excipients and further optimizing the preparation conditions to enhance NP dispersion and distribution in the skin. Alternatively to PLGA NPs and HA, other MN matrix materials or nanoparticles, such as liposomes [15] and nanogels [37,38], or use of antigen-adjuvant conjugates [39,40] could be examined, and thus more suitable formulations could be discovered. Conclusions HA-based dissolving MNs loaded with PLGA NP were developed for minimally invasive intradermal vaccination. The optimal ratio of NPs and HA in MNs was a critical parameter for MN preparation and dissolution in the ex vivo human skin. The importance of the drying conditions in the preparation of dissolving MNs was shown by AF4/DLS analysis of the NP size after in vitro dispersion of MNs. Intradermal immunisation with PLGA NP-loaded dissolving MNs was able to evoke humoral but practically no detectable cellular immune responses, whereas hollow MN mediated delivery of identical PLGA NPs resulted in robust humoral and cellular immune response. Finally, these results are a valuable foundation for the future development of MN-based delivery of particulate vaccines.	v3-fos-license
2020-10-17T13:06:27.797Z	2020-10-15T00:00:00.000	222837191	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.nature.com/articles/s41598-020-74467-1.pdf", "pdf_hash": "146021678001b3555a42370c201dc6e7d1b9b6ab", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2369", "s2fieldsofstudy": [ "Biology" ], "sha1": "507e462f1ccaf5e224b41ec580724049738ad3c0", "year": 2020 }	pes2o/s2orc	Cyclodextrin inclusion complex inhibits circulating galectin-3 and FGF-7 and affects the reproductive integrity and mobility of Caco-2 cells Galectin-3 (Gal-3) is a carbohydrate-binding protein, that promotes angiogenesis through mediating angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). There is strong evidence confirming FGF involvement in tumor growth and progression by disrupting cell proliferation and angiogenesis. In this study, we investigated the effect of β-cyclodextrin:everolimus:FGF-7 inclusion complex (Complex) on Caco-2 cell migration, cell motility and colony formation. In addition, we examined the inhibitory effect of the Complex on the circulating proteins; Gal-3 and FGF-7. Swiss Target Prediction concluded that Gal-3 and FGF are possible targets for β-CD. Results of the chemotaxis cell migration assay on Caco-2 cell line revealed that the Complex has higher reduction in cell migration (78.3%) compared to everolimus (EV) alone (58.4%) which is possibly due to the synergistic effect of these molecules when used as a combined treatment. Moreover, the Complex significantly decreased the cell motility in cell scratch assay, less than 10% recovery compared to the control which has ~ 45% recovery. The Complex inhibited colony formation by ~ 75% compared to the control. Moreover, the Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for β-CD and EV, respectively. Additionally, β-CD and β-CD:EV were able to bind to FGF-7 and decreased the level of FGF-7 more than 80% in cell supernatant. This confirms Swiss Target Prediction result that predicted β-CD could target FGF. These findings advance the understanding of the biological effects of the Complex which reduced cell migration, cell motility and colony formation and it is possibly due to inhibiting circulating proteins such as; Gal-3 and FGF-7. One of the most common cancer in the United State is colorectal cancer (CRC) 1 . About 20% of patients which are newly diagnosed with CRC found to have metastatic disease. Moreover, around 30% of early stage CRC patients develop metastatic disease 2 . Blood supply is crucial for tumor growth, solid tumor cannot spread more than 2 mm 3 in diameter (100-300 cells) without blood circulation. Nevertheless, through angiogenesis tumor have the ability to develop their own blood supply. Angiogenesis starts when tumor cells start secreting angiogenic growth factor to stimulate endothelial cells close to blood vessels, this allow the formation of new vessels to grow towards the tumor. The small tumor has more oxygen and nutrients supply that is able to grow and metastasize to different parts of the body 3 . Recently, inhibiting circulating proteins like galectin-3 (Gal-3), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) have gained interest due to their important role in pathophysiological diseases such as cancer 4,5 . Galectins are carbohydrate-binding proteins that specifically bind to β-galactosides of different types of large glycan 6 . Galectin/glycan complex are able to connect to other galectin/glycan complex forming lattices-like structure on the plasma membrane. This can influence several Scientific Reports \| (2020) 10:17468 \| https://doi.org/10.1038/s41598-020-74467-1 www.nature.com/scientificreports/ biological functions among the cells such as cell signaling, cell adherence and cell migration 7 . Gal-3 promotes angiogenesis by numerous mediators such as VEGF and FGF. Lactose; a Gal-3 inhibitor reduces VEGF and FGF-mediated angiogenesis in vitro which is similar to the effect when Gal-3 is knockdown 8 . Additionally, inhibiting circulating angiogenic factors VEGF and FGF have a beneficial effect by reducing cancer cells survival, migration, and proliferation 2 . FGF and VEGF are angiogenic growth factors that stimulates vascular repair, blood vessel sprouting, and regeneration 9 . FGF and VEGF mediate their effects through binding specifically to their receptors and promote receptor dimerization, this activates receptor intercellular tyrosine kinase domain followed by auto-phosphorylation to allow specific intracellular molecules to propagate the signal from the cell surface. Activation of FGF and VEGF receptors regulate differentiation, proliferation and migration. Molecules that inhibit FGF and VEGF signaling are currently considered to be promising drug-like molecules with some already reached clinical trial 10 . Elevated levels of FGF, VEGF and Gal-3 are detectable in patients with CRC 11,12 . The Gal-3 and FGF expression correlate with the genesis, progression and development of the malignant tumors [11][12][13] . In our previous work, we have designed a new class of a potential anticancer complex to enhance the antiproliferative efficacy of everolimus (EV) on Caco-2 cell line. We revealed that the β-cyclodextrin:everolimus:FGF-7 inclusion complex (Complex) improved the anticancer effect of the mammalian target of rapamycin inhibitor EV by enhancing its cellular uptake, intracellular retention, therefore preventing cell proliferation. Also, we showed that the Complex has less toxicity to normal human cell line FHs 74 Int, the selectivity index of the Complex was found to be 18.24 14 . Bioactive molecules can exert a phenotypic effect by targeting one or, frequently, several target proteins. Practically it would be impossible to test each drug-like compound for every possible molecular target. For that reason, computational methods such as structure similarity search and molecular docking can be used to fill this gap and identify the possible targets for each individual molecule. In this study, we investigated the effect of the Complex on Caco-2 cell migration, cell motility, colony formation. In addition, we assessed the ability of the Complex to inhibit circulating proteins; Gal-3 and FGF-7. Structure similarity search and molecular docking were used to identify the molecular target of β-cyclodextrin (β-CD). Results Target Identification. Structure similarity search, which is based on the idea that molecule-possessing similar 2D/3D structure may target similar protein targets 15,16 . The obtained results were processed via R Software using (Pheatmap package) (Fig. 1). Swiss predicted that basic FGF and Gal-3 as possible targets for β-CD based on the 2D similarity to CHEMBL198643 (score 0.787) and CHEMBL1669628 (score 0.870), respectively. In comparison, Swiss predicted that lactose (standard galectin-specific sugar) might target basic FGF based on its 3D similarity to CHEMBL198643 with a score of 0.959. In addition, lactose can target Gal-3 based on 2D similarity to CHEMBL1669628 with a score of 0.870. The structure of lactose, β-CD, CHEMBL198643 and CHEMBL1669628 are available in Supplementary Fig. S1. Real-time chemotactic cell migration analysis. The effect of the Complex on metastatic process that dependent on cells migration was investigated. Caco-2 cell line was subjected to a real-time migration assay using xCELLigence system integrated electronically with cell migration (CIM) Boyden chamber (CIM-Plate 16). Over a period of 24 h, starving cells were treated with β-CD, EV, lactose and the Complex. The changes in electrical impedance were recorded as cell index ( Fig. 2A), and the slope represents the degree of cells migration over time (Fig. 2B). Cell motility measurement by scratch assay. The effect of the lactose, β-CD, EV and the Complex on inhibiting Caco-2 cell motility was investigated by the scratch assay over 8 h. Cell motility was significantly (P < 0.05) decreased by the Complex (sub-lethal concentrations treatment) with only ~ 10% recovery compared to the control which has ~ 45% recovery (Fig. 3). Colony formation assay. The effect of lactose, β-CD, EV and the Complex on Caco-2 colony formation was measured by clonogenic assay. Figure 4 shows the sub-lethal concentrations treatment of the Complex for 9 days led to ~ 75% inhibition in the growth which is better than the EV (sub-lethal concentrations treatment) alone which has inhibition of ~ 65% (Fig. 4A). The Complex significantly (P < 0.01) reduced colony formation compared to the control (Fig. 4B). Agglutination inhibition assay. Hemagglutination inhibition assay was utilized to evaluate Gal-3 inhibitory activity of β-CD, EV and the Complex. The galectin-mediated agglutination inhibition of red blood cells was determined as a minimum inhibitory concentration for each sample. Results were compared with the activity of standards galectin-specific sugars (galactose and lactose). The Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for β-CD and EV respectively (Fig. 5). Competitive ELISA. Finally, to invistigate the possible interaction between β-CD and FGF-7, we performed two separate tests: in the first test, FGF-7 (4 µg/mL) only was dissolved in PBS followed by the addition of β-CD, lactose and β-CD:EV and incubated for one hour. Afterwards, the concentration of FGF-7 was measured. In the second test, Caco-2 cells were treated with β-CD, lactose, and EV:β-CD in culture media containing FGF-7 (4 µg/mL). After 24 h, the concentration of FGF-7 was measured in culture media. β-CD, lactose and β-CD-EV interacted with FGF-7 and prevented the antibody to bind to FGF-7 and decreased the level of FGF-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ significantly (P < 0.001) both in PBS and culture media. β-CD signifcantly decreased the level of β-CD in PBS and cell supernatant which confirms the structure similarity results that β-CD could target basic FGF (Fig. 6). Discussion Searching for a better delivery system for the known anticancer drug EV ( Supplementary Fig. S2), encouraged us to discover and form an inclusion complex (by adding β-CD and FGF-7 to EV) to enhance the antiproliferation effect of EV. In this work, structure similarity search was utilized to identify the possible molecular target of β-CD to shed light on the mechanism behind the Complex activity. Target prediction results revealed that β-CD could possibly target circulating proteins such as; Gal-3 and FGF (Fig. 1). Gal-3 is a carbohydrate-binding protein promotes angiogenesis through mediating angiogenic growth factors such as VEGF and FGF. There is strong evidence confirming FGF involvement in tumor growth and progression by disrupting cell proliferation and angiogenesis. Dalton et al. 17 have reported that by blocking the carbohydrate recognition domain of Gal-3 with lactose, a significant reduction in cell fusion was observed. Inhibiting Gal-3 could reduce FGF-mediated angiogenesis, which is similar to the effect that associated to Gal-3 knockdown. In our previous work, we reported that β-CD could compete with heparin and prevent heparin from binding to FGF, which inhibits FGF oligomerization and FGF receptor dimerization. This could suppress the intracellular cascades of FGF receptors by preventing its activation and inhibit cell growth 14 . Molecular docking was used to explore the interaction at the binding site between β-CD and the two circulating proteins; Gal-3 and FGF (Supplementary Table S1). Molecular docking insights suggested that β-CD mimicking some of the interaction between lactose (a strong Gal-3 inhibitor) and Gal-3: Glu-184 H-donor, Arg-162 H-acceptor and Trp-181 H-pi ( Supplementary Fig. S3). Similarly, β-CD mimicked some of the interactions between the native ligand; heparin tetramer fragment and FGF-7: Lys-130 H-acceptor, Asn-28 H-acceptor, Arg-121 H-acceptor and Lys-130 H-acceptor ( Supplementary Fig. S4). Tumour metastasis is a multistep cascade that usually starts locally with the invasion of the primary tumour into the adjacent tissue, accompany with cancer cells dissemination and secondary tumours formation at distant sites 18 . O'Driscoll et al. 19 have reported that endogenous Gal-3 could regulate cell migration, since Gal-3 overexpression in lung cancer cell has increased cell motility and invasiveness in vitro. In this study, we measured the effect of EV alone and the Complex on cell migration by chemotaxis cell migration method on Caco-2 cell line. It was observed that EV reduced the cell mobility by 58.47% compared to the control and by adding β-CD and FGF-7 to EV (forming the Complex), they enhanced the effect of EV and decreased the cell mobility by 78.36% compared to the control (Fig. 2). This is possibly due to the synergistic effect of these molecules when used as a combined treatment. According to Dogan et al. 20 , modified rapamycin (as inclusion complex with β-CD or conjugated with polyethylene glycol) outperformed rapamycin in lower concentrations for inhibiting fibroblast proliferation and wound closure of PT-K75 porcine mucosal fibroblasts. In this study, we assessed the effect of the Complex on inhibiting Caco-2 cell motility by using the scratch assay over 8 h. Before conducting the test, sub-lethal concentration of the Complex was tested for its cytotoxic effect and was found to have no effect on the cell proliferation and therefore was used for the scratch assay. The Complex treatment of Caco-2 cells has possessed a significant decrease with ~ 10% recovery compared to the control which has a ~ 45% recovery (Fig. 3). The findings of the scratch assay revealed that the effects of sub-lethal concentration of the Complex on Caco-2 cells is not due to its cytotoxic activity, but simply due to its inhibition of cell motility. Results suggested that the Complex is a possible anti metastatic agent, which supported by its inhibitory effect on cell migration that mentioned previously. To assess the long-term effects of the Complex on Caco-2 cell line, clonogenic assay was used. Gamage et al. 21 have used the clonogenic assay in vitro to evaluate the tumorigenicity of deoxypodophyllotoxin on Caco-2 cell line. In this work, it was found that colony formation in Caco-2 cells treated with sub-lethal concentration of the Complex over 9 days was suppressed by ~ 75% compared to the control (Fig. 4). These results suggested that the Complex could target the reproductive integrity of cancer cells and prevent clonal expansion. Hemagglutination inhibition assay was utilized to evaluate the Gal-3 inhibitory activity of the Complex. Stegmayr et al. 22 used this assay to investigate the inhibition of the Gal-3 canonical galactoside-binding site by the bioactive pectic samples. Gal-3 is known to play a crucial role in events related to metastasis. According to the results presented in Fig. 5, the Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for β-CD and EV, respectively. Competitive ELISA test was used to measure the FGF-7 interaction with β-CD, lactose and β-CD-EV both in PBS and culture media containing FGF-7. Results confirmed Figure 6. The effect of β-CD, lactose and the Complex on the FGF-7 protein expressing in PBS and Caco-2 cells culture media (cells were treated with β-CD, lactose, and EV:β-CD in culture media containing FGF-7 (4 µg/ mL) and incubated for 24 h). The FGF-7 levels in PBS and culture media was measured using FGF-7 ELISA Kit. FGF-7 fibroblast growth factor 7, ELISA enzyme-linked immunosorbent assay (*P < 0.001). Scientific Reports \| (2020) 10:17468 \| https://doi.org/10.1038/s41598-020-74467-1 www.nature.com/scientificreports/ that β-CD interacted with FGF-7 and decreased the FGF-7 level in PBS and culture media by 82.4% and 82.3%, respectively. Lactose decreased the FGF-7 level by 75.2% in PBS which suggested that lactose possessed weaker interaction to FGF-7 when compared to β-CD. On the other hand, lactose decreased the FGF-7 level in culture media with 10% more than in PBS which could be due to the inhibition of Gal-3 thus inhibiting FGF-7 production in cell supernatant. Similarly, the Complex decreased the FGF-7 level in culture media (83.8%) more than in PBS (80.2%). This indicated that the Complex decreased FGF-7 level in culture media more than in PBS due to the inhibition of Gal-3, therefore inhibiting FGF-7 production (Fig. 6). Conclusion Results revealed that the Complex significantly decreased cell motility, cell migration and colony formation. The Complex treatment has possessed stronger agglutination inhibitory effect compared to β-CD and EV alone. Target identification and molecular modeling insights demonstrated that β-CD could target Gal-3 and FGF-7. The findings of the present work advance the understanding of the biological effects of the Complex which reduced cell migration and motility and it is possibly due to inhibiting circulating proteins such as Gal-3 and FGF-7. Target identification. Swiss target prediction was utilized to estimate the most probable macromolecular targets of the tested molecules, which were assumed as bioactive. The targets prediction is obtained by the combination of their 2D and 3D similarity with a library of 370,000 known actives on more than 3000 proteins from three different species. SMILES of β-CD and EV were obtained from PubChem and were entered into Swiss target prediction server to predict their molecular targets 16 Cell motility measurement by scratch. Caco-2 cells were seeded (in triplicates) in 24-well plate and incubated under a humidified atmosphere of 5% CO 2 and 95% air at 37 °C until a confluent monolayer of cells was formed. Uniform scratches were implemented on the cell-monolayer surface using a p-10 pipette tip. Cells were monitored over the course of 8 h while filling the scratched area. Images were taken using light microscope (Axio Vert.A1, Carl Zeiss, Germany). Cell motility was quantified by measuring the distance between the migrating cells boundaries using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA). Cell motility was expressed as a relative percentage of the changes in the distance between the migrating cells boundaries from the start-point to the end-point of each treatment. Materials Colony formation assay. Colony forming (or clonogenic) assay was used as a tool to evaluate the effects of novel chemotherapeutic drugs that target the reproductive integrity of cancer cells in a dose-dependent manner as described by Franken et al. 23 . Briefly, Caco-2 cells were seeded in 6-well plate at 1 × 10 3 cells/well (in triplicates) in 4 mL DMEM supplemented with 3% FBS and with or without treatment and incubated for 9 days in a humidified incubator with 95% air and 5% CO 2 at 37 °C. Thereafter, cells were fixed with methanol and stained with 0.2% crystal violet (v/v in water). Colonies (≥ 50 cells) were scored using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA). Assay agglutination inhibition assay. The evaluation of potential galectin-inhibitors was performed using Microplate agglutination assay according to the protocol that introduced by Nowak et al. 24 www.nature.com/scientificreports/ and the Complex in 0.15 M NaCl. The bioefficacy of β-CD and the Complex as potential galectin-inhibitors was evaluated by determining the Minimum Inhibitory Concentration (MIC) of the tested samples. Competitive ELISA. For the binding measurement, ELISA test was used for quantitative determination of rHuKGF using KGF (FGF7) ELISA kit. Briefly, β-CD, lactose and β-CD:EV were diluted at the desired concentration in the sample buffer. rHuKGF (4 µg/mL) was prepared in sample buffer and added to samples well in equal amount. 100 µL of the samples solutions were added to the wells and incubated at 37 °C for 1 h. The wells were washed 5 times with 1 × PBS/0.05% Tween 20 and 100 µL of horseradish peroxidase (HRP)-conjugated secondary antibody and added to each well. After incubation for one hour at 37 °C and washing as above, the bound HRP conjugate was detected by adding 100 µL of tetramethyl benzidine (TMB). The peroxidase reaction was stopped after 15 min by the addition of 50 µL 0.5 M H 2 SO 4 . Optical densities at 450 nm were measured using an ELISA reader. The assay was conducted in triplicates 25 . Data analysis. The results are expressed as mean ± SD (n = 6), and the statistical analysis was performed with GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). The significance of any differences between experimental groups was evaluated by the one-way ANOVA followed by a Turkey-Kramer multiple comparisons test. MIC values were expressed as the mean (M) ± S.E.M. (n = 6). The 95% confidence intervals (CIs) of the binding affinity (according to their mean and standard errors) were estimated with 2.5 and 97.5 percentile as the lower and upper bounds. Error bars represent the standard error of the mean.	v3-fos-license
2016-05-12T22:15:10.714Z	2012-04-30T00:00:00.000	610011	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0035443&type=printable", "pdf_hash": "66e68069112e3112a7565ccf92c1f5901635de80", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2376", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "66e68069112e3112a7565ccf92c1f5901635de80", "year": 2012 }	pes2o/s2orc	The Influence of Spin-Labeled Fluorene Compounds on the Assembly and Toxicity of the Aβ Peptide Background The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species. Introduction Alzheimer's disease (AD) is characterized by the deposition of various amyloid b (Ab) aggregates forming amyloid in the brain. Evidence from a variety of studies has established that the oligomeric species of Ab (AbO) carries the greatest toxicity, triggering a variety of downstream effects resulting in neurotoxicity and cognitive deficits [1,2,3,4]. A major impediment to the development of effective anti-Ab compounds for AD therapy is that essentially 100% of large-molecule drugs and .98% of smallmolecule drugs fail to cross the blood-brain barrier (BBB) [5]. Recently [6], we explored a series of compounds based on a highly rigid tricyclic fluorene ring that were developed as amyloid imaging agents [7]. These compounds contain a tertiary amine electron-donating group attached to one aromatic ring and display excellent pharmacokinetics properties and brain bioavailability. In that work, we reported on the ability of two fluorene compounds to disrupt AbO assemblies and reduce Ab toxicity [6]. These compounds (K01-162 and K01-186) were identified based on their ability to block cell death secondary to intracellular AbO production. Both fluorene compounds bind and destabilize AbO, and are capable of penetrating the brain and reducing the cerebral amyloid burden in APP transgenic mice. Fluorenes therefore have a potential use in AD therapy by targeting AbO toxicity at both intraneuronal and extracellular sites [6,8]. In AD, accumulating evidence points to oxidative stress as the ultimate downstream component of Ab-induced toxicity [9,10]. For example, Ab increases NMDA receptor activation, and one of the newer drugs for the treatment of AD (Memantine) targets NMDA receptors in order to block glutamate excitotoxicity. Among other pathways, over-stimulation of NMDA receptors activates phospholipase A, leading to elevated arachidonic acid levels, which in turn generates oxygen free radicals and further activation of phospholipases [11]. Thus the excitotoxicity involves a feedback loop that ultimately leads to neuronal self-digestion via increased Ca 2+ levels, protein breakdown, free radical formation and lipid peroxidation [10]. As shown previously [6], the antiamyloid fluorenes have antioxidant properties. Furthermore, because nitroxides such as the pyrroline species can cycle within a redox cascade via a relatively stable non-damaging N-oxyl (nitroxyl) radical intermediate [12,13], compounds carrying this moiety are likely to have the added potential for decreasing oxidative stress and attenuating the damage caused by reactive oxygen species. In this study, we apply electron paramagnetic resonance (EPR) spectroscopy to a novel fluorene compound containing a pyrroline nitroxide. This spin-labeled fluorene (SLF) exerts similar potency in AbO disruption and protection against AbO-induced toxicity, while also having superior free radical scavenging compared to the model fluorene compounds. Furthermore, the nitroxide moiety provides an intrinsic reporter group that can be probed by EPR spectroscopy, which may provide a sensitive diagnostic tool for in vivo detection of Ab plaques in patients with AD [14]. Thus, in addition to its potential as a novel bifunctional candidate to address AbO toxicity, the SLF compound may also help as a diagnostic and research tool in elucidating fluorene mechanism of action. SLFs protect against AbO toxicity in cultured neurons As demonstrated earlier [6,8] K01-162 attenuates intracellular AbO accumulation and protects against AbO-induced toxicity in cultured neurons. To test the efficacy of SLFs to block Ab toxicity in cultured neurons, we examined the potential of a recently described class of SLF molecules to influence the survivability of MC65 neuroblastoma cells [16] containing conditional expression of the C-terminal region (C99) of the amyloid precursor protein (APP). In the MC65 model system, expression of the APP fragment is turned on in the absence of the transgene suppressor, tetracycline (TC). Upon APP-C99 induction, Ab is generated after proteolysis by cellular c-secretase [16]. As shown in Figure 2, the viability of cells is severely diminished when the expression of APP-C99 is turned on (2TC), compared to the control cells (+TC) lacking Ab generation. However, increasing levels of SLF HO-4160 restore cell viability to near control levels, with an EC 50 of 30 nM. This response is superior to the K01-162 model fluorene (Figure 2, inset). At concentrations above 1 mM, the viability of both APP-induced and un-induced cells declines, reflecting a tolerance limit of the cells to the SLF. As mentioned above, HO-4160 is one of a group of related SLF compounds that we have recently described [15]. We also measured the ability of the other related SLF analogs (see Fig. S1 in Supporting Information) to block Ab toxicity. As shown in Table 1, except for compound 5 in this series, each of the compounds offers protection with potency comparable or superior to K01-162. Because HO-4160 provides the greatest amount of protection against Ab toxicity, we therefore selected this SLF for more detailed analysis of its molecular effects on AbO. SLF HO-4160 reduces AbO accumulation in cultured neurons Our results clearly demonstrate the protective effect of SLF HO-4160 on MC65 cells expressing APP-C99. To determine whether this protective effect corresponds to a reduction in the AbO burden of the cells, we used both Western blot and immunofluorescent staining to analyze the AbO levels in SLFtreated cells. Western blot analysis of MC65 cellular extracts reveals decreased levels of AbO assemblies of varying sizes in SLFtreated cells expressing APP-C99 (2TC) compared to control cells (+TC). As shown in Figure 3, the Ab fragments produced by csecretase action fail to form any readily apparent oligomers when the cells are treated with 300 nM HO-4160. Complementary results were achieved by using immunofluorescence to probe for AbO using the oligomer-specific antibody A11 [17]. Figure 4 shows greatly decreased levels of AbO assemblies in MC65 cells expressing APP-C99 and treated with SLF ( Fig. 4D), compared to untreated cells (Fig. 4B). These results indicate that not only does SLF HO-4160 block the toxic effects associated with Ab production in cultured neuronal cells, but also directly attenuates the formation of oligomeric Ab species in the same model. Disruption of AbO aggregates by SLF To test whether SLF HO-4160 blocks the formation of AbO assemblies that can be imaged by atomic force microscopy (AFM) [6], we acquired AFM images of Ab preparations (50 mM peptide) incubated in PBS buffer for 1 hour, with and without 50 mM SLF treatment. As shown in Figure 5 (A&B), there is a lack of particles .5 nm when Ab is incubated with the SLF. We also tested whether SLF decreases the formation of particles rich in betastrand secondary structure using the amyloidophilic dye thioflavin T (ThT). When SLF is included in a 24-hour incubation of Ab, the ThT fluorescence is decreased by nearly one-half (Fig. 5C). Both the AFM and ThT observations are consistent with a mechanism where the protective activity of HO-4160, like fluorene K01-162, is related to its ability to bind and disaggregate Ab. EPR spectroscopy detects the binding of SLF to Ab Due to its small size, the rotational diffusion of the SLF molecule when free in solution will average the hyperfine anisotropy of its EPR spectrum, producing the narrow, time-averaged line shape seen in Figure 6 (black line) indicative of subnanosecond diffusion. However, in the presence of AbO, substantial broadening of the SLF (10 mM) signal is apparent (Fig. 6, red line). The most likely cause of the spectral broadening is a reduction in the rate of rotational diffusion by the SLF molecule in solution upon binding to the Ab peptide. While a small broad peak appears to arise uniquely in the sample containing AbO (see arrow, Fig. 6), we are unable to resolve this feature sufficiently above background. Therefore it is difficult to conclude with certainty whether the resulting spectrum of the SLF in the presence of Ab represents the compound docked to a mixture of monomers and higher oligomers, or a mixture of bound and unbound SLF. Given the high affinity of fluorenes for AbO [6], we expect that the SLF compound is entirely occupied by the excess Ab peptide. Oligomerization of Ab as reported by peptide containing the TOAC spin label at position 26 We recently [18] reported on Ab synthesized with a TOAC spin label positioned in the central region of the peptide, at position 26 (Ab (26TOAC) ). TOAC is a backbone-restricted nitroxide that offers improved detection of the dynamics arising from movement of a peptide's backbone and/or global rotational diffusion. We have previously shown that the local backbone at position 26 is sufficiently ordered such that the EPR spectrum of Ab (26TOAC) is sensitive to changes in the peptide's rate of global rotational diffusion [18]. Since oligomerization of Ab (26TOAC) will have profound effects on the rate of global rotational diffusion, this modified peptide provides direct insights into the oligomeric state of Ab in solution. As shown in Figure 7A, there is a timedependent broadening of the Ab (26TOAC) EPR line shape, consistent with an increasing molecular volume resulting from oligomerization. Because of the close proximity of peptides in AbO, the samples in Figure 7A contained a 25% molar fraction of Ab (26TOAC) that was spin-diluted with wild-type Ab. Thus the increased line-broadening in these samples can be attributed to changes in spin-label correlation time, and not spin coupling. However, the strong influence of dipolar coupling in the oligomer is evident if spin dilution of Ab (26TOAC) is not carried out ( Figure 7B). Effects of SLF observed by spin labels located within Ab As shown previously [6], the dynamics of Ab containing a spin label near its N-terminus serve to indicate the disruption of AbO by active fluorene compounds. Because AbO disruption should be accompanied by increased rates of global rotational diffusion in solution, the spin labels attached to Ab provide an additional means of observing AbO disruption by the SLF. Furthermore, if the proximity of the nitroxide moieties located on the fluorene ligand and the Ab peptide are close enough to interact magnetically, the dipolar broadening may be evident in the composite spectrum. As shown in Figure 8, 2 hours after addition of the compound to AbO harboring a spin label at position 2 or 26, the spectrum greatly increases in amplitude (red trace). This demonstrates that the SLF is able to greatly disrupt the AbO after 2 hours. Since spin labels are only attached to 25% of the Ab peptides, the level of broadening suggests the nitroxides on the oligomeric peptides can interact with more than one docked fluorene. This is supported by the observation that after complete disruption of AbO, there is a large increase in the spectral amplitude. Some increase in the amplitude is expected due to faster diffusion of monomeric Ab [18]. Our investigation of the ability of 10 mM SLF to disrupt oligomers formed after 24 hours produced varied results that most likely reflect the heterogeneity of assembly size and structure, some of which appear to be resistant to SLF disruption. . C99 is subsequently cleaved by cellular c-secretase to generate Ab. TP17, an inactive tricyclic pyrone, serves as a negative control [31], while vitamin E (Vit E), a potent antioxidant that was shown to also block AbO formation in MC65 cells [30], serves as a positive control. p8 is an unresolved band that could be an Ab homodimer, or a heterodimer of Ab and APPD31 [31], a caspase cleavage product involving residues just downstream from the Ab origin on APP. The presence of this band does not correlate with MC65 cell death [31]. Blotting was carried out using the Ab antibody 6E10 (upper panel) and the loading control (lower panel) was probed using an antibody directed against b-actin. The blot shown is representative of 3 replicates. doi:10.1371/journal.pone.0035443.g003 Interaction between the nitroxides on the SLF ligand and Ab Figure 9 compares EPR spectra from the following conditions using Ab spin-labeled at either position 2 or 26: a sample containing spin-labeled AbO alone, a sample containing both spin-labeled AbO and SLF, and a composite spectrum generated from the individual SLF and spin-labeled AbO spectra. From these comparisons, it is evident that both of the spin-labeled positions in AbO show at least some interaction with the nitroxide moiety on the SLF. This is based on the observation that the spectrum of the sample with both species labeled is lower in amplitude than the sample containing labeled AbO alone. Position 26 in Ab (Fig. 9B) experiences a stronger interaction with the SLF than position 2 of Ab (Fig. 9A). The blue trace in Figure 9A or 8B is a sum of the individual spectra of the two spin-labeled species (Ab and SLF,) and indicates what the actual mixed sample spectrum would look like if no interaction were present. However, the observed spectrum of the mixed sample (black trace) is lower than the composite spectrum. The broadening of the mixed spectrum is slight when Ab contains the spin label at position 2, indicating the nitroxide of SLF approaches within 2 nm of the Nterminal region. However, the broadening of the sample containing a spin label at position 26 is more substantial, demonstrating the nitroxide of SLF is in closer proximity (1.5 nm or less). Circular dichroism analysis of SLF influences on Ab Circular dichroism (CD) spectroscopy provides a global indicator for the increase in beta-strand content that accompanies the oligomerization of the Ab peptide [19]. To determine whether SLF affects the transition of Ab monomers into secondary structures, CD spectra of Ab were collected immediately after its initial introduction into aqueous buffer and then throughout a time course of 24 hours. In the absence of SLF, the CD spectra of Ab reflect a transition from an unstructured random coil to a spectrum characteristic of beta-strand secondary structure (Fig. 10) [20]. The most substantial effect of SLF on the CD spectrum of Ab is seen at 1 hour, where in the absence of SLF the spectrum shows a distinct beta absorption pattern. However in the presence of SLF, the peptide maintains a largely disordered structure after 1 hour. Although SLF does slow the development of beta secondary structure, by 24 hours the CD spectrum of Ab in the presence of SLF shows strong absorption in the wavelength range that interacts most efficiently with a beta-sheet fold. As shown in Figure 10, however, the 24-hour spectra for Ab in the presence and absence of SLF are significantly different, suggesting a unique conformation for the peptide in the presence of SLF. This is not unexpected, as the peptide's structure likely adopts a distinct fold as it incorporates into larger assemblies. As the neurotoxicity of Ab correlates more strongly with aggregative ability than secondary structure [21], compounds that influence the former provide better candidates for intervening in the molecular pathology of AD. SLF scavenges free radicals Sterically hindered alpha, alpha9-tetrasubstituted 5-membered cyclic secondary amines are sensitive to oxidation by ROS to Noxyls. In turn, the N-oxyls can be oxidized to the N-oxoimmonium, or reduced to diamagnetic N-hydroxylamines [22,23,24]. A major advantage of the N-oxyl radical is that it represents a stable free radical that does not induce damage to DNA, proteins, lipids or sugars. Thus, this added feature of the fluorene should improve its ability to lower oxidative stress by either donating or accepting electrons with radicals (NR), such as reactive oxygen species (NROS). To measure the free-radical scavenging potential of candidate compounds, spin-trapping in the presence and absence of SLF was measured by EPR spectroscopy to look for the depletion of superoxide and hydroxyl radicals. The EPR spectra of BMPO adducts with superoxide and hydroxyl radicals (generated via horseradish peroxidase/H 2 O 2 and ironsulfate/H 2 O 2 , respectively) are shown in green (Fig. 11). The spectrum in the presence of SLF is shown in red, reflecting a decrease in the amount of BMPO adduct formed. The antioxidant activity of the SLF further extends to cellular systems. In the same MC65 neuroblastoma cell model described previously, addition of SLF attenuates the production of hydrogen peroxide in response to APP-C99 expression (2TC) compared to control cells (+TC) lacking APP-C99 (Fig. 12). The reduced hydrogen peroxide production approaches control levels and is similar to the reduced levels achieved by treatment of Ab-generating cells with the antioxidant vitamin E. Summary We have previously shown that selected fluorene compounds can rapidly disrupt AbO in solution, and dramatically attenuate AbO toxicity in vivo [6]. The results reported here demonstrate how SLFs help elucidate the mechanism of fluorene action and, more importantly, that SLFs have superior potency in alleviating AbO-induced toxicity. Simulations of the SLF parent K01-162 and Ab show preferential interaction of the compound with a hydrophobic core region of the peptide constituted by residues 17-21 [25]. Figure 13 illustrates one potential mode of interaction between AbO and SLF consistent with our findings regarding substituted fluorenes [6], where the binding of SLF to Ab occludes a hydrophobic interface that facilitates peptide oligomerization. Preparation of Ab oligomers Solid Ab peptide (Bachem Cat # 1194.0001, Torrance, CA) was dissolved in Hexa-Fluoro-Iso-Propanol (HFIP, Sigma, St. Louis, MO). The solution was incubated at room temperature for 1 day until the solution became clear and colorless. HFIP is a strong reducing reagent that can break hydrogen bonds and keep Ab in the monomeric form. All the HFIP was removed by SpeedVac Concentrator (Savant, SV100H, Thermo Scientific, Waltham, MA). To generate Ab oligomers, a 100% DMSO stock solution of 1 mM Ab was diluted in cold PBS buffer pH 7.4 to a total concentration of 100 mM. For EPR experiments using spin-labeled Ab, the AbO preparations contained a mixture of 25% spin-labeled peptide to 75% native Ab . This dilution of labeled peptide in the AbO sample minimizes the broadening of the EPR spectrum that arises from the dipolar interaction of spins in close proximity (,2 nm) [28]. Cell culture models exhibiting intraneuronal AbO The cell culture model used for these studies was the human neuroblastoma cell line (MC65) equipped with conditional expression of the carboxyl-terminal 99 residues of the amyloid-b precursor protein (APP-C99). Ab is generated from APP-C99 after proteolysis by cellular c-secretase. To induce cellular Ab production, the transgene suppressor, tetracycline (TC), was removed from the media, as described previously [3]. Intracellular AbO started to accumulate as early as 4 hours after TC removal. The cytotoxicity was determined on day 3 using a colorimetric MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay, the results of which were comparable to data obtained using counts of viable cells based on trypan blue exclusion and the LIVE/DEAD assay (Invitrogen, Grand Island, NY). To test the fluorene compounds, the compounds were added immediately after TC removal, and the cells were maintained for 3 days without media change before the MTT assay. Western blotting The preparation of cell homogenates and Western blotting were performed as previously described [29,30]. Immunofluorescence staining Immunofluorescent labeling of AbO in cultured MC65 cells by the A11 anti-oligomer antibody (Chemicon-Millipore, Billerica, MA) was performed according to our previously published protocols [16]. Atomic force microscopy (AFM) AFM was employed to analyze the oligomer formation of wildtype AbO. All surface scans employed a Dimension 3100 Scanning Probe Microscope with a Hybrid closed-loop XYZ head and Nanoscope IVa controller (Vecco, Santa Barbara, CA). All samples were prepared on freshly-cleaved mica (Ted Pella, Redding, CA) and imaged in tapping mode in air by a phosphorous-doped silicon cantilever with a nominal spring constant of 40 N/m. Particle dimension measurements and image enhancement were performed with the Nanoscope software supplied by Veeco, version 6.14. For each measurement, an aliquot of AbO was removed from the 1 mM DMSO stock solution and diluted to 50 mM of AbO in PBS pH 7.4, and immediately spotted on freshly cleaned mica. After 2 minutes the samples were washed with 200 mL distilled water and then partially dried by compressed air and completely dried at room temperature [6]. Thioflavin T (ThT) binding assay Ab (1-40) (40 mM) in the presence or absence of SLF (10 mM) was incubated at room temperature for 24 hours. Ab (1-40) (20 mL) with or without SLF was incubated in 170 mL PBS buffer and 10 mL ThT (ThT stock: 0.1 mM stored in the dark at 4uC) for 15 minutes in the dark. Then ThT fluorescence was measured by a VICTOR3 Multilabel Plate Counter (PerkinElmer, Waltham, MA) spectrofluorometer at an excitation of 450 nm, with excitation and emission slit widths of 10 nm. Circular dichroism spectroscopy CD measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier set to 25uC (Jasco, Easton, MD). Ab was diluted to 40-80 mM in phosphate buffer (25 mM, pH 7.4). SLF in DMSO (final concentration 10-20 mM) or DMSO alone (final concentration 0.01-0.02%) was added to the Ab, and the samples incubated for 0, 1, 2, 4, 6 and 24 hours at room temperature. After each incubation time point the samples were placed in a 0.1 mm quartz cuvette and, after extensive purging with nitrogen, scanned in the 200 to 260 nm region (scan speed was 20 nm/min). Averages of five scans were baseline-subtracted (25 mM phosphate with 0.01-0.02% DMSO, or 25 mM phosphate with 10-20 mM of SLF in DMSO). EPR spectroscopy EPR measurements were carried out in a JEOL TE-100 Xband spectrometer fitted with a loop-gap resonator as described previously [23] (JEOL USA, Peabody, MA). SLF (10 mM) was added to the spin-labeled AbO (40 mM) at a final concentration of 32 mM for 0 and 2 hours prior to EPR measurements. Appropriate vehicle controls were used for all samples. Approx- Figure 13. Model of action of SLFs on AbO assemblies. The illustration highlights the bifunctional properties of the SLF, including its ability to block the formation of nm particles and disrupt small oligomers, as well as its antioxidant activity. The ability of SLF to disrupt fibrils or more mature fibrillar oligomers [32] is undetermined. doi:10.1371/journal.pone.0035443.g013 imately 5 ml of the protein, at a final concentration of 32 mM was loaded into a sealed quartz capillary tube. The spectra were obtained by averaging two 2-minute scans with a sweep width of 100 G at a microwave power of 4 mW and modulation amplitude optimized to the natural line width of the attached spin probe. All the spectra were recorded at room temperature. Antioxidant activity Measurement of superoxide and hydroxyl free radicals by EPR. The free radical scavenging activity of SLF compounds was determined by measuring the adduct levels accumulated by the spin trap BMPO. Briefly, a mixture of horseradish peroxidase (100 ng) and 0.03% hydrogen peroxide in PBS pH 7.4 was used to generate superoxide radicals. Hydroxyl radicals were generated by mixing ferrous ammonium sulfonate (0.1 mM) and hydrogen peroxidase (0.1 mM) in PBS. All EPR measurements were performed in PBS buffer pH 7.4 which contained BMPO (1 mM) in the presence or absence of SLFs at varied concentrations. The superoxide and hydroxyl radicals were measured as BMPO-OOH and BMPO-OH adducts, respectively. Scavenging of peroxyl and hydroxyl radicals measured by fluorescence. Fluorescence detection was determined using the Radical Absorbance Capacity Assay (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer's instructions. Briefly, the indicator is oxidized by ROS species resulting in a loss of fluorescence. Thus ROS scavenging activity is determined by the intensity of fluorescence following addition of the hydroxyl or peroxyl challenge. Measurement of cellular hydrogen peroxide levels. MC65 cells were plated onto 12-well plates at 2610 5 cells per well in Opti-MEM with and without tetracycline (1 mg/ ml). Compounds, such as HO-4160 (0.3 mM) or a-Tocopherol (referred to as vitamin E, 100 mM, Sigma, St. Louis, MO) were added to the cultures immediately after plating. Culture medium was collected after a 24-hour incubation at 37uC. Hydrogen peroxide in the conditioned medium was analyzed by the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit following the instructions of the manufacturer (Invitrogen, Grand Island, NY). Figure S1 Structures of SLF compounds [15] evaluated in Table 1. Supporting Information (TIF) Author Contributions	v3-fos-license
2018-04-03T02:10:58.325Z	2016-06-24T00:00:00.000	262272702	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://journals.iucr.org/e/issues/2016/07/00/hb7593/hb7593.pdf", "pdf_hash": "b0975820a6e96c555a12a9d0e8bfa28f6217373a", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2419", "s2fieldsofstudy": [ "Chemistry", "Materials Science" ], "sha1": "61529b2985d106399625b0cdf5be22b462669a02", "year": 2016 }	pes2o/s2orc	N′-[(1E)-(5-Nitrofuran-2-yl)methylidene]thiophene-2-carbohydrazide: crystal structure and Hirshfeld surface analysis The title molecule is curved as seen in the dihedral angle [27.4 (2)°] between the outer rings. Supramolecular chains about a 41 screw axis are formed by amide-N—H⋯O(carbonyl) hydrogen bonding. In the title carbohydrazide, C 10 H 7 N 3 O 4 S, the dihedral angle between the terminal five-membered rings is 27.4 (2) , with these lying to the same side of the plane through the central CN 2 C( O) atoms (r.m.s. deviation = 0.0403 Å ), leading to a curved molecule. The conformation about the C N imine bond [1.281 (5) Å ] is E, and the carbonyl O and amide H atoms are anti. In the crystal, N-HÁ Á ÁO hydrogen bonds lead to supramolecular chains, generated by a 4 1 screw-axis along the c direction. A three-dimensional architecture is consolidated by thienyl-C-HÁ Á ÁO(nitro) and furanyl-C-HÁ Á ÁO(nitro) interactions, as well asinteractions between the thienyl and furanyl rings [intercentroid distance = 3.515 (2) Å ]. These, and other, weak intermolecular interactions, e.g. nitro-N-OÁ Á Á(thienyl), have been investigated by Hirshfeld surface analysis, which confirms the dominance of the conventional N-HÁ Á ÁO hydrogen bonding to the overall molecular packing. Structural commentary In (I), Fig. 1, the conformation about the C6 N2 bond [1.281 (5) Å ] is E. A 5-nitrofuran-2-yl ring is connected at the C6 atom. The furanyl ring is almost planar [r.m.s deviation = 0.006 Å ] and the nitro group is almost co-planar with its attached ring as seen in the O3-N3-C10-O2 torsion angle of À1.7 (5) . The thienyl ring is also planar within experimental error [r.m.s. deviation = 0.005 Å ] and orientated so that the sulfur atom is syn to the carbonyl-O1 atom. Overall, the molecule is curved with the rings lying to the same side of the plane through the bridging CN 2 C( O) atoms, r.m.s. deviation = 0.0403 Å , with twists noted in both the S1-C1-C5-O1 and N2-C6-C7-O2 torsion angles of À9.8 (5) and 5.4 (6) , respectively; the dihedral angle between the fivemembered rings is 27.4 (2) . Supramolecular features The anti relationship between the carbonyl-O and amide-H atoms enables the formation of directional N-HÁ Á ÁO hydrogen bonds leading to supramolecular chains, generated by a 4 1 screw-axis propagating along the c-axis direction, Fig. 2a and Table 1. The chains are connected into a threedimensional architecture by thienyl-C-HÁ Á ÁO(nitro) and furanyl-C-HÁ Á ÁO(nitro) interactions, involving the same nitro-O4 atom, Table 1. In addition,interactions are formed between the two five-membered rings with the intercentroid distance being 3.515 (2) Å , and the angle of inclination is 3.9 (2) for symmetry operation: (i) 1 À y, 1 2 À x, À 1 4 + z. A view of the unit-cell contents is shown in Fig. 2b. Hirshfeld surface analysis Crystal Explorer 3.1 (Wolff et al., 2012) was used to generate Hirshfeld surfaces mapped over d norm , d e , shape-index, curvedness and electrostatic potential. The latter were calculated using TONTO (Spackman et al., 2008;Jayatilaka et al., 2005) integrated into Crystal Explorer, wherein the experimental structure was used as the input geometry. In addition, the electrostatic potentials were mapped on Hirshfeld surfaces using the STO-3G basis set at Hartree-Fock level of theory over a range AE0. The molecular structure of (I), showing displacement ellipsoids at the 70% probability level. Symmetry codes: (i) Ày þ 1 2 ; x; z À 1 4 ; (ii) x þ 1 2 ; y À 1 2 ; z À 1 the Hirshfeld surface to the nearest atom inside and outside, respectively, enable the analysis of intermolecular interactions through the mapping of d norm . The combination of d e and d i in the form of a two-dimensional fingerprint plot (McKinnon et al., 2004) provides a useful summary of intermolecular contacts in the crystal. Two views of Hirshfeld surfaces calculated for (I), mapped over d norm in the À0.1 to 1.2 Å range are shown in Fig. 3. The bright-red spots near the amino-N-H and carbonyl-O atoms, labelled as '1' in Fig. 3, indicate their roles as respective donor and acceptor sites in the dominant N-HÁ Á ÁO hydrogen bonding in the crystal. These also appear as blue and red regions, respectively, corresponding to positive and negative electrostatic potentials, respectively, on the Hirshfeld surface mapped over electrostatic potential in Fig. 4. The light-red spots labelled as '2' and '3' in Fig. 3, and light-blue and lightred regions in Fig. 4, represent the intermolecular thienyl-C-HÁ Á ÁO(nitro) and furanyl-C-HÁ Á ÁO(nitro) interactions involving the nitro-O4 atom as described above in Supramolecular features. The immediate environment about the molecule within d norm mapped Hirshfeld surface mediated by the above interactions is illustrated in Fig. 5. Figure 4 A view of the Hirshfeld surface mapped over electrostatic potential for (I). The red and blue regions represent negative and positive electrostatic potentials, respectively. Figure 5 A view of Hirshfeld surface mapped over d norm for showing intermolecular interactions about a reference molecule of (I). Table 2 Summary of short interatomic contacts (Å ) in the crystal of the title compound. The overall two-dimensional fingerprint plot is shown in Fig. 6a and those delineated into OÁ Á ÁH/HÁ Á ÁO, HÁ Á ÁH, NÁ Á ÁH/HÁ Á ÁN, CÁ Á ÁH/HÁ Á ÁC, CÁ Á ÁC, CÁ Á ÁO/OÁ Á ÁC and SÁ Á ÁH/ HÁ Á ÁS contacts (McKinnon et al., 2007) are illustrated in Fig. 6b-h, respectively; their relative contributions to the overall Hirshfeld surface are summarized in Table 3. In the fingerprint plot delineated into OÁ Á ÁH/HÁ Á ÁO contacts, which make the greatest contribution to the Hirshfeld surface, i.e. 36.4%, arises from the N-HÁ Á ÁO hydrogen bond and is viewed as a pair of spikes with tips at d e + d i $2.1 Å in Fig. 6b. The C-HÁ Á ÁO interactions, which are masked by the above interactions, appear as the groups of green points appearing in pairs in the plot. However, a forceps-like distribution of points in the fingerprint plot delineated into CÁ Á ÁO/OÁ Á ÁC contacts, In the fingerprint plot corresponding to HÁ Á ÁH contacts, which make the next most significant contribution to the surface, Fig. 6c, the points are scattered in the plot at (d e , d i ) distances greater than their van der Waals separations with the comparatively low contribution, i.e. 13.6%, due to the relatively low hydrogen-atom content in the molecule. The absence of characteristic wings in the fingerprint plot delineated into CÁ Á ÁH/HÁ Á ÁC and the low contri- Figure 7 Two views of Hirshfeld surface mapped with shape-index property for (I). The pairs of red and blue regions identified with arrows indicatestacking interactions. bution to the Hirshfeld surface, Fig. 6e and Table 3, clearly indicate the absence of C-HÁ Á Á interactions in the crystal. However, a pair of thin edges with their ends at d e + d i $2.9 Å belong to short interatomic CÁ Á ÁH contacts, Fig. 6f. The presence ofstacking interactions between the symmetryrelated thienyl and furanyl rings is also indicated by the appearance of red and blue triangle pairs on the Hirshfeld surface mapped with the shape-index property identified with arrows in the images of Fig. 7, and in the flat region on the Hirshfeld surface mapped over curvedness in Fig. 8. Finally, although the SÁ Á ÁH/HÁ Á ÁS contacts in the structure of (I) make a 8.9% contribution to the surface, and also show a nearly symmetrical distribution of points in the corresponding fingerprint plot, Fig. 6h, they do not have a significant influence on the molecular packing as they are separated at distances greater than the sum of their van der Waals radii. The final analysis based on the Hirshfeld surfaces is an evaluation of enrichment ratios (ER) (Jelsch et al., 2014); a list of the ER values is given in Table 4. The low content of hydrogen in the molecular structure of (I) yields a very low ER, 0.72, indicating no propensity to form intermolecular HÁ Á ÁH contacts. The ER value of 1.55 from OÁ Á ÁH/HÁ Á ÁO contacts is in the expected 1.2-1.6 range and confirm their involvement in the N-HÁ Á ÁO and C-HÁ Á ÁO interactions. The presence of intermolecular C-HÁ Á ÁO interactions is also confirmed through the ER value near to unity i.e. 0.99, corresponding to the CÁ Á ÁO/OÁ Á ÁC contacts. The high propensity to formstacking interactions between the thienyl and furanyl rings is reflected from the high enrichment ratio 2.66 for CÁ Á ÁC contacts. The ER value of 1.26 resulting from 6.75% of the surface occupied by nitrogen atoms and a 7.5% contribution to the Hirshfeld surface from NÁ Á ÁH/HÁ Á ÁN contacts is due to the presence of short NÁ Á ÁH contacts in the structure, Table 2. The ER values < 1 related to other contacts and low % contribution to the surface indicate their low significance in the crystal. The relative dispositions of the heteroatoms in the two structures are the same but, the twist in (II) is significantly less as seen in the dihedral angle of 10.2 (6) between the fivemembered rings. This is highlighted in the overlay diagram in Fig. 9. The molecular structure of the all thienyl analogue of (I) has been described recently (Cardoso et al., 2016b). There are two almost identical, near planar molecules in the asymmetric unit and each adopts the conformation indicated in Scheme 2, which might be described as having the thienyl-S atoms syn. The intramolecular SÁ Á ÁS separations of 3.770 (4) and 3.879 (4) Å , are beyond the sum of their van der Waals radii. The conformational differences found for the thienyl molecules is consistent with our NMR studies that indicate multiple conformations exist in solution for these compounds. Figure 9 Overlay diagram of molecules of (I) (red image) and (II) (blue). The molecules have been overlapped so that the five-membered rings are coincident. Figure 8 A view of Hirshfeld surface mapped over curvedness for (I). The flat regions highlight the involvement of rings instacking interactions. Synthesis and crystallization The title compound was prepared following a procedure outlined in Fig. 10. Yellow rods of (I) were grown by slow evaporation of a methanol solution held at room temperature. Refinement details Crystal data, data collection and structure refinement details are summarized in Table 5. The C-bound H atoms were geometrically placed (C-H = 0.95 Å ) and refined as riding with U iso (H) = 1.2U eq (C). The N-bound H atom was located from a difference map and refined with (N-H = 0.88AE0.01 Å ), and with U iso (H) = 1.2U eq (C). The slightly elongated displacement ellipsoid for the C2 atom in the thienyl ring is likely due to unresolved disorder in the ring where the second, co-planar orientation related by 180 to that modelled is present. However, this was not modelled as the maximum residual electron density peak was only 0.46 e Å À3 , 0.61 Å from the C2 atom. It is also noted that the relevant S-C and C-C bond lengths show the expected values. Preparation of the title compound. Reagents: i = SO 2 Cl 2 , MeOH; ii = N 2 H 2 ÁH 2 O, EtOH; iii = 5-nitrofurancarbaldehyde, EtOH. program(s) used to refine structure: SHELXL2014 (Sheldrick, 2015); molecular graphics: ORTEP-3 for Windows (Farrugia, 2012), QMol (Gans & Shalloway, 2001) and DIAMOND (Brandenburg, 2006); software used to prepare material for publication: publCIF (Westrip, 2010). N′-[(1E)-(5-Nitrofuran-2-yl)methylidene]thiophene-2-carbohydrazide Crystal data Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.	v3-fos-license
2019-04-05T03:34:04.529Z	2005-03-08T00:00:00.000	94920295	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://zenodo.org/record/5829941/files/627-638.pdf", "pdf_hash": "76a7784cfa6421e89f49d4e95bce6303e456da4d", "pdf_src": "ScienceParsePlus", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2440", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "738605292d34e1d0323ed12cdf0c8128dec96f38", "year": 2004 }	pes2o/s2orc	Aminomethylation reactions of nitrogen and sulfur five membered heterocyclic compounds \If Five membered nitrogen and sulfur heterocyclic compounds such as isatins, benzimidazole, benzimidazolin-2-thi one, bcnzoxazoli none- 2, bcnzoxazoli n-2-th ione, henzotriazole, benzoth iazoli n-2 -th ione, 1 ,3,4-oxadiazoli n-5-thione and 1,2,4-triazolin-5-thiones have been prepared and subjected to aminomethylation reactions in pres ence of formaldehyde and amines. Secondary as well as primary aromatic amines bearing different substituents have been successfully utilized in the aminomcthylation reaction. The aminomethylated products have been tested for antibacterial, antifungal, antiviral, anticancer, antileishmanial and antifilarial activity. A number of such products have exhibited promising antifungal and antileishmanial activities. and sulfur five membered heterocyclic com pounds active hydrogen atom to nitrogen 4-Chloroisatin 70% pH 4.5 The isomeric mixture 5 of 4 and 6 ch\oro isatins was dissolved in N/2 NaOH. The solution was filtered. To the filtrate N/2 HCl was gradually added. As the pH dropped below 8 precipitation of 4-chloroisatin commenced which was completed at pH 4. 5 be due to extensive sulfonation of the aromatic ring due to strong activating influence of methoxy group. With a view to moderating the electron donating influence of methoxy group to a considerable extent a bromine/chlorine atom has now been introduced at position-3 of panisidine and position-4 of m-anisidine. The 3-bromo/ chloro-4-methoxy and 4-bromo/ch !oro-3-methox y anilines, thus obtained, were converted to the corresponding isonitrosoacetanilides, which underwent smooth cyclization in cone. sulphuric acid furnishing the mixtures of isomeric pairs consisting of 4-bromo/chloro-5methoxy-, 6-bromo/chloro-5-methoxy-and 5-bromo/ chloro-4-methoxy-, 5-bromo/ch loro-6-methoxy-isatins in 80-85% yields. Thus, mild electron withdrawing or mild electron donating groups give isatins in excellent yields and strong electron donating groups have to be suitably moderated in order to obtain better results (Scheme 4). During dissolution of isomeric pairs of 4-and 6-substituted isatins in sodium hydroxide solution, the isatin ring opens up resulting in the formation of the sodium salt of the corresponding isatinic acid which on acidification with HCl immediately cyclizes back to isatin (Scheme 4). The isatinic acid anion corresponding to the 4-substituted isatin is a relatively strong conjugate base, and, therefore, more inclined to accept a proton. It stabilizes at pH 8.0 or above. The lowering of pH value below 8.0 destabilizes the isatinic acid anion resulting in the liberation of isatinic acid which, immediately gets transformed into the 4-substituted isatin. The continuous removal of isatinic acid in the form of 4-substituted isatin prevents the establishment of isatinic acid anion!isatinic acid equilibrium resulting in almost total conversion of isatinic acid anion to the 4-substituted isatin as the pH is progressively brought down to 4.5. The isatinic acid anion corresponding to the 6-substituted isatin is a relatively weak conjugate base and, therefore, less inclined to accept a proton. It continues to remain stable at pH as low as 4.0. The anion gets destabilized when the pH value is lowered below 4.0 leading to the formation of the corresponding isatinic acid which immediately undergoes cyclisation yielding the 6-substituted isatin. The precipitation is complete as the pH is brought down to 2.0. ¢' -~ It is obvious that the precipitation of 4-and 6-substituted isatins from alkaline solution of their isomeric mixtures over widely differing pH values affords a novel and efficient method for their separation. The method, indeed is of wide applicability as it can be used to separate iso-meric binary mixtures of organic acids with a marked difference in their relative acidity. This has been experimentally verified by effecting the separation of ochlorobenzoic acid and p-chlorobenzoic acid from the alkaline solution of their mixtures. As expected the pchlorobenzoic acid, being relatively week acid, precipitated out at a higher pH while the o-chlorobenzoic acid separated out at a lower pH value. The substituents at 4/6 position of isatin acquire positions ortho!para to the carbonyl group in the conesponding isatinic acid, respectively. The acidity of isatinic acids is determined to a large extent by the proximity of the carbonyl group to the carboxylic group. The relative acidity of an isatinic acid with a substituent ortho to the carbonyl group is apparently less than its isomer with substituent para to the carbonyl group. This is in contrast to the substituted aromatic acids where the orthosubstituted acids are invariably stronger than their paraisomers. The relative proximity of the substituent ortho to the carbonyl group, greatly reduces ·its inductive effect, thus rendering the isatinic acid relatively week. The substituent para to the carbonyl group, on the other hand, exerts its inductive effect in a normal manner, thereby making the isatinic acid relatively strong. Aminomcthylation of 4-bromo-5-rncthox.yisati 11 and 6-bromo-5mctbox.yisatin 630 isati ns 6 ( 19) thus separated in a pure state were aminomethylated with morpholine in the presence of formalin to yield corresponding !-ami nomethylated products (20,21). It is interesting to note that the is<itin N-Mannich bases appeared to be more promising as antiviral agents. Next a series of 4-arylthiosemicarbazides were prepared and condensed with isatins in acidic medium. The 3-arylthiosemicarbazono-2-indolinones thus prepared were then treated with formalin and secondary amines leading to aminomethylated-2-indolinones (28). A number of benzoyl hydrazines, anilino-acetyl hydrazines and phenoxy acetyl hydrazines were prepared by the standard procedures. These hydrazine derivatives were then condensed with isatins as nucleophilesl4-17. The products obtained after condensation were subjected to a1ninomethylation reactions. The aminomethylated products were obtained in excellent yields (32)(33)(34)41) compound (39) was prepared via a number of steps starting from (35) as indicated in the Scheme 5. Aminomethylation of benzimidazole Benzimidazole(s) (48) contain an active hydrogen atom attached to nitrogen which can be easily replaced by an aminomethyl group 18 -22 (49) in the presence of formalin and an amine. Many benzimidazoles arc available commercially. If desired they arc easily prepared from appropriately substituted o-phcnylcnc diamine (47) and formic acid. One can also take acetic acid in place of formic acid to get 2 methylbenzimidazolc. Thus by taking appropriate carboxylic acid one can get appropriately 2-substituted benzimidazoles. 2-Substitutents in benzimidazole bulkier l!CS-2 Aminomethylation of benzimidazolin-2-thione Benzimidazolin-2-thione (55) unlike benzimidazole has two active hydrogen atoms attached to each nitrogen. Both these hydrogens have been replaced with aminomethyl groups 23 -26 (57) in the presence of formalin and an amine. Secondary as well as primary aromatic Aminomethylation of benzoxazolinone-2 Benzoxazolinone-2 (61) is one of the versatile five membered heterocyclic system which undergoes aminomethylation with ease. In order to prepare benzoxazolinone-2, o-aminophenol and urea are fused 28 together at elevated temperature. The crude product is repeatedly recrystallized from water to get pure material. 632 During fusion with urea considerable decomposition takes place resulting in the lower yields of the material. We have developed a preparative method for benzoxazolinone-2. In this procedure o-aminophenol and urea are heated in dry pryridine. After pyridine is distilled off the residue is poured into water thereby pure benzoxazolinone-2 is obtained in high yields 29 . Aminomethylation of benzoxazolinone-2 has been done in ethanolic medium in the presence of formalin and an amine. Different types of amines have been utilized to obtained the required amino methylated products 30 -4 Aminomethylation of benzoxazolin-2-thione Next we have taken a closely related system benzoxazolin-2-thione (64) to be studied for amino-methylation reaction. Its method of preparation is described in organic synthesis 41 . The procedure involves treatment of o-aminophenol (63) with potassium ethylxanthate in ethanol followed by acidification of the reaction mixture with acetic acid. The benzoxazolin-2-thione (64) thus obtained has been treated with formalin and amines. The reaction takes place both with secondary as well as with primary aromatic amines 42 -4 4 (69) (Scheme 9). Aminomethylation of benzotriazole Benzotriazole, a five membered heterocyclic system with three nitrogen atoms at 1.2,3 positions contains an active hydrogen atom attached to nitrogen atom. It was subjected to aminomethy1ation reaction 45 benzotriazoles (72) can also be obtained by treating l-hydroxymethylbenzotriazole46 (71) with primary aromatic amines, when 1-dimethylaminomethylbenzotriazole (73) was treated with aniline, the dimethylamino group got displaced with aniline (Scheme 10). Microwave mediated aminomethylation Microwave mediated synthesis of heterocyclic compounds have attracted more attention of chemists because of shorter reaction time, simple reaction conditions, higher yields and purer products. Many reactions like oxidation, reduction, alkylation, 0-benzylation, hydrazinolysis, esterification, aromatic substitution and decarboxylation proceed smoothly under microwave conditions. Aminomethylation 57 was the further addition to the microwave mediated reactions. Mechanism of aminomethylation reaction The aminomethylation reaction 61 -6 7 is believed to tak.-: place in two steps. Initially the amine reacts with formaldehyde to generate an aminomethylol (A). The aminomethylol (A) then reacts with active hydrogen containing substrate [say for instance benzoxazolinone-2 (B)] to furnish amino methylated product (C). Antileishmanial activity against Aminomethylated compounds showing promising antifungal and antileishmanial activity are listed in Tables 1 and 2. I.	v3-fos-license
2019-03-22T17:11:04.356Z	1987-01-01T00:00:00.000	85505397	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://downloads.hindawi.com/journals/apec/1987/058065.pdf", "pdf_hash": "80508bba18cf80ab517ca6fe60f5d59c435460c2", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2450", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Physics" ], "sha1": "80508bba18cf80ab517ca6fe60f5d59c435460c2", "year": 1987 }	pes2o/s2orc	THE GROWTH AND ASSESSMENT OF GaAs EPITAXIAL LAYERS OBTAINED FROM GaAs-Bi SOLUTIONS X-Ray investigations of GaAs epitaxial layers obtained from Ga-As-Bi solutions with different amounts of bismuth are presented. An equilibrium cooling and two phase technique for the deposition of the GaAs epitaxial layers on semi-insulating GaAs:Cr(100) substrates has been used. INTRODUCTION The usage of bismuth as a solvent instead of gallium in the liquid phase epitaxy (LPE) of GaAs has been recently reported and associated with a decrease of the carrier concentration and a considerable increase of carrier mobility in the deposited GaAs layers [1,2].It is well known that silicon is a residual impurity which is always present in GaAs deposited by PLE techniques and therefore this may be explained by a high coefficient of bismuth and silicon interaction Si-Bi in the solutions [1].In depositing GaAs from the Bi-solution we have in fact a three component system GaAs-Bi.The phase diagram of GaAs-Bi system in the area of Ga-Bi-GaAs has been experimentally defined by Evgenev and Ganina [3].It is also known that the GaAs compound is the only interphase of a constant composition and that bismuth is an isoelectronic dopant in the analysed system [4].Its segregation coefficient and solubility in GaAs are very low [1,2,3].By depositing the GaAs epitaxial layers from stoichiometrical solution of Ga and As in bismuth, the greatest solubility Bi in GaAs should be obtained. The physical properties of epitaxial layers obtained from Ga-As-Bi solution over a wide range of compositions of liquid phase have not been described in the literature so far.In this paper preliminary assessment of such layers obtained from Ga-As-Bi solutions with different amounts of bismuth are presented. EXPERIMENTAL 2.1 Growth of Specimens The epitaxial layers were grown on Cr-doped semi-insulating (100)m oriented GaAs substrates with > 107ff2cm and dislocation density 5.104/cm supplied by the CEMI (a local vendor).The substrates were cut to dimensions 10 x 11.5 mm and cleaned in organic solvents followed by an etch in a H2SO4:HzOz:H20 solution (12:1:1 volume ratio) and finally rinsed in propan-2-ol.Bismuth, qallium and polycrystalline GaAs of 6N purity were used for preparing of solutions.The mass of the initial composition of liquid phase, i.e. mGa + mBi was constant and equal to 2 gm.This corresponded to 3.2 mm (for 0 at.% Ga) height of the melt in the graphite boat. The epitaxial deposition of GaAs layers were performed in horizontal open system quartz reactor, which contained a conventional graphite sliding boat [5] in a Pd purified H2 ambient atmosphere. The growth processes were performed in two different ways: 1.A two phase technique where the melt was saturated by arsenic from polycrystalline GaAs. 2. An equilibrium cooling technique where the melt was saturated with help of a dummy GaAs:Cr substrate which was identical to a proper substrate.(In this case of course the melt would also contain some chromium, which was present in the dummy substrate.) The saturation of the melt or solution was made for 2 hours at 1073 K. 1073 K is also the normal deposition temperature regardless of melt composition.The compositions of the solutions used in this work are presented in Table 1. Following saturation the melt was brought into contact (by moving the slider of the boat) with the substrate.Simultaneously, the program of the linear cooling with the rate, vn 0.25 K/min was initiated.The layer was deposited for 30 min.Growth times of subsequent layers also 30 mins.Taking into account composition of the solutions used, the epitaxial layers obtained were classified into six technological groups.The compositions of the used solutions and the thicknesses of the obtained layers are shown in Table 1. Electrical evaluations of the GaAs Epilayers Hall and C-V measurements were used to characterise the GaAs epitaxial layers and results are summarised in Table 2. The measurements were carried out at 300K.The layers of IV to VI groups were not measured. .3 X-Ray Investigations X-Ray diffraction patterns for the epitaxial layers taken from the technological groups presented in Table 1 were obtained.They were identical in spite of differences in the solution compositions used.(i.e.The same as for the monocrystalline GaAs with cry- stallographic orientation (100).)X-ray photographs obtained by the Laue method confirm the monocrystallinity of each layer.The X-ray photographs of the GaAs:Cr substrate and the epitaxial layers deposited on the substrate for the most representative groups I-VI are presented in Figure 1.Additionally, these layers were tested using X-ray microprobe. FIGURE Lane photographs of the GaAs:Cr substrate with and without epitaxial layers from the Groups shown in Table 1. Besides Ga and As no other elements were identified in the range of sensitivity (0.1 at.%) of the X-ray microprobe used (Figure 2). DISCUSSIONS AND CONCLUSIONS The following conclusions can be drawn from the investigations: the epitaxial layers are GaAs monocrystalline with the crystallographic orientation (100) independent of the used solution composition.Bi and Cr were not identified in the investigated layers in the sensitivity range (0.1 at %) of the X-ray microprobe used. These preliminary results suggest that GaAs of good crystallinity and a carrier density as low as 1014cm -3 can be grown by LPE from Ga-As-Bi solutions and that such a process may offer the potential for producing materials for device fabrication. As K [keV] FIGURE 2 ( FIGURE 2(b) Layer from Group TABLE 2 Electrical parameters of the GaAs epilaycrs Table 1 . L FIGURE 2(c) Layer from Group IIA FIGURE 2(d) Layer fromGroup IV	v3-fos-license
2018-04-03T00:29:28.739Z	1997-04-11T00:00:00.000	12300069	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "http://www.jbc.org/content/272/15/10279.full.pdf", "pdf_hash": "5354c880635b46cb3cde4803763a82734d7c0185", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2464", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "sha1": "65a7324e031b3a2e4fb10d4652af85d0fb2899d3", "year": 1997 }	pes2o/s2orc	Folding of the amino-terminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein. The initial assembly of apolipoprotein B100 (apoB) into lipoprotein particles occurs cotranslationally. To examine steps required to initiate this process, the intracellular folding and assembly of the amino-terminal 28% of apoB (apoB28) was examined using several criteria including nonreducing gel electrophoresis, sensitivity to dithiothreitol (DTT)-mediated reduction, and buoyant density gradient centrifugation. In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, labeled apoB28 migrated during gel electrophoresis in the folded position and was resistant to reduction in vivo with 2 mM DTT. A similar rate and extent of folding was observed in Chinese hamster ovary cells, a microsomal triglyceride transfer protein (MTP)-negative cell line that can neither lipidate nor efficiently secrete apoB28. Amino-terminal folding of apoB28 was essential for its subsequent intracellular lipidation as apoB28 synthesized in hepatoma cells under reducing conditions remained lipid poor (d > 1.25 g/ml) and was retained intracellularly. Upon DTT removal, reduced apoB28 underwent a process of rapid (t1/2 approximately 2 min) post-translational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assembly pathway, post-translational lipidation of apoB28 displayed a strict dependence upon amino-terminal folding. We conclude that: 1) folding of the amino-terminal disulfide bonded domain of apoB is achieved prior to the completion of translation and is independent of MTP and events associated with buoyant lipoprotein formation and 2) domain-specific folding of apoBs amino-terminal region is required to initiate MTP-dependent lipid transfer to nascent apoB in the hepatic endoplasmic reticulum. In addition to the protein and lipid biosynthetic capacities present in most cell types, the biogenesis of very low density lipoprotein requires at least two dedicated gene products: apoB, the major protein component of very low density lipoprotein (1)(2)(3)(4)(5)(6), and the microsomal triglyceride transfer protein (MTP), 1 a soluble lipid transfer protein localized to the ER of hepato-cytes and intestinal epithelial cells (7). As the initial assembly of apoB with lipids occurs cotranslationally (8), it was predicted that the amino-terminal domain may play a critical role in initiating the process of lipoprotein particle assembly (7,9). Indeed, the amino-terminal ϳ18% of apoB (apoB18) is atypical relative to the rest of the protein: (i) it is globular and highly disulfide bonded (6 of the 8 disulfide bonds in apoB100 are positioned within the amino-terminal 11% of the protein (10)), (ii) it demonstrates a relatively lower affinity for plasma low density lipoprotein particles than internal and carboxyl-terminal domains (2,(11)(12)(13), and (iii) in transfected cells, apoB18 lacks the capacity to recruit a significant amount of lipid and its secretion can be achieved independently of MTP (14 -17). While the amino-terminal domain of apoB is incapable of forming core-containing lipoproteins on its own, several lines of evidence suggest that it nonetheless plays an essential role in lipoprotein secretion. When disulfide bond formation was disrupted in the amino-terminal domain of apoB by preincubating HepG2 cells with 2 mM DTT, the ability of apoB to form a secretable lipoprotein was irreversibly blocked (9). In addition, internal domains of apoB, which are otherwise incapable of undergoing secretion, could be rendered secretion competent by appending them to the carboxyl-terminal end of apoB17 (18). While proper folding of the amino-terminal domain of apoB appears essential to promote its secretion, due to apoB's large size (4,536 amino acid residues) and hydrophobicity, it has not yet been experimentally established which stage of the lipoprotein assembly and secretion process is dependent upon this amino-terminal folding event. In the current report, this and other mechanistic aspects of the assembly and secretion of apoB-containing lipoproteins were explored by focusing on the biogenesis of the aminoterminal 28% of apoB (apoB28). This form of apoB has been shown previously to be of sufficient size to undergo secretion in the form of a buoyant, lipid core-containing lipoprotein particle (8,14,15,19). Furthermore, the secretion of apoB28 is MTPdependent, a hallmark of apoB biogenesis (7, 20 -22). While capable of MTP-dependent lipoprotein assembly, the apoB28 polypeptide chain is sufficiently small (1270 amino acid residues) to enable resolution of reduced and folded forms by nonreducing SDS-PAGE. This allowed us, using techniques that are not applicable to full-length apoB48 or apoB100, to directly monitor the relationship between intracellular folding of apoB and its capacity to undergo MTP-dependent lipidation in the ER. The studies described here indicate that folding of the aminoterminal domain of apoB28 occurs either during or shortly after translation and can be achieved independently of MTP. Furthermore, the data indicate that folding of this domain is essential for apoB28's capacity to undergo subsequent MTP-dependent assembly with lipids. The requirement for amino-terminal folding was observed for both the normal cotranslational assembly pathway as well as during the posttranslational folding and lipidation that occurs when apoB28 was artificially reduced in the ER and then allowed to refold. These studies provide compelling evidence that the initiation of lipoprotein formation in the hepatic ER proceeds via two distinct stages: the amino-terminal globular domain of apoB undergoes a cotranslational folding event that occurs autonomously and prior to appreciable lipid recruitment. Once completed, this folding event facilitates the capacity of apoB's downstream sequences to undergo MTP-dependent assembly with lipids. The requirements for the production of small, dense apoB28-containing lipoprotein particles described here, may also be necessary to initiate the formation of native lipoproteins containing apoB48 or -100. EXPERIMENTAL PROCEDURES Materials-Restriction and DNA modification enzymes were from New England Biolabs. Tissue culture media and supplements were obtained from ICN (Irvine, CA) or MediaTech (Washington, D.C.). Cycloheximide, DTT, and IAA were from Sigma. Tran 35 S-label (an ϳ5:1 mixture of [ 35 S]Met and Cys) was from ICN. Anti-FLAG M2 monoclonal antibody was from Eastman Kodak Scientific Imaging Systems (New Haven, CT). Polyclonal antibodies to human apoB100 and albumin were obtained from Boehringer Mannheim. Centricon-10 and Centriprep-10 centrifugal concentrators were from Amicon (Beverly, MA). Construction of an ApoB28-containing Expression Plasmid-A form of apoB28 containing an 8-amino acid carboxyl-terminal epitope tag and stop codon was constructed. The SacII/XhoI fragment from the apoB53containing plasmid pB53LII (obtained from Dr. Zemin Yao, University of Ottawa) was ligated to SacII and XhoI-digested pBluescript IISKϩ (Stratagene, La Jolla CA) to form plasmid SK28. A double-stranded oligonucleotide encoding the 8-amino acid epitope tag (DYKDDDDK) (23,24), stop codon, and flanking restriction ends was ligated to XhoI and KpnI digested SK28 to form SK28F. The fusion protein was excised from SK28F using the EcoRI and KpnI restriction enzyme sites flanking the apoB coding region and subcloned into EcoRI and KpnI-digested pCMV5 (25) to form plasmid apoB28F. Tissue Culture and Transfection Conditions-COS-1 and HepG2 cells were cultured as described previously (9,26). McA-RH7777 cells were maintained in low glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. CHO cells were maintained in a 50:50 mixture of low glucose Dulbecco's modified Eagle's medium and Ham's F-12 containing 10% fetal bovine serum. Transient transfections of COS-1 cells by the DEAE dextran method and HepG2 cells by the calcium phosphate method were performed as described (27,28). To control for possible plate to plate variability in transient transfection efficiencies, HepG2 cells were trypsinized 24 h after transfection, mixed, and replated. Experiments were performed 16 -24 h after replating. For stable transfections, McA-RH7777 or CHO cells were cotransfected with apoB28F and pSV2neo (29) at a mass ratio of 20:1 as described (28). Stably transfected CHO cells were selected in 700 g/ml G418 and McA-RH7777 cells in 400 g/ml G418. Individual clones were expanded and screened for apoB28F expression and secretion by Western blot analysis. For routine propagation of transfected McA-RH7777 and CHO cell lines, G418 concentration was reduced to 350 g/ml. Metabolic Labeling of Cells-Cells were washed with phosphatebuffered saline and preincubated in serum free, Met/Cys-deficient high glucose Dulbecco's modified Eagle's medium (COS-1 cells) or minimal essential medium (McA-RH7777, CHO, and HepG2 cells) for 20 min prior to labeling. For CHO cells, the Cys/Met-deficient minimal essential medium was supplemented with 35 g/ml L-proline. Labeling was initiated by addition of 100 Ci/ml [ 35 S]Met/Cys (total volume of labeling media was 0.4 ml/60-mm dish, 2 ml/100-mm dish, and 5 ml/150-mm dish). Chase was initiated by diluting labeling media with 2.5 volumes of chase media (minimal essential medium containing 2.5 mM additional Met and 1 mM additional Cys). Media was removed and replaced with 2 ml (100-mm dishes) or 5 ml (150-mm dishes) fresh chase media. In cases where post-translational lipidation and secretion were monitored (Figs. 6 and 7), cycloheximide was included in the chase to block new protein synthesis. After the pulse in the presence of DTT, chase media containing 100 M cycloheximide was added and incubation was continued for an additional 5 min. Media was then replaced with chase media containing 100 M cycloheximide but lacking DTT. Control experiments (not shown) revealed that a 5-min preincubation with 100 M cycloheximide reduced protein synthesis to less than 0.5% of control values. Immunoprecipitation of apoB28F from cells and media was performed as described (26). Generation of Reduced ApoB28F-In instances where cells were labeled in the presence of DTT (Figs. 4, 6, and 7), DTT was added 1.5 min prior to addition of label. Under these conditions, approximately 80% of labeled apoB28F was in reduced form, primarily because the DTT present during the preincubation blocks the disulfide bond formation that normally occurs during translation. Because DTT is an inhibitor of protein synthesis (9, 30 -32), which in HepG2 cells occurs predominantly at the level of initiation (9), DTT preincubation reduces incorporation of radiolabeled amino acids into total protein (9,30,31). In Fig. 4, where a comparison is made between apoB28F labeled in the presence and absence of DTT, twice as many cells were employed when labeling was performed in the presence DTT. Gradient Fractionation of Intracellular ApoB28F-Post-nuclear membranes were prepared by Dounce homogenization as described previously (26). Membranes were suspended in 300 l of membrane buffer (26) and adjusted to 0.1 M sodium carbonate, pH 11.5, by addition of a 1 M stock. Samples were diluted to 2 ml with 100 mM sodium carbonate, pH 11.5, and incubated on ice for 60 min with occasional low speed vortexing. Membranes were pelleted by centrifugation at 100,000 rpm (412,000 ϫ g) for 15 min at 4°C in a Beckman TL100 tabletop ultracentrifuge using the TLA 100.3 rotor. Supernatant fractions were transferred to clear polystyrene tubes and adjusted to 0.005% phenol red, 200 mM NaCl, 50 mM Tris-HCl, pH 7.5, 2.5 mg/ml bovine serum albumin and sufficient dilute HCl (ϳ108 l of a 1:5 dilution of concentrated HCl) to bring the pH to 7.5. The neutralized sample was adjusted to 1.25 g/ml with solid KBr and a final volume of 3 ml. Samples were transferred to polyallomer quickseal tubes, and centrifuged in the TLA 100.3 rotor at 100,000 rpm for 12-15 h at 15°C. The top 1-ml (d Ͻ 1.25 g/ml) and bottom 2-ml (d Ͼ 1.25 g/ml) fractions were recovered with a tube slicer (Beckman Instruments). Fractions were concentrated to ϳ50 l using Centricon 10 centrifugal concentrators (Amicon) and diluted to 2 ml with phosphate-buffered saline. After another round of centrifugation/concentration, samples were diluted into 1 ml of lysis buffer (1% Triton X-100, 25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 2.5 mg/ml bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml pepstatin) and subjected to immunoprecipitation with anti-FLAG M2 monoclonal antibody as described (26). Pellets from carbonate extraction were washed 2 ϫ with 1 ml of TBS and resuspended in 200 l of 1% SDS, 25 mM Tris, pH 7.4, and boiled for 10 min. Samples were diluted with 800 l of lysis buffer and subjected to immunoprecipitation as described (26). Density Gradient Centrifugation of Secretory ApoB28F-Eight ml of 1.25 g/ml KBr in phosphate-buffered saline was added to an SW41 polyallomer centrifuge tube (Beckman Instruments) and overlaid with 1 ml of conditioned media that had been adjusted to 1.20 g/ml with solid KBr. The 1-ml sample was then overlaid with 3 ml of 1.05 g/ml KBr. Tubes were centrifuged at 37,000 rpm (160,000 ϫ g) for 48 h at 20°C in an SW41 rotor. In Fig. 7, 9 ml of conditioned media were first concentrated to 1 ml using a Centricon Centriprep-10 concentrator prior to adjusting to 1.20 g/ml. Tubes were fractionated from the top using an Autodensiflow Gradient Fractionator (Labconco Industries) into 1-ml samples. The density of each fraction was determined gravitometrically. For immunoprecipitation, gradient fractions were exchanged into phosphate-buffered saline using Centricon-10 centrifugal concentrators as described above. After diluting into lysis buffer, fractions were immunoprecipitated with anti-FLAG M2 monoclonal antibody. ApoB28F: A Model System for Examination of Early Stages of Hepatic Very Low Density Lipoprotein Assembly-Because of the cotranslational kinetics of lipoprotein assembly, steps required to initiate small particles formed by the amino-terminal ϳ25% of apoB may be the same as those required for apoB48 and apoB100-containing lipoproteins. We therefore investigated whether an epitope-tagged form of apoB28 could serve as an appropriate model system for analyzing the relationship between apoB folding and subsequent steps required for the initiation of lipoprotein particle assembly. A plasmid encoding the amino-terminal 28% of apoB with the 8-amino acid FLAG epitope (23, 24) and termination codon appended to its carboxyl terminus (apoB28F; "Experimental Procedures") was transiently transfected into HepG2 cells (a human hepatoma cell line). Cells were pulse-labeled for 10 min with [ 35 S]Met/Cys, and chased for 90 min in media containing an excess of unlabeled amino acids. Chase media was collected and subjected to density gradient centrifugation as described under "Experimental Procedures." As shown in Fig. 1A, apoB28F was secreted as a buoyant lipoprotein particle with a peak density of ϳ1.175 g/ml, a value similar to that reported by others for apoB28 (14,19). A similar peak density was also observed for apoB28F secreted from stably transfected rat hepatoma cells (McA-RH7777 cells; not shown). Furthermore, the secretion of apoB28F from the kidney fibroblast cell line, COS-1, was induced by ϳ5-fold upon coexpression with the large subunit of MTP (Fig. 1B, compare amount of apoB28F in media (M) in the presence (ϩ) and absence (Ϫ) of MTP expression). These results indicate that apoB28F undergoes MTP-dependent secretion as a buoyant lipoprotein particle. We next examined whether the folding state of apoB28F could be monitored by nonreducing SDS-PAGE, a common means of examining disulfide bond formation in intact cells (33). Labeled apoB28F secreted from McA-RH7777 cells was immunoprecipitated and boiled in the absence or presence of 50 mM DTT. After alkylation of free sulfhydryls with IAA, samples were analyzed by nonreducing SDS-PAGE. As observed in Fig. 1C, a subtle but reproducible mobility shift was observed upon DTT-mediated reduction of secretory apoB28F (Fig. 1C, compare lanes 1 and 2, 3 and 4). ApoB28F Undergoes Rapid Folding into a DTT Resistant Form-Utilizing both mobility changes during nonreducing SDS-PAGE as well as onset of DTT resistance as criteria, the folding kinetics of apoB28F were examined in transfected McA-RH7777 cells. The underlying basis for the ability of proteins to undergo a transition from DTT sensitivity to DTT resistance in the ER is not fully understood. The transition may reflect conformational changes in the protein and/or different extents or stages of chaperone association (34,35). Nonetheless, for several model proteins previously examined, the rate of onset of DTT resistance in the ER closely parallels the time required to achieve a native conformation competent for anterograde transport in the secretory pathway (34,36). Stably transfected McA-RH7777 cells were pulse-labeled for 1 min with [ 35 S]Met/Cys and chased for various times prior to adjusting the chase media to 2 mM DTT (30). After incubating with DTT for 5 min (a condition that allows elongation of nascent polypeptide chains (37)), cells were placed on ice and treated with an excess of IAA (30). ApoB28F immunoprecipitated from cell lysates was resolved by nonreducing SDS-PAGE. As observed in Fig. 2A, lanes 1-7, the predominant form of apoB28F migrated with the folded standard (lane R) and at no time after the pulse did addition of DTT cause appreciable reduction. Since proteins labeled during the 1-min pulse were, by definition, nascent polypeptides at the time that [ 35 S]Met/ Cys was added to the cells, and it is estimated that ϳ3-4 min is required to synthesize apoB28F (ϳ300 -400 amino acids per minute (37)), these results indicate that the disulfide bonds in apoB28F progressed to the DTT resistant form either before or shortly after (within 1 min) translation was completed. Although the anti-FLAG antibody allowed us to monitor only full-length protein, use of a polyclonal antibody with the potential to recognize amino-terminal epitopes of apoB28F (38) failed to detect an appreciable spectrum of nascent polypeptide chains under our labeling conditions (not shown). Furthermore, it may be difficult to detect and interpret SDS-PAGE mobility shifts based on differences in disulfide bond formation within a spectrum of nascent polypeptide chains. The rapid onset of DTT resistance observed for apoB28F is in marked contrast to all other disulfide bonded proteins examined to date which undergo a time-dependent post-translational transition from DTT sensitivity to DTT resistance in the ER (9,34,36,39,40). As an example, the onset of DTT resistance of albumin in HepG2 cells was examined. Although 11 of the 17 disulfide bonds in albumin form cotranslationally, with the remainder forming almost immediately after translation (41,42), the disulfide bonds in albumin remained sensitive to DTT-mediated reduction well after completion of synthesis ( Fig. 2A, lane 1). Approximately 3 min was required for half of the labeled albumin to achieve DTT resistance after a 1-min pulse (Fig. 2B, lane 4). Other disulfide bonded secretory proteins examined in this way often display considerably longer periods of DTT sensitivity in the ER (34,36,40). Rapid Folding of the Amino-terminal Domain of ApoB Occurs Independently of MTP and Buoyant Lipoprotein Formation-We investigated whether the rapid folding of apoB28F, as measured by its almost immediate onset of DTT resistance, was an autonomous property of the apoB protein or whether folding was dependent upon the presence of liver specific factors such as MTP. For this purpose, apoB28F was stably transfected into CHO cells, an MTP negative cell line that lacks the capacity to form secretable lipoproteins (17). Although CHO cells cannot efficiently secrete apoB28F (not shown), we also explored whether or not CHO cells were capable of lipidating apoB28F by the MTP-independent pathway that may exist in some nonhepatic cell lines (43). McA-RH7777 and CHO cells, stably transfected with apoB28F, were labeled for 2 h with [ 35 S]Met/Cys. Post-nuclear membranes were prepared and subjected to extraction with sodium carbonate, pH 11.5, to release lumenal contents (44,45). After centrifugation, pellet fractions were solubilized and immunoprecipitated with anti-FLAG M2 monoclonal antibody. Supernatant fractions were adjusted to 1.25 g/ml with KBr and subjected to density gradient centrifugation as described under "Experimental Procedures." The top 1-ml fractions (d Ͻ 1.25 g/ml) and bottom 2-ml fractions (d Ͼ 1.25 g/ml) were immunoprecipitated and analyzed along with the carbonate pellets by SDS-PAGE and fluorography. As shown in Fig. 3A, in McA-RH7777 cells, the carbonate extractable apoB28F was distributed in an ϳ2:1 ratio between the top (T) and bottom (B) gradient fractions. In contrast, CHO cells were incapable of converting apoB28F into a form with sufficient lipid to float in the d Ͻ 1.25 g/ml fraction. To examine the contribution of MTP and events associated with buoyant lipoprotein formation on apoB28F folding, the experiment in Fig. 2 was repeated in CHO cells. When 2 mM DTT was added to transfected CHO cells immediately after the 1-min pulse, the folded form of apoB28F represented ϳ70% of total (Fig. 3B, lane 2). This result was somewhat different from that obtained in McA-RH7777 cells where ϳ90% of apoB28F resisted DTT immediately after the 1-min pulse ( Fig. 2A, lane 1). Both of these results, however, were distinct from the behavior of albumin (Fig. 2B) and other proteins whose disulfide bonds remain completely sensitive to DTT-mediated reduction for a characteristic period of time after completion of translation. That the majority of apoB28F in CHO cells achieved DTT resistance immediately after the pulse indicated that its capacity to fold rapidly occurred independently of MTP, and independently of events necessary for buoyant lipoprotein assembly. This conclusion was further supported by the finding that, when apoB28F was reduced and then allowed to refold, the rate and extent of post-translational folding in CHO and McA-RH7777 cells was also similar (see below). Reduced ApoB28F Remains Lipid Poor in the Hepatic ER-To examine the intracellular consequences of blocking amino-terminal folding, the buoyant density of apoB28F extracted from cells that were labeled under control and reducing conditions was examined. For these experiments transiently transfected HepG2 cells were used because, unlike McA-RH7777 cells, they express detectable amounts of serum albumin which is useful as an endogenous disulfide bonded control protein (not shown). Forty-eight hours after transfection, HepG2 cells were pulse-labeled for 10 min in the absence or presence of 2 mM DTT. Where DTT was used, it was added 1.5 min prior to addition of label. Under these conditions, ϳ80% of the labeled pool of apoB28F is reduced ("Experimental Procedures"). After metabolic labeling, post-nuclear membrane fractions were prepared from control and DTT-treated cells and extracted with sodium carbonate, pH 11.5, to release their lumenal contents. Pellet and supernatant fractions were analyzed as described for Fig. 3A. As shown in Fig. 4, when cells were labeled in the absence of DTT, the majority of apoB28F was recovered in the carbonate supernatant (S) which in turn was distributed in an ϳ2:1 ratio between the top and bottom density gradient fractions. In DTT-treated cells, a greater proportion of apoB28F remained associated with the membrane pellet (ϳ50%). This material copelleted with membranes due either to membrane binding and/or because of DTT-induced aggregation (31,46). Of the carbonate releasable material, however, greater than 90% was recovered in the bottom (d Ͼ 1.25 g/ml) gradient fraction (compare lanes 5 and 6). A portion of each gradient fraction was also analyzed for albumin content. Under both reducing and nonreducing conditions, all of the albumin was recovered in the bottom fraction and was FIG. 4. Reduced apoB28F is unable to form of a buoyant lipoprotein. Transiently transfected HepG2 cells were pulse-labeled for 10 min in the absence or presence of 2 mM DTT as indicated. Where DTT was included, it was added 1.5 min prior to addition of label. For cells incubated without DTT, three 100-mm dishs of cells were employed. For cells incubated with DTT, six 100-mm dishes were used ("Experimental Procedures"). A post-nuclear membrane fraction was prepared and extracted with sodium carbonate, pH 11.5. Supernatant fractions (S) were subjected to density gradient centrifugation as described for Fig. 3A. ApoB28F contained in the top 1-ml (T) and bottom 2-ml (B) gradient fractions as well as carbonate pellets (P) were immunoprecipitated with anti-FLAG M2 monoclonal antibody. ApoB28F was analyzed by 6% nonreducing SDS-PAGE and fluorography. The position of reduced and folded forms of apoB28F are indicated. either fully reduced or folded, depending upon the labeling condition (not shown). These results indicate that folding of the amino-terminal domain is an essential prerequisite for apoB28F's ability to recruit lipid in the hepatic ER. Post-translational Folding of ApoB28F-While disulfide bond formation is in many cases cotranslational, proteins artificially reduced in the ER by DTT can fold post-translationally and undergo efficient secretion (30,39,47). It was previously shown that reduced apoB100 was incapable of achieving secretion competence post-translationally (9). However, because of its large size it was impossible to assess whether this was due to an inability to achieve post-translational disulfide bond formation or whether subsequent steps required for its assembly and secretion were blocked. To examine this question, transiently transfected HepG2 cells were pulse-labeled for 10 min in the presence of DTT to produce reduced apoB28F. After the pulse, the cells were chased for various periods of time in the absence of DTT. After each chase period, cells were placed on ice, treated with IAA, and prepared for immunoprecipitation. As observed in Fig. 5A, upon removal of DTT, reduced albumin underwent a process of post-translational folding as evidenced by its almost immediate (within 0.5 min) conversion to a diffuse series of electrophoretic forms representing intermediates in folding. Within 4 min, the diffuse grouping of bands was predominantly converted to a more rapidly migrating species corresponding to the folded form. This form has been shown previously to undergo efficient secretion (9,30). When apoB28F was examined in the same extracts, it also underwent a timedependent conversion from the reduced to the folded form which, based on nonreducing SDS-PAGE mobility, was complete within 4 min. In addition to its comigration with native folded apoB28F by nonreducing SDS-PAGE, ϳ70% of the posttranslationally folded apoB28F resisted reduction with DTT within 4 min of DTT removal (not shown). This post-translational folding reaction was not facilitated by MTP or events associated with apoB28F lipidation as similar results were observed when the experiment was repeated in CHO cells (not shown). It appears, therefore, that the amino-terminal domain of apoB28F is capable of relatively rapid post-translational folding in the ER. Post-translational Lipidation of ApoB28F Is Dependent Upon Folding of the Amino-terminal Domain-Since apoB28F can fold post-translationally, we examined whether post-translational folding was accompanied by a process of post-translational lipidation. Transiently transfected HepG2 cells were pulse-labeled in the presence of DTT for 10 min and then chased for 0, 5, or 15 min in the absence of DTT. At the end of the labeling period, post-nuclear membranes were prepared and treated with sodium carbonate, pH 11.5, to extract lumenal contents. After centrifugation, pellet and supernatant fractions were analyzed as described for Figs. 3A and 4. As observed in Fig. 6A, lanes 2 and 3, cells labeled for 10 min in the presence of DTT contained predominantly lipid-poor apoB28F in the carbonate supernatants (i.e. most was recovered in the bottom fraction of the gradient). Although it was previously demonstrated that post-translational folding was essentially complete within about 4 min of DTT removal (Fig. 5), this folding was not accompanied by appreciable post-translational lipidation as the percentage of d Ͻ 1.25 g/ml apoB28F was not significantly changed after the 5-min chase (compare ratio of apoB28F in lanes 2 and 3 versus lanes 5 and 6). During this time frame, a greater proportion of apoB28F became carbonate extractable. Only ϳ35% of apoB28F was extracted after the 0-min chase whereas ϳ65% was extracted after the 5-min chase (compare ratio of apoB28F in supernatant and pellet fractions in lanes 1-3 and 4 -6). This result has been consistently observed and indicates that, as apoB28F undergoes posttranslational folding (Fig. 5B), some of it is converted from carbonate-resistant to carbonate-extractable form, perhaps reflecting disaggregation and/or release from the ER membrane. However, virtually none of the carbonate-resistant apoB28F that was released into the supernatant after 5 min of chase was lipidated. After 15 min of chase, the proportion of carbonateextractable apoB28F was unchanged. During this same time frame, however, the proportion of the apoB28F in the d Ͻ 1.25 g/ml fraction was increased by ϳ4-fold (compare lanes 2 and 3 with 8 and 9). This increase in the percentage of buoyant apoB28F formed post-translationally was dependent upon amino-terminal folding as it was not observed if DTT was present throughout the 15-min chase period (lanes 10 -12). To quantitate the extent of post-translational lipidation of apoB28F following DTT removal, the experiment in Fig. 6A was performed in triplicate and mean Ϯ S.D. values were calculated. As shown in Fig. 6B, apoB28F consistently underwent a characteristic post-translational lipidation reaction that resulted in an ϳ4-fold increase in d Ͻ 1.25 g/ml apoB28F within 15 min of DTT removal. Taken together, the data in Fig. 5B and Fig. 6 indicate that the post-translational lipidation of apoB proceeds through two distinct stages. DTT removal from cells containing reduced apoB28F results in a process of posttranslational folding (Fig. 5B) and possible disaggregation which, however, is not accompanied by appreciable lipidation (Fig. 6A, lanes 5 and 6). After folding has been completed, apoB28F appears to undergo a relatively slow (as compared with the usual cotranslational pathway) process of lipidation that is easily detected within 15 min of DTT removal. The lipidation that occurs after the completion of folding is MTPdependent as it was not observed in CHO cells (not shown). Characterization of ApoB28F-containing Lipoproteins Formed by the Post-translational Assembly Pathway-To determine if the apoB28F-containing lipoprotein particles formed post-translationally were secretion competent, transfected HepG2 cells were pulse-labeled with [ 35 S]Met/Cys in the presence of DTT to produce predominantly reduced apoB28F and then chased for either 0 or 60 min in the presence or absence of DTT. At the end of the 60-min chase in the presence of DTT, less than 2% of the apoB synthesized was recovered in the medium (Fig. 7A). When the chase was performed in the absence of DTT the percentage of apoB28F recovered in the medium increased by greater than 4-fold, confirming that the lipoproteins formed post-translationally underwent secretion. To further characterize the nature of the apoB28F-containing lipoproteins formed post-translationally, their buoyant density gradient profile was examined. Transiently transfected HepG2 cells were pulse-labeled for 10 min in the presence of DTT and chased in the absence of DTT. After the chase, media was recovered, concentrated, and subjected to density gradient centrifugation as described for Fig. 1A. Although approximately 80% of the labeled apoB28F in media from such an experiment is due to post-translational folding and assembly (Fig. 7A), no evidence was obtained to indicate that the buoyant density profile of this lipoprotein population differed from that observed in control cells (Fig. 1A). This result, along with the secretion data (Fig. 7A), indicates that, although the usual pathway for apoB assembly occurs cotranslationally, apoB28F is capable of undergoing a post-translational folding and assembly process which gives rise to a native, secretion-competent lipoprotein particle. As is the case for normal cotranslational assembly (Fig. 4), the post-translational pathway also demonstrates that amino-terminal folding is a prerequisite for apoB's capacity to undergo MTP-dependent assembly with lipid. DISCUSSION Recent studies in hepatoma cells indicate that translation of the amino-terminal ϳ25% of apoB is sufficient to produce a small lipid core-containing lipoprotein particle (14,19). Once formed, this small dense particle appears to undergo further MTP-dependent enlargement and maturation concomitant with the ongoing translation of apoB (8). Additional lipid may also be added to nascent lipoprotein particles post-translationally (48 -50). While a conceptual framework exists for understanding how a small dense lipoprotein formed by the aminoterminal ϳ25% of apoB can be enlarged by both co-and posttranslational mechanisms, little is known about how the process of lipoprotein formation is initiated. Considering the cotranslational nature of lipoprotein assembly we assumed that steps required to initiate the formation of a small dense lipoprotein particle (e.g. that formed by apoB28) should be the same as those required for native forms of apoB48 and -100. We, therefore, examined the biogenesis of apoB28F with a particular focus on events that precede and, therefore, may be responsible for its capacity to function as an acceptor for MTPmediated lipid transfer in the hepatic ER. Using both electrophoretic mobility changes during nonreducing SDS-PAGE and the onset of DTT resistance as criteria for protein folding in vivo, as used extensively by others (33-36, 39, 47), the rate and extent of folding of the amino-terminal domain of apoB28F was examined in both hepatoma and CHO cells. As with many disulfide bonded proteins, these studies revealed that apoB28F and, we presume by analogy apoB100, forms the bulk of its amino-terminal disulfide bonds during the process of translation. Unlike other proteins, however, apoB28F did not display a distinct temporal window in which its disulfide bonds were sensitive to DTT-mediated reduction in the ER (9,34,36,39,40). Interestingly, this uniquely rapid onset of DTT resistance was not dependent upon MTP or lipoprotein formation, as the rate and extent of folding was similar in CHO cells, a cell line that can neither lipidate nor secrete apoB28F (Fig. 3). Hence, the rapid folding of the aminoterminal domain is an autonomous property of the apoB protein and not a consequence of its extensive assembly with lipids. The underlying basis for the rapid onset of DTT resistance of apoB28F is not fully understood. The ability of DTT to reduce newly disulfide bonded proteins in the ER is an ATP-requiring event suggesting the involvement of ER-localized chaperones (34,35). That the amino-terminal domain of apoB28F progresses so rapidly to a form that resists DTT may reflect its rapid rate of folding and/or its unusually short-lived interactions with ER chaperones. Irrespective of its underlying basis, it is clear that the disulfide bonds in the amino-terminal domain of apoB achieve DTT resistance cotranslationally. The amino terminus of apoB may, therefore, represent a modular domain whose folding occurs during its translation and whose function is essential for subsequent stages of lipoprotein assembly. This latter possibility was explored by examining the intracellular fate of apoB28F translated under conditions that blocked folding of its amino-terminal domain. When folding of the amino-terminal domain was disrupted by preincubation of transfected HepG2 cells with 2 mM DTT, apoB28F was unable to recruit sufficient lipid to float at d Ͻ 1.25 g/ml. In contrast, ϳ50% of apoB28 was present in d Ͻ 1.25 g/ml fraction under control labeling conditions. This implies that, the strong lipid binding sequences in the form of predicted amphipathic ␤-sheet domains positioned between apoB20 and -28 (11) are not alone capable of sequestering appreciable lipid in vivo without the participation of the amino-terminal domain. This also indicates that the primary defect in apoB100 secretion caused by misfolding of the amino-terminal domain, which we observed in an earlier study (9), is most likely the result of an inability to recruit lipid and not due to production of a lipoprotein particle that is structurally aberrant and, therefore, incapable of anterograde sorting within the secretory pathway. Nevertheless, it is a formal possibility that, unlike apoB28F, longer forms of apoB with their more extensive lipid binding sequences may have the capacity to overcome the assembly block caused by misfolding of the amino terminus. To further examine the relationship between amino-terminal folding and MTP-dependent lipid transfer to apoB, we explored the possibility that apoB28-containing lipoproteins could be assembled post-translationally. Although in many cases disulfide bond formation occurs cotranslationally, studies in DTTtreated cells have demonstrated that artificially reduced proteins readily undergo post-translational folding once the normal oxidizing conditions within the ER are re-established (30,39,40,47,51). When DTT was removed from transfected hepatoma cells, reduced apoB28F underwent a process of posttranslational folding, similar to that observed for albumin (Fig. 5) and other disulfide bonded proteins. Although the posttranslational folding of apoB28F into a DTT-resistant form was nearly complete within 5 min of DTT removal, very little posttranslational lipidation was observed within this time frame (Fig. 6). Hence, the folding of the amino-terminal domain of apoB into the DTT-resistant form is not, in and of itself, accompanied by extensive lipidation. However, after 15 min in the absence of DTT, a considerable proportion of apoB28F was chased from the d Ͼ 1.25 fraction to the d Ͻ 1.25 fraction. This result provided further compelling evidence that lipoprotein assembly cannot proceed until a critical function performed by the amino-terminal domain has been completed. An alternative explanation for the inhibitory effects of DTT on apoB28F assembly is that, rather than inhibiting apoB folding, DTT may reversibly inhibit one or more factors necessary for its assembly with lipids. This possibility is unlikely for several reasons: the activity of the most likely such factor, MTP, is unaffected in vitro by 2 mM DTT (not shown). Furthermore, the secretion and buoyant density of apoB100 secreted from HepG2 cells is completely unaffected by DTT if it is added after the translation of the amino-terminal ϳ20% of the protein has been completed (9). Therefore, any cellular processes required for the cotranslational enlargement of apoB20-containing particles is unaffected by DTT. Most critically, however, preliminary results indicate that, within the amino-terminal 11% of apoB, substitution of single cysteine pairs involved in disulfide bond formation with serine residues, profoundly inhibits apoB28F secretion and intracellular processing. 2 In contrast to the apoB28F results reported here, previous studies in HepG2 cells indicated that once reduced, apoB100 was incapable of undergoing post-translational assembly into a secretion competent form. At least two possible reasons may be responsible for the difference in behavior between apoB100 and apoB28F. First, apoB28F was found to be quite stable in reduced form. After a 10-min pulse in the presence of DTT, followed by a 60-min chase in the absence of DTT, 75 Ϯ 7% of the apoB28F synthesized under reducing conditions was recovered from cell pellets and media. When DTT was maintained throughout the chase the recovery was 89 Ϯ 14% (both values are mean Ϯ S.D., n ϭ 3). This stability is in contrast to apoB100 where only 16% of apoB synthesized under reducing conditions was recovered in cells and media after a 60-min chase (9). Considering this dramatic difference in intracellular stability, it is possible that the rate of degradation for reduced, unlipidated apoB100 far exceeds its potential rate of post-translational folding and lipidation. The other feature that distinguishes apoB28F from apoB100 is the extent of their amphipathic lipophilic domains. It is possible that the capacity of the ER protein folding machinery to mediate the disaggregation and/or refolding of apoB's lipophilic domains, once translocated into the ER in unlipidated or underlipidated form, may be inversely related to the length of these domains and hence their degree of aggregation. Although the present results demonstrate a distinct stage in apoB folding which precedes and appears to be essential for its MTP-dependent lipidation, the results do not directly demonstrate the specific role played by the amino-terminal domain of apoB. We had previously hypothesized that folding of the amino-terminal domain of apoB may be accompanied by an autonomous phospholipid recruitment step which would provide a lumenal lipid surface for MTP-mediated lipid transfer in the ER (9). This prediction is based, in part, on the fact that the amino-terminal domain of apoB can solubilize dimyristoylphosphatidylcholine vesicles in vitro (16) and that enrichment of hepatic membranes with the phosphatidylcholine/ethanolamine analog, phosphatidylmonomethylethanolamine, reduces the secretion of even small amino-terminal forms of apoB (52). In the present studies, folding of the amino-terminal domain of apoB was not accompanied by substantial lipid recruitment. However, depending upon the amount of lipid associated with amino-terminal folding, density gradient centrifugation may not be a sufficiently sensitive means of detecting such proteinlipid interactions. Recent results of Wu et al. (53) indicate that during the assembly of apoB100 in HepG2 cells, MTP and apoB100 form a complex which can be coimmunoprecipitated. It has since been shown that apoB-MTP interactions occur for forms of apoB containing as little as the amino-terminal 13% of the protein (54). These results indicate that a direct apoB-MTP interaction may be necessary for the selective transfer of lipid from the ER membrane or some other donor site to nascent apoB. The strict dependence of amino-terminal folding on the capacity of apoB to initiate lipid sequestration indicates that this domain may either directly (via a protein-protein interaction) or indirectly (via a protein-lipid interaction) mediate a critical apoB-MTP interaction necessary for lipoprotein formation. In conclusion, it appears that while much of apoB is composed of domains with avid and in some cases irreversible lipid binding properties, these sequences alone may not be sufficient to drive the normal process of lipoprotein assembly. In addition to apoB's lipophilic sequences, a relatively soluble amino-terminal domain appears to play an essential role in the initiation and perhaps subsequent stages of lipoprotein particle formation. Future studies of the structure and function of this domain will likely reveal important mechanistic insights into the process of apoB-containing lipoprotein assembly. Finally, the development of the post-translational assay reported here, which uncouples the assembly of apoB with lipids from the process of translation, may provide an additional valuable system to further dissect steps required for apoB's unique form of assembly in the hepatic ER.	v3-fos-license
2018-04-03T02:58:48.763Z	2012-05-05T00:00:00.000	208917668	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "http://journals.iucr.org/e/issues/2012/06/00/bg2457/bg2457.pdf", "pdf_hash": "1d669c6c5203910d074d6e42f7e279733a9fa85d", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2482", "s2fieldsofstudy": [ "Chemistry" ], "sha1": "4796971542d0b07bb8b690661c46456d21d03e9d", "year": 2012 }	pes2o/s2orc	6-Benzyloxy-2-phenylpyridazin-3(2H)-one In the title compound, C17H14N2O2, the central pyridazine ring forms dihedral angles of 47.29 (5) and 88.54 (5)° with the benzene rings, while the dihedral angle between the benzene rings is 62.68 (6)°. In the crystal, molecules are linked by two weak C—H⋯O hydrogen bonds and three weak C—H⋯π interactions. In the title compound, C 17 H 14 N 2 O 2 , the central pyridazine ring forms dihedral angles of 47.29 (5) and 88.54 (5) with the benzene rings, while the dihedral angle between the benzene rings is 62.68 (6) . In the crystal, molecules are linked by two weak C-HÁ Á ÁO hydrogen bonds and three weak C-HÁ Á Á interactions. As a continuation of our studies on the crystal structures of Pyridazinone analogues (Ju et al., 2011), we report here the synthesis and crystal structure, an ellipsoid plot of which is shown in Fig. 1. The central pyridazine ring forms dihedral angles of 47.29 (5)° and 88.54 (5)° with the two benzene rings, while the dihedral angle between the two benzene rings is 62.68 (6)°. The structure is stabilized by two weak C-H···O and three C-H···Cg intermolecular hydrogen bonds (Cg's: centroids of the benzene rings) (Table 1). Experimental 3-hydroxyl-1phenyl-6-pyridazone(0.94 g, 5 mmol), benzyl chloride(0.63 g, 5 mmol) and K 2 CO 3 (0.69 g, 5 mmol) were added to absolute ethanol(30 ml). The mixture was stirred in the room temperature for 10 h. The suspension was filtered and the residue was washed with absolute ethanol. The title compound was recrystallized from the mother solution and single crystals of (I) were obtained by slow evaporation. Refinement All H atoms were placed in calculated positions, with C-H = 0.95 Å and C-H = 0.99 Å, and included in the final cycles of refinement using a riding model, with U iso (H) = 1.2Ueq(C). Computing details Data collection: CrystalClear (Rigaku/MSC, 2005); cell refinement: CrystalClear (Rigaku/MSC, 2005); data reduction: CrystalClear (Rigaku/MSC, 2005); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: CrystalStructure (Rigaku/MSC, 2005 The asymmetric unit of the title compound, (I), with displacement ellipsoids drawn at the 30% probability level. where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.29 e Å −3 Δρ min = −0.20 e Å −3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.	v3-fos-license
2019-04-07T13:07:55.457Z	2014-01-10T00:00:00.000	101406833	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://doi.org/10.9734/bjast/2014/11152", "pdf_hash": "8246a2667e2b84276c0d2d0e331ec9adb8281ab6", "pdf_src": "MergedPDFExtraction", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2517", "s2fieldsofstudy": [ "Engineering", "Environmental Science" ], "sha1": "0957f1259004ca7e8f016496fc2570a345c18775", "year": 2014 }	pes2o/s2orc	Preliminary Study of an Efficient OTEC Using a Thermal Cycle with Closed Thermodynamic Transformations The research work is focused on thermal engine structures undergoing isobaric expansion-compression based thermal engines powered by ocean thermal energy. The isobaric expansion-compression based thermal cycles referred to in this paper, differs from the conventional quadrilateral Carnot based thermal cycles in that the conversion of heat to mechanical work is performed assuming a load reaction driven path function, where as heat is being absorbed (isobaric expansion process) and rejected (isobaric compression process), mechanical work is simultaneously performed without the conventional quasi-entropic expansion, contrary to what happens in conventional quadrilateral based Carnot engines. An analysis of the ideal thermal cycle performed by means of an isobaric expansioncompression based cylinder is carried out and results compared with some previously achieved results of conventional technology including that of a Carnot cycle operating under the same ratio of temperatures. Into the range of its inherent low operating temperatures high ideal thermal efficiency is achieved for hydrogen and helium as working fluids. The achieved results associated with a simple and compact machine Original Research Article British Journal of Applied Science & Technology, 4(26): 3840-3855, 2014 3841 envisage the way towards a new generation of ocean thermal energy convertor (OTEC) power plants operating with the isobaric expansion-compression based cylinder. INTRODUCTION All the known thermal cycles under use so far are derived from the Carnot engine, which means quadrilateral cycles in which ideally heat is absorbed at constant temperature (top temperature) and work is delivered when temperature decreases from the top temperature to approach the bottom temperature under a quasi entropic transformation. The power cycles that obey to this model are classified in two main groups based on the nature of the working fluid: gas power cycles and vapour power cycle. The difference between the two groups is that in the first case the working fluid is gaseous and does not experience any phase change, while for the second group, there is a liquid-vapour phase change process of the working fluid within the cycle. In Fig.1 a simple classification of heat engines based cycles is depicted. Thermal cycle based on load reaction driven path functions. Isobaric expansion based path functions at constant load, [19] Non Carnot based heat engine (a) (b) Renewable energy conversion techniques contribute on reducing carbon dioxide emissions, global warming, and environmental pollution. The ocean thermal energy conversion (OTEC) procedure uses the temperature difference between the surface warm seawater and the deep cold seawater to drive Rankine cycle based power plants as power source [1][2][3]. OTEC techniques exhibit a very small environment impact. However the low net efficiency of OTEC as consequence of its inherent low difference of operating temperatures restricts the implementation of this technology [4]. Consequently, improving the thermal efficiency of OTEC based power plants has becoming an important objective, for instance by selecting suitable working fluids for use in the Rankine cycles. In [5,6] the major components of an OTEC plant theoretically and experimentally have been studied. In their simulation results, the temperatures of warm and cold seawater were 26ºC and 4ºC, for optimising a 100-MW OTEC system. The authors reported also that R717 was one of the suitable working fluids for a closed Rankine cycle OTEC power plant. Using R717 as the working fluid, the authors of the work referenced as [7] studied theoretically the effects of temperature and flow rate of cold seawater on the net output of an OTEC power plant. They concluded that the network has a maximal output at a specific cold seawater flow rate. In order to enhance thermal efficiency while reducing system costs, the organic Rankine cycle (ORC) is used with working fluids that have a low boiling temperature. In this way, in [8] and [9] suitable working fluids for an ORC have been investigated to convert low-grade heat. The design for an OTEC based ORC with R717 and R134a has been optimised in [10]. Various applications aiming to increase the thermal efficiency of the OTEC system uses solar heat energy [11,12] and the waste heat of nuclear power condenser [13] to increase the temperature of warm seawater contribution on the thermal efficiency enhancement. In [14] an experimental study measured the temperature and pressure before and after each component of a demonstration OTEC system was reported. According to the report, a maximum thermal efficiency of approximately 1.5% was obtained in the OTEC system when R134a was used as the working fluid. They also determined that both the thermal efficiency and the power output of the system increased upon increasing the operating temperature difference, which is the temperature difference between warm and cold seawater. In [15] a regenerative ORC in which R123 was used as working fluid, and reported that the performance of basic ORC system and the regenerative system were 6.15% and 7.98%, respectively, with a 130ºC geothermal heat source has been experimentally investigated. These results showed that the regenerator in the ORC system improved by 1.83% as the power output was 6kW. From the points of view of performance and economy, finding the optimal operating temperature of the heat exchanger is critical for making the heat exchanger as small as possible when using an available temperature difference of only approximately 20ºC. The heat transfer performance in exchangers of the Rankine cycle has been considered widely to evaluate OTEC systems from an economic standpoint. An objective function, which represented the ratio of total heat transfer area to the net power output of the OTEC system, was presented in [16], who analyzed the optimization for a 1-MW OTEC power system operated in a simple closed Rankine cycle with R717 being used as the working fluid. In [17] A Preliminary assessment of OTEC resources has been performed. Calculations indicated a long-term heating of the tropical water column if deep cold seawater were used at flow rates per unit area of the order of the average abyssal upwelling. The author concluded that this phenomenon would correspond to a transient cooling of the tropical mixed layer, and that such predictions should be further evaluated with a three-dimensional model of the oceanic circulation since a one-dimensional representation does not allow any potential adjustment from convective horizontal currents. According to the mentioned study, about 5 TW of steady-state OTEC power may be available at most. This is slightly more than a recent estimate based on conservative standardised OTEC conditions but it remains much smaller than values generally available in the technical literature. However, the present OTEC resource estimates still largely exceed today's worldwide electricity consumption. It is unlikely that a possible lack of sustainability of OTEC resources at very large scales will ever be tested in practice. Taking into consideration the studies carried out in [17], a limited amount of OTEC based power should be installed and exploited in order to assure long time sustainability. The work has been organised into 5 main sections, where section is 2 is devoted to the description of the combined OTEC power plant, section 3 describes the new thermodynamic concepts regarding the thermal cycle options, in section 4 a case study is included in which the performance analysis and discussion of results have been carried out on a feasible structure of the projected plant, and finally in section 5 some relevant conclusions are highlighted. DESCRIPTION OF THE OTEC POWER PLANT OPERATING WITH CLOSED ISOBARIC EXPANSION-COMPRESSION BASED TRANSFORMATIONS The core of the proposed OTEC power convertor is the isobaric expansion-compression based cylinders (IECC), which is schematically depicted in Fig. 2. The OTEC-IECC system shown in Fig. 2 uses four operating fluids: -the thermal working fluid (WF) confined into the cooler and heater heat exchangers and the cylinder, -the cooling working fluid consisting of cool deep seawater necessary to cool the WF acting as heat rejection device which transfers heat from cylinder to the environment, and -the heating working fluid pumped from the hot seawater surface by means of the HSW necessary to heat the WF, and -the hydraulic fluid responsible for transferring hydraulic energy to the hydraulic motor-generation set. The proposed OTEC-IECC depicted schematically in Fig. 2, is composed of the following components: actuator cylinder, coolers, heaters, I/O cooler valves, I/O heater valves, regenerator valve, hot sea water pump, cold sea water pump, reciprocating hydraulic pump equipped with inlet and outlet three way-two position valves, and a hydraulic motorgenerator set. Description of the IECC Functioning The IECC is an active part of the OTEC responsible for converting the thermal energy of a gaseous working fluid into mechanical work. According to the structure of the IECC, two general modes of converting thermal energy to mechanical work are feasible: -Conversion of thermal energy to mechanical work by cooling a gaseous working fluid and, -Conversion of thermal energy to mechanical work by heating a gaseous working fluid. Fig. 2. Structure of the OTEC implemented with an actuator cylinder as power power convertor The processes of cooling and heating a working fluid, which are responsible for converting heat into work (performing mechanical work), are depicted in Fig. 3. The process of converting partially the thermal energy contained into a gaseous working fluid to mechanical work can be carried out by rejecting heat to the environment for which the condition that the cooling environment temperature is colder than the temperature of the working fluid must be met. To show such paradigm, a double effect cylinder equipped with two coolers and two heaters is depicted in Fig. 3(a). The closed thermodynamic transformations carried out during the cooling process of the cylinder chamber X is referred to the T-s diagram shown in the Fig. 3(b), where the state changes (4)-(5)-(6)-(1) are performed while cooling the cylinder chamber X. In the same way, the process of converting partially the thermal energy contained into a gaseous working fluid to mechanical work can be carried out by absorbing heat from a heat source for which the condition that the heating environment temperature is hotter than the temperature of the working fluid must be met. Thus, the closed thermodynamic transformation carried out during the heating process of the cylinder chamber X is referred to Performing mechanical work by cooling a fluid Considering the piston located at position Y and the two position three way valves positioned so that cooler X and heater Y are active, with the rest of heat exchangers inactive, the energy balance for the cooling transformation yields ) ( has been assumed. Therefore the mechanical work delivered during the cooling process is defined as From equations (1) and (2) follows that mechanical work is being developed by rejecting heat as qo. Performing mechanical work by heating a fluid Considering the piston located at position X and the two position three way valves positioned so that the heater X and the cooler Y are active, with the rest of heat exchangers inactive, the energy balance for the heating transformation yields has been assumed. As consequence, the mechanical work delivered while heating process is defined as From equations (3) and (4) follows that mechanical work is being developed by absorbing heat as q i . Description of the OTEC Thermal Cycle The structure of the thermal cycle composed by IECCs (isobaric expansion-compression based cylinders) which exhibits certain similarity with the plant structure described in [18] is shown in Fig. 2 and Fig. 4 respectively. The cycle functioning consists of four main operating steps described according to the plant structure depicted in Fig. 4 (a) and the T-s diagram of the Fig. 4 (b). The status of the valves is described in Table 1 for non regenerative and regenerative cycles. Non-regenerative IECC The non regenerative cycle is composed by the following steps according to the information depicted in Fig. 4(a) and Fig. 4(b): where hot WF expands at constant pressure p 3 =p 2 into the side (X) of the cylinder coming through the valve (HV) at high temperature, and simultaneously, cooled WF is being compressed at constant pressure p 4 = p 1 into the side (Y), leaving the cylinder through the valve (CV) at low temperature. -step (3)-(4)-(1)-(2) where hot WF expands at constant pressure p 2 = p 3 into the cylinder through the valve (HV), and simultaneously cooled WF is being compressed at constant pressure p 4 = p 1 into the side (X) leaving the cylinder through the valve (CV), completing a cycle. Regenerative IECC The regenerative IECC is composed by the following steps according to the information depicted with Fig. 4(a) and Fig. 4(c): -step (6)-(1) corresponding to the regeneration phase, where the regeneration valve (RV) remains open and the pressure in the side (X) of the cylinder increases from p 6 to p 1 =p 4 and in the side (Y) decreases from p 3 to p 4 = p 1 , so that the pressure in both sides is equal p 4 = p 1 . -step (1)-(2)-(3) where hot WF expands at constant pressure p 3 =p 2 into the side (X) of the cylinder coming through the valve (HV) at high temperature, and simultaneously, cooled WF is being compressed at constant pressure p 5 = p 6 into the side (Y) at low temperature. -step (4)-(5)-(6) where hot WF expands at constant pressure p 2 = p 3 into the cylinder , and simultaneously cooled WF is being compressed at constant pressure p 5 = p 6 into the side (X), completing a cycle. MODELLING THE CLOSED TRANSFORMATION BASED NON REGENERATIVE IECC This section introduces a class of thermal engines characterised by its ability to develop mechanical work simultaneously during the heat absorbing and heat rejection processes. In order to show the behaviour of a generic IECC to which the Carnot statement does not apply, the case of a double acting cylinder designed to develop mechanical work under a constant load-based path function (isobaric path function at constant load) along its active strokes is described. The Non Regenerative IECC In order to apply a technique on closed system based transformations in which the cooling phase (4)-(1) responsible for heat rejection is carried out by performing simultaneously useful mechanical work, the thermal cycle depicted in Fig. 4(a) and 4(b) is analysed under ideal assumptions [19]. The supplied heat from an external power source is ( 2 3 2 2 3 2 3 2 3 2 2 3 23 32 The rejected heat to the heat sink is ) )( The specific work is then given as Thus the ideal thermal efficiency is Taking into account that the term ) ( (8) is almost the unity, in practical applications follows that equation (8) (9) which means that the cycle temperatures has very little influence on the ideal thermal efficiency. THE NON-REGENERATIVE CASE STUDY Cycle modelling is carried out with data extracted from [20] (REFPROP) and the tool Engineering Equation Solver (EES). The REFPROP consists of an up-to date data base used for property calculations and is available in commercial software packages whose library makes use of Helmholtz fundamental equation correlations to determine fluid properties from the highest-accuracy published data available in literature. Furthermore, the database can be linked with software based tools such Visual Basic, Fortran, C, and even spread sheets to help the automation of data processing. In the proposed case study the data necessary to carry out the performance analysis is presented in Tables A1 and A2 shown in appendix A. For the analysis of the cycle with hydrogen and helium as working fluids, several tests have been carried out to study it's the cycle performance and behaviour into a range of top temperatures of (294-300 K). Results and Discussion The results of the study achieved using the EES yield the values of Table 2 and Fig. 5 using the data shown in Table A1 of the appendix A. The data used in the case study for the study of the hydrogen and helium as working fluids is taken from reference [20]. Cycle computation is referred to the T-s diagram of Fig. 4(a) and 4(b) for the non regenerative IECC. The values of the state variables corresponding to each cycle state point for the two working fluids are shown in appendix A, where for every working fluid a range of top temperatures from 294K to 300K has been considered. The seawater temperatures are assumed into a range of 279 K for the cold seawater near to the seabed and 293-299 K for the warm seawater at the sea surface. According to the results shown in Table 2, as the top temperatures increases from 294 K to 300 K it is observed that the thermal efficiencies remain almost constant approaching 58.5% for hydrogen and 80.5% for helium. =280 K and T 3 = 298 K It is observed also that while the ideal thermal efficiency exhibits almost no temperature dependence, specific work depends strongly on temperature according to the results shown in Table 2. However, the pressure ratio (defined as the ratio of the highest (top) pressure to the lowest (bottom) pressure into the cylinder) exerts a strong influence on the specific work as depicted in Fig. 5. Consequently, the specific work depends on the top temperature and pressure ratio. From the commented premises follows that high operating pressure with low pressure ratios yield good performance on the basis of a low specific volume, which means low structural masses and sizes while keeping high constant thermal efficiencies. Restrictions on the thermal efficiency imposed by the Carnot factor cannot be applied to the IECC because of its very low dependence on the operating temperatures. As consequence of the analysis of the achieved results shown in Table 2 regarding the Carnot factor, follows that it isn't comparable to the results rendered by the IECC based OTEC because of the inherent differences of the proposed cycle. However the OTEC power plant investigated in [18] will be taken as reference to compare performance results. Thus, in [18] (10) Assuming the losses for the IECC based OTEC similar to the losses of the OTEC described in [18], because of its structural similarities with respect to its heat exchangers, the overall efficiency for a non regenerative IECC can be estimated as Therefore, the net IECC thermal efficiency has been computed for the same top temperatures assumed for the Table 1, which are within the range of 294 K and 300 K. As noted from Table 3, net efficiencies are yet substantially higher than both, the Carnot factor and the CAPILI engine studied in [18]. With the aim of scaling up the proposed model, the results achieved as consequence of the studied case have been extrapolated to a 10 MW OTEC. Thus, using hydrogen and helium as working fluids and using the results depicted in the Table A2 of the appendix A, the main operating parameters for the 10 MW OTEC are shown in Table 4. In this way, Table 4 shows the amount of required working fluid (WF mass kg), the volume at 11 bar (WF vol. m 3 ), including the volume of the heat exchangers located into the cylinder, the total heat flow (heat flow kJ/s), and the total seawater flow (seawater flow kg/s), which includes the cold seawater and the warm seawater. CONCLUSIONS A preliminary design study of an OTEC based power plant using the IECC characterised by its ability to delivering mechanical work while rejecting and absorbing heat under load reaction based path functions has been proposed, and the IECC operating optionally with hydrogen or helium as working fluids has been analysed. According to the characteristics of the new IECC, expressions for the ideal thermal efficiency have been achieved. The performance results have been compared with the results obtained for the Carnot cycle operating with the same range and ratio of temperatures, and compared also with that of reference [18]. The most important conclusion is related to the IECC thermal efficiency which largely exceeds the Carnot efficiency under appropriated operating conditions. The reason obeys to the fact that in a IECC, the following conditions are met: -The fact of performing mechanical work by rejecting heat to the environment means a relevant contribution to the thermal efficiency of the IECC. -The conventional quasi-isentropic expansion process is avoided in the IECC, so that the degradation of heat energy due to the isentropic efficiency is neglected. Consequently, the main reasons for the efficiency enhancement with respect to conventional OTECs operating under quadrilateral Carnot based cycles (conventional ORCs) are due to the association of the following contributions: -The cycle absorbs heat and simultaneously converts a fraction of the absorbed heat into mechanical work avoiding the conventional quasi-isentropic expansions which contributes on the heat degradation. -The selected working fluid due to its inherent characteristics and behaviour. On the basis of the feasible structure proposed for the IECC as well as the ideal thermal efficiency which is significantly increased in comparison with the conventional OTECs under realizable and viable conditions a new family of OTEC convertors is expected. Furthermore, the estimated net efficiency is accordingly high. Finally the widespread use of ocean thermal energy as power sources as well as the fact of increasing the thermal efficiency, apart from the fact of contributing on reducing the massive use of fossil fuels and consequently the global warming potential, including contaminant fossil fuels based emissions, supposes a relevant fact that needs to be experimentally researched. Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=592&id=5&aid=5353	v3-fos-license
2017-12-15T18:02:29.148Z	2017-12-15T00:00:00.000	7484871	{ "extfieldsofstudy": [ "Chemistry", "Medicine" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fncel.2017.00390/pdf", "pdf_hash": "855a0f323b7ce2ab4d77d692b6621bbef9c4ed67", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2526", "s2fieldsofstudy": [ "Biology" ], "sha1": "855a0f323b7ce2ab4d77d692b6621bbef9c4ed67", "year": 2017 }	pes2o/s2orc	Preferential Initiation and Spread of Anoxic Depolarization in Layer 4 of Rat Barrel Cortex Anoxic depolarization (AD) is a hallmark of ischemic brain damage. AD is associated with a spreading wave of neuronal depolarization and an increase in light transmittance. However, initiation and spread of AD across the layers of the somatosensory cortex, which is one of the most frequently affected brain regions in ischemic stroke, remains largely unknown. Here, we explored the initiation and propagation of AD in slices of the rat barrel cortex using extracellular local field potential (LFP) recordings and optical intrinsic signal (OIS) recordings. We found that ischemia-like conditions induced by oxygen-glucose deprivation (OGD) evoked AD, which manifested as a large negative LFP shift and an increase in light transmittance. AD typically initiated in one or more barrels and further spread across the entire slice with a preferential propagation through L4. Elevated extracellular potassium concentration accelerated the AD onset without affecting proneness of L4 to AD. In live slices, barrels were most heavily labeled by the metabolic level marker 2,3,5-triphenyltetrazolium chloride, suggesting that the highest metabolic demand is in L4 when compared to the other layers. Thus, L4 is the layer of the barrel cortex most prone to AD, which may be due to the highest metabolic demand and cell density in this layer. INTRODUCTION The brain is highly metabolically active and particularly vulnerable to metabolic insults. During global or focal ischemia, the limited supply of oxygen and glucose causes a fall in ATP levels, arrest in sodium-potassium pump activity and depolarization of neurons in the metabolically deprived brain regions (Lipton, 1999;Somjen, 2001). Release of potassium and glutamate into the extracellular space accelerates depolarization of the adjacent neurons igniting an avalanchelike wave of collective Anoxic Depolarization (AD), which shares many common features with the spreading depression (SD) described by Leao (Leao, 1947;Nedergaard and Hansen, 1993;Somjen, 2001;Pietrobon and Moskowitz, 2014;Dreier and Reiffurth, 2015;Hartings et al., 2017). Near total collective neuronal depolarization during AD is associated with large DC shifts of the extracellular local field potential, and with an increase in tissue light transmittance as a result of cellular swelling (Aitken et al., 1999;Joshi and Andrew, 2001;Somjen, 2001). AD is an initiator of the ischemic damage and irreversible loss of activity in the ischemic core in vivo and oxygen-glucose deprivation (OGD) induced injury in the submerged brain slices in vitro (Rader and Lanthorn, 1989;Tanaka et al., 1997;Joshi and Andrew, 2001) (for reviews, Martin et al., 1994;Lipton, 1999;Dreier, 2011). Factors that increase metabolic activity such as increased neuronal activity or elevated temperature accelerate AD onset and ischemic neuronal death, whereas reduction in metabolic demand is neuroprotective against ischemic damage (Dzhala et al., 2000;Joshi and Andrew, 2001;Tyzio et al., 2006). Different neuronal populations and structures display different sensitivities to ischemia that may involve different metabolism in different cell types (Kawai et al., 1992;Lipton, 1999). In the hippocampus, CA1 pyramidal cells display the highest vulnerability to ischemia, which correlates with the preferential initiation and spread of AD in the CA1 region of the hippocampus (Aitken et al., 1998;Basarsky et al., 1998). The neocortex also displays heterogenous incidence and propagation of SD and AD between different cortical regions and layers (Bogdanov et al., 2016;Kaufmann et al., 2017). Previous studies using slices of non-identified neocortical areas revealed AD and SD "tropism" for the superficial layers 2/3 (Basarsky et al., 1998;Joshi and Andrew, 2001;Kaufmann et al., 2017). The somatosensory cortex and particularly its whisker-related barrel region are highly sensitive to ischemia (Lin et al., 1990), and SD and AD preferentially arise in and propagate through the whisker barrel region of the parietal sensory cortex (Bogdanov et al., 2016;Kaufmann et al., 2017). However, the initiation and spread of AD across the layers of the somatosensory cortex remain largely unknown. Here, we addressed initiation and propagation of AD induced by OGD to mimic ischemia-like conditions in slices of the rat barrel cortex. We found that AD was specifically initiated in L4 barrels and its initial front preferentially propagated along layer 4. Preferential initiation and spread of AD in L4 correlated with the most intense L4 staining with TTC, a histological metabolic activity marker. We propose that sensitivity to metabolic insult is non-uniform across layers of the barrel cortex: it is highest in L4 barrels, where AD is preferentially initiated, that may involve the highest metabolic demand and cell density in this layer. Ethical Approval All animal-use protocols followed the guidelines of the French National Institute of Health and Medical Research (INSERM, protocol N007.08.01) and the Kazan Federal University on the use of laboratory animals (ethical approval by the Institutional Animal Care and Use Committee of Kazan State Medical University N9-2013). Electrophysiological Recordings Extracellular recordings of the local field potentials (LFP) were performed in the barrel cortex using single or 16-site electrodes. Single site glass pipette electrodes were pulled from borosilicate glass capillaries (BF150-86-10, Sutter Instrument, Novato, CA, USA) and had resistances of 2-3 M when filled with ACSF. Electrodes were connected via chlorided silver wire to the headstage of a MultiClamp700B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Recordings were performed in voltage-clamp mode and currents were inverted and voltage calibrated using 5 mV steps. 16-channel recordings were performed using Menendez-de La Prida style 16 shank silicone probes with a separation distance of 100 µm between electrodes (NeuroNexus, Ann Arbor, MI, USA). The signals from extracellular recordings using silicone probes were amplified and filtered (1,000×; 0-9 kHz) using a Digital Lynx SX amplifier (Neuralynx, Inc., Bozeman, MT, USA), digitized at 32 kHz and saved on a PC for post-hoc analysis. Stimulating bipolar electrodes were placed in the white matter or L6 above the recorded cortical column. Voltage pulses (10-50 V, 50 µs duration, 0.1 Hz) were applied to evoke LFP responses of 100-300 µV in L4. Optical Intrinsic Signal Recordings Optical intrinsic signal (OIS) recordings were performed using slice transillumination as described in Aitken et al. (1999). The slice was illuminated by a halogen lamp with a 775 nm bandpass filter and visualized using a BX51WI upright microscope equipped with a 4×/0.10 Plan N objective (Olympus, Tokyo, Japan). Images were acquired using a QIClick-R-F-M-12 CCD camera (QImaging, Surrey, BC, Canada) usually at 174 × 130 pixel resolution and 5 frames/s acquisition rate. In some experiments a higher resolution of 348 × 260 or 696 × 520 was used. TTC-Staining Brain slices were stained with 1% TTC (2,3,5triphenyltetrazolium chloride) in phosphate-buffered solution (PBS) for 2-3 min at 38 • C. Then slices were rinsed in PBS (for 1 min, 3 times). Microphotographs of TTC-stained slices were FIGURE 1 \| Oxygen-glucose deprivation induced anoxic depololarization in barrel cortex. (A) Scheme of the experimental setup. Submerged cortical slices is exposed to the ACSF in which oxygen is replaced by nitrogen and glucose is replaced by sucrose (oxygen-glucose deprivation, OGD) to mimic ischemic conditions. Local field potential (LFP) recordings and optical intrinsic signal (OIS) imaging in transmittance mode is performed to record anoxic depolarization (AD). (B) Microphotograph of the cortical slice. LFP is recorded from a cortical barrel, regions of interest (ROIs) in the supragranular, granular, and infragranular layers of are indicated by color boxes. Data Analysis Data were analyzed using custom-written procedures in Matlab (MathWorks, Inc., Natick, MA, USA). OIS was calculated using the first-frame subtraction approach: OIS(t) = (I(t) -I 0 )/I 0 , where I(t) -pixel intensity at the moment t, I 0 -time-averaged pixel intensity in the preconditioned baseline period (100 s). Resulting frames were filtered with a 10 × 10 median filter. Regions of interest (ROI) were selected as square areas near recording sites. OIS traces were calculated as the average OIS signal within selected ROIs. LFP signals were downsampled to 1 kHz. Continuous running line fit was removed using local linear regression in 300 s windows with a 10 s overlap [locdetrend function from the Chronux toolbox (http://chronux.org/)]. Amplitude of the LFP evoked response was calculated as a negative peak value of the LFP in the 100 ms after the stimulus relative to baseline level (average of the LFP in the 10 ms before the stimulus). Data were smoothed by the 1,000-point moving average filter and the first derivatives were calculated. Local negative peak time of the first LFP derivative was calculated within the 20 s window preceding the negative AD peak. The value of the LFP at this time was taken as 100% and the previous time corresponding to 30% considered as AD onset. Velocity of vertical AD propagation was calculated from onset values as a distance between neighboring recording sites (100 µm) divided by AD onset delays between corresponding channels. The baseline level was calculated for each recording site as the mean value of the LFP in the −20 to −10 s time window preceding AD onset. AD amplitude was calculated as the maximal negative LFP peak from the baseline. Data from different slices were aligned by L4 and average amplitude and onset depth profiles were calculated. Depth profiles of amplitude were smoothed by the 2-point moving average filter. OIS onsets and amplitudes were calculated in same manner. Microphotographs of TTC-stained slices were analyzed as follows. Pixel intensities were calculated along the barrel cortex and along the perpendicular direction intersecting the barrel and averaged in a 100 µm wide bar. Intensity was converted to a percentage (intensity value of each pixel divided by maximal intensity). Staining efficiency (opacity) was calculated as the value inverse to the calculated intensity. Statistical Analysis Statistical analysis was based on the nonparametric Wilcoxon (paired samples) or Mann-Whitney (independent samples) signed rank sum test with the significance level set at p < 0.05. Results are given as means ± SEM. Electrophysiological and Optical Intrinsic Signals during Anoxic Depolarization In the present study, we explored spatial-temporal dynamics of the OGD-induced AD in slices of the barrel cortex using extracellular recordings of LFP, and OIS recordings (Figures 1A,B). AD was initiated within 6-13 min (9.5 ± 0.5 min; n = 17 slices from 8 rats) and manifested as a sharp increase of light transparency attaining 29.2 ± 3.0% dI/I and negative LFP shift of 8.9 ± 0.6 mV in L4 (n = 17; Figure 1C). LFP signals were typically biphasic with an initial sharp negative transient followed by a secondary negative wave. The increase in OIS during AD started in L4 and further spread to L2/3 and L5/6 ( Figure 1D). The responses evoked in L4 by stimulation of the white matter or L6, progressively decreased during OGD and were completely and irreversibly abolished during and after AD (Figure 1C), while the evoked responses could recover following shorter OGD episodes without AD ( Figure 1E) that is in keeping with the results of previous studies (Rader and Lanthorn, 1989;Tanaka et al., 1997;Joshi and Andrew, 2001). In the experiment illustrated in Figure 1, OIS recordings revealed that AD unilaterally propagated through the slice with the leading front in L4 and delayed fronts in the supraand infragranular layers ( Figure 1F). The increase in light transmittance was followed by a decrease in light transmittance probably reflecting cellular swelling followed by dendritic beading (Aitken et al., 1999;Joshi and Andrew, 2001;Somjen, 2001). After AD in the barrel cortex, AD was also observed in the hippocampus and striatum after a several minute delay ( Figure 1F). Vertical AD Propagation in a Cortical Barrel Column We also performed simultaneous OIS and multisite LFP recordings from a cortical barrel column using 16-shank silicone probes (Figures 2A,B). In keeping with the results described above, AD was initiated in L4 and spread to L2/3 and L5/6 Red circles indicate the OIS-AD onset. (F) Group data on the onsets and amplitudes of OIS associated with OGD-induced AD as a function of cortical depth (mean ± SE, n = 7). Note that AD first occurs in L4 and further spreads to L2/3 and L5/6 and that AD amplitude is maximal in L2/3. with a velocity of 4.0 ± 0.1 mm/min (n = 7 slices from 4 rats). The maximal amplitude of negative LFP shift during AD was observed in the superficial layers (13.8 ± 0.8 mV at a depth of 400 µm from the cortical surface; n = 7; Figures 2C,D). In L4 and L5/L6 the amplitude of AD was 12.0 ± 0.6 and 10.7 ± 0.7 mV, respectively (n = 7). The OIS profile of AD was remarkably similar to that of the electrophysiological response including an initial onset in L4 and vertical delays in the supraand infragranular layers, the speed of vertical propagation, and a maximal amplitude of light transmittance increase of 38.5 ± 10.3% (n = 7) attained in L2/3 and smaller change in deeper layers (Figures 2E,F). Patterns of AD Initiation and Propagation OIS imaging revealed variability of the AD initiation and propagation patterns, which could be classified in four main groups (Figure 3 and Videos 1-4): 1. Single-barrel AD initiation (Figure 3A; Video 1). AD emerges in one barrel within the imaging window and spreads concentrically. The AD spread is often anisotropic with a preferential horizontal propagation along L4 forming a characteristic "bird head" OIS image. This pattern was observed in 11 of 30 slices. (Figure 3B; Video 2). AD emerges in two (or more) barrels within the imaging window. AD fronts move concentrically and collide first in L4 and then in the superficial and deep layers (n = 6 of 30 slices). Multiple-barrel AD initiation 3. One side propagating AD (Figure 3C; Video 3). AD originates on one side of slice but outside of the imaging window and spreads through the slice with a preference to L4 (n = 8 of 30 slices). 4. Double-side propagating AD (Figure 3D; Video 4). AD emerges on two sides of slice outside of the imaging window. Two AD waves move toward each other with a preference to the L4 and collide similarly to the multi-barrel initiation pattern (n = 5 of 30 slices). These results indicate that despite variety in the site of OGD-induced AD initiation in a slice, preference of AD to L4 is a hallmark of all initiation and propagation AD patterns. The rate of AD propagation along L4 was 1.7 ± 0.1 mm/min (n = 18 slices from 8 rats) which is consistent with the rate of AD and SD propagation in slices and in the intact brain in vivo (Nedergaard, 1996;Basarsky et al., 1998;Joshi and Andrew, 2001). In the cases of single-barrel AD initiation the rate of medial AD propagation along L4 was of 1.8 ± 0.1 mm/min that was not different from the rate of lateral AD propagation of 1.7 ± 0.1 mm/min (n = 11 slices from 4 rats, p = 0.76). We next addressed a question of whether AD in L4 is a necessary condition for emergence of AD in the supragranular and infragranular layers. In this aim, we explored OGD-induced AD after surgical cuts made above, below and through the L4 (Figure 4 and Videos 5-7). We found that AD efficiently invaded supragranular and infragranular layers even after disconnection from L4, with an AD front moving horizontally around the cuts thus indicating that both superficial and deep layers are capable of generating AD independently from L4. We further calculated the time difference between AD in the surface and deep layers FIGURE 4 \| AD propagation after surgical cut above, below and through L4. (A-C) Example microphotographs of the barrel cortex slices in DIC-IR (left) with the cuts made above (A), below (B), and through (C) the layer 4 and OIS snapshots at different time points after OGD induction. Below, the corresponding AD fronts are presented as the OIS first derivative. Bottom left panel shows time color coded SD front contours plotted at 3 s intervals. Note that AD waves invade supragranular and infragranular layers in all cases but AD emerges earlier if connection with L4 is preserved. AD propagation to L2/3 above the cut on panel (A) was too slow and is truncated on the contour map. See Video 5 for the entire AD wave in this experiment. at the areas vertically aligned to the middle of the cut. When the cut was made above L4, AD arrived to L2/3 88 ± 19 s later than to L5/6 (n = 4). When the cut was made below L4, AD in L2/3 emerged 49 ± 6 s earlier than in L5/6 (n = 5). Thus, AD was generated earlier in the layers maintaining connection with L4 than AD in the layers disconnected from L4. With the cut made through L4, the time delay between AD in L2/3 and L5/6 reduced to 12 ± 4 s (n = 4). Together, these results indicate that AD in L4 is not a necessary condition for AD in the surface and deep layers, where AD can propagate horizontally. However, vertical AD vector originating from L4 accelerates AD in the supragranular and infragranular layers maintaining their connection with L4. Elevated Extracellular Potassium Concentration Accelerates the AD Onset Hyperactivity compromises the metabolic state of the tissue under OGD-conditions and accelerates the AD onset in hippocampus (Dzhala et al., 2000). With the aim of exploring the effect of increased activity on the OGD-induced AD in the barrel cortex, we elevated extracellular potassium concentration in ACSF from 3.5 to 8.5 mM. The high-potassium solution itself induced a slight increase in light transmittance and a negative shift in the LFP baseline in L4 (Figure 5A). Further superfusion with high-potassium/OGD solution evoked AD with a delay of 5.9 ± 0.4 min (n = 13 slices from 5 rats), that was almost twofold quicker (p < 0.001) than AD evoked by OGD in normal potassium conditions (9.5 ± 0.5 min; n = 17 slices from 8 rats) (Figure 5C). In the high-potassium/OGD solution the negative LFP shift of 8.5 ± 0.6 mV (n = 13) in L4 was similar to those in normal potassium conditions (p > 0.05) while the increase in light transparency of 19.8 ± 1.0% dI/I (n = 13) was less than in normal conditions (p < 0.05), due to the progressive increase of dI/I before AD which occurs in elevated potassium conditions ( Figure 5A). OIS imaging revealed that preference of AD initiation and propagation in L4 was maintained under conditions of elevated potassium (Figure 5B). Highest Metabolic Activity in L4 Preferential initiation and propagation of AD in the barrels may involve the higher metabolic demand of barrels and therefore their higher vulnerability to metabolic deprivation. We explored this hypothesis using 2,3,5-triphenyltetrazolium chloride (TTC) staining of live slices of the barrel cortex. As shown on Figures 6A,D, TTC most intensively stained L4. Quantification (left) and OIS snapshots at different time points after OGD induction. (C) Group data on AD onsets in control ACSF (n = 17) and in the ACSF with potassium concentration elevated to 8.5 mM (n = 13). Each white circle corresponds to one slice and black circles show the mean ± SE. Note that AD onset is accelerated almost two-fold after elevation of extracellular potassium concentration. ***p < 0.001. of TTC-staining along the horizontal projection in L4 revealed peaks in TTC-staining corresponding to neighboring barrels ( Figure 6B). In the vertical projection across cortical depth, TTC staining peaked at the L4 depth ( Figure 6C). A second, less intense peak was found ∼0.5 mm deeper at the L5B/L6A border ( Figure 6C). Cross-layer comparisons revealed significantly higher TTC-staining of L4 compared to L2/3 (p < 0.05) and L5/6 (p < 0.05) (n = 7 slices from 4 rats; Figure 6E). Slices that had been exposed to OGD for 30 min and reperfused with normal ACSF for 2 h displayed only weak non-specific staining ( Figure 6F). TTC-staining of L4 in OGD-exposed slices revealed no difference with L2/3 (p > 0.05) and L5/6 (p > 0.05) staining and was significantly lower compared to control slices (p < 0.01; n = 9 slices from 4 rats) (Figure 6E). DISCUSSION The principal conclusion emerging from the present study is that different layers of the barrel cortex differ in their propensity to AD and that L4 is the most prone to AD. This conclusion is supported by electrophysiological recordings and OIS imaging indicating that OGD-induced AD is preferentially initiated in, and preferentially spreads through L4. We also found that enhanced L4 susceptibility to OGD correlates with the highest metabolic activity, in L4, revealed with TTC staining of live slices. Anisotropy is a characteristic feature of the heterogeneous incidence and horizontal spread of cortical SD in vivo (Kaufmann et al., 2017). Previous studies in non-identified neocortical areas revealed anisotropy of AD and SD across cortical layers with a "tropism" to the superficial layers 2/3 (Basarsky et al., 1998;Joshi and Andrew, 2001;Kaufmann et al., 2017). However, our findings indicate that in the barrel cortex, which contains large barrels and the thickest L4 of all the cortical regions, AD is initiated and preferentially propagates via L4. Onset and preferential propagation of AD in L4 was evidenced by the earliest onset of the negative LFP DC shift and the earliest increase in optical transparency in barrels during the OGD-induced AD. Multisite LFP recordings and simultaneous OIS imaging revealed vertical spread of AD from the L4 to the superficial and deep layers within a column. OIS recordings also enabled us to assess two-dimensional spatial-temporal AD dynamics in the barrel cortex slices revealing a variety of AD initiation and propagation patterns, yet with a common delimiter of the highest proneness of L4 to AD. While AD primarily originated in L4 in the barrel cortex, L4 appeared to be not necessary for the emergence of AD in the supragranular and infragranular layers, however. Indeed, our experiments with surgical cuts above, below and through the L4 revealed that AD may propagate through these layers horizontally around the cuts, although at lower speed. This indicates that supra-and infragranular layers are capable of generating AD independently from the L4. Yet, early ignition of AD in L4 is important for driving AD in the supra-and infragranular layers in the intact slice. Various factors have been suggested to explain anisotropy of SD and AD (Herreras and Somjen, 1993;Somjen, 2001;Canals et al., 2005;Kaufmann et al., 2017). High neuronal density in L2/3 has been hypothesized to promote a mutual promotion of depolarization and potassium release and accumulation, making these layers more prone to AD (Joshi and Andrew, 2001). In the barrel cortex, the highest neuronal density is observed in L4, where it attains 124 thousand neurons/mm 3 compared to 102 and 86 thousand neurons/mm 3 in L3 and L2, respectively (Meyer et al., 2010). Thus, our findings of the preferential initiation and spread of AD in L4 of the barrel cortex are consistent with the "neuronal density" hypothesis. Interestingly, SD and AD preferentially arise in and propagate through the whisker barrel region of parietal sensory cortex in vivo (Bogdanov et al., 2016;Kaufmann et al., 2017). Because the barrel cortex contains large barrels and the thickest L4 of all the somatosensory cortical regions, the elevated proneness of L4 to AD as revealed in the present study may also explain high proneness of the barrel cortex to AD. Our observations of heterogeneous TTC-staining in different cortical layers with the maximum in L4 suggest that elevated metabolic demand could also be a factor contributing to the particular susceptibility of this layer to AD. TTC staining intensity is determined by the metabolic activity of mitochondrial dehydrogenases, which enzymatically convert colorless TTC to red formazan (Goldlust et al., 1996). The elevated L4 metabolic activity revealed with TTC-staining is consistent with the highest density of a mitochondrial enzyme cytochrome oxydase and elevated number of mitochondria in L4 of the barrel cortex where they reside mainly in dendrites and axonal terminals (Wong-Riley and Welt, 1980). Considerable evidence indicates that elevation of metabolic debt strongly aggravates ischemic insults. Indeed, various factors increasing the metabolism such as an increase in neuronal activity caused by adenosine A1 receptor antagonists, blockers of GABA(A) receptors and potassium channels (Dzhala et al., 2000), elevation of extracellular potassium as in the present study or elevated temperature (Joshi and Andrew, 2001) strongly accelerate the AD onset. Thus, due to elevated metabolic activity, L4 neurons are most likely to quickly lose ATP, depolarize and ignite AD in metabolically-compromised conditions. The question then arises: why the metabolic activity is highest in L4 barrels? Although the underlying mechanisms are unknown, it could be suggested that it involves a particular cytoarchitectonic and synaptic barrel organization. Indeed, densely packed excitatory and inhibitory neurons form a highly interconnected network in L4 barrels (Feldmeyer et al., 1999;Lefort et al., 2009;Valiullina et al., 2016) that may impose a higher metabolic charge to equilibrate the ionic disturbances caused by the activity in this layer. Thus, in the present study we have shown that different cortical layers differ in their sensitivity to metabolic insult with L4 being the most prone to AD initiation and preferential propagation. Elevated ongoing metabolic demand of L4 could be a factor contributing to this enhanced sensitivity of L4 to OGD. Our findings also support rationale of the strategies aimed to reduce the metabolic demand as an approach to alleviate ischemic brain damage. AUTHOR CONTRIBUTIONS RK conceived the project. EJ and MM performed the experiments. AN, MM, EJ, AG, and MS analyzed the data. RK wrote the paper. ACKNOWLEDGMENTS This work was supported by RSF (17-15-01271) and performed in the framework of the Program of Competitive Growth of Kazan Federal University. We thank D. Suchkov for providing the code for the OIS acquisition.	v3-fos-license
2020-02-06T09:02:54.258Z	2020-01-29T00:00:00.000	238085136	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GREEN", "oa_url": "https://www.researchsquare.com/article/rs-12780/v1.pdf", "pdf_hash": "179a5c3e8475df9391efb8c9abb886065fd678d7", "pdf_src": "Adhoc", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2569", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "sha1": "22159ccb748e66aca76847d085abed076cecbd77", "year": 2021 }	pes2o/s2orc	Alleviating effects of silicon on cadmium toxicity in ginger ( Zingiber officinale Roscoe) Background: This study assessed the effects of varying concentrations of silicon (Si) on the reduction of cadmium (2mg/kg Cd) toxicity in Zingiber officinale Rosc. via growth modulation, photosynthetic efficiency, and antioxidant defense. Result: As the Cd level increased, the physiological indexes of ginger exhibited a decreasing trend. This trend was especially noticeable when the Cd level was 2mg/kg. Next, the effect of different content of soil conditioner Si on ginger under 2mg/kg Cd soil background was explored. This effect was assessed under 2mg/kg Cd soil background. The three treatment Si0 (0), Si1 (1g/kg), Si2 (2g/kg) were examined. Morphology indexes, Cd content, Cd transfer and absorption coefficient, photosynthesis, and antioxidant enzyme activities under Cd stress were determined. On day 120 of the experiment, plant height had increased by 18.8% and 24.7% under the Si1 and Si2 treatments compared with the CK treatment. Meanwhile, the fresh weight of the rhizomes in Si1 and Si2 had increased by 14.3% and 19.5% in comparison with the Si0 treatment. Also, the yield of ginger improved dramatically under the Si1 and Si2 treatments. These treatments had increased by 14.3% and 19.5% compared with the CK treatment. The growth of ginger increased after the addition of Si under Cd stress, especially under the Si2 treatment. The Cd content of the mother-ginger, son-ginger, and grandson-ginger decreased by 27.6%, 35.5%, and 51.2%, respectively, under 2mg/kg Cd stress in the Si2 treatment. The Cd transfer was inhibited from the underground to the aboveground in the high-concentration Si treatment (Si2). When 1 g/kg and 2 g/kg Si was added to the soil, the leaf photosynthetic rate increased by 15.1% and 26.9% compared with the CK treatment at 11:00, respectively. Conclusion : Above all, soil conditioner Si could alleviate the negative effects of Cd stress on ginger by improving growth parameters, photosynthetic efficiency and the antioxidative defense. Si2 (2g/kg) oxide contributes to cadmium toxicity cadmium up-20 3 Background Cadmium (Cd) is one of the most poisonous heavy metals and widespread environmental pollutants [1]. The concentration of Cd in soil has been increasing in recent years. It is ranked as number seven among the top 20 toxins [2]. Its absorption coefficient is higher compared with other heavy metals such as copper and zinc. Absorption of Cd relies on the Cd concentration in soil [3]. As a result of industrial wastewater, exhaust emissions, and inappropriate use of pesticides and fertilizers, Cd pollution in the soil has become increasingly common. It thus poses a serious health risk to humans. Cd can immobilize in soil by binding to organic matter, and it is easily taken up and accumulated by plants [4]. Thus, dietary intake of the biotoxic crops could be a severe threat to humans, and the most likely risk is chronic toxicity in humans [5]. Besides being carcinogenic, Cd inhalations could harm the lungs, liver, kidneys, and bones [6]. In taller plants, Cd could accumulate in any part of the plant since it is highly mobile in phloem tissue. Cd toxicity changes chloroplast ultrastructure, decreases the net photosynthetic rate, leaf transpiration, and stomatal conductance [7]. Cd toxicity is usually accompanied by oxidative stress and DNA breakdown [8] and, ultimately, causes cellular damage or apoptosis [9, 10]. Recent studies indicate that a variety of strategies have been developed to lessen the toxic effects of Cd pollution. Nitric oxide (NO), as a signaling molecule, has a pivotal role in plant resistance to various stresses, including heavy metal stresses, such as Cd [11]. NO could interact with ROS, especially O 2− , because of the presence of an unpaired ewithin the NO molecule [12]. NO improved tolerance to Cd toxicity by reducing oxidative stress that is considered as the leading cause of cell death [13]. In Lupinus, when roots were grown in 50 µM Cd, NO could stimulate SOD activity to counteract the overproduction 4 of O 2− [ 14]. One study found that salicylic acid alleviates Cd-induced photosynthetic damage and cell death by inhibiting reactive oxygen overproduction [15]. The improvement of signal transduction also increased plant tolerance to Cd, and decreased cell death resulted from Cd [10,16]. The soil amendment, Biochar, has been known to protect plants against heavy metal stress [17]. In wheat, Biochar reduced cadmium toxicity for plants growing in Cd-contaminated saline soil [18].Biochar application decreased the oxidative stress in plants and recovered the antioxidant enzyme activities. Silicon is the second-most abundant element in the world, and it could promote plant growth and weaken both biological and non-biological stresses [19,20]. It has been reported that Si plays a vital role in the transfer and accumulation of Cd in plants [21]. Si alleviates Cd toxicity in several ways. It activates the antioxidant system in plants [22], forms Si and Cd precipitants, and restrains Cd translocation, which weakens its biological activity [23]. In rice, 120 mg L − 1 Si decreased Cd accumulation and also reduced the ratio of Cd, transferring from roots to shoots, which lessened the Cd toxicity [24]. In Pisum sativum L, the application of Si decreased Cd accumulation and increased the absorption of macronutrients and micronutrients in shoots and roots, which alleviated Cd toxicity Result The effect of cadmium stress on the growth and quality of ginger There is an impact on ginger growth when 1, 2, and 4 mg/kg of Cd are applied during the growth process of ginger (Table 1). At 40d, the plant height and fresh weight were significantly lower than the CK at four mg/kg Cd level. At 80d and 120d after the treatment, the physiological indexes of ginger decreased as Cd levels increased. Above all, the physiological indexes under 2 mg/kg and 4 mg/kg of Cd stress were significantly lower than those of the CK. This indicates that the root growth was significantly inhibited, the number of branches was reduced, and the biomass of the above-ground parts was reduced when the Cd level was higher than 2 mg/kg ( Table 1). The quality indexes of ginger rhizomes indicated there were significant differences among the treatments. The yield and dry weight of ginger showed a decreasing trend with the increasing of Cd levels. The yield decreased by 9.1%, and dry weight decreased by 7.8% 6 compared with the CK at 2 mg/kg of Cd. Gingerols act as the primary indicators when evaluating ginger flavor and quality. As seen in Table 2, the content of gingerols significantly decreased as the Cd level increased. The content of gingerols was reduced by 12.1%, 31.0%, and 38.0% compared with the CK. This showed that the ginger quality was significantly reduced at 2 mg/kg Cd stress ( Table 2). The effect of silicon on the growth and quality of ginger under Cd stress Based on the effect of Cd stress on the growth and quality of ginger, the physiology indexes significantly decreased compared with the CK from the 2 mg/kg of Cd. Because of this, 2 mg/kg of Cd was chosen for the background value in the subsequent research. To observe the effect of silicon on the growth and quality of ginger under Cd stress, we used 0 g/kg, 1 g/kg, 2 g/kg Si to explore the alleviation effect of varying Si concentrations under 2 mg/kg of Cd stress. The growth of ginger was promoted after the addition of Si under Cd stress (Fig. 1). There is no significant difference among different treatments at 40d, but the difference was significant at the rhizome stage. (Table 3). Also, the yield of ginger improved dramatically under the Si1 and Si2 treatments, which showed an increase of 14.3% and19.5% compared with the CK. Soluble sugar, crude cellulose, soluble protein, free amino acid, and vitamin C increased as the application of Si increased. The effect was the greatest in the Si2 treatment. Application of Si could improve ginger flavor quality. The content of gingerols was improved by 36.8% and 63.2% under Si1 and Si2, compared with the Si0 (Table 4). 7 The effect of silicon on Cd content, Cd accumulation in different organ of ginger under Cd stress Cd content was significantly reduced after the addition of Si under 2 mg/kg Cd stress (Table 5). At 40d and 80d, the Cd content of the rhizome and the root was significantly reduced after the addition of Si. At the same time, the Cd content of the stem and the leaves had reduced, but not significantly so. At 120d, Cd content of each part was dramatically decreased after applying Si. The Cd content had reduced from 0.2566, 0.1070, and 0.0580 µg/g, respectively, to 0.1861, 0.0686, and 0.0277 µg/g, respectively, in mother-ginger, son-ginger, and grandson-ginger under the Si2 treatment (Table 5). As Table 6 shows, Cd accumulation in the root and rhizome was less than the CK under the Si1 treatment. The Cd accumulation in the aboveground part changed insignificantly and showed a slightly increasing trend, indicating that Cd accumulation in the aboveground part was promoted after applying Si. The Cd accumulation under the Si2 treatment significantly decreased compared with the CK, indicating that high-concentration Si treatment significantly inhibited Cd accumulation. In conclusion, Si in ginger could promote or inhibit Cd accumulation, depending on the concentration of Si in the soil (Table 6). The effect of silicon on cadmium absorption coefficient and transfer coefficient under Cd stress The primary transfer coefficient under the Si2 treatment was significantly lower than the Si0 and Si1 treatments, implying that higher concentrations of Si inhibited Cd migration from the root to the rhizome ( Table 7). The secondary transfer coefficient under the Si1 treatment was significantly higher than in the Si0 and Si2 treatments, which indicates that the lower concentration of silicon promotes the transfer of Cd from the rhizome to the ground. The root absorption coefficient of the Si2 treatment was significantly lower than in 8 the Si0 and Si1 treatments, which indicates that a high concentration of silicon could dramatically inhibit the root from absorbing Cd from the soil. The aboveground absorption coefficient of the Si2 treatment (0.023) was significantly lower than the Si0 (0.040) and Si1 (0.038) treatments, indicating that a high concentration of silicon inhibited the plant from absorbing Cd from the soil ( Table 7). The ROS level exhibited a rising trend as the treatment days increased (Fig. 4) (Fig. 4). The SOD, POD, and CAT increased as the Si content increased under two mg/kg Cd (Fig. 5). There was a significant difference in the three treatments, and the Si2 treatment was the highest. With the extension of time, the SOD activity of the Si0 treatment gradually decreased. The SOD activity of the Si1 treatment and the Si2 treatment increased and then decreased. At 120d, the SOD activity of the Si2 treatment was the highest, the Si1treatment was second, and the Si0 treatment was the lowest, suggesting that the addition of Si inhibited the SOD activity declining (Fig. 5). The most minor damage to the membrane system in the ginger leaf was in the Si2 treatment, while the Si1 treatment was second. The Si0 treatment showed the most critical damage to the membrane system. During the whole growth stage, the extent of damage was increasingly severe as time went on. At 120d, the MDA content reached the maximum. The MDA of the Si1 treatment and the Si2 treatment was reduced by 14.6% and 20.0% compared with the Si0 treatment which indicates that the damage of the membrane system in the ginger leaf was lower when silicon-containing fertilizer was applied. The Proline content significantly decreased from the Si0 treatment to the Si1 treatment, to the Si2 treatment during the whole growth period (Fig. 6). Silicon augmented plant growth and biomass Heavy metal stress can destroy the physiological and biochemical processes in plants Silicon restored photosynthetic efficiency Cd could reduce chlorophyll synthesis, the photochemical quantum yield of ΦPSII, and the CO 2 fixation rate. It was reported that in maize, chlorophyll synthesis and the photochemical quantum yield of ΦPSII decreased under Cd stress [42]. In durum wheat, Cd affected chlorophyll fluorescence [43]. However, the exogenous addition of Si under Cd stress had powerful effects on chlorophyll synthesis and photosynthetic machinery. In our study, the Pn was the least at 13:00, and the Pn of the Si1 and the Si2 treatments were improved by 14.4% and 24.6%, respectively, compared with the CK. Therefore, Si addition under Cd stress alleviated the damage to the ginger plants by weakening the decreasing trend of the Pn under Cd stress (Figure 2). The results were similar in peas, cotton, and maize. In the pea plant, Si addition promoted the contents of chlorophyll pigment and carotene [44]. The same condition occurred in cotton seedlings; the contents of photosynthetic pigments were increased with the addition of exogenous Si [45]. The positive effects of Si on photosynthesis could be due to the destroyed uptake of heavy metals, which could enhance PSI and PSII activation [46]. In maize, Si alleviates Cd toxicity by increasing photosynthetic rate in the modified bundle sheath cells [47]. Silicon modulated antioxidant activity Plants could use a series of strategies against the toxic effects of heavy metals under heavy metal stress conditions. The activities of SOD, POD, and CAT are of considerable significance to scavenge the ROS caused by heavy metals [48,49]. Previous studies have reported that Si mediates up-regulation of the antioxidant defense system by increasing the SOD, POD, CAT, and GR activity [50,51].Alleviation of heavy metals toxicity by Si was correlated with protection against oxidative damage. In a previous report, Si could alleviate Cd stress because of a noticeable increase in antioxidant activity and a decrease in MDA in pakochi [52]. In cotton, Si addition could significantly improve the plant's defense capacity against oxidative damage caused by Cd stress. MDA, H 2 O 2 , and electrolyte leakage were reduced, and SOD, POD, APX, and CAT activities were enhanced under Cd stress [45]. In cucumber, the application of Si could eliminate heavy metal Mn toxicity by improving antioxidant activity, according to Shi et al. [53]. Our study showed that SOD, POD, and CAT activities were increased as the Si content 13 increased under Cd stress (Fig.5). At 120d, MDA of the Si1 treatment and the Si2 treatment was reduced by 14.6% and 20.0% compared with the Si0 treatment, indicating that the damage of the membrane system in the ginger leaf was lower when siliconcontaining fertilizer was applied. Proline content was markedly decreased from the Si0 treatment to the Si1 treatment to the Si2 treatment during the whole growth period (Fig.6). At 120d, after applying Si, the Cd content in various organs of the ginger plant was reduced. The Pn of ginger leaves appeared in double peaks in one day, and the diurnal variation trend of each treatment was similar, ranked as Si2 > Si1 > Si0. Overall, our results suggest that Si could alleviate the negative effect of Cd stress on ginger, and the Si2 treatment was the most effective. Experimental materials The experiment took place at Shandong Agricultural University at the experimental 14 horticulture station. The ginger variety used was "Laiwudajiang" (WanXing Food company, LaiWu, Shandong Province). The soil tested was pH=7.3, with 100.5 mg/kg alkalihydrolyzed nitrogen (N), 63.4 mg/kg available phosphorus (P 2 O 5 ), and 127.8 mg/kg available potassium (K 2 O). The background value of soil Cd was 0.14 mg/kg. The soil used for the study was air-dried and put into a plastic basin with a 30 cm diameter and a height of 28 cm. Each basin contained 8.0 kg of air-dried soil. The ginger was planted when it germinated to 1cm. Two plants were grown per pot. Experimental design The experiment followed the environmental quality standard GB15618-2009.The Cd level was set at zero, one, two, and four mg/kg (soil). There were 10 basins per treatment, and each treatment had three replicates. Varying concentrations of cadmium chloride (CdCl2·2.5 H2O) solution was added to the soil via sewage irrigation once the ginger emerged. The CdCl2·2.5 H2O was applied in this way to ensure the uniform distribution of heavy metals in the soil and to prevent loss from the plastic containers. Other aspects of the experiment were conducted according to the standard method. Samples were collected at the seedling stage, trilling stage, and rhizome expansion stage, respectively (i.e., 40 d, 80 d, and 120 d), after the sewage irrigation treatment, and relevant indexes were determined. Based on the experiment done in the first year, the stress level of Cd was set at two mg/kg (soil), and the amount of silicon fertilizer was zero, one, and two g/kg (soil), respectively. There were 10 pots in each treatment, and each treatment had three replicates. When sowing ginger, the silicon fertilizer mixed with the 10 cm topsoil in a basin. Two mg/kg Cd (in terms of Cd 2+ ) were applied to the soil using the sewage irrigation method when the ginger emerged. The sewage irrigation method was used to 15 ensure that the heavy metals were evenly distributed in the soil and not lost from the pot. Samples were taken at 40 d, 80 d, and 120 d after treatment, and relevant indexes were measured. Determination of growth indexes The plants were taken and rinsed with water. Plant height, stem diameter, branch number, leaf number, root, stem, leaf and fresh weight of rhizome were measured. After 20 minutes in the drying oven at 105 ℃, the samples were dried to constant weight at 75 ℃. The dry matter of each organ was measured. Determination of quality The volume of soluble proteins, soluble sugars, free amino acids, crude fiber, and vitamin C were measured using various methods. Soluble proteins were measured by staining with Coomassie Brilliant Blue. Soluble sugar content was determined using the anthrone colorimetry technique [54]. Free amino acids were measured using the ninhydrin method [55]. Crude fiber volume was determined by the acid-wash method. And vitamin C was measured using the standard of 2,6-dichloro indophenol [56]. To measure the content of gingerols, 1 g of ginger powder was added to the 100-ml volumetric flask, then 70 ml acetone was mixed into the mixture and shaken for 1 h at 50°C. The mixture was then cooled and diluted with acetone to volume. Filter liquor was used for determining the content of gingerols [57]. Ethics approval and consent to participate Not applicable Consent for publication Not applicable. Availability of data and material Not applicable Competing interests The authors declare that they have no competing interests. Funding This study was supported by the National Characteristic Vegetable Industry Technology System Project (Grant No. CARS-24-A-09) and the Shandong Province's dual-class discipline construction project (Grant No. SYL2017YSTD06).The funding organizations provided the financial support to the research projects, but were not involved in the design of the study, data collection, analysis of the data, or the writing of the manuscript. Authors' Contributions: As designed and performed the experiment, ZC and JZ did the experiment. Values followed with the same letter was not significant at P = 0.05. Error bars stand for the standard errors. Tables.pdf	v3-fos-license
2017-09-15T22:55:27.654Z	2014-12-01T00:00:00.000	40189029	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://doi.org/10.5935/0103-5053.20140270", "pdf_hash": "47f137dd3001b91d26b8770d03dc6fb85ad4c277", "pdf_src": "ScienceParseMerged", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2589", "s2fieldsofstudy": [ "Chemistry", "Engineering", "Environmental Science" ], "sha1": "47f137dd3001b91d26b8770d03dc6fb85ad4c277", "year": 2014 }	pes2o/s2orc	Flash Pyrolysis of Oleic Acid as a Model Compound Adsorbed on Supported Nickel Catalysts for Biofuel Production A pirólise rápida do ácido oleico foi estudada sobre catalisadores com 10% Ni suportados em sílica e alumina. Os catalisadores foram impregnados com 10% m/m de ácido oleico. Os precursores secos e os catalisadores contendo ácido oleico foram caracterizados por análise termogravimétrica. Os catalisadores calcinados foram analisados por difração de raios X (XRD) e redução à temperatura programada (TPR). As amostras com ácido oleico adsorvido foram submetidos à pirólise rápida a 650 °C. A pirólise de ácido oleico puro levou a 10% de conversão, enquanto a pirólise catalítica resultou em praticamente completa conversão. O catalisador NiO/alumina produziu mais hidrocarbonetos do que o NiO/sílica. Os principais produtos obtidos com NiO/sílica foram 1-alcenos, enquanto que os principais produtos obtidos com NiO/alumina foram isômeros de alcenos e aromáticos, e pequenas quantidades de compostos oxigenados, principalmente álcoois. A pirólise rápida de ácido oleico adsorvido em catalisadoras representa um método útil para distinguir as propriedades dos catalisadores e suas diferentes atividades. Introduction Upgrading of vegetable oils and related compounds to obtain liquid fuels has been extensively studied since the end of the 70's.][3][4][5][6] Transesterification is the process usually employed at the industrial level.In this case, vegetable oils react with a short chain alcohol to form esters of fatty acids.These esters are usually named biodiesel.Industrially, methyl esters of fatty acids can be added to petroleum diesel up to 7-8 wt.%.These mixtures, unlike pure petroleum diesel, show a slightly lower energetic power due to their oxygen content and they have limited chemical stability. 7herefore, stabilizers must be added to biodiesel to render it useful. 8,9The thermochemical routes can lead to highly deoxygenated compounds fully miscible with liquid fuels of fossil origin. 5Companies, such as UOP LLC, Neste Oil, Petrobras and Eni S. p. A., have studied hydrocracking of triglycerides with catalysts similar to the ones used in the hydrotreatment processes.For example, Eni S. p. A. and UOP LLC are using the Ecofining™ technology to obtain green diesel, a qualitative biofuel. 10When carried out in the presence of hydrotreating-type catalysts, at hydrogen pressures higher than 3 MPa and at temperatures between 300-400 °C, hydrocracking of triglycerides leads to important amounts of saturated linear hydrocarbons, with a high selectivity towards C16-C18 molecules. 5,11During hydrocracking two main routes have been observed: decarboxylation and decarbonylation (DCO).In both of these reactions the triglycerides are initially transformed into fatty acids that can lose CO 2 and CO + H 2 O, producing hydrocarbons which have a carbon chain with one less C atom than the fatty acid from the initial feed. 5A third reaction is also observed in the presence of hydrogen and is considered as hydrodeoxygenation (HDO).In this case, oxygen is eliminated from the intermediate fatty acid as H 2 O and the main hydrocarbon molecules formed have the same carbon number as the starting fatty acid chain. 5Because the HDO reaction consumes important amounts of hydrogen, studies aiming at favoring DCO over HDO are important.Kubicka and Kaluza 12 showed that hydrotreating-type catalysts containing only Mo species favor HDO during rapeseed oil hydrocracking, whereas the addition of Ni favors DCO.Kubicka et al. 13 also showed that Ni-Mo catalysts with the same composition and the same method of preparation have HDO activity varying with the nature of the support.Therefore, modification of the composition of catalysts appears as an effective way to direct hydrocracking of triglycerides towards DCO. In order to limit hydrogen consumption, cracking of triglycerides and/or model compounds in the absence of added hydrogen has been considered.5][16][17] When using catalysts, the degree of deoxygenation is generally enhanced. 180][21] When the catalysts have strong acid sites, the amount of liquid alkenes and alkanes is decreased whereas important amounts of aromatic compounds are formed. 22umerous studies have considered the use of model compounds to help understand some mechanistic aspects of the decomposition of triglyceride molecules due to their high complexity.2][23][24][25][26][27][28][29] A recent study on the pyrolysis of oleic acid in an autogeneous atmosphere, between 350 and 450 °C, confirmed that both decarboxylation and decarbonylation took place in the reactor, but also revealed internal cracking at the allylic C position, leading to a predominance of C6-C10 hydrocarbons in the liquid products and the formation of C9 and C10 fatty acids. 30The amount of fatty acids decreased when the temperature of pyrolysis was increased, whereas the amount of mono-and poly-aromatic compounds increased.A recent study from our group described the flash pyrolysis of micro amounts of fatty compounds adsorbed on different solid catalysts, NaZSM-5, HZSM-5, g-alumina, SAPO-5 and NiMo/SAPO-5, as a rapid way to screen some catalyst properties and confirm the presence of even minute amounts of products, especially primary reaction intermediates. 31he present work studies the flash pyrolysis of oleic acid, as it is the main fatty acid in most triglycerides taken from vegetable oils.Supported nickel catalysts were used to increase the deoxygenation process.We pre-adsorbed a very small amount of oleic acid on the surface of the catalysts in order to limit the influence of pyrolysis without catalyst.Thus, we could study the role of the catalyst in the initial steps of the decomposition of oleic acid. Experimental Preparation of the catalysts Supported nickel catalysts with 10 wt.% Ni (as NiO) were prepared by impregnation with an excess of aqueous solution of nickel(II) nitrate hexahydrate (Merck PA).The supports used were a transition alumina (Pural Sasol), and a commercial silica (Kali Chemie AF125), both in powder form.The solids obtained after evaporation in a rotating device were dried at 110 °C in static air, manually ground for homogenization and calcined under air at 650 °C (heating rate of 10 °C min -1 ) to generate the calcined catalysts precursors.Supports impregnated with pure water were treated in the same way as the supported catalysts in order to have a true reference carrier when necessary. Addition of oleic acid onto the catalysts NiO/silica and NiO/alumina after a new drying at 150 °C were mixed with small amounts of pure oleic acid (OA) (Sigma Aldrich > 93%), in a mass proportion of 1 g of catalyst for 0.1 g of OA, under permanent manual agitation to allow complete spreading of the OA.At the end of this "pseudo" impregnation, the catalysts maintained their powder form.They are referred to as OA/NiO/support in the following sections.The use of a high catalyst:reactant ratio was to provide high availability of catalytic sites and minimize the influence of thermal pyrolysis. Characterization of the catalysts Pure nickel nitrate and nickel nitrate deposited on both supports at the end of the drying treatment, as well as OA/NiO/silica and OA/NiO/alumina, were characterized by thermogravimetric/differential thermal analysis (TG/DTA) using a Perkin Elmer STA 6000 instrument.The experiments were conducted under a synthetic airflow rate of 20 mL min -1 between 30 and 600 °C at a heating rate of 10 °C min -1 .The reduction of supported NiO was done using a homemade temperature programmed reduction (TPR) equipment, at a heating rate of 10 °C min -1 up to 800 °C, using a mixture of H 2 /argon (1.5 vol.% of hydrogen) with a gas flow rate of 70 mL min -1 .The supports, the calcined NiO supported samples and the reduced catalysts at the end of TPR were characterized by X-ray diffraction (XRD) (Shimadzu diffractometer, model 6000) using the CuKα radiation, between 10 and 80° at a scanning rate of 2° min -1 .The accelerating voltage and the current employed were 40 kV and 30 mA, respectively.The acidity of the catalysts was determined by using pyridine as probe molecule in the same TG Perkin Elmer equipment.An amount of 3 mg of catalyst was pre-treated from 35 to 110 °C for 30 min to remove humidity and then heated to 550 °C at 20 °C min -1 to remove chemisorbed water.After cooling the sample an amount of 1 µL of pyridine per mg of catalyst was carefully added to the catalyst at 120 °C.The desorption of pyridine excess was carried out at the same temperature until reaching equilibrium, after about 60 min.The chemisorbed pyridine was desorbed by heating up to 550 °C at 10 °C min -1 and the loss of mass was recorded with temperature.Specific surface area of the catalysts was determined by the Brunauer-Emmett-Teller (BET) method in a Quantachrome NOVA-2000 equipment at 77 K (-196 °C) with nitrogen adsorption.Samples were pre-treated at 250 °C for 2 h under vacuum before measurement. Flash pyrolysis experiments The flash pyrolysis of pure OA and OA adsorbed on both NiO/supported samples were performed in a Pyroprobe CDS-5200 micropyrolysis set up, linked to a gas chromatography/mass spectrometry (GC/MS) analysis system (Shimadzu GC-MS QP 2010 Plus).The powdered sample of the OA/catalyst was placed in a 2 mm × 25 mm quartz tube between quartz wool plugs, in an amount of around 1.0 mg of catalyst with 0.1 mg of impregnated oleic acid.The quartz tube was placed inside a resistive platinum coil heater.The flash pyrolysis was conducted at 650 °C for 0.25 min, at a heating rate estimated as 1000 °C min -1 with a helium flow rate through the sample of 150 mL min -1 .After pyrolysis, the vapors and gases flowed to the GC injector through a transfer line heated at 170 o C, as shown in Figure 1.The chromatographic analysis was performed in a DB-5MS analytical column (30 m × 0.25 mm × 0.25 µm), with a helium flow rate through the column of 1 mL min -1 .The column program was 5 min at 45 °C, heating to 280 °C at a heating rate of 4 °C min -1 , with a 10 min stay at 280 °C.The ion source was maintained at 280 °C and the interface at 290 °C.The m/z data were measured between 40 and 400.The chromatographic peaks of the pyrolysis products were identified using the National Institute of Standards and Technology (NIST) library standards as well as comparing with data from the literature.The probability of products identification was better than 90% for the great majority of the peaks.The standard deviations of the main peaks in some replicated experiments were smaller than 25%.are attributed to water loss, whereas the high temperature event is due to NO x release. 32,33Above 650 °C, no further mass loss was observed suggesting a complete decomposition of pure nickel nitrate to nickel oxide.In the presence of both supports, a first mass loss ending between 150 and 170 °C is attributed to water release from the support.Further mass losses vary with the nature of the support, a clear mass loss process appearing only with nickel nitrate deposited onto silica, with a maximum rate at around 270 °C.The other steps are not well resolved.Table 1 shows the mass loss percentage from the decomposition of pure or supported nickel nitrate on silica and alumina, obtained from the TG curves.Figure 2 and Table 1 show that the decomposition temperature of the nickel nitrate for the release of NOx follows the order: NiO/alumina > NiO/silica.This suggests that the interaction of the nickel with alumina is higher than with silica. Characterization of the catalysts The DTA curves presented in Figure 3 show some similarities to the DTG curves, all thermal processes being endothermic.For pure nickel nitrate, there are five main peaks, the first one with a maximum at 50 °C, due to both a fusion process of the hydrated salt and the loss of a first molecule of water.The second, third and fourth events at around 100, 200 and 250 °C are essentially due to water release, whereas the fifth one, with a maximum at around 320 °C is due to NO x elimination.For the supported materials, the low temperature endotherm is due to water elimination from the supports, the process ending at 180 °C.The endothermic peaks observed at around 270 °C for both nickel/silica and nickel/alumina precursors are attributed to NO x elimination.In this latter case a stronger interaction of the nickel precursor with alumina decreases the decomposition rate of the precursor.TG curves in Figure 2a show that the decomposition of nickel nitrate on silica is faster than on alumina.In both Figures 2 and 4, no further mass loss and/or clear thermal event occurs above 500 °C, suggesting that the precursor salt of nickel is fully decomposed at this temperature.The difference in decomposition behavior between both supported materials suggest that the interaction of the impregnated nickel salt is different on both supports, a situation that may lead to different interactions with the support of the NiO particles formed at the end of heat treatment, in agreement with data from the literature. 34able 2 gives some properties of the supported catalysts either in oxidized or reduced form such as temperature reduction interval, crystallite mean size, specific surface area and acidity.This table shows that the reduction of NiO/silica is completed at 460 °C, whereas the reduction of NiO/alumina is completed at 750 °C.The rather low reducibility of the present supported NiO is essentially due to the experimental conditions used, where the partial pressure of hydrogen is low.As the reduction rate of unsupported nickel oxide presents a positive reaction order regarding the hydrogen pressure, the low pressure used in the present TPR experiments does not favor the metal reduction, but it may allow possible NiO-support interactions during the heating ramp, increasing the temperature of the reduction process.Such situation may affect the final state of the reduced nickel. 35The reduction temperature of nickel oxide on alumina is higher than on silica suggesting that the interaction of NiO with alumina is higher than with silica.A limited formation of nickel aluminate, at the interface between alumina and supported NiO particles, can also be advocated to explain the lower reducibility of alumina supported nickel oxide.This situation will be further discussed during the analysis of XRD data (Figure 4).Table 2 also shows that the specific surface area of NiO/silica is twice as much the area of NiO/alumina.The acidity of NiO/alumina (0.15 mmol of pyridine g -1 ) is almost three time higher than the acidity of NiO/silica. Figure 4 presents the XRD diffractograms for silica, NiO/silica, reduced Ni/silica after TPR, alumina, NiO/alumina and reduced Ni/alumina.The XRD results of the silica supported catalyst present a wide peak at 2θ = 22°, due to the quasi amorphous structure of the silica and diffraction lines at 2θ = 37, 43, 63° typical of NiO. 36iffraction lines attributed to metallic nickel Ni 0 (2θ = 45, 52 and 76°) are observed after TPR, the amorphous line of silica support being unaltered.Therefore, on silica, supported NiO is obtained at the end of the calcination at 650 °C of the supported nickel nitrate, and metallic nickel at the end of TPR. In Figure 4, the XRD diagram of alumina is very typical of transition alumina.The lines at 2θ = 19, 32, 37, 39, 45, 60 and 67° are close to those described in the case of g-alumina. 37In this case, peaks due to alumina and NiO are partially merged.Only the diffraction lines of NiO at 43 and 63° are clearly observed.This observation confirms that with alumina, NiO is also obtained at the end of the calcination at 650 °C.However, Figure 3 shows that the presence of nickel aluminate NiAl 2 O 4 cannot be ruled out since its XRD lines overlap with the lines characteristic of transition alumina. 38After TPR, Ni 0 diffraction lines are clearly present at 2θ = 52 and 76°.Therefore, although the reduction process of the catalyst supported on alumina requires higher temperature under TPR conditions than the reduction of the catalyst supported on silica, the reduction mainly transforms NiO to metallic nickel in both cases.We were able to estimate a crystallite size of 15 nm for Ni 0 /silica and smaller than 9 nm in the case of Ni 0 /alumina using the Scherrer equation with the diffraction line at 52 and 76° (Table 2). 39Therefore, after reduction, the Ni crystallite size is different on silica and alumina, probably as a consequence of the differences of NiO interaction with both supports.These differences of interaction probably started during impregnation/drying and/or decomposition processes of the nickel precursor as suggested by the different decomposition profiles observed in both DTG (Figure2) and DTA (Figure 3).Table 2 also shows that the particle size of NiO on silica is bigger than the size of NiO on alumina. DTG of oleic acid, either pure or adsorbed on NiO/support catalysts Figure 5 presents the TG and DTG curves obtained under nitrogen atmosphere for OA/NiO/silica, OA/NiO/alumina and pure OA as reference.For pure OA, a single mass loss is observed with the maximum rate at around 260 °C.In the case of OA adsorbed on both catalysts, after a mass loss before 150-200 °C attributed essentially to water desorption from the catalyst surface, the main mass loss occurs at higher temperatures by comparison with pure OA mass loss.Thus, the adsorption of OA onto the catalysts increases the temperature of the mass loss due to OA release and/or decomposition.Furthermore, the differences in mass loss profiles for pure OA and OA adsorbed on both NiO/alumina and NiO/silica catalysts, as well as the differences in the temperature of maximum mass loss rate, indicate that the strength of OA adsorption varies with the nature of the catalyst.Consequently, it can be supposed also that the pyrolysis of OA, either unsupported or supported on both catalysts will generate different families of products, both catalysts retaining more strongly some pyrolysis products when compared with pyrolysis without catalyst.One further point must be added: above 600 °C, no clear mass loss event is observed, justifying in part, together with literature data, 17 the choice of 650 °C as the final flash pyrolysis temperature used in the next section. Flash pyrolysis of oleic acid, either pure or adsorbed on NiO/support catalysts Figure 6 shows the peaks of products (pyrogram) obtained during the flash pyrolysis at 650 °C of pure OA and OA adsorbed on both NiO/silica and NiO/ alumina.Table 3 summarizes the main classes of products identified.During the pyrolysis of pure OA, the percentage of the products formed up to the retention time of 41.1 min represents less than 10% of the whole area of the pyrogram.The main peaks on the right side of the pyrogram, with retention times equal and higher than 41.1 min are due to C14 and C16 fatty acids and to untransformed C18 oleic acid (retention time of 52 min).That part of the pyrogram represents more than 90% of the area of the whole pyrogram.Therefore, during the flash pyrolysis of pure OA at 650 °C, fatty acids with shorter C chain are obtained before DCO can occur.At retention times lower than 41.1 min, many other oxygenated products are found: among them, dodecanoic (0.25%), undecylenic (0.16%), decanoic (0.41%), octanoic (0.31%), 7-octenoic (0.07%), heptanoic (0.10%) and acetic (0.09%) acids are identified.Aldehydes, alcohols and ethers are also identified.Finally, the amount of deoxygenated compounds, mainly monounsaturated alkenes, does not represent more than 3.8% of the whole pyrogram.Hence, the flash pyrolysis of pure OA in the present experimental conditions is limited and does not favor deoxygenation.The two pyrograms obtained when OA is adsorbed on both NiO/silica and NiO/alumina hardly show the presence of residual unconverted OA: the contact between the catalyst surface and the adsorbed OA allows a complete transformation of oleic acid.Contrary to pyrolysis without catalyst, the pyrolysis in the presence of catalysts reveals the formation of a very important amount of light products.Among these products it is possible to observe peaks due to homologous compounds like 1-alkenes (peaks 1, 3, 4, 5, 6), as shown in Figure 7, where the names of organic compounds are attributed to some peaks between the retention times of 5 and 25 min. Table 4 gives the semi-quantitative distribution of the deoxygenated compounds (area percentage of saturated, monounsaturated, polyunsaturated and aromatic compounds).Whereas Table 3 indicated that the amount of deoxygenated compounds (hydrocarbons) is practically similar for both OA/NiO/alumina and OA/NiO/silica, Table 4 shows important differences between the distribution of the hydrocarbon families obtained after pyrolysis at 650 °C of OA adsorbed onto both catalysts: in the case of NiO/alumina, the pyrolysis of OA leads to an important amount of aromatic products, a family of compounds produced in a much lower amount with OA/NiO/silica.On the other hand, OA/NiO/silica produces more alkanes, alkenes and polyunsaturated hydrocarbons, such as dienes, trienes and alkynes than OA/NiO/alumina. Table 5 identifies the main aromatic products formed during pyrolysis with both catalysts.In the case of pyrolysis of OA without catalyst, no aromatic compound was detected analyzing peaks with a percentage area equal to or higher than 0.06%.For OA/NiO/silica, together with benzene, linear alkylbenzenes with lateral chain containing 1, 2, 3, 4 and 6 C were found, as well as one dialkyl benzene.For OA/NiO/alumina, more than 50 aromatic compounds were detected, among them linear alkylbenzenes, with lateral carbon chain between 1 and 11 C, representing more than 50% of all the aromatic compounds detected.A few number of alkenyl benzenes, an important number of di-and trialkyl benzenes and a rather large number of polyaromatic compounds, such as indane/indene, naphthalenes and fluorene, either unalkylated or with limited alkyl chains, were also identified.Table 6 summarizes the amounts of monoalkyl benzenes formed with both OA/NiO/silica and OA/NiO/alumina.Monoalkyl benzenes have been observed in preceding studies dealing with cracking/hydrocracking of fatty compounds, but up to the time of our study, a general sequence of alkyl benzenes as observed with OA/NiO/alumina Table 5. Main aromatic compounds formed during the pyrolysis of oleic acid adsorbed on both NiO/alumina and NiO/silica.The second column is the retention time t R (min), the third is the name of the aromatic compound, the fourth is the chemical formula, the fifth and sixth are the area percentage from the pyrogram, respectively on NiO/alumina and NiO/silica has not been reported. 30,40In the industrial production of linear alkyl benzenes for detergent applications, the alkylation of olefins is conducted in the presence of a strong acidic medium, either with HF, or more recently with zeolite-type heterogeneous catalysts. 41,42The mechanism of this type of catalytic alkylation implies the participation of carbocations and acidic sites.Such a mechanism is rather unexpected under present conditions, although the carboxylic moieties may form acidic OH groups on metallic or support surface sites after adsorption of the acidic function under the form of carboxylate species. 43,44However, alumina-supported nickel catalysts have shown very high activity and selectivity in the alkylation of benzene with propene to form cumene. 45 Therefore, alkylation with supported nickel catalysts is possible, although the conditions of the present study are different from the conditions used by Jian et al.. 45 Maybe under the present experimental conditions, the linear alkyl benzenes have essentially been formed through an internal aromatization after or during decarboxylation.Molecules, such as linear alkyl cyclopentenes or linear alkyl cyclohexenes able to lose hydrogen on nickel sites to transform the alkylated cycloolefins to alkylated benzenes, are probably used as intermediate molecules.In fact, small amounts of cyclopentenes with linear alkyl chain containing 3, 4, 5, 7 and 8 C, and cyclohexenes with linear alkyl chain with 4 and 6 C have been identified.However, the exact mechanism of formation of the family of alkylbenzenes during the pyrolysis of OA adsorbed on NiO/alumina is still unkown.Table 7 shows the amounts of linear 1-alkenes in the 3 experiments.Whereas this amount is very limited for the pyrolysis of OA without catalyst (1.8%), both NiO/ alumina (20.7%) and principally NiO/silica (33.5%) decomposed OA towards 1-olefins with satisfactory selectivity.Although both catalysts help to decompose the adsorbed OA fully, differences in product distribution is clearly observed.It seems evident that the higher acidity of NiO/alumina compared with NiO/silica (Table 2) must play an important role in the formation of the isomers of linear olefins and in the formation of aromatic compounds, these latter compounds being probably formed also due to the dehydrogenating properties of nickel sites.Hydrogen transfer pathways, advocated for example during the decomposition of saturated fatty acids on activated alumina may probably occur in the present experimental conditions, together with the participation of adsorbed hydrogen on the metallic nickel surface, as hydrocarbons are able to reduce nickel oxide to metallic nickel at temperatures in the 400-500 °C range. 19,46lthough the amount of oxygenated compounds is not very high, it is important to indicate that CO 2 and acetates were observed with the NiO/alumina sample, but not with NiO/silica.Such a situation is probably linked to the partial adsorption of oleic acid through carboxylate species on the alumina surface, such species being practically absent when the adsorption occurs on the silica surface. 43Among the other oxygenated compounds, carboxylic acids are observed in greater amount with NiO/silica (1.15%) than with NiO/alumina (0.41%), in agreement with the better deoxygenation properties of this latter catalyst.In the same way, more alcohols are formed when pyrolyzing OA on NiO/silica (5.12%) than on NiO/alumina (2.91%).But a large majority of these alcohol and acid molecules are susceptible to transformation into unsaturated hydrocarbons when the experimental condition is slightly changed, DCO and dehydration being rather frequent reactions.A last point can be mentioned, dealing with the identification of some ketones in the condensable pyrolysate.In this case, the amount of ketones is lower with OA/NiO/silica (0.27) than with OA/NiO/alumina (0.70).This is probably linked to the fact that ketones have been shown to be important intermediate species during the cracking of saturated fatty acids in the presence of activated alumina. 19,20he present results confirm that during the decomposition of oleic acid adsorbed on the catalysts, cooperative processes occur between the support surface and the active phase surface.On the one hand, the products are different from the products obtained during cracking without catalysts and therefore, the thermal decomposition has limited importance; on the other hand, both catalysts also generate not always similar products, indicating that the adsorption properties on both catalysts are different.Therefore, as was advocated in a preceding publication, it is confirmed that the present experimental conditions using flash pyrolysis of adsorbed species can be seen as a "pseudo" catalytic test, helping a description of the potential properties of a catalyst before its use in more classical flow or batch reactors. 31 Conclusions The flash pyrolysis of oleic acid adsorbed on supported nickel catalysts generates complete decomposition of the fatty acid whereas the pyrolysis without catalyst allowed a decomposition lower than 10%.The products of pyrolysis with supported nickel catalysts were highly deoxygenated, and hydrocarbon content close to 80% was observed in both cases.The selectivity to hydrocarbons was different for both catalysts: an important amount of 1-alkenes was obtained with oleic acid adsorbed on NiO/silica, whereas NiO/alumina generated more alkene isomers, more polyunsaturated hydrocarbons and more aromatic compounds than NiO/silica.The differences of selectivity can be linked on one hand to hydrogen transfer occurring when alumina is used as support, and on the other hand to different adsorption modes of oleic acid on both catalysts, with the carboxylate species probably being more important with alumina support than with silica support.The flash pyrolysis of adsorbed fatty compounds can be proposed as a quick "pseudo" catalytic test to select catalysts before long term reactions are initiated. Figure 2 presentsFigure 1 . Figure 2 presents TG and derivative thermogravimetry (DTG) curves obtained during the decomposition of nickel nitrate hexahydrate either pure or impregnated on both alumina and silica.The decomposition of pure nickel nitrate shows four main mass losses, with maxima at around 100, 180, 240 and 320 °C.The three low temperature mass losses Figure 5 . Figure 5. TG (a) and DTG (b) curves under nitrogen atmosphere for pure OA and OA adsorbed on both NiO/silica and NiO/alumina, up to 600 °C (10 °C min -1 ). Figure 6 . Figure 6.Total ion chromatograms showing the products from flash pyrolysis for pure oleic acid (OA) (a) and of OA adsorbed on both NiO/silica (b) and NiO/alumina (c). Table 1 . Mass loss percentage from the decomposition of pure or supported nickel nitrate on silica and alumina, from the TG curves Table 2 . Reduction temperature of NiO to metalic nickel from TPR, NiO or Ni crystallite particle size and specific surface area and acidity of NiO/silica and NiO/alumina Table 3 . General distribution of compounds analyzed at the end of flash pyrolysis of pure OA, and OA adsorbed on both NiO/silica and NiO/ alumina Table 4 . Area percentage of the deoxygenated products formed during the flash pyrolysis of AO either pure or adsorbed on both NiO/silica and Table 6 . Area percentage of alkylbenzenes formed during the flash pyrolysis of pure OA, and OA adsorbed on both NiO/silica and NiO/alumina as a function of the number of C in the alkyl chain Table 7 . Distribution of 1-alkenes formed during the flash pyrolysis of oleic acid (OA) and OA adsorbed on both NiO/alumina and NiO/silica	v3-fos-license
2019-03-31T13:42:52.340Z	2016-03-11T00:00:00.000	55253853	{ "extfieldsofstudy": [ "Chemistry" ], "oa_license": "CCBY", "oa_status": "HYBRID", "oa_url": "https://ccsenet.org/journal/index.php/jas/article/download/56597/31037", "pdf_hash": "be4a402571489fc691786adb716cebdeef8164a3", "pdf_src": "Anansi", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2733", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "sha1": "800734cc7940522f38837b0e8c118ce49485d787", "year": 2016 }	pes2o/s2orc	Comparison of Antibacterial Activity of the Spent Substrate of Pleurotus ostreatus and Lentinula edodes Nowadays, the uncontrolled use of antibiotics has created the problem of bacterial resistance to them, what has motivated the search for new alternatives of drug for the treatment of bacterial diseases. Here, we compare antimicrobial activity of spent substrate of mushroom Pleurotus ostreatus and Lentinula edodes, against Escherichia coli, Salmonella tiphymorium, Staphylococcus aureus and Micrococcus luteus. We designed two mixtures, barley straw to be used as a substrate of cultivation of mushroom Pleurotus ostreatus and oats or cedar for the cultivation of mushroom Lentinula edodes; and were obtained aqueous extracts from spent substrates; extracts were tested for antibacterial activity. The protocol was a completely randomized assay with a factorial arrangement design. The data were analyzed with PROC GLM, SAS. The results showed that in the case of Escherichia coli the greatest inhibition zone was of 12.66 mm at a concentration of 6 mg mL, with treatment of Lentinula edodes/Cedar; Salmonella tiphymorium showed a greatest inhibition zone of 31.10 mm to a concentration of 5.12 mg mL, with treatment of Pleurotus ostreatus/Barley straw; Staphylococcus aureus showed a greatest inhibition zone of 9.33 mm to a concentration of 100 mg mL, with the treatment of Lentinula edodes/Cedar and finaly, Micrococcus luteus showed a greatest inhibition zone of 15.00 mm to a concentration of 50 mg mL, with the treatment Lentinula edodes/Oats. In conclusion, the results suggest that it is possible to use indistinctly the spent substrate of Pleurotus ostreatus and Lentinula edodes as source of extracts with antibacterial activity. Introduction The uncontrolled use of antibiotics has caused serious problems in human and animal health, causing that bacterias develop resistance to them, so World Health Organization considered to infections caused by bacteria resistant to drugs as a public health problem; therefore, it is necessary to find new pharmacological strategies, among which we can find natural products such as plants and fungi (Roca et al., 2015). Spent mushroom substrate containing carbohydrates as cellulose and hemicellulose, lignin, remnant of edible fungi, is a byproduct of mushroom production industry.Substrate used as growth media to produce mushroom is composed of maize cobs, wheat straw, grass straw, sugarcane bagasse, field hay, corn cobs, cotton seed hulls and some other.After several mushroom harvesting cycles, the productivity of the substrate could decrease so that the substrate is declared as spent (Guoa & Choroverb, 2006;Onyango, Palapala, Arama, Wagai, & Gichimu, 2011).One of the main problems in the production of mushrooms still the treatment and disposal of spent mushroom substrate; many studies have already been carried out for the use of such substrates, among which we mention feeding and/or antimicrobial activity (Zhu, Sheng, Yan, Qiao, & Lv, 2012). The fungi have an important role in the degradation of organic matter (Chang & Miles, 1984), in addition to being a source of bioactive substances to produce antibiotics or pharmaceutical drugs, such as functional food and additives in feeding stuffs (Santoyo, Ramírez-Anguiano, Reglero, & Soler-Rivas, 2009).Crop of mushrooms (Pleurotus ostreatus) it is a source of products agricultural, their organic waste generated can be used as a source of food with high protein content and as an alternative pharmaceutical treatment; Pleurotus ostreatus is a fungus that has the ability to grow on agricultural wastes, accelerates the biodegradation and recycling them, avoiding its burning and the subsequent environmental pollution (Varnero, Quiroz, & Álvarez, 2010).Lentinula edodes, fungi edible, is the most studied, has been shown that fruiting body and mycelium have antimicrobial properties; likewise, the lentine inhibits mycelium growth of other fungi as Physalospora piricola, Mycosphaerella arachidicola and Botrytis cinerea (Rojas, 2013;Romero-Arenas, Martínez, Damian, Ramírez, & López-Olguín, 2015). The production of mushroom has been used with a large number of substrates; one of the main is straw, used as a source of carbon to increase the nutritional characteristics and palatability of the fruiting body, getting a better nutritional quality (Sánchez, 2010).The crop of fungi and the quick growth in mushroom production worldwide has resulted in large quantities of spent substrate mushroom (about 13.6 million t year -1 ).The massive amounts of waste can cause environmental problems; this causes, led more research to develop technologies for its treatment or use (Lin et al., 2014). In recent years, there has been a need study antimicrobial phytochemicals with potential to generate new pharmacological options.Our group previously demonstrated that the use of spent substrate of Pleurotus ostreatus mixed or not with medicinal plants, has antibacterial activity (Ayala et al., 2015).Therefore, the objective of this study was to determine the antibacterial activity of spent substrate of Pleurotus ostreatus and/or Lentinula edodes against Escherichia coli, Salmonella tiphymorium, Staphylococcus aureus and Micrococcus luteus at different concentrations. Strain, Substrates and Cultivation Mushrooms The blocks (1 Kg) were obtained from Centro de Investigaciones Biológicas of the Universidad Autónoma del Estado de Hidalgo, México.The blocks were formed by a mixture of barley straw; which were purchased in Central Abastos in Pachuca Hidalgo, Mexico, the taxonomic identification did it the botanist Dr. Miguel Angel Villavicencio Nieto; the specimens were deposited at the Herbarium of the Centro de Investigación de Ciencias Biológicas, of the Universidad Autónoma del Estado de Hidalgo, México.To form the substrate, barley straw was colonized with mycelium of Pleurotus ostreatus UAEH-004 in solid substrate fermentation; mushrooms were harvested at 23 d and was obtained spent substrate; on the other hand, oats or cedar were colonized with mycelium of Lentinula edodes UAEH-015 in solid substrate fermentation; mushrooms were harvested at 90 d and were obtained spent substrate of each one. Preparation of Organic Extracts The extracts were obtained by mixing 100 g of spent substrate mushroom of each treatments and 300 mL distilled water in case of Pleurotus ostreatus and 600 mL distilled water in case of Lentinula edodes.Then, the mix was macerated in blender during 1 min.Subsequently, mix macerated was filtered using gauze, to separate solid and liquid parts, was filtered through filter paper Whatman® #41.The extract was placed in a water bath to 70 °C for 48 h, to obtain the sample dry; for each 25 ml of liquid extract were obtained 0.472 g of Pleurotus ostreatus and 0.451 g of Lentinula edodes of dry extract. Antimicrobial Assay The antibacterial activity of the spent substrate of Pleurotus ostreatus and/or Lentinula edodes extracts were studied by the method of paper disc diffusion assay with slight modification (Kil et al., 2009).The bacterial pathogens strain were grown in liquid medium for 24 h to yield a final concentration of Escherichia coli 7.7 × 10 6 CFU/200 µL, Salmonella tiphymorium 1.11 × 10 7 CFU/200 µL, Staphylococus aureus 1.0 × 10 7 CFU/200 µL and Micrococcus luteus 1.04 × 10 7 CFU/ 200µL.Next, aliquots of 0.1 ml of the test microorganisms were spread over the surface of agar plates.Sterilised filter paper discs of 6 mm diameter (paper Whatman® #41) were saturated with 50 μl of different concentrations (0, 6, 12.5, 25, 50 and 100 mg mL -1 ) spent substrate of Pleurotus ostreatus and/or Lentinula edodes extracts.The soaked discs were then placed in the middle of the plates and incubated for 24 h at 37 °C (Forma Series II Water Jacket CO 2 , Incubator, Model 3100, Thermo Scientific, USA), after which the diameter (in mm) of each inhibitory zone was measured (scalimeter).Negative control was prepared with distilled water; as positive control was used commercial antibiotic (Penicillin G sodium salt, Sigma-Aldrich, St. Louis, MO, USA) to a concentration 100 mg mL -1 on medium culture Mac Conkey and Estafilococos No. 110. Statistical Analysis Data were analyzed using factorial design 3 × 3 and blocked by extract type and extract concentration and bacterial strain as factors.A PROC GLM procedure and LSMEANS option were used (SAS, 2002). Results Treatment with spent substrate of Pleurotus ostreatus and Lentinula edodes extracts were effective against four bacterial strains tested; antibacterial activity at different concentrations is showed in Table 1; the tested bacteria were quantitatively assessed by measuring the diameter of inhibition generated for each sample; each result is the mean of three replicates.The results showed that the spent substrate Pleurotus ostreatus/Barley straw extracts presented highest inhibitory effect against Escherichia coli (7.7 × 10 6 CFU/200 µL) at a concentration of 12.5 mg mL -1 with 9.86 mm inhibition halo; Staphylococus aureus (1.0 × 10 7 CFU/200 µL) at a concentration of 25 mg mL -1 with 9 mm inhibition halo and Micrococcus luteus (1.04 × 10 7 CFU/ 200µL) at a concentration of 50 mg mL -1 with 9.66 mm inhibition halo.Salmonella tiphymorium (1.11 × 10 7 CFU/200 µL) at a concentration of 12.5 mg mL -1 with 31.10 mm inhibition halo, showing significant differences with the concentrations 6, 25, 50 and 100 mg mL -1 (P < .05). When comparing the largest zone of inhibition of the three treatments tested (Pleurotus ostreatus/Barley straw, Lentinula edodes/Oats y Lentinula edodes/Cedar) against each bacteria (Table 2), the results showed in the case of Escherichia coli the largest zone of inhibition was at a concentration of 6 mg mL -1 (12.66 mm) with treatment of Lentinula edodes/Cedar; Salmonella typhimurium showed the largest zone of inhibition at a concentration of 5.12 mg mL -1 (31.10 mm) with treatment of Pleurotus ostreatus/Barley straw; Staphylococcus aureus showed the largest zone of inhibition at a concentration of 100 mg mL -1 (9.33 mm) with the treatment of Lentinula edodes/Cedar and Micrococcus luteus showed the largest zone of inhibition at a concentration of 50 mg mL -1 (15.00 mm) with treatment of Lentinula edodes/Oats.Salmonella typhimurium and Staphylococcus aureus no showed significant differences between each treatments; Escherichia coli treated with Lentinula edodes/Oats is significantly different to the treatment Lentinula edodes/Cedar (P < 0.05), but without showing difference significant with treatment Pleurotus ostreatus/Barley straw, while Micrococcus luteus treated with Lentinula edodes/Oats is significantly different with treatment of Lentinula edodes/Cedar and Pleurotus ostreatus/Barley straw (P < 0.05). Discussion In recent years the bacteria have acquired the ability of multi-resistance to antibiotics (Nehra, Meenakshi, & Yadav, 2012) which has generated that recent research are focus in the search for alternative treatments, such as fungi.Edible fungi such as Pleurotus ostreatus, have shown a high nutritional value as food (Patel, Naraian, & Singh, 2012) and anti-inflammatory, antidiabetic, antiviral, antioxidant, anticancer, antitumor, inmunomodulatory and antibacterial activity; however, most of the research has been based on the study of the fruiting body and not in spent substrate (Hearst et al., 2009;Deepalakshmi & Mirunalini, 2014). A water-soluble polysaccharide named PL was isolated and purified from spent mushroom substrate, the polysaccharide contained two fractions (PL1 and PL2), composed of glucose, rhamnose and mannose; the antibacterial activity of polysaccharide against E. coli was the strongest, while the weakest against Sarcina lutea, the minimal inhibition concentrations of PL2 were 12.5, 25 and 100 μg/mL, respectively (Zhu et al., 2012).We show that aqueous extract of spent substrate of Pleurotus ostreatus/Barley straw has antibacterial activity against Escherichia coli (9.86 mm), Staphylococcus aureus (9.00 mm), Micrococcus luteus (9.66 mm) and Salmonella tiphymorium (31.10 mm), similar to that obtained using extracts of mushroom Pleurotus ostreatus obtained with different organic solvents (24.56 and 14 mm) for Gram positive and Gram negative bacteria (Nehra et al., 2012). For the cultivation of Lentinula edodes for many years have used various agricultural and industrial wastes, among which we mention sorghum, sugar cane, sawdust, oak, cedar (Grodzínskaya et al., 2002) as carbon source; shiitake mushrooms (Lentinus edodes) is of great importance, due to its attributed not only to its nutritional value, but also potential applications in industrial food and medicine as antibacterial (Hearst et al., 2009) and/or antitumor, among other features; this activity is due to the lentina (one polysaccharide isolated from fruiting body), which acts as an enhancer of host defense; has shown action against Staphylococcus aureus, Bacillus subtilis and Escherichia coli (Hatvani, 2001).Chowdhury, Kubrai, and Ahmed (2015) mentioned antimicrobial activity of 3 edible mushrooms (Pleurotus ostreatus, Lentinula edodes, Hypsizigus tessulatus) methanolic extracts, indicated considerable activity against bacteria and fungi, reveling zone of inhibition ranged from 7 ± 0.2 to 20 ± 0.1 mm; Kazue, Megumi, and Dantas (2001) found that the mycelium of 35 different strains of Lentinus edodes, has antibacterial activity against B. subtilis, with inhibition halos 5-20 mm in diameter similar to our findings. This work is novel because for the first time is studied the use of spent substrate Lentinula edodes as antibacterial, since only been shown this activity in the fruiting body; they have been used different extraction techniques: high-pressure operations and low-pressure methods.The high-pressure technique was applied to obtain Lentinus edodes extracts using pure CO 2 and CO 2 with co-solvent or organic solvents such as n-hexane, ethyl acetate and dichloromethane (Kitzberger, Smânia Jr., Pedrosa, & Ferreira, 2007); here it is included barley straw as a substrate for the cultivation of Pleurotus ostreatus and oats or cedar for the cultivation of Lentinula edodes, in order to obtain aqueous extracts and determine its antibacterial activity; the findings suggest that it is feasible to use these substrates in the future for obtain antibacterial pharmaceutical compounds and at the same time reduce the pollution by their accumulation. Table 1 . Antibacterial activity of spent substrate of Pleurotus ostreatus and Lentinula edodes extracts at different concentrations in vitro Table 2 . Comparison of antibacterial activity of spent substrate of Pleurotus ostreatus and Lentinula edodes extracts against Escheriquia coli, Salmonella tiphymorium, Staphylococus aureus and Micrococcus luteus in vitro.Note.ab Literal different ranks indicate significant difference between treatments of each bacteria (P < 0.05) with the Tukey test.	v3-fos-license
2017-12-19T18:09:41.248Z	2017-12-19T00:00:00.000	21227087	{ "extfieldsofstudy": [ "Medicine", "Chemistry" ], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fnmol.2017.00427/pdf", "pdf_hash": "f411fbca15b00826693e4907c89b84a4a9483ef2", "pdf_src": "PubMedCentral", "provenance": "20241012_202828_00062_s4wg2_d7ea2846-036c-420f-8433-dd5c6d8a39fe.zst:2735", "s2fieldsofstudy": [ "Medicine", "Biology", "Environmental Science" ], "sha1": "f411fbca15b00826693e4907c89b84a4a9483ef2", "year": 2017 }	pes2o/s2orc	Involvement of Astrocytes in Alzheimer’s Disease from a Neuroinflammatory and Oxidative Stress Perspective Alzheimer disease (AD) is a frequent and devastating neurodegenerative disease in humans, but still no curative treatment has been developed. Although many explicative theories have been proposed, precise pathophysiological mechanisms are unknown. Due to the importance of astrocytes in brain homeostasis they have become interesting targets for the study of AD. Changes in astrocyte function have been observed in brains from individuals with AD, as well as in AD in vitro and in vivo animal models. The presence of amyloid beta (Aβ) has been shown to disrupt gliotransmission, neurotransmitter uptake, and alter calcium signaling in astrocytes. Furthermore, astrocytes express apolipoprotein E and are involved in the production, degradation and removal of Aβ. As well, changes in astrocytes that precede other pathological characteristics observed in AD, point to an early contribution of astroglia in this disease. Astrocytes participate in the inflammatory/immune responses of the central nervous system. The presence of Aβ activates different cell receptors and intracellular signaling pathways, mainly the advanced glycation end products receptor/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, responsible for the transcription of pro-inflammatory cytokines and chemokines in astrocytes. The release of these pro-inflammatory agents may induce cellular damage or even stimulate the production of Aβ in astrocytes. Additionally, Aβ induces the appearance of oxidative stress (OS) and production of reactive oxygen species and reactive nitrogen species in astrocytes, affecting among others, intracellular calcium levels, NADPH oxidase (NOX), NF-κB signaling, glutamate uptake (increasing the risk of excitotoxicity) and mitochondrial function. Excessive neuroinflammation and OS are observed in AD, and astrocytes seem to be involved in both. The Aβ/NF-κB interaction in astrocytes may play a central role in these inflammatory and OS changes present in AD. In this paper, we also discuss therapeutic measures highlighting the importance of astrocytes in AD pathology. Several new therapeutic approaches involving phenols (curcumin), phytoestrogens (genistein), neuroesteroids and other natural phytochemicals have been explored in astrocytes, obtaining some promising results regarding cognitive improvements and attenuation of neuroinflammation. Novel strategies comprising astrocytes and aimed to reduce OS in AD have also been proposed. These include estrogen receptor agonists (pelargonidin), Bambusae concretio Salicea, Monascin, and various antioxidatives such as resveratrol, tocotrienol, anthocyanins, and epicatechin, showing beneficial effects in AD models. 1 Grupo de Investigación en Neurociencias (NeURos), Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia, 2 Biomedical Sciences Research Group, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia Alzheimer disease (AD) is a frequent and devastating neurodegenerative disease in humans, but still no curative treatment has been developed. Although many explicative theories have been proposed, precise pathophysiological mechanisms are unknown. Due to the importance of astrocytes in brain homeostasis they have become interesting targets for the study of AD. Changes in astrocyte function have been observed in brains from individuals with AD, as well as in AD in vitro and in vivo animal models. The presence of amyloid beta (Aβ) has been shown to disrupt gliotransmission, neurotransmitter uptake, and alter calcium signaling in astrocytes. Furthermore, astrocytes express apolipoprotein E and are involved in the production, degradation and removal of Aβ. As well, changes in astrocytes that precede other pathological characteristics observed in AD, point to an early contribution of astroglia in this disease. Astrocytes participate in the inflammatory/immune responses of the central nervous system. The presence of Aβ activates different cell receptors and intracellular signaling pathways, mainly the advanced glycation end products receptor/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, responsible for the transcription of pro-inflammatory cytokines and chemokines in astrocytes. The release of these pro-inflammatory agents may induce cellular damage or even stimulate the production of Aβ in astrocytes. Additionally, Aβ induces the appearance of oxidative stress (OS) and production of reactive oxygen species and reactive nitrogen species in astrocytes, affecting among others, intracellular calcium levels, NADPH oxidase (NOX), NF-κB signaling, glutamate uptake (increasing the risk of excitotoxicity) and mitochondrial function. Excessive neuroinflammation and OS are observed in AD, and astrocytes seem to be involved in both. The Aβ/NF-κB interaction in astrocytes may play a central role in these inflammatory and OS changes present in AD. In this paper, we also discuss therapeutic measures highlighting the importance of astrocytes in AD pathology. Several new therapeutic approaches involving phenols (curcumin), phytoestrogens (genistein), neuroesteroids and other natural phytochemicals have been explored in astrocytes, obtaining some promising results regarding cognitive improvements and attenuation of neuroinflammation. Novel strategies comprising astrocytes and aimed to INTRODUCTION The loss of cognitive abilities induced by the development of dementia represents one of the main pathological burdens in humans, critically interfering with social and occupational activities. According to the World Alzheimer Report, over 46 million people live with dementia worldwide, totaling an estimated cost of US $818 billion in 2015, expecting to rise up to $1 trillion by 2018 (Prince et al., 2015). The elevated economic and social impact of dementia has been considered as a public health priority by the World Health Organization (Frankish and Horton, 2017). Many types of dementia with varied pathophysiological mechanisms have been described, but the most frequent in humans is AD accounting for 50-70% of all cases (Querfurth and LaFerla, 2010). Characteristically, AD has been divided in early-onset AD (<65 years) and lateonset AD, with the latter representing around 90% of ADaffected individuals (Mendez, 2017;Pierce et al., 2017). The development of early-onset AD has been related to an altered genetic background, explained primarily by autosomal dominant mutations in APP (MIM #104760), Presenilin 1 (PSEN1) (MIM #104311), and Presenilin 2 (PSEN2) (MIM #600759) genes (Lanoiselée et al., 2017). Whereas a complete explanation for the development of late-onset AD (also commonly referred to as sporadic AD) remains obscure, despite the many risk factors associated with this pathology. Among these factors are included: genetic, such as the presence of the APoE ε4 allele, environmental, and several modifiable lifestyle factors (Killin et al., 2016;Van Cauwenberghe et al., 2016;Vos et al., 2017). The main pathological hallmarks of AD are the presence of extracellular Aβ plaques, intraneuronal neurofibrillary tangles primarily composed of hyperphosphorylated tau, and brain atrophy, together with increased brain neuroinflammation (Raskin et al., 2015;Bronzuoli et al., 2016). Although many theories have been proposed to explain the pathogenesis of AD, the most widely accepted is the amyloid hypothesis, which states that Aβ dyshomeostasis is responsible for the cognitive phenotype of the disease, acting upstream and contributing to other molecular and cellular alterations observed in this condition (Selkoe and Hardy, 2016). Aβ peptide is obtained from the serial cleavage of APP, first through the action of BACE-1 also referred to as beta-secretase, and posteriorly through the gamma-secretase complex (Carroll and Li, 2016;Yan, 2017). The gamma-secretase complex, which also acts on the notch pathway, is composed of four subunits: presenilin (1 or 2), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN2); with presenilin being the most actively studied and related to AD, as it contains the catalytic subunit of the complex (Ahn et al., 2010). Alterations in the cleaving process of APP produce abnormal lengthy species of the Aβ peptide which are deleterious to the brain cellular environment. These Aβ species have been reported to exhibit different profiles of toxicity, and among them, the soluble forms seem to be more neurotoxic than the fibrillary (aggregated) forms. In particular, the oligomeric form of the soluble Aβ 1−42 is considered to be highly harmful . The pathological study of brains from individuals with AD has revealed the presence of both neuroinflammation and OS (Lue et al., 1996;Ansari and Scheff, 2010). The precise mechanistic basis leading to the development of these changes in AD is not clear, and the debate of whether they are a causative factor or a consequence of the disease is still open. Despite the discussion, accruing evidence support a direct relationship between Aβ abnormal production and the development and/or maintenance of neuroinflammation and OS (Guerriero et al., 2016). Plenty receptors and carriers have been reported to interact with the different presentations of Aβ, although it seems that depending on the structure of Aβ (monomer, oligomer, fibrillary), some promote clearance or degradation while others mediate the neurotoxic effects through uptake and accumulation (Jarosz-Griffiths et al., 2016). Aβ interacts and binds to several cellular-expressed pattern recognition receptors in astrocytes and microglia, initiating an innate immune response (Minter et al., 2016). Accordingly, components of innate immunity and complement cascade have been considered risk factors for the development of AD and have been associated with abnormal clearing or deposition of Aβ; in particular, variants in the genes complement receptor 1 (CR1) , CD33 (Walker et al., 2015), and TREM2 (Yeh et al., 2016). Also, it has been shown that Aβ species, such as Aβ 1−42 , are able to induce the release of several proinflammatory cytokines and agents, including IL-1β, IL-6, NO, and TNFα, from glial cells (Lindberg et al., 2005;Hou et al., 2011). The precise intracellular signaling pathways involved in the proinflammatory and OS responses in neuronal and non-neuronal cells in AD are still not clear, although the NF-κB pathway has been reported to become activated in both settings (Shi et al., 2016). Astrocytes are important CNS resident cells involved in numerous physiological aspects. Similar to neurons, astrocytes represent a heterogeneous population of cells depicting diverse functional and morphological characteristics (Ben Haim and Rowitch, 2017). Astrocytes express several markers that allow them to be distinguished from neurons and other glial cells, including GFAP, calcium-binding protein S100B, glutamine synthetase, and Aldh1L1 (Sofroniew and Vinters, 2010). Astrocytes are key for the maintenance of homeostatic balance and participate in processes such as neurotransmitter uptake and recycling, gliotransmitter release, neuroenergetics, inflammation, modulation of synaptic activity, ionic balance, and maintenance of BBB, among others (Magistretti and Allaman, 2015;Iglesias et al., 2017;Vasile et al., 2017). Precisely, due to this wide array of functional properties, astrocytes have become interesting targets for the study and treatment of numerous brain pathologies. In AD, several reports have shown that astrocytes contribute to cellular and functional degeneration, disrupting glial-neuronal and glial-vascular signaling (Acosta et al., 2017). The aim of this paper is to review the relevant aspects concerning a possible role of astrocytes in the neuroinflammatory and OS changes observed in AD. As well, we will discuss novel neuroprotective and therapeutic measures highlighting the importance of astrocytes in AD pathology. ASTROCYTES AND ALZHEIMER'S DISEASE Different studies have shown that the cooperative activity between glia and neurons results in the modulation of cognitive functions (Perea et al., 2009;Fields et al., 2014). Neuron-glial interactions actively control synaptic plasticity and neurotransmission. The concept of "tripartite synapse" refers to this cellular network involving both presynaptic and postsynaptic neurons, as well as astrocytes (Araque et al., 1999;Perea et al., 2009). Numerous gliotransmitters released from astrocytes control synaptic plasticity in different brain structures (Yang et al., 2003;Pascual et al., 2005;Panatier et al., 2006) such as cortex (Ding et al., 2007) and hippocampus (Araque et al., 1998;Jourdain et al., 2007), and are involved in the modulation of memory and learning processes. The interruption of astrocyte's functions and hence in glia transmission, may result in different neuropsychiatric disorders (Rajkowska et al., 1999;Cohen-Gadol et al., 2004;Fellin et al., 2004;Webster et al., 2005), as well as neurodegenerative diseases, including AD (Forman et al., 2005;Halassa et al., 2007). Calcium Dysregulation A pathological increase in the amount of Aβ can induce functional and morphological changes in glial cells, including calcium dysregulation. In fact, microglia and astrocytes are activated close to senile plaques to internalize and break down Aβ (Mohamed and Posse de Chaves, 2011). This cellular activation may result in an inflammatory response and OS, playing a dual role in the pathophysiology of AD with both detrimental and neuroprotective results. Inflammatory mediators (i.e., bradykinin) may increase intracellular calcium concentration via nicotinic receptors and PI3K-Akt pathway in cultured astrocytes (Makitani et al., 2017). Tau has also been connected with astrocytes in AD, as Aβ was shown to bind the CaSR in human astrocytes, activating intracellular signaling which induced the production and release of phosphorylated Tau (Chiarini et al., 2017). Amyloid beta has been shown to disrupt gliotransmission by enhancing calcium signaling in astrocytes . This calcium/gliotransmission alteration could underlie an important role of astrocytes in AD pathology. Actually, astrocytic calcium levels are abnormal in several models of AD as both acute and chronic exposure to Aβ elevates baseline calcium levels in cultured astrocytes (Haughey and Mattson, 2003;Alberdi et al., 2013;Lim et al., 2013). This calcium is partially released from intracellular sources such as the endoplasmic reticulum (Toivari et al., 2011). In addition, Aβ interacts with several types of surface receptors in astrocytes which leads to calcium entry, including purinergic receptor P2Y1 (Delekate et al., 2014), nicotinic receptors (α7-nAChRs) (Xiu et al., 2005;Lee et al., 2014), and glutamate metabotropic receptor mGluR5 Ronco et al., 2014). For instance, hippocampal astrocytes exposed to Aβ increased the frequency of NMDA receptor-mediated slow inward currents, together with calcium elevations mediated through α7nAChR activation (Pirttimaki et al., 2013). Aβ-induced dysfunction of NMDA receptors in astrocytes disrupts neuronglial signal transmission with dramatic consequences on neuronal homeostasis, synaptic transmission, and plasticity. Therefore, neurotoxicity and selective neurodegeneration may be explained by Aβ simultaneous interaction with several receptors and neurotransmitter systems in the context of astrocyte calcium dysregulation. Glutamatergic Dysfunction and Excitotoxicity In AD, it has been shown that Aβ can interrupt glutamate uptake capacity and astrocytic calcium signaling (Vincent et al., 2010;Matos et al., 2012). Also, an increase in the expression of astrocytic Tau from aged transgenic animals leads to a decline in GLT activity and therefore in subsequent neurodegeneration (Komori, 1999;Dabir et al., 2004). Some studies have demonstrated in ex vivo astrocyte preparations that Aβ 1−42 decreases the expression of GLT-1 and GLAST, two major GLTs in astroglia, via adenosine A2A receptors (de Vivo et al., 2010;Matos et al., 2012). Therefore, disruption in the clearance of excitatory neurotransmitters and increased levels of Aβ and Tau from astrocytes seem to be involved in the neuronal excitotoxicity observed in AD. Glutamate NMDA and AMPA receptors have been related to the physiopathology of AD (Parameshwaran et al., 2008). Different studies have identified the expression of functional NMDA receptors in astrocytes (Kommers et al., 2002;Lalo et al., 2006) involved in cerebral vasodilatation, synaptic transmission, and neuronal-glial signaling (Verkhratsky and Kirchhoff, 2007;Palygin et al., 2010;Parfenova et al., 2012). Hence, Aβ-induced dysfunction in the expression and function of glutamate receptors in astrocytes, mainly in NMDA receptors, can interfere with neuronal-glial communication (Mota et al., 2014). The cellular excitotoxicity produced by the excessive stimulation of NMDA receptors in neurons and astrocytes has been shown to be reduced with the use of MK801 and memantine (NMDA receptor antagonists) (Lee et al., 2010). Furthermore, due its possible therapeutic role in neurodegenerative diseases including AD, a recent antagonist (UBP141) with preferential effects on astroglial NMDA receptors has been developed (Palygin et al., 2010(Palygin et al., , 2011. A better comprehension of the differences between neuronal and glial NMDA receptors may provide key elements for the development of novel therapeutics which primarily or selectively target astrocytic function. As well, Aβ can induce glutamate release from astrocytes resulting in an extrasynaptic activation of NMDA receptors. In this case, nitromemantine, which selectively inhibits extrasynaptic NMDA receptors, may protect against Aβ-induced synaptic dysfunction in the hippocampus (Talantova et al., 2013). Additionally, nitromemantine may prevent the synapse-destroying effects of Aβ/α7-nAChR signaling (Dal Prà et al., 2015). Therefore, using astrocytic signaling as a possible target for drug development may have a therapeutic function in AD's prevention and control. The antiepileptic drug levetiracetam has shown to reverse synaptic dysfunction as well as memory and learning deficits in human APP (hAPP) transgenic mice (Sanchez et al., 2012). Moreover, a retrospective observational study has shown clinical benefits of levetiracetam in early AD (Vossel et al., 2013). One way this drug may act is increasing glutamate and GABA transporters in astrocytes (Ueda et al., 2007). Chronic administration of levetiracetam may attenuate glutamate excitotoxicity and increase inhibitory neurotransmission. This molecular mechanism involving astrocytes may result in a reduction of cognitive abnormalities in AD. Aβ Clearance Astrocytes also participate in the degradation and removal of Aβ as they express different types of proteases involved in the enzymatic cleaving of Aβ. The metalloendopeptidases NEP, IDE, and ECE1 and ECE2 have been reported to be expressed in astrocytes, and are involved in the degradation of monomeric Aβ species (although NEP also hydrolyze oligomeric forms) (Mulder et al., 2012;Ries and Sastre, 2016). It has been proposed that the modification from "natively foldedactive" to "aggregated-inactive" form of IDE and NEP may be a relevant pathological mechanism in late-onset AD (Dorfman et al., 2010). Astrocytes also express and secrete several MMPs, including MMP-2 and MMP-9, which degrade both monomeric and fibrillar extracellular forms of Aβ (Ries and Sastre, 2016). Furthermore, it was found in APP/presenilin 1 transgenic mice that astrocytes surrounding Aβ plaques increased the expression of both MMP-2 and MMP-9 Yin et al., 2006). Apolipoprotein E is primarily produced by astrocytes in the CNS and has been proposed to play a major role in AD. In mice, ApoE( −/− ) astrocytes have been shown to fail to respond or internalize Aβ deposits to the same extent as do wild-type astrocytes (Koistinaho et al., 2004). As well, mice astrocytes expressing the ApoE ε4 allele were less effective eliminating Aβ plaques than those astrocytes expressing the ApoE ε3 allele (Simonovitch et al., 2016). Astrocytes derived from human induced pluripotent stem cells (iPSC) which expressed the ApoE ε4 allele failed to support neuronal neurotrophic functions such as survival and synaptogenesis (Zhao et al., 2017). As the presence of ApoE ε4 allele is considered a major risk factor in AD while the presence of ApoE ε2 allele is considered a protective factor, a differential regulation of these isoforms regarding the presence of Aβ and associated responses such as neuroinflammation has been proposed (Dorey et al., 2017). AD and Astrocyte Imaging One of the most important research areas in AD is related to the development of biomarkers. Although several types of biomarkers have been explored, there is still not one that specifically diagnose, differentiate, and predict the rate of decline between populations of cognitively healthy/preclinical dementia, mild cognitive impaired and AD individuals (Fiandaca et al., 2014;Salvatore et al., 2015;Huynh and Mohan, 2017). Due to their fundamental role in CNS homeostasis, astrocytes could be considered as possible targets for tracking and studying in vivo changes in AD as well as serving as a biomarker for the disease. L-Deprenyl is a selective inhibitor of the enzyme MAO-B, predominantly found on astrocytes (Levitt et al., 1982). This compound has been successfully used in vitro and in vivo to study the distribution and activity of MAO-B through different techniques including quantitative autoradiography and positron emission tomography (PET) (Kumlien et al., 1992;Arakawa et al., 2017). In postmortem samples from individuals with AD, the activity of both MAO-B and the binding of [3H]L-deprenyl was found to be increased in many brain regions (Jossan et al., 1991). In addition, MAO-B has been found to be increased during reactive astrocytosis in neurodegenerative conditions (Ekblom et al., 1993(Ekblom et al., , 1994. As astrocytes produce MAO-B and this enzyme is increased during reactive astrocytosis, which is a process observed in AD, it seemed plausible to use L-dyprenyl as a marker of astrocytosis in this condition. Accruing evidence seems to support the use of L-deprenyl in AD. A comparative study using PET between one of the currently accepted biomarkers for AD, the 11C-Pittsburgh Compound B, and (11)C-deuterium-L-deprenyl, concluded that the latter provided non-redundant information on both functional and pathologic aspects of the disease (Rodriguez-Vieitez et al., 2016a). Furthermore, L-deprenyl has provided valuable information about the stage of progression of AD. In a human study, the highest binding for (11)C-deuterium-L-deprenyl was observed in Braak I-II (initial AD stages), whereas it decreased with the most advanced Braak stages (Gulyás et al., 2011). Similar results have been obtained in other studies using (11)C-deuterium-Ldeprenyl, where astrocytosis is prominent at the initial phases, even at preclinical stages, and then declines as the disease progresses (Carter et al., 2012;Schöll et al., 2015;Rodriguez-Vieitez et al., 2016b). In addition, a similar laminar binding pattern for tau and [3H]L-deprenyl at the temporal lobe was recently demonstrated, suggesting tau deposits and astrocytic inflammatory processes are closely related in AD (Lemoine et al., 2017). All these results point to an early contribution of astrocytes in AD pathology. GABA-Glutamine Cycle Neurons and astrocytes work in a coordinated way throughout different metabolic pathways to synthesize and release glutamate and GABA (Bak et al., 2006). At inhibitory synapses this pathway is called the GABA-glutamine cycle and it depends on GABA transporters and a multi-enzyme machinery that coordinates this process (i.e., GABA transaminase, glutamate decarboxylase, and glutamine synthetase) (Bak et al., 2006;Hertz, 2013). In AD, the processes related to GABA-glutamine cycle and GABA release from astrocytes seem to be altered. The glutamine-glutamate/GABA cycle consists of the transfer of glutamine from astrocytes to glutamatergic and GABAergic neurons. This process depends on glutamine synthetase and the tricarboxylic acid cycle (Walls et al., 2015). A reduction in pyruvate carboxylation, glutamine levels, and tricarboxylic acid cycle turnover in GABAergic neurons and astrocytes was shown in the transgenic rat AD model, McGill-R-Thy1-APP (Nilsen et al., 2014). Similarly, reduced expression of glutamine synthetase in postmortem AD brain samples indicates a profound alteration in neurotransmitter and protein synthesis, as well as metabolic dysfunction (Robinson, 2000). Astrocytes may produce and release GABA, with a main role on hippocampal synaptic plasticity function during memory processing. Increased activity of glutamate decarboxylase enzyme was found in glial synaptosomes obtained from the cortex of APP/TS1 transgenic mice, suggesting Aβ plaques stimulate GABA synthesis from astrocytes (Mitew et al., 2013). Reactive astrocytes from APP/PS1 transgenic mice have also been shown to produce GABA involving MAO-B, and release it through the bestrophin 1 channel, in an aberrant manner (Jo et al., 2014). In the same study, the suppression of GABA production or release from astrocytes completely restored the cognitive deficits and impairments in synaptic plasticity observed in the mice. Under physiological conditions, astrocytic GABA exerts a disinhibitory action at the perforant path to dentate gyrus neurons via GABA B receptors on interneurons. However, in the APPswe/PSEN1dE9 mice, it has been shown an inhibitory action of astrocytic GABA by targeting GABA A receptors in glutamatergic terminals (Yarishkin et al., 2015). These results provide a useful specific GABAergic target aimed at memory impairment reduction in AD. Alterations in the metabolic functions of astrocytes and consequently in glutamate and GABA-glutamine cycles may help explain cognitive disorders in AD (Le Prince et al., 1995;Robinson, 2000;Nilsen et al., 2014). Neurotransmitter transporters and effectors together with GABA-metabolizing enzymes are of special interest in drug development regarding therapeutical options for GABA-related neurological dysfunctions such as AD (Sarup et al., 2003;Nava-Mesa et al., 2014;Mutis et al., 2017;Sánchez-Rodríguez et al., 2017). Although special attention should be taken regarding the differential functional roles of neuronal and glial neurotransmitter transporters and overlying GABA/glutamate metabolic pathways in the development of high selective cellspecific drugs, in order to avert pharmacological interactions and unexpected side effects. Metabolic Compromise The metabolic cooperation between astrocytes and neurons is essential to the brain functioning. The energy metabolism of neurons depends on blood oxygen supply but also on astrocytic glucose transporters, mainly GLUT1 (Morgello et al., 1995). In addition, astrocytes may convert glycogen to lactate during periods of higher activity of the nervous system (Falkowska et al., 2015). Both in vivo and in vitro studies indicate that astrocytes participate in the regulation of cerebral blood flow according to neuronal activity and metabolic demand (Magistretti and Pellerin, 1999;Magistretti, 2006). Therefore, astrocytes are key to guarantee an adequate coupling between brain activity and metabolic supply. Several studies have shown reduced cerebral glucose metabolism in early stages of AD and correlation with symptoms severity (Desgranges et al., 1998;Mosconi et al., 2005Mosconi et al., , 2006Mosconi et al., , 2008. As mentioned early, Aβ affects neuronal excitability and it also may reduce astrocytic glycolytic capacity (Soucek et al., 2003;Schubert et al., 2009) and reduce the neurovascular unit function (Acosta et al., 2017;Kisler et al., 2017). Moreover, reductions in GLUT1 and lactate transporters in astrocyte cultures derived from transgenic AD mice have been reported (Merlini et al., 2011). In AD, the resulting metabolic dysfunction may alter the overall oxidative neuronal microenvironment (Mosconi et al., 2008). The chronic sustained effect of diminished lactate supply, increased neuronal activity, and reduced neurovascular coupling, underlines the OS increase during AD. Therefore, astrocytes are crucial players either acting as protectors against OS or participating in the progression of AD. The specific role of astrocytes on inflammatory response and OS damage will be reviewed in the next sections. NEUROINFLAMMATION, ALZHEIMER'S DISEASE, AND ASTROCYTES Inflammation is a protective physiological response necessary to regulate processes associated with damage mechanisms in the organism. Several actions related to general inflammatory activities include protection against microorganisms, tissue repair, and removal of cellular debris. The CNS possesses some characteristics that differentiate the immune and inflammatory activities of the brain and spinal cord from those occurring in the rest of the body. Mainly, these differences arise through the presence of the BBB, which restricts the pass of leukocytes into the brain parenchyma, and also due to the cellular interactions of microglia and astrocytes, responsible for most of the immune/inflammatory CNS responses (Ransohoff et al., 2015). Although neuroinflammation arises innately as a protective mechanism when injury is present in the CNS, alteration in any of the components of this response may compromise the cellular microenvironment and become noxious to the brain. Many neurodegenerative conditions, including AD, have been associated with the presence of abnormal neuroinflammation (Ransohoff, 2016). The role of astrocytes in neuroinflammation has been highlighted in the past years with many observations both in vivo and in vitro depicting the importance of these glial cells in this process (Colombo and Farina, 2016). In fact, an increase in the expression of GFAP is commonly considered as a hallmark of neuroinflammation in many neurodegenerative conditions, including AD (Millington et al., 2014). Astrocytes, together with microglia, react to a diverse range of pro-and anti-inflammatory agents (Sofroniew, 2014). Depending on the cytokine, astrocytes modify their phenotype to either activated or deactivated state. Increased levels of INF-γ, IL-1β, IL-6, and TNFα induce astrocytes to adopt a classical activation state (increased activation of NF-κB pathway, production of ROS and NO, and release of IL-1β, IL-6, and TNFα), while increased levels of IL-4 and IL-13 induce an alternative activation (increased secretion of IL-4 and decreased production of ROS and NO); oppositely, high levels of IL-10 and TGF-β induce astrocytic deactivation (reduced immune surveillance and proinflammatory signaling) (Dá Mesquita et al., 2016). Furthermore, the reactive state of astrocytes may also depend on the source of injury (neuroinflammation or ischemia), indicating the complex range of responses these cells are capable to produce (Zamanian et al., 2012). In a recent paper, a new classification of reactive astrocytes was proposed, designating A1 those astrocytes that developed a neurotoxic phenotype and A2 those that depicted neurotrophic and neuroprotective characteristics (Liddelow et al., 2017). The authors also reported that the presence of IL-1α, TNFα, and C1q (all three released from microglia) promoted the appearance of A1 astrocytes and that this phenotype was found to be predominant in brain tissue from AD patients. These findings raise a number of questions regarding the manner in which the brain deals with different types of injuries and specifically how astrocytes and astrocyticcellular interactions induce either a protective or harmful profile. An increase in A1 astrocytes seems to occur in AD, but still is not clear if Aβ induces this phenotype or if another specific agent is involved in this reaction. Nonetheless, it has been reported that the interaction of Aβ with astrocytes induces a pro-inflammatory profile and even astrogliosis (Batarseh et al., 2016). RAGE, Astrocytes, and Amyloid Beta Amyloid beta has been reported to interact with numerous cellular receptors and astrocytes express a large amount of them, including TLRs, scavenger receptors, glycoprotein receptors, lipoprotein receptors, RAGE, acetylcholine receptors, complement and chemokine receptors, T-cell receptors, and mannose receptor, among others (Farfara et al., 2008). Binding of Aβ to different types of receptors seems to depend on the Aβ peptide form (monomer or fibrillar). For example, IRand SEC-R bind monomeric forms of Aβ, scavenger receptor CD36 and glycoprotein receptors lactadherin, and CD47 prefers fibrillary Aβ, while RAGE, ApoE, and nAChR α7nAChR bind both monomer and fibrillar forms (Verdier et al., 2004). The specific outcome of all these complex Aβ-astrocytic interactions is still under research, as the precise intracellular and intercellular communication changes prompted by the different types of Aβ acting on these receptors is yet to be elucidated. Despite the gaps in knowledge, the activation of some receptors, in particular RAGE, has been reported to induce proinflammatory changes in astrocytes when exposed to Aβ (González-Reyes and Graciela Rubiano, 2016). Advanced glycation end products receptor is a multiligand pattern-recognition receptor, member of the immunoglobulin superfamily with a variety of isoforms present in brain cells (Ding and Keller, 2005). In addition to Aβ and several AGE, RAGE can bind DNA-binding protein HMGB1/amphoterin (Hori et al., 1995) and S100/calgranulins (Hofmann et al., 1999). The main intracellular pathway activated through RAGE is the NF-κB pathway (Tóbon-Velasco et al., 2014), although it can also activate other downstream pathways including Cdc42-Rac, p21ras and MAPK, JNK, and ERK (González-Reyes and Graciela Rubiano, 2016). Furthermore, astrocytes have been reported to adopt a phagocytic profile capable of engulfing Aβ, mediated by CD36, CD47, and RAGE receptors (Jones et al., 2013). Apart from Aβ, the interaction of astrocytic RAGE with other ligands, such as S100B, may also be involved in AD neuroinflammation (Cirillo et al., 2015). These findings point to the interaction of RAGE/NF-κB pathway in astrocytes as an important factor in the development or maintenance of inflammation in AD. Astrocytes and NF-κB Pathway The transcription factor NF-κB is currently considered as an important agent related to neuroinflammation in AD (Shi et al., 2016). NF-κB is known to be mainly activated by two pathways, the canonical (or classical) and the noncanonical (or alternative) (Nakajima and Kitamura, 2013). The canonical pathway involves activation of various receptors including RAGE and cytokine receptors, such as TNF receptor, IL-1 receptor, and the TLR family. These will induce the further activation of many downstream agents, in special IKKs alpha (IKKα) and beta (IKKβ), and NEMO (in charge of the degradation of the cytoplasmic inhibitor IKBα), and the subsequent complexes (mainly RelA) that act as transcription factors in the nucleus (Marcu et al., 2010;Wan and Lenardo, 2010). The non-canonical pathway, also known as the NEMO-NF-κB-independent pathway, occurs when NF-κB is activated by specific recruitment of TRAF2 and TRAF3, and involves p52 and RelB (Morgan and Liu, 2011). Both canonical (Wang et al., 2013) and non-canonical (Akama and Van Eldik, 2000) activation of NF-κB has been observed in astrocytes stimulated with Aβ, but still is not clear which type predominates in AD or if a differential NF-κB activation is related to the stage of the disease. It has been reported that most of the cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the NF-κB pathway, suggesting a central role of this factor in the proinflammatory (neurotoxic) and immunoregulatory (neuroprotective) actions of astrocytes in the CNS (Choi et al., 2014). Also, NF-κB is involved in other functions such as neuronal survival, differentiation, apoptosis, neurite outgrowth, and synaptic plasticity, all found to be altered in AD (Mémet, 2006). In astrocytes and microglia, the activation of NF-κB due to Aβ stimulation leads to the production of the pro-inflammatory cytokines IL-1β, IL-6, iNOS, and TNFα (Bales et al., 1998;Akama and Van Eldik, 2000;Hou et al., 2011). In rats treated with Aβ 1−42 oligomers, it was shown that COX-2, IL-1β, and TNFα were expressed in reactive astrocytes surrounding the Aβ-injection site and in nearby blood vessels, as well was found co-localization of NF-κB proteins with GFAP and COX-2 (Carrero et al., 2012). In primary astrocytic and mixed astrocytic-neuronal cell cultures from rats, the use of minocycline, an anti-inflammatory agent, reduced astrocytic inflammatory responses together with a decrease in neuronal loss, caspase-3 activation, and caspase-3-truncated Tau species in neurons (Garwood et al., 2011). Minocycline has been shown to inhibit the NF-κB signaling pathway in spinal rat astrocytes (Song et al., 2016). Other reports of beneficial outcomes due to regulation of the NF-κB signaling pathway in astrocytes were reviewed by Colombo and Farina (2016). Although an exaggerated neuroinflammatory response is observed in AD, an absolute suppression of the NF-κB signaling pathway may be undesirable and even worsen the pathological condition. In APPswe/PS1dE9 transgenic mice, the suppression of NF-κB attenuated astrogliosis in the hippocampus and cortex of the animals but increased the amount of Aβ 1−42 , suggesting a role of astrocytic-mediated neuroinflammation in the clearance of Aβ (Zhang et al., 2009). As well, the clinical evidence for the use of non-steroidal anti-inflammatory drugs (NSAIDS) in AD patients has not proven to be of benefit (Aisen et al., 2008;Szekely et al., 2008;Beeri et al., 2012; Alzheimer's Disease Antiinflammatory Prevention Trial Research Group, 2013). Inflammatory Induction of Aβ in Astrocytes Astrocytes not only are activated and induced to release chemokines and cytokines in the presence of Aβ, these cells also react to the presence of pro-inflammatory cytokines and even increase the production of Aβ in response. Therefore, the presence of inflammation is capable of increasing the production of Aβ. In addition, the development of neuroinflammation has also been related to cognitive changes in AD (Westin et al., 2012;Echeverria et al., 2016;Laurent et al., 2017). Neuroinflammation in AD is characterized by the accumulation of cytokines such as IL-1β, IL-6, TNF-α, or TGF-β, which can contribute with cerebral amyloid deposition, augmentation of APP expression, Aβ formation, and subsequent recruitment and activation of microglial cells (Esler and Wolfe, 2001). In general, TNF-α, IL-1β, IFN-γ, L-6, and TGF-β are able to stimulate β-secretase and γ-secretase enzymatic activity through a JNK-dependent MAPK pathway, which cleaves APP and initiates Aβ formation (Liao et al., 2004). Astrocytes express and respond to a large scope of cytokines and chemokines suggesting a central role in the inflammatory-induced production of Aβ. A study reported that a systemic immune challenge in wildtype mice during late gestation induced the development of ADlike pathology during aging, with animals displaying increased levels of hippocampal APP and altered Tau phosphorylation, together with microglia and astrocytic activation (Krstic et al., 2012). Additionally, it was shown that LPS-induced systemic inflammation in mice could contribute to cognitive impairment and increased expression of APP and Aβ 1−42 , associated with increased production of inflammatory mediators such as COX-2, IL-1, and iNOS (Lee et al., 2008). The same paper reported that these changes were accompanied with astrocytic activation. Other studies have found evidence, both in human and murine models, that inflammation induces the expression of Aβ. Primary astrocytes from mice, treated with a combination of TNFα and INF-γ, significantly increased levels of BACE1, APP, and Aβ 1−40 (Zhao et al., 2011). In human primary astrocytes, treatment with INF-γ in combination with either TNFα or IL-1β induced the secretion of Aβ 1−40 and Aβ 1−42 (Blasko et al., 2000). Furthermore, TGF-β1 was found to induce overexpression of APP in astrocytes but not in neurons (Lesné et al., 2003). Cytokines seem to act on the 5 -untranslated region (5 -UTR) of the APP gene in astrocytes . On the one hand, it seems that the presence of Aβ is able to induce the production and release of pro-inflammatory cytokines and chemokines from astrocytes, which could as well act in an autocrine manner to further induce the production of Aβ from astrocytes and possibly other cells. On the other hand, these results suggest that inflammation may be present at early stages (pre-clinical) of the disease or even that inflammation may be responsible for the appearance of pathological Aβ production and accumulation. Under both circumstances, astrocytes appear to be deeply involved in inflammatory changes observed in AD. Astrocytes and Other Mechanisms of Neuroinflammation Other factors related to astrocytes may contribute to the appearance or enhancement of neuroinflammation in AD. For example, the presence of elevated glucose levels (as found in diabetes) has been shown to increase neurotoxicity and the release of pro-inflammatory cytokines from primary human astrocytes (Bahniwal et al., 2017). A relation between AD and diabetes/metabolic syndrome has been explored previously , as both Aβ and AGE bind to RAGE in astrocytes. As well, pro-inflammatory signaling in astrocytes may involve changes in the expression of the calcium-dependent phosphatase CaN, which has been shown to interact with another transcription factor involved in inflammatory responses, the NFAT (Acosta et al., 2017). Enhanced nuclear accumulation of CaN/NFAT was observed in human AD hippocampus and astrocytic cultures treated with Aβ (Abdul et al., 2009). In addition, it has been reported that Aβ deregulates calcium homeostasis via CaN and its downstream target NF-κB, as well as increasing NF-κB-dependent expression of mGluR5 and IP3R2 in astrocytes (Lim et al., 2013). Changes in mGluR5 and IP3 receptor expression have been reported in astrocytes surrounding amyloid plaques in a genetic mouse model of AD (Norris et al., 2005). Another possible factor contributing to the presence of neuroinflammation is the BBB, as in AD, it has been reported that the BBB occasionally loses its integrity (Chakraborty et al., 2017). This may be explained in part thanks to the accumulation of Aβ in brain blood vessels and also due to the associated vascular inflammation, allowing crossed communication between the peripheral immune system and the brain (Takeda et al., 2014). As astrocytes have a very important interaction with the BBB and its functional components are plausible to consider their involvement in BBB-associated neuroinflammatory changes in AD. Uncontrolled neuroinflammation is a critical element in the progression of AD, impairing the normal function of the CNS. Astrocytes, together with microglia, are the main cells involved in the inflammation/immune responses of the CNS. The presence of Aβ activates different astrocytic cell receptors, mainly RAGE, inducing the activation of the inflammatory pathway NF-κB responsible for the transcription of numerous pro-inflammatory cytokines and chemokines in astrocytes. In addition, the presence of pro-inflammatory cytokines such as IL-1β can act on astrocytes stimulating the production of Aβ and perpetuating a proinflammatory profile in astrocytes. Astrocytes are key in the maintenance of the homeostatic balance of the CNS and use the mechanism of reactive astrogliosis as a defensive reaction (Pekny et al., 2016), therefore is fundamental to understand the pathophysiological process that causes astrocytes to convert from a protective agent into a cell that produces a maladaptive astrogliosis response in AD (Table 1). OXIDATIVE STRESS, ALZHEIMER'S DISEASE, AND ASTROCYTES Oxidative stress is the result of a dysregulation between the amount of free and non-free radicals produced, including ROS and RNS. This can be attributed to the loss of homeostasis due to mitochondrial overproduction of oxidants over the production of antioxidants (Swomley and Butterfield, 2015). Among the most important ROS are the peroxyl radicals (ROO·), NO, the superoxide radical anion (O − 2 ), the hydroxyl radical OH· and some other non-radical species such as peroxynitrite (ONOO − ), single oxygen (O 2 ), and hydrogen peroxide (H 2 O 2 ) (Dasuri et al., 2013). ROS, as well as RNS, are produced under physiological conditions during the common metabolic pathways. These reactive species act on second messengers TABLE 1 \| Summary of studies reporting effects of Aβ or AD on pro-inflammatory and anti-inflammatory cytokines and chemokines in astrocytes. Frontiers in Molecular Neuroscience \| www.frontiersin.org and subsequently may influence several signaling pathways in a positive or negative form, depending on the regulatory mechanism of its concentration, called redox regulation (Valko et al., 2007). Likewise, mitochondria are able to produce antioxidants which counteract the harmful effects of OS to maintain the balance between the production and detoxification of ROS. These antioxidants are classified in two main groups: enzymatic antioxidants such as SOD, catalase, antioxidant GSH, GPX, GSH reductase, and GSH-S-transferase, and nonenzymatic antioxidants such as GSH, thioredoxin, vitamins A, E, and C, flavonoids, and proteins like albumin and metallothionein (Valko et al., 2007;Halliwell, 2012). Oxidative Stress and Alzheimer's Disease The development of OS in AD has been related to mitochondrial dysfunction, leading to superoxide overproduction ending in synaptic damage (Friedland-Leuner et al., 2014;Bhat et al., 2015). Mitochondrial dysfunction in AD seems to be linked to the increased presence of ROS and RNS (Islam, 2017). Müller et al. (2010) observed a decreased mitochondrial potential in transgenic Thy1-APP751SL mice. The same authors reported that increased intracellular Aβ production might trigger mitochondrial dysfunction quite early and independently of Aβ plaques and, that the accumulation of these alterations with aging lead to disruption of respiratory chain complexes (mainly III and IV) and significant reduction in the generation of NADH. The authors suggested that progressive increase in oxidant production together with a decrease in antioxidant components may conduce to the loss of brain homeostasis observed in AD. However, in AD, it has been demonstrated that prior to the appearance of senile plaques, brains present glucose hypometabolism due to abnormal oxidative metabolic routes in the mitochondria, which also induce increased ROS production and subsequent oxidative cell damage (Maruszak andŻekanowski, 2011). Additionally, variants of gene expression profiles in AD have shown downregulated expression of NeuroD6, which encodes a transcription factor involved in triggering antioxidant responses and the maintenance of the production of mitochondrial antioxidants (Uittenbogaard et al., 2010;Fowler et al., 2015). Also, RNA-Seq and microarray data analysis indicated a consistent downregulation of NeuroD6 in brains of individuals with AD, suggesting downregulation of NeuroD6 as a possible biomarker for AD . The role of ROS and OS in neurodegenerative diseases, including AD, is not entirely clear, although it has been observed that a modest level of oxidative RNA damage occurs during the process of aging in brain neurons, but a prominent level of oxidative RNA damage is present in vulnerable neurons which correspond to the earliest stage of cognitive decline in the transition from cognitively normal aging to AD (Nunomura et al., 2012). Furthermore, DNA bases are vulnerable to OS damage involving hydroxylation, protein carbonylation, and nitration in AD (García-Blanco et al., 2017). Changes in oxidative markers have been reported in brain regions such as hippocampus and inferior parietal cortex, which are also compromised in AD (Floyd and Hensley, 2002). Brain is considered to be especially vulnerable to OS and susceptible to lipid peroxidation because of its high lipid and poly-unsaturated fatty acids content, and its low concentrations of antioxidants (Butterfield et al., 2013). In AD, neurotoxic effects of Aβ induce OS through lipid peroxidation, protein degradation, and amino acid oxidation, which in turn increase the production of ROS and RNS by positive feedback (Swomley and Butterfield, 2015). Alkenals, 4-hydroxynonenal (HNE), and 2-propenal (acrolein) are reactive products obtained from lipid peroxidation induced by Aβ. These agents can modify covalently some amino acids residues or change protein conformation, which in turn affects its function. Thus, a coupling between increased lipid peroxidation and structural modification of GLT-1 has been proposed, explained by increased HNE binding due to excessive Aβ 1−42 (Butterfield et al., 2002). These events can compromise astrocyte function, inducing glutamate transport inhibition and increasing excitotoxicity to neurons in AD. Astroglia Role in Oxidative Stress Astrocytes seem to be involved in the processes leading to the appearance or maintenance of OS in AD. Abramov et al. (2004a), working in rat astrocytes, have shown that Aβ treatment increases intracellular-free calcium influx from the extracellular space, and induces changes in mitochondrial functions. These changes are associated with the activation of NOX due to Aβ interaction on the membrane, which, in turn, induces [Ca 2+ ]i changes. The main changes leading to mitochondrial dysfunction in astrocytes are associated with mitochondrial depolarization, increased conductance, and presence of mitochondrial permeability transition pores (Duchen, 2000). A possible mechanism which explains why calcium influx is induced by Aβ into astrocytes could be related with the formation of calcium selective channels on the membrane, which seem capable of generating a different conductance (Abramov et al., 2004b). These channels have been shown to be formed by insertion of Aβ peptides in the membrane, and also are arranged in a structural configuration which requires a lesser content of cholesterol on the lipidic membrane (Kawahara and Kuroda, 2001;Arispe and Doh, 2002;Arispe et al., 2007). Additionally, it has been found that Aβ 1−42 oligomers are key factors on the induction of OS stress by astrocytes. Aβ 1−42 oligomers binding to RAGE on astrocytes induce ROS production via NOX complex activation (Askarova et al., 2011). However, astrocytes are also able to trigger ERK1/2 pathways and cytosolic phospholipase A2 phosphorylation, independent of NOX activation, which in turn causes mitochondrial dysfunction by decreasing mitochondrial membrane potential, enhancement of NOX activity, and overproduction of ROS (Zhu et al., 2006). Astrocytes seem to be a primary target of Aβ, as this peptide induces various effects related to OS such as altered intracellular calcium signaling and calcium-dependent reduction in astrocytic GSH (Abramov et al., 2004b). Although this GSH depletion affects astrocytes, neurons are the cells that show a higher rate of cell death, suggesting that neurotoxicity reflects the neuronal dependence on astrocytes for antioxidant support. This could better be explained by the fact that astrocytes are the producers of the primary elements required for the production of GSH in neurons, such as glycine and cysteine (Abramov et al., 2003;Gandhi and Abramov, 2012). Regarding GSH, it has also been shown that in cultured astrocytes, a prolonged incubation with Aβ reduces induction of the transporter ABCC1 which is the main pathway for GSH release (Ye et al., 2015). Astrocytes play a central role in maintaining the neuronal integrity, nevertheless cytokines and neurotransmitters released by damaged or activated astrocytes may increase the neurotoxicity and vulnerability of neurons. During chronic OS, as observed in AD, the crosstalk communication between astrocytes and neurons is impaired resulting in disrupted memory consolidation. This compromise in memory formation is probably due to calcium overload and activation of MAPK pathways in astrocytes, which involve as well the JNK/SAPK pathways, and may conduce to anomalous deleterious signaling, including autophagic astroglial signals and apoptosis (Ishii et al., 2017). Oxidative Stress and the Connection with Neuroinflammation During neuroinflammation, increased concentrations of ROS/RNS may lead to the activation of the transcription factor NF-κB which induces the overexpression of NO synthases in astrocytes and microglia, in particular NOX2 and iNOS, resulting in peroxynitrite production by superoxide and NO reaction producing neuronal damage (Saha and Pahan, 2006;Brown, 2007;Morgan and Liu, 2011). Moreover, NF-κB activation induces the expression of COX-2 and cytosolic phospholipase A2, which in turn stimulate the generation of prostaglandins, promoting inflammation and OS (Hsieh and Yang, 2013). Castegna et al. (2003) reported that the formation of peroxynitrite ONOO − leads to protein nitration in enzymes, such as alpha and gamma enolases, implicated in brain glucose metabolism. Thus, the signaling pathway NF-κB, which is also heavily involved in inflammatory reactions, has been proposed to be involved in OS, as a direct crosstalk between ROS and NF-κB has been reported (Turillazzi et al., 2016). In AD, the presence of chronic OS alters the protective physiological role of the NF-κB transcription pathway, which normally promotes cell survival and prevents apoptosis and necrosis, through modulation of the JNK signaling pathway (Morgan and Liu, 2011). In astrocytes, it was reported that under certain conditions, IL-1β may act stimulating astrocytic GSH production, and potentially, augmenting total antioxidant capacity in the brain, via an NF-κB-dependent process (He et al., 2015). In this way, NF-κB pathway has been associated with both pro-oxidant and antioxidant roles. In AD, an alteration of this pro-and anti-oxidant role of NF-κB in astrocytes seems to be present, tending toward a pervasive pro-oxidative and pro-inflammatory profile ( Table 2). Novel anti-Alzheimer's drugs will need to consider the selective modulation of astrocyte activity in order to reduce pro-inflammatory signaling as well as to attenuate OS and diminish excitotoxicity (Figure 1). Taking into account the complex physiopathology of AD, a deep knowledge about dysfunctional astrocyte intracellular pathways evoked by Aβ opens the possibility for the design of new effective multi-target directed drugs. NOVEL NEUROPROTECTIVE AND THERAPEUTIC MEASURES IN ALZHEIMER'S DISEASE Alzheimer's disease is a major neurodegenerative disease affecting millions worldwide without a known curative treatment. Currently, only five drugs have been approved by the Food and Drug Administration (FDA) of the United States, three cholinesterase inhibitors (donepezil, galantamine, and rivastigmine), an NMDA receptor antagonist (memantine) and a combined donepezil-memantine drug. The cholinesterase inhibitors are approved for symptomatic treatment of mild-tomoderate stages of AD, while the NMDA antagonist is used for moderate-to-late stages. Regrettably none of these drugs is able to halt the progression of the disease and its uses are aimed at maximizing the quality of life of patients though broad symptom management (Caselli et al., 2017). Is therefore of great importance to design and develop new treatments which offer better therapeutic outcomes and disease-modifying responses to the patients with AD. Epidemiologic studies indicated that prolonged treatment with anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs could delay AD onset, as well as reduce disease rate progression (McGeer et al., 1990;Pasinetti, 2002;Cudaback et al., 2014). The inhibition of COX-mediated signaling pathways may reduce some inflammatory cytokines related with the physiopathology of AD. In addition, human studies confirm that OS plays a main role in the physiopathology of AD (Schrag et al., 2013;Chang et al., 2014). However, there are some controversies between observational studies and randomized controlled trials about the efficiency of antioxidative agents and anti-inflammatory drugs to reduce AD risk (Viña et al., 2004;Cudaback et al., 2014;Wang et al., 2015;Jiang et al., 2016). Many research groups and pharmaceutical companies have been developing new strategies to overcome the disease, but so far none of the Aβ-targeted phase three clinical trials reported has shown statistically significant benefit on its pre-specified clinical endpoints (Selkoe and Hardy, 2016). Many explanations may be offered for this lack of success, ranging from poorly designed trials to late interventions (irreversible modification of the disease due to advanced stage) but also, to the incomplete knowledge about the basic pathophysiological mechanisms of AD. As astrocytes have been shown to be involved in a diverse range of pathological changes observed in AD, they have been proposed as an interesting novel therapeutic target (Finsterwald et al., 2015). Because increased proinflammatory cytokines induced by Aβ are associated with enhanced production of free radicals in the astrocytes (Masilamoni et al., 2005a), new compounds with antioxidative and anti-inflammatory properties could reduce the effects of neurodegeneration in AD. Acute ↑GSH after monomeric Aβ. Induction of ABCC1 was reduced in 6-month old 5xFAD mice. primary cortical rat astrocytes, the use of a light-generating nanoparticle attenuated Aβ-induced OS and inflammatory responses, through a reduction in the superoxide anion production and a lowering of IL-1β and iNOS expression (Bungart et al., 2014). Curcumin, a natural phenol obtained from plants and commonly used as a spice, has been proposed to be of benefit in AD, reducing Aβ formation and decreasing neurotoxicity in the brain (Lim et al., 2001;Yang et al., 2005;Cole et al., 2007). In a recent study using APP/PS1 transgenic mice and primary rat mixed neuronal/glial cultures, curcumin was reported to improve spatial memory deficits and promote cholinergic neuronal function in vivo, and in vitro, attenuated the inflammatory response of both microglia and astrocytes, acting through PPARγ, which inhibited the NF-κB signaling pathway in these cells . Activation of PPARγ with the use of the isoflavone phytoestrogen genistein showed an increase in the release of ApoE from primary astrocytes in an in vivo mouse model of AD (Bonet-Costa et al., 2016). In the same paper, the authors reported that treatment with genistein improved several cognitive features (hippocampal learning, recognition memory, implicit memory, and odor discrimination) as well as a reduction in the number and area of Aβ plaques. Neuroesteroids, such as progesterone, have been proposed to offer neuroprotection in neurodegenerative diseases including AD (Liu et al., 2013). In primary cultures of rat astrocytes, treatment with progesterone reduced Aβ-induced inflammatory responses (decreasing the production of IL-1β and TNFα), and also suppressed endoplasmic reticulum stress activation together with attenuation of PERK/elF2a signaling (Hong et al., 2016). In addition to polyphenols, many other natural phytochemicals have shown anti-inflammatory and immunosuppressive efficacy in AD models. For example, triptolide extract inhibit activation of microglia and astrocytes in the APP/PS1 transgenic mouse model of AD . Punicalagin, a compound derived from pomegranate, not only may reduce neuroinflammation (lowering TNFα and ILβ) but also prevents OS through the reduction of iNOS, COX-2, and the production of ROS . several cannabinoid receptor agonists such as WIN, 2-AG, and methanandamide (Gajardo-Gómez et al., 2017). Pantethine (B5 vitamine precursor) was able to modulate the astrocytic metabolic changes and inflammatory patterns induced by Aβ 1−42 in astrocytes derived from the 5xFAD transgenic mouse model of AD (van Gijsel-Bonnello et al., 2017). In cultured cortical astrocytes, donepezil was shown to reduce inflammatory responses via nAChR and PI3K-Akt pathway, and to decrease intracellular ROS levels (Makitani et al., 2017). As mentioned early, donepezil is a cholinesterase inhibitor commonly used in AD patients. CONCLUSION Neuroinflammation and OS are part of the functional changes frequently observed in the brains of individuals with AD. Aβ has been shown to alter the normal dynamics of both inflammatory and antioxidant and prooxidant balance, promoting an unhealthy state for the brain and neuronal-glial communication networks. Astrocytes are involved in both inflammation and OS regulation in the CNS, and seem to have a central role in the basic pathophysiological aspects that surround this neurodegenerative disease. Although the precise relation between neuroinflammation, OS, astrocytes, and AD is still not clear, the evidence points toward an important participative role of the Aβ/NF-κB interaction in astrocytes as a critical agent in the pathological mechanism of AD. Despite the continuous efforts to develop a successful treatment for AD, there is still a gap in the knowledge of the precise etiological aspects of this disease which difficult the advance of therapeutics. Therefore, and due to the evidence presented in this review, is important to start considering astrocytes as a valuable novel therapeutic and neuroprotective target for future studies related to the treatment and mechanistic comprehension of AD. FUNDING The review was funded in part by Universidad del Rosario.	v3-fos-license

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