The N-terminal domain (NTD) of the GluN1 subunit (GluN1-NTD) is important for NMDA receptor structure and function, but the interacting proteins of the GluN1-NTD are not well understood. Starting with an unbiased screen of ~ 1,500 transmembrane proteins using the purified GluN1-NTD protein as a bait, we identify Protocadherin 7 (PCDH7) as a potential interacting protein. PCDH7 is highly expressed in the brain and has been linked to CNS disorders, including epilepsy. Using primary neurons and brain slice cultures, we find that overexpression and knockdown of PCDH7 induce opposing morphological changes of dendritic structures. We also find that PCDH7 overexpression reduces synaptic NMDA receptor currents. These data show that PCDH7 can regulate dendritic spine morphology and synaptic function, possibly via interaction with the GluN1 subunit. NMDARs are critical modulators of synapse formation and synaptic transmission. Dysfunction of NMDARs (hyperactivity or hypofunction) have been linked to various central nervous system (CNS) diseases3, including epilepsies4, schizophrenia5, and Alzheimer’s disease8. Each NMDAR subunit contains an extracellular N-terminal domain (NTD), ligand binding domain (LBD), transmembrane domain (TMD) and a cytoplasmic tail9. The N-terminal domain (NTD) of GluN1 has been reported to regulate NMDAR subunit oligomerization and assembly10. Functionally, GluN1 NTD is involved in modulating NMDA receptor gating and pharmacological properties11. EphB receptors have been reported to bind GluN1-NTD and regulate excitatory synapse formation via this interaction12. Other binding partners of GluN1-NTD remain to be discovered.NMDA receptors (NMDARs) are a subclass of glutamate-gated ion channels that are heterotetramers consisting two GluN1 subunits and two GluN2A-D/GluN3A-B subunits13. Protocadherins comprise the largest family within the cadherin superfamily, and consist of two major groups, depending on if the genes are clustered on the chromosome: clustered protocadherins and nonclustered14. Non-clustered protocadherins (PCDHδ family) are predominantly expressed in the nervous system, and multiple members from this group have been shown to modulate axon/dendrite structure and function and have been reported to be possibly related to human neurological disorders17. For instance, PCDH8 and PCDH10 (δ2 subfamily) co-localize with PSD95 and regulate dendritic spine density19. PCDH9 (δ1 subfamily) has been linked to autism spectrum disorder20, PCDH17 (δ2 family) to schizophrenia22, and PCDH19 (δ2 family) to epilepsy in females with mental retardation (EFMR)23.The cadherin superfamily of cell adhesion molecules consists of more than 100 members24. In the brain, based on the RNA-seq data from sorted cells, PCDH7 is highly expressed in neurons and astrocytes25. Despite the expression pattern, most studies on PCDH7 have focused on its role in cancer16. In recent years, a few studies have implied the role of PCDH7 in brain function: MeCP2 can bind to the promoter region of PCDH7 and down regulate its mRNA level, suggesting a possible link between PCDH7 and Rett syndrome26; genome-wide association studies (GWAS) show that PCDH7 is linked to epilepsies, is a risk factor for shorter sleep27 and is associated with antipsychotic treatment response in schizophrenia patients28. On the molecular level, in a proteomic analysis of synaptic clefts, PCDH7 was found in the excitatory synaptic cleft29. All the data point to a potential role of PCDH7 in the brain and perhaps in synapses, but the underlying mechanisms remain to be elucidated.PCDH7 belongs to the PCDHδ1 subfamily and is also called BH -protocadherin because of its predominant expression in brain and heartPcdh7 gene expression is colocalized with Grin1 in neurons and find PCDH7 enriched in postsynaptic densities (PSDs). When overexpressed, PCDH7 causes “collapse” of dendritic spines and reduces NMDAR currents. In contrast knockdown of PCDH7 results in elongation of spines. Taken together, our findings reveal that PCDH7 is a synaptically localized, GluN1-interacting protein that regulates synapse morphology and function.Here we discover that PCDH7 is a potential binding partner of GluN1-NTD via an unbiased screen. We find that N-Cadherin) and 29 protocadherins included in the library of this screening and none showed binding to GluN1-NTD except for PCDH7. The only other proteins with significant signal were Fc receptors and ARMCX3 and TIE1 which were all considered non-specific because these proteins appear repeatedly as hits in screens with other unrelated bait proteins  to measure the FITC signal. The intensity of the FITC signal reflected the amount of Fc-tagged protein bound to the cell surface. Consistent with the screening results, GluN1-NTD bound to the cell surface of PCDH7-transfected cells, but not the untransfected cells. In addition, Fc-tagged PD-L1 protein did not bind to the PCDH7 overexpressing cells, suggesting the binding was specific between GluN1-NTD and PCDH7  experiments from CHO cells overexpressing GluN1/GluN2A together with PCDH7, or from mouse brain lysates (data not shown). It is possible that the interaction between PCDH7 and GluN1 is of too low affinity to be detected by these methods, or that the interaction depends on some quaternary structure that is disrupted by detergent solubilization of CHO cells or brain tissue.2+ inhibition of NMDA current. In the presence of NMDA receptor agonists glutamate and glycine, GluN1/GluN2A receptors exhibit a biphasic Zn2+ inhibition curve with IC50 values in nanomolar and micromolar ranges for the high affinity and low affinity binding sites, respectively. Deletion of GluN1-NTD results in significantly reduced Zn2+ inhibition of GluN1/GluN2 receptors30. Given that PCDH7 can bind the GluN1-NTD, we tested whether PCDH7 affects Zn2+ inhibition using a CHO cell line that stably expresses GluN1 and GluN2A31. We transfected these GluN1/GluN2A expressing CHO cells with either GFP or PCDH7 + GFP, and recorded NMDAR currents from the transfected cells in response to application of glutamate/glycine and a series of increasing concentrations of Zn2+ ions. The baseline currents from GFP and PCDH7 transfected cells before Zn2+ application were comparable  and low affinity IC50 shifted from 79.5 to 165.3 μM (p = 0.04)   with a non-isotopic assay. In adult mouse brain, using dual labeling ISH, we found that Pcdh7 and Grin1 were broadly expressed and co-localized in neurons, including in cell layers composed predominantly of excitatory neurons , somatostatin (Sst), vasoactive intestinal peptide (Vip), and Neuropeptide Y (Npy)  Fig. B. Thus, N-Cadherin. Protein fractionation experiments of mouse forebrain showed that PCDH7 was enriched in the postsynaptic density (PSD) fractions, together with N-Cadherin, PSD95 and GluN1 , and about a threefold increase in the density of longer spines (length > 2 μm)  that suppressed PCDH7 protein levels by more than 60%  and simultaneously from a neighboring untransfected cell. Picrotoxin was added to the solution to block inhibitory current. NMDAR currents were measured 50 ms after stimulation to exclude contamination of AMPAR currents. Comparison of NMDAR currents from transfected and untransfected cells showed that knockdown of endogenous PCDH7 by either of two distinct shRNAs had no significant effect on the amplitude of the evoked NMDA current, although there was a trend towards a slight reduction in current , and some of them are filopodia-like, reminiscent of the report showing the elongation of dendritic protrusions after blockade of classic cadherins38. Since dendritic filopodia usually represent transient dynamic structures that may turn into spines, this phenotype suggests that there might be more immature synapses, or higher spine motility in PCDH7-knockdown neurons, pointing to a stabilization role of PCDH7 in spines. Time-lapse live imaging of the neurons would help clarify these questions. The elongation of the spines was also significant in cultured slices, but the effect size was smaller, which could be due to either the difference in knockdown efficiency or difference in resilience to partial loss of PCDH7 in a more physiological environment. Theoretically, rescue experiments with shRNA-resistant PCDH7 would provide additional evidence supporting the specificity of the knockdown phenotype. However, as described above, overexpressing PCDH7 had strong effect on dendritic spine morphology in neurons, even with 10% of the amount of DNA normally used for transfection (data not shown). We believe that the effects of overexpression of the shRNA insensitive PCDH7 would overwhelm the effects of knockdown and thus give inconclusive answers. On the other hand, the observations that two distinct shRNAs both induced elongation of spines in dissociated neurons and slice cultures and that overexpression of PCDH7 caused the somewhat opposite “collapse” of spines suggests that the PCDH7 targeting shRNAs effects were most likely specific.Both overexpression and knockdown of PCDH7 significantly changed the morphology of dendritic structures. Overexpression of PCDH7 resulted in collapse of spines and abnormal dendritic swelling in which PCDH7 and synaptic proteins like SHANK3 were concentrated. In addition, when we transfected cultured hippocampal slices with GFP tagged PSD95, together with PCDH7 and DsRed, overexpressed PSD-95 was also localized to the dendritic dilatations  in the postsynaptic site. Moreover, PCDH7 has been reported to bind to phosphatase PP1α via its cytosolic region CM316, and PP1α plays an important role in synaptic plasticity39. Therefore, PCDH7 might play a more important role in regulating synaptic plasticity than basal synaptic transmission. On the other hand, overexpression of PCDH7 changed Zn2+ inhibition sensitivity of NMDA current in a heterologous system, supporting a role of PCDH7 in direct modulation of NMDAR function and perhaps synaptic function under certain circumstances. Besides neurons, PCDH7 mRNA is also highly expressed in astrocytes. Previous reports have shown that astrocytic γ-Pcdhs are important for synaptogenesis and dendrite complexity40. Whether astrocytic PCDH7 plays a role in regulating synapse formation awaits to be examined.Knockdown of PCDH7 did not affect NMDAR current in our experiments despite changing morphologies of dendritic spines. Since we were only recording from transfected neurons while stimulating presynaptic fibers impinging on those cells, and transfection was sparse in slice cultures, we were only evaluating the potential function of While our data do not distinguish whether the effects of PCDH7 on synaptic function are due to the interaction with GluN1 that we identified, the demonstration of this potentially physiologically relevant interaction, and the characterization of synaptic effects of PCDH7 should provide the basis for future studies exploring mechanisms NMDAR protein interactions and regulation of synaptic function.Guide for the Care and Use of Laboratory Animals.All rodent experiments were approved by the Genentech Animal Care and Use Committee and followed the National Institutes of Health Pcdh7 were in pCAGGS vector; SHANK3-tdTomato was in pcDNA vector; PCDH7 rat shRNA constructs (both scrambled and targeted) were ordered from Origene.Human PCDH7 and PCDH7-GFP were in pRK5 vector; EGFP (GFP), DsRed and rat N-Cadherin, Cell Signaling 4061, rabbit polyclonal antibody used for immunoblot 1:1,000; Actin, Cell Signaling 5125 clone 13E5, rabbit monoclonal HRP conjugated antibody used for immunoblot 1:2,000; Cy5, Invitrogen 81-6716, rabbit anti-mouse IgG conjugated to Cy5 secondary antibody used for FACS 1:50; Fc-FITC, Novus NBP1-74956, goat anti-human IgG conjugated to FITC secondary antibody used for FACS 1:50.PCDH7, Abcam ab139274 clone 2G6, mouse monoclonal mouse antibody used for immunoblot at 1:1,000 and FACS at 1:50; NMDAR1, Novus NB300-118 clone R1JHL, mouse monoclonal antibody used for immunoblot 1:1,000; NMDAR1, Cell Signaling 5704 clone D65B7, rabbit monoclonal antibody used for FACS 1:50; PSD-95, Cell Signaling 3409 clone D74D3, rabbit monoclonal antibody used for immunoblot 1:1,000; 41.Human GRIN1 (D23-Q393) fused to the Fc portion of human IgG1 was cloned into a vector derived from pAcGP67A (BD biosciences) that included a gp67 signal sequence for protein secretion in insect cells. hGRIN1-Fc was expressed for 72 h in Sf9 cells at a multiplicity of infection of 0.5. It was affinity purified using a MabSelect Sure column , and subsequent purification was done by size-exclusion chromatography with a Superdex 200 column  according to the manufacturer’s instructionsA clone library containing ~ 1,500 genes representing single-pass transmembrane proteins was assembled with the goal of having each gene represented by at least one full-length sequence verified cDNA clone. Clones were obtained from in-house collection (Genentech) or purchased from external vendors. Complete list of genes is provided in Supplementary Table ® ViewRNA ISH tissue assay kits (Affymetrix) for single and 2-plex probes were used following the manufacturer's protocol43. Deparaffinization was carried out on a Leica XL stainer and after the boiling step in a Thermo Scientific PT module the slides were briefly dipped in ethanol and dried before assembling the ISH chamber for the Tecan platform. A probe set to Bacillus subtilis dihydropicolinate reductase (dapB) (L38424) was used as a negative control. A probe set to murine ACTB (NM_007393.1) was used as a positive control. Gene-specific probe sets for detection of PCDH7 (NM_018764), GRIN1 (AF146569), PVALB (NM_013645), SST (NM_009215), VIP (NM_001313969), and NPY (NM_023456) were used on tissue samples. Horseradish peroxidase (HRP)-conjugated label probe was used, followed by tyramide signal amplification (TSA) to increase sensitivity (Perkin Elmer). Briefly, TSA plus DIG stock solution (digoxigenin) was diluted 1:50 in 1 × Plus Amplification Diluent, applied to sections, and incubated for 10 min at room temperature. This was followed by incubation with anti-DIG-AP (Roche) diluted 1:500 in TNB blocking buffer with 4% lamb serum (Gibco) for 30 min at room temperature. Vulcan Fast Red and Ferangi Blue chromogens  were used for chromogenic detection. Hybridized target mRNAs were visualized using bright field microscopy.Non-isotopic in situ hybridization (ISH) was performed on 4 µm formalin-fixed paraffin embedded tissue sections on a Tecan platform equipped to carry out non-isotopic ISH. QuantiGene45. Briefly, hippocampi were dissected in ice-cold PBS, homogenized, spun to remove debris, snap frozen in liquid nitrogen, and stored at − 80 °C until blotting. Protein extracts of 50 μg were run on SDS-PAGE followed by immunoblotting.Homogenization of mouse forebrain was performed in ice-cold RIPA buffer with protease and phosphatase inhibitors46, embryos from timed pregnant Sprague–Dawley rats were removed at E18 and decapitated into cold HBSS/HEPES buffer. The cortex including hippocampus was dissected and placed into 5 mL of HBSS/HEPES buffer. Tissue was washed 4 times before digestion at 37 °C for 20 min with papain (0.5 mL at 200 U/mL into 4.5 mL HBSS/HEPES buffer with tissue). Tissue was washed four times with warm HBSS/HEPES buffer and once with neurobasal media. Dissociated tissue was triturated and passed through a 70 μm filter before plating on poly-d-lysine/laminin coated plates or coverglass with neurobasal media supplemented with B27, Glutamax, and Pen/Strep. Cells were plated at a density of 120,000 cells/well in 24 well plates with coverglass for imagining. Media was exchanged 100% 3–5 h after initial plating and then exchanged 50% once per week.As described previously46. Primary neuronal cultures were transfected between DIV12-14 with lipofectamine 2000. Neurons were transfected by incubating cells with 2 μL lipofectamine, 400–1,000 ng DNA or shRNA, and 500 μL neurobasal for 45 min. Cells were fixed 1–3 days post-transfection, as indicated in figure legends, using 4% paraformaldehyde in PBS for 12 min at room temperature. Cells were permeabilized and blocked using GDB buffer  and incubated with primary antibodies overnight at 4 °C. Antibodies were visualized using Alexa dye-conjugated secondary antibodies. Images were acquired by a single experimenter blinded to transfection or treatment using a Zeiss LSM780 laser scanning confocal microscope with a 20× or 100× oil objective . For spine quantifications, images were quantified by a single experimenter blinded to transfection or treatment using ImageJ software. Image z-stacks were collapsed into maximum projections and thresholded prior to analysis. Dendritic spines from secondary dendrites were analyzed. Spine density was calculated by counting the total number of dendrite protrusions using the GFP channel along a ~ 20 μm segment of dendrite. Spine length was also measured. The cumulative frequency of spine length was calculated using Prism6 software.Neuron transfection and staining were done as previously described47. When co-transfecting PCDH7 or shRNA with GFP, gold particles (1.6 μm) were coated with PCDH7 or shRNA with GFP at the ratio of 9:1. Live imaging of transfected neurons was taken using Olympus confocal microscope, with a water-immersion 40× objective  in ACSF solution (see below) and data analysis was done by MATLAB software. For both the experiments and data analysis, the investigator was blinded to the conditions.Organotypic slices were cultured from P7 Sprague Dawley rats. Biolistic transfection with gene gun (Biorad) was done at DIV3-748, the recording solution was oxygenated artificial CSF (ACSF) containing the following (in mM): 127 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 4 MgSO4, and 4 CaCl2, along with 100 μM picrotoxin and 2 μM 2-chloroadenosine. NMDAR mediated EPSCs were recorded at holding membrane potential at + 40 mV in the presence of NBQX (10 μM) or measured 50 ms after stimulation artifact. Pipette solution contained the following (in mM): 140 Cs methanesulfonate, 10 HEPES, 2.5 MgCl2, 10 EGTA, and 5 QX-314 Cl.For patch clamp recordings of hippocampal CA1 neurons from organotypic slices31. CHO cells were transfected with either GFP or PCDH7-GFP using lipofectamine 2000. Whole-cell recordings from transfected CHO cells were performed using Dynaflow Resolve rapid solution exchange system (Cellectricon) as described31. The pipette solution contained (in mM) 120 CsF, 10 NaCl, 2 MgCl2, and 10 HEPES, adjusted to pH 7.2 with CsOH. The extracellular solution to test Zn2+ inhibition contained (in mM): 140 NaCl, 2.8 KCl, 1 CaCl2, and 10 HEPES, adjusted to pH 7.4 with NaOH. In order to make solution containing free Zn2+ (in nM): 1, 3, 10, 30, 100, 300, 1,000, 10 mM Tricine was added to buffer Zn2+, and the added Zn2+ concentration was (in μM) accordingly: 0.26, 0.78, 2.6, 7.8, 26, 77.5, 25430. To make solution containing free Zn2+ higher than 1,000 nM, the corresponding Zn2+ was directly added to the extracellular recording solution without tricine. 100 µM glutamate and 50 μM glycine were added to the faxt exchanging solution to elicit the currents. Currents were recorded at holding potential of -50 mV.Doxycycline-inducible CHO cell line that express GluN1 and GluN2A was as previously describedRat PCDH7, along with shRNA against rat PCDH7 were transfected into HEK293 cells for 72 h. Four potentially PCDH7-targeted shRNA constructs were tested as well as a control scrambled shRNA. To determine the extent of PCDH7 knockdown, cell lysates were blotted for PCDH7 or ACTIN loading control. PCDH7_A and PCDH7_C were the most effective and chosen to test on primary neuronal cultures.49. Briefly, the forebrain was dissected and mechanically homogenized using a glass–Teflon homogenizer in 5 mM HEPES pH 7.4, 1 mM MgCl2, 0.5 mM CaCl2 with phosphatase, and protease inhibitors. Samples were spun for 10 min at 1,400×g to clear non-homogenized tissue, and an aliquot of the supernatant was taken as S1. Next, the supernatant was spun for 10 min at 13,800×g and the pellet was resuspended in 320 mM sucrose, 6 mM Tris. Aliquots of the supernatant S2 and resuspended pellet P2 were collected. The P2 resuspension was further fractionated via a stepwise sucrose gradient, 1,200 mM sucrose, 1,000 mM sucrose, 850 mM sucrose, and P2 in 320 mM sucrose. The gradient was spun at 82,500×g for 120 min. The synaptosome fraction (SY) sits between the 1,200 mM and 1,000 mM gradients. An aliquot SY was collected from this fraction and the rest was treated with 0.5% triton for 15 min rotating at 4C. Sample was spun at 200,000×g for 20 min and supernatant was collected as final PSD fraction. 10 µg of protein per fraction was loaded onto SDS-PAGE followed by immunoblotting.Mouse forebrain homogenates were fractionated via sucrose gradientHEK293 cells transfected with full length human PCDH7 using lipofectamine 2000. Approximately one million cells in a final volume of 500 μL were used for each experiment. Cells were washed with PBS and then trypsinized to create a single cell suspension. Cells were washed 1× with FACS Buffer  and then blocked for 45 min at 4 °C in 1% BSA. After washing cells 3× with 1% BSA, cells were incubated in 2 mL volume of 1% BSA with 5 μg/mL of purified protein  for 1 h at 4 °C. After incubation, cells were washed 3× with FACS buffer. HEK293 cells were stained for PCDH7 followed by Cy5 to confirm transfection and surface expression. Cells were stained with human-Fc-FITC conjugated to detect if GluN1-NTD-Fc, or PD-L1-Fc was bound to cells. All antibody incubations were performed for 10 min rotating at 4 °C. Cells were washed 3× in FACS buffer before final resuspension in 500 µL. Flow cytometry was done using FACSCalibur (BD Biosciences). The FITC geometric mean was calculated using FlowJo software and normalized first to the background (PCDH7 transfected cells stained for Fc-FITC). The final values are plotted normalized to control (untransfected cells + 5 μg/mL GluN1-NTD-Fc stained for Fc-FITC.Supplementary file1 (XLSX 32 kb)Supplementary file2 (PDF 7004 kb)


Gordonia sp. strain NB4-1Y. To identify genes involved in the breakdown of these compounds, the transcriptomic response of NB4-1Y was examined when grown on 6:2 FTAB, 6:2 FTSA, a non-fluorinated analog of 6:2 FTSA (1-octanesulfonate), or MgSO4, as sole sulfur source. Differentially expressed genes were identified as those with ± 1.5 log2-fold-differences (± 1.5 log2FD) in transcript abundances in pairwise comparisons. Transcriptomes of cells grown on 6:2 FTAB and 6:2 FTSA were most similar (7.9% of genes expressed ± 1.5 log2FD); however, several genes that were expressed in greater abundance in 6:2 FTAB treated cells compared to 6:2 FTSA treated cells were noted for their potential role in carbon–nitrogen bond cleavage in 6:2 FTAB. Responses to sulfur limitation were observed in 6:2 FTAB, 6:2 FTSA, and 1-octanesulfonate treatments, as 20 genes relating to global sulfate stress response were more highly expressed under these conditions compared to the MgSO4 treatment. More highly expressed oxygenase genes in 6:2 FTAB, 6:2 FTSA, and 1-octanesulfonate treatments were found to code for proteins with lower percent sulfur-containing amino acids compared to both the total proteome and to oxygenases showing decreased expression. This work identifies genetic targets for further characterization and will inform studies aimed at evaluating the biodegradation potential of environmental samples through applied genomics.Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are environmental contaminants of concern. We previously described biodegradation of two PFAS that represent components and transformation products of aqueous film-forming foams (AFFF), 6:2 fluorotelomer sulfonamidoalkyl betaine (6:2 FTAB) and 6:2 fluorotelomer sulfonate (6:2 FTSA), by The online version of this article (10.1007/s10532-020-09917-8) contains supplementary material, which is available to authorized users. Perfluoroalkyl and polyfluoroalkyl substances (PFAS) represent a diverse group of anthropogenic compounds of concern due to their widespread use, potential toxicity  for hydrocarbon firefighting had been used over 40 years, suggesting that this genus may be actively metabolizing PFAS in soil and groundwater environments. While multi-omic studies examining DNA, RNA, protein and metabolite profiles of microbial communities in PFAS-impacted environments will be valuable for guiding bioremediation, in order to make sense of these complex datasets, fundamental knowledge of the biochemical mechanisms of specific PFAS metabolic steps is needed.Microbial community metabarcoding tools and quantitative polymerase chain reaction have been applied in an effort to correlate changes in microbial community structures to PFAS contamination in order to form hypotheses about the main microbial players involved in PFAS transformations  and 6:2 fluorotelomer sulfonate (6:2 FTSA)  enables the quantitative analysis of transcription across all genetic elements in a genome. This genome-wide perspective is useful for finding genes that are differentially expressed in response to compounds such as 6:2 FTAB or 6:2 FTSA. To find coordinated metabolic programs in NB4-1Y, global gene expression in the presence of 6:2 FTAB or 6:2 FTSA was compared to NB4-1Y cultures growing on either MgSO4 as a sulfate rich control, or the sodium salt of 1-octanesulfonate as a non-fluorinated structural analogue of 6:2 FTSA. Through these analyses we identified, for example: 20 genes associated with the transport and metabolism of sulfur compounds more highly expressed with 6:2 FTAB, 6:2 FTSA and OCT compared to MgSO4; three genes associated with carbon–nitrogen bond cleavage more highly expressed with 6:2 FTAB; three alcohol dehydrogenases, three monooxygenases, and three genes associated with acetyl-CoA metabolism, as being more highly expressed with 6:2 FTAB and 6:2 FTSA. These data are being used to identify target genes that will be cloned and expressed so that the activity of purified enzymes against 6:2 FTAB, 6:2 FTSA, and breakdown products can be characterized.Here we examine the transcriptomic responses of −1; Shanghai Kingpont Industrial Co. Ltd., Shanghai, China), sodium 1-octanesulfonate and magnesium sulfate  were prepared at 4 mM in a 50:50 mixture of anhydrous ethanol  and 18 MΩ water. The 6:2 FTSA solution was heated to 55 °C and stirred overnight to dissolve. All other chemicals were from Millipore Sigma.The ammonium salt of 6:2 FTSA , a technical grade solution of 6:2 FTAB (0.27 g mL2PO4), 1.60 g potassium monobasic phosphate (K2HPO4), 1.55 g ammonium chloride (NH4Cl), 5.00 g sodium acetate (NaCH3COO), 0.165 g magnesium chloride (MgCl2), 0.090 g calcium chloride dihydrate (CaCl2·2H2O), 5.00 mL Wolfe’s minerals, and 1.00 mL Pfenning’s vitamin solution. KH2PO4, K2HPO4, NH4Cl and NaCH3COO were autoclaved in 18 MΩ water at 121 °C for 20 min, cooled to room temperature, and a 0.22 µM pore sized filter sterilized solution of MgCl2, CaCl2·H2O, Wolfe’s minerals and Pfenning’s vitamin solution was added aseptically. Difco Nutrient Agar was used for the preparation of solid medium .Van Hamme et al.  was follGordonia sp. strain NB4-1Y was isolated from vermicompost for its ability to cleave subterminal alkyl carbon–sulfur bonds, and has been shown to metabolize 6:2 FTAB and 6:2 FTSA . Sterile controls for each sulfur source were also prepared. All cultures were incubated on a tissue culture roller drum  set to 150 rotations per minute at 30 °C.Next, single isolated colonies from nutrient agar plates were used to prepare inoculum cultures in sterile 20-mL culture tubes containing 5 mL SFA medium and 75 µL of sulfur source stock solution  and inoculated with 250 µL of the respective inoculum culture . These were plugged with foam stoppers and covered with aluminum foil, and incubated in an orbital shaker at 30 °C with 150 rotations per minute.g to pellet biomass. Supernatants were removed, pellets flash frozen in liquid nitrogen, and shipped on dry ice to the University of Regina for RNA extraction and sequencing.Once cultures reached the mid-log phase, 10 mL of freshly prepared 5% (vol/vol) phenol in absolute ethanol was added to each 25 mL culture in order to neutralize cellular activity and stabilize RNA prior to extraction. These were incubated on ice for 1.5 h and, following this, 1.4 mL aliquots were centrifuged in microcentrifuge tubes for 5 min at 8200×Gordonia sp. strain NB4-1Y, an organism which is difficult to lyse. Protocols developed for Rhodococcus sp. strain RHA1, TRIzol, RNA Powersoil Total RNA Isolation Kit , and EZNA Bacterial RNA Kit  methods did not yield sufficient RNA for sequencing. In the end, sample pellets stored at – 80 °C were thawed on ice and all subsequent steps were performed at 4 °C. Thawed pellets were resuspended in 1.2 mL of RLT buffer (Qiagen); RLT buffer was prepared fresh by addition of 10 µL of β-mercaptoethanol to 1 mL of RLT buffer. To prepare lysis beads, 500 µL of RLT buffer was added to Lysing Matrix B  in 2-mL tubes, followed by vortexing to remove trapped air, then addition of 1.2 mL of bacterial suspension. Bacteria were lysed using a Fastprep-24 classic , beating at 4 m s−1 for 40 s a total of three times with incubation of tubes on ice for 5 min between each bead beating step. The samples were centrifuged for 4 min at 21,130×g and supernatant transferred to a fresh 2-mL microcentrifuge tube. It is important to note that the cell debris from lysis of Gordonia behaves oddly; cell debris is buoyant and settles between beads and the supernatant. Thus, removal of the supernatant requires very careful pipetting to not disturb the pellet. In the clean tube, an equal amount of pre-chilled 70% ethanol was added to the supernatant and mixed well by pipetting. RNA was isolated and cleaned on an RNeasy Mini (Qiagen) spin column as per manufacturer’s guidelines (RNeasy Mini Kit. ID: 74104); if a sample volume was > 700 µL, the total sample volume was passed serially through a column until all sample was purified. Total RNA was eluted in 30 µL of RNase free water. The concentration of RNA was measured using the Qubit RNA BR kit (Thermo Fisher Scientific).A number of methods were tested, with limited success, for extracting RNA from RNA samples were treated with Ambion Turbo DNA free kit (Thermo Fisher Scientific) to remove contaminating DNA, following the manufacturer’s instructions. Extracted RNA was stored at − 80 °C. Ribosomal RNA (rRNA) depletion was carried out as per manufacturer’s guidelines using the Gram positive Bacterial Ribo Zero rRNA depletion kit (Illumina MRZGP126). Post depletion the quantity and quality of RNA was checked using Qubit RNA BR kit and the RNA Nano 6000 LabChip kit  following the manufacturer’s instructions.4 samples were sequenced on the Illumina MiniSeq using the 150-cycle paired-end cartridge and MiniSeq 75 cycles protocol, respectively.After rRNA depletion, RNA was converted to DNA for sequencing using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760). FTAB and OCT samples were sequenced on the Illumina MiSeq platform using the V3 150 cycle paired-end cartridge; 6:2 FTSA and MgSOFor all libraries, forward read sequence data was processed with Trimmomatic  or MgSO4 was carried out. In each case, after NB4-1Y was revived from storage on nutrient agar plates, inoculum cultures were first grown to early stationary phase  on 6:2 FTAB, 6:2 FTSA, OCT and MgSO4 prior to inoculating experimental cultures and incubating to mid-logarithmic phase . Transcriptional output from each open reading frame was calculated as transcripts per million (TPM) as this is the most robust method for normalizing between culture conditions  between all combinations of culture conditions show that gene expression patterns were most similar between cells provided with 6:2 FTAB and 6:2 FTSA  genes were expressed ± 1.5 log2FD.To examine how 4 conditions , dehalogenation, formation of acetyl-CoA adducts, and oxidation of the shared C8 backbone in 6:2 FTAB and 6:2 FTSA. While changes in gene expression relating to stress in response to PFAS were not detected, putative desulfonation genes that were more highly expressed when NB4-1Y was exploiting 6:2 FTAB and 6:2 FTSA for sulfur were found to code for proteins with reduced cysteine and methionine contents.As described below, examination of the putative function of genes with higher levels of expression in PFAS-containing cultures compared to OCT and MgSOHighly expressed genes in the 6:2 FTAB and 6:2 FTSA conditions includes 23 hypothetical proteins and three proteins annotated as ‘domain of unknown function’. Similarly, of the 79 genes more highly expressed upon exposure to 6:2 FTAB, 15 were annotated to code for hypothetical proteins, and one was annotated as ‘domain of unknown function’; for the 36 genes more highly expressed under 6:2 FTSA conditions, there were nine annotated to code for hypothetical proteins and three annotated as ‘domain of unknown function’. These data imply that more detailed analyses in the context of PFAS metabolism may allow us to assign function to genes that are not well annotated in the NB4-1Y genome.2FD compared to MgSO4 being between − 0.14 and 1.47 .As a control for consistency of core metabolic functions between the four culture conditions, the expression levels of 23 genes associated with a complete citric acid cycle and glyoxylate shunt were examined; these genes would be expected to be similar across treatments given that all cultures had access to the same major carbon sources (acetate and ethanol) under all conditions. There were no striking differences between conditions , with all log4  and a sulfurtransferase (RS22035) were more highly expressed when compared to other conditions, while for 6:2 FTSA a cysteine desulfurase (RS04970) and an alkyl sulfatase (RS13185) were more highly expressed. Bacterial sulfate ABC transporters consist of sulfate binding proteins, membrane components, and an ATPase to drive the transport reactions. In  Kessler , and alk Kessler . Given t Kessler .E. coli) in the presence of O2 and FMNH2 generates an aldehyde and sulfite  in NB4-1Y cultures, either as a product of the 6:2 FTOH that is generated, or as a consequence of an alkanesulfonate monooxygenase-like desulfonation of 6:2 FTSA  and a sulfurtransferase (RS18310) that were more highly expressed when NB4-1Y was provided either 6:2 FTAB or OCT, the remaining 12 sulfur metabolism genes were more highly expressed with all three substrates when compared to MgSO4. These include four sulfurtransferases, a methionine ABC transporter ATP-binding protein, a sulfite oxidase-like oxidoreductase, and a cysteine dioxygenase. An aliphatic sulfonate ABC transport protein (RS20390) was more highly expressed in 6:2 FTAB, 6:2 FTSA and OCT cultures with TPM > 10 . The alkanesulfonate monooxygenase (RS14690) that appears in Fig. 2FD threshold, but in all cases the TPM was < 10, suggesting that the gene may not be significantly transcribed and may not be involved in desulfonation of any of the substrates.With the exception of a cysteine synthase gene . Two FMN-dependent monooxygenases, RS09755 and RS09775 (discussed further below), were more highly expressed in 6:2 FTAB, 6:2 FTSA and OCT containing cultures, and the TPM values were higher than for the alkanesulfonate monooxygenase. In addition to these genes, one taurine dioxygenase gene was expressed under all conditions, although the log2FD compared to MgSO4 was only above 1.5 for 6:2 FTAB, and not for 6:2 FTSA or OCT. Taurine dioxygenases are 2-oxoglutarate-dependent enzymes that cleave 2-aminoethane sulfonate (taurine) to release sulfite, aminoacetaldehyde, succinate and CO2, and are active against other organosulfonates including butane-, pentane- and hexanesulfonic acids , 0.88 (n = 10) and 1.33 (n = 11) percent sulfur-containing amino acids, respectively, compared to the overall proteome average of 3.01% (n = 4351). Furthermore, oxygenases that were expressed at lower levels for these three conditions averaged 4.41 (n = 4), 3.48 (n = 8) and 3.82 (n = 7) percent, respectively.We previously observed in two-dimensional differential in-gel electrophoresis (2D DIGE) experiments that two FMN-dependent monooxygenases (RS09755 and RS09775) that were produced when NB4-1Y was provided with 6:2 FTSA as a sole added source of sulfur, had a lower percentage of sulfur-containing amino acids  than the overall predicted proteome -piperazine-2-tert-butylcarboxamide to form (R)-piperazine-2-carboxylic acid  and free fatty acids, and have been isolated from Pseudomonads associated with dermatitis in humans , and the log2FD for 6:2 FTAB, 6:2 FTSA and OCT were 5.37, 4.91 and 5.85, respectively. RS14155 was not expressed with OCT or MgSO4 (TPM < 10 for both), and the log2FD for 6:2 FTAB and 6:2 FTSA were 4.76 and 4.86, respectively. Nitrilotriacetate monooxygenases convert nitrilotriacetate to iminodiacetate and glyoxylate, and these enzymes are in a subclass of flavoprotein monooxygenases that includes alkanesulfonate monooxygenases (e.g. SsuD) capable of desulfonating a broad range of sulfonated compounds  were more highly expressed in the presence of 6:2 FTAB compared to MgSO4  was uniquely expressed in 6:2 FTAB grown cultures, this could be attributed to oxidative attack of the methyl groups in the functional group or to oxidation of the 6:2 FTOH detected as a metabolic intermediate  class flavin-dependent oxidoreductase genes  were expressed in 6:2 FTAB and 6:2 FTSA cultures. LLMs are flavin-dependent monooxygenases, a group of enzymes that catalyze a diversity of reactions including hydroxylations, epoxidations, and Baeyer–Villiger rearrangements  was expressed in 6:2 FTAB and 6:2 FTSA cultures. This may indicate that acetyl-CoA adducts are formed during PFAS metabolism, but are not detected using standard PFAS analytical methods.During beta oxidation of fatty acids, coenzyme A (CoA) is added to the carboxyl group to yield a fatty acyl-CoA ester from which acetyl-CoA is eventually liberated for entry into the citric acid cycle. While CoA adducts of PFAS have not been reported, other conjugates such as glutathione-8:2 FTUAL have been reported , suggesting that NB4-1Y is well suited to thrive in PFAS-contaminated environments. Overall, as for genes associated with the citric acid cycle, no obvious differences between the culture conditions emerged for known stress response genes.4. Alkyl hydroperoxide reductases and peroxiredoxins are part of a robust reactive oxygen species defence system found across Bacteria; the former converts damaging hydroperoxides to alcohols, the latter reduces metabolically generated hydrogen peroxide to water  when NB4-1Y was exposed to 6:2 FTSA for 2D DIGE experiments, a protein that was not produced when NB4-1Y was grown on MgSO Kertesz . While, Gordonia sp. strain NB4-1Y in response to being provided with 6:2 FTAB or 6:2 FTSA as sole sources of added sulfur for growth. These changes are driven by sulfate starvation conditions that are the norm in natural soil environments Supplementary file2 (XLSX 80 KB)Supplementary file3 (XLSX 210 KB)Supplementary file4 (PDF 576 KB)Below is the link to the electronic supplementary material.


Hoechst staining and flow cytometry also revealed that omentin markedly prevented H2O2-induced apoptosis. Moreover, omentin not only significantly inhibited ROS production (p < 0.01) but also significantly (p < 0.01) increased GPx activity in HUVECs. In conclusion, our data suggest that omentin may protect HUVECs from injury induced by H2O2.Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of cardiovascular diseases. Omentin, an adipocytokine that is abundantly expressed in visceral fat tissue, has been reported to possess anti-inflammatory and antidiabetic properties. However, endothelial protective effects of omentin against oxidative stress remain unclear. This study aimed to evaluate the protective effect of omentin against hydrogen peroxide (H2O2)-induced cell injury in human umbilical vein endothelial cells (HUVECs). Cytotoxicity and cytoprotective effects of omentin were evaluated using 3--2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic activity of HUVECs was detected using Annexin-V/PI and Hoechst 33258 staining methods. Antioxidant activity of omentin was evaluated by measuring both reactive oxygen species (ROS) levels and glutathione peroxidase (GPx) activity. No cytotoxicity effect was observed in HUVECs treated with omentin alone at concentrations of 150 to 450 ng/ml. MTT assay showed that omentin significantly prevented the cell death induced by H The blood vessel wall consists of a single layer of endothelial cells (EC), which plays important roles in hemostasis, vasodilatation, angiogenesis, vascular permeability, and the response of blood vessels to other physiological and pathological stimuli. EC possess both metabolic and synthetic functions and serve as the mechanical barrier between the circulating blood and vascular smooth muscle cells. By secreting a large variety of mediators such as growth factors, anti-thrombotic factors, inflammatory mediators, and others, EC are able to control a vast array of cellular functions throughout the body . Alterat2O2) penetrates freely through the plasma membrane and damages neighboring cells as well as H2O2-producing cells. In view of this feature, H2O2 is extensively used to induce oxidative stress in many in vitro models [2O2-induced oxidative stress and injury in endothelial cells has been considered as a potential therapeutic strategy for cardiovascular diseases [Oxidative stress is a result of an imbalance between the pro-oxidant and the antioxidant defense mechanisms in the body. An excessive amount of reactive oxygen species (ROS) causes severe damage to cellular structures and components including nucleic acids, proteins, and lipids, thereby leading to cell death . It has o models ,8. Therediseases ,10. 2O2 is eliminated by glutathione peroxidase biochemically where glutathione peroxidase neutralizes H2O2 to water using two hydrogen atoms from two molecules of reduced glutathione (GSH) to form oxidized glutathione (GSSG) [In physiological conditions, the level of intracellular ROS is tightly controlled by the antioxidant defense system in cells. ROS is directly detoxified or reduced into nontoxic substances through the action of nonenzymatic radical scavengers  and antioxidant enzymes , and superoxide dismutase (SOD)). High concentration of He (GSSG) . Thus, te (GSSG) .Omentin is an adipokine produced by visceral adipose tissue with insulin-sensitizing effects. Omentin is detectable in human plasma from 100 to 800 ng/ml . The pla2O2 from 100–800 µM for 4 h, compared to the control group  to 90% after 1 h. On the other hand, no cytotoxicity was observed in HUVECs treated with 150 and 300 ng/ml of omentin. In another set of experiments, 400 μM of H2O2 significantly lowered HUVEC viability (p < 0.001) to 78.6%  significantly increased the viability of the H2O2-treated HUVECs where the survival rates were 97.9%, 99.7%, and 107.3%, respectively. These results suggest that omentin protects HUVECs from oxidative stress-related cellular injury.As presented in to 78.6% C. As no 2O2 for 4 h, HUVECs showed condensed, fragmented, and small nuclei, which is a typical apoptotic morphology, in comparison to the nuclear size of the control group . There was no significant difference between the effects of all the concentrations of omentin.Nuclear morphological changes were evaluated using a fluorescence microscope and apoptotic cells are indicated with arrows in ol group a. This wol group d–f. The 2O2 group (14 ± 0.7%), compared to the control group (3.9 ± 0.15%). However, pre-treatment with different concentrations of omentin  for 1 h significantly decreased the percentage of early apoptosis to 5.3 ± 1.23%, 4.5 ± 0.55% and 5.3 ± 0.52%, respectively. Meanwhile, treatment with omentin (450 ng/ml) alone had no significant effect on cell apoptosis. These results demonstrate that omentin protects HUVECs from oxidative stress-induced apoptosis in vitro.To further confirm this observation, flow cytometry with Annexin V-FITC/PI double staining was performed A,B. In l2O2 induces intracellular ROS production, which affects cell metabolism [2O2 was increased, compared to the control group. Pre-treatment with omentin evidently attenuated the DCF fluorescence elicited by H2O2. Spectrofluorometry showed that the production of ROS in HUVECs exposed to H2O2 alone was increased to about 20-folds, compared to the control group (Exogenous addition of Htabolism . To inveol group B. Pre-tr2O2 group (19.74 U/ml ± 1.76), compared to the control group (50.57 U/ml ± 0.773). Pre-treatment with omentin at concentrations of 150, 300, 450 ng/mL significantly elevated the activity of GPx to 55.44 U/ml ± 0.76, 58.5 U/ml ± 0.99, 44.40 U/ml ± 0.76, respectively. Simvastatin, at 5 µM, also augmented GPx activity to 54.71 U/ml ± 1.1. In addition, omentin alone was found to increase the activities of the antioxidant enzyme, compared to the H2O2 group.To protect cells from the damage caused by ROS, excessive ROS is removed by several intracellular defense mechanisms that involve antioxidant enzymes such as GPx. The activity of GPx is shown in 2O2, superoxide anions (O2−), and hydroxyl radicals (·OH). In physiological conditions, ROS are continuously produced in cells as products of cellular oxidation-reduction processes and they are crucial for biophylaxis. At high concentrations, ROS cause severe damage to cellular structures and components including nucleic acids, proteins, and lipids, thereby leading to cell death [Oxidative stress is mainly caused by excessive accumulation of ROS that include Hll death . Omentin is a novel adipose tissue-derived adipokine and is highly expressed in visceral adipose tissue. Previous studies showed that omentin stimulates vasodilation in isolated blood vessels and suppresses cytokine-stimulated inflammatory responses in cultured endothelial cells ,17. More2O2 to induce a stable response of oxidative stress in HUVECs and this enables us to reproduce similar responses in all the experiments. H2O2 diffuses freely into and out of the cells and modulates multiple cellular processes such as cell proliferation, signal transduction, gene expression, DNA damage, apoptosis, and necrosis [2O2-induced decreased cell viability  were purchased from Merck Millipore . 3- -2,5-dephenyltetrazolium bromide (MTT) and Hoechst 33358 were purchased from Sigma Co. . 2.5% Trypsin-EDTA solution 10× was purchased from Santa Cruz Biotechnology . Annexin V/PI assay kit and GPx assay kits were purchased from Trevigen . All the other chemicals used were of analytical grade.Recombinant human omentin was purchased from Biovendor . EndoGRO Basal Medium supplemented with kit, H2 flasks. Cells were maintained in a humidified incubator with 5% CO2 at 37 °C and the medium was changed every two days till the cultures reached 90% confluence. The cells were trypsinized using 2.5% Trypsin-EDTA solution 10x, centrifuged (1200 rpm for 10 min) and subcultured into five 25 cm2 flasks. Passages 3–4 were used in all the experiments to maintain cell morphology and biological properties.Human umbilical vein endothelial cells (HUVECs) were purchased from Merck Millipore  and the cells were cultured with EndoGRO-LS Complete Culture Media Kit. The cell suspension from the newly purchased vial were grown into eight 25 cm2O2 (400 μM) for 4 h. Following exposure to H2O2, the cells were harvested for further analysis.For all experiments, otherwise specified in the protocol, HUVECs were cultured to confluence in 96-well plates or 6-well plates. The cells were pre-treated with omentin  for 1 h before exposure to H4 cells per well onto a 96-well plate. In order to determine the cytotoxic effects of omentin and H2O2, HUVECs were treated with 150–450 ng/ml of omentin for 1 h or 0–800 µM H2O2 for 4 h. All treatments were prepared in 100 µL of media. Following treatments, 50 µL of MTT solution (2 mg/mL in PBS) was added into each well which contained 100 µL of treatment and incubated at 37 °C for another 4 h. The MTT solution was removed and 100 µL of Dimethyl Sulfoxide (DMSO) was added to dissolve the purple formazan. The optical density (OD) was measured using a microplate reader at 570 nm . In another set of experiments that was designed to confirm the cytoprotective effect of omentin, HUVECs were treated with omentin (150–450 ng/mL) for 1 h before stimulated with H2O2 (400 µM) for 4 h. 100 µL of omentin was first added to the well and incubated for 1 h. Then, the media was removed and 100 µL of H2O2 was added into the well for 4 h in the cytoprotective assay. Each set of experiments was performed in triplicate. The cell viability was expressed in percentage.Cell viability was assessed using MTT as previously described with some modifications . Briefly4 cells per well onto a 96-well plate and grown for overnight. After treatment, the cells were washed twice with 100 µL of cold PBS and fixed with 100 µL cold methanol and acetic acid at a ratio of 3:1 (v/v) for 30 mins in dark. The fixed cells were stained with 20 µL of Hoechst 33358 (1 mg/ml) for another 30 mins in dark [HUVEC were seeded at 2 × 10 in dark . The cel6 per well in a 6-well plate and incubated for 24  h. The cells were treated with omentin  for 1 h and induced with H2O2 (400 µM) for another 4 h. After treatments, cells were washed once with 1X PBS, trypsinized and centrifuged at 1000 rpm for 5 minutes. The cell pellets were washed twice with cold PBS and resuspended in 100 µL of 1× Annexin-binding buffer. The suspension was transferred to a 1.5-mL tube, and 5 µL of Annexin V and 5 µL of PI were added to 100 µL of cell suspension. The cells were incubated in dark at room temperature for 15 minutes and 400 µL of 1× Annexin-binding buffer was added. After mixing gently, the fluorescence intensity was detected with a flow cytometer . Data were analyzed using BD FACSDiva Software version 6.1.3. Approximately 1–5 × 105 cells were analyzed in each sample.Annexin-V/PI assay was performed using BD Annexin V-FITC Assay kit according to the manufacturer’s instructions. Briefly, HUVECs cells were seeded at 1  ×  104 cells/well in a black and clear bottom 96-well plate containing 100 µL of culture medium and incubated at 5% CO2 and 37 °C overnight. The cells were pretreated with omentin (150–450 ng/ml) or 2 positive controls ((simvastatin (5µM) and Trolox (100µM)) for 1 h. Next, the media were removed, and the cells were washed twice with PBS, and this was followed by stimulation with 400 uM H2O2 for 1 h. The cells were then incubated with 5 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) for 30 min at 37 °C [The formation of ROS was measured using a ROS-sensitive dye, 2,7-dichlorodihydro-fluorescein diacetate , as an indicator. Cells were seeded at 2 × 10at 37 °C . The flug for 10 min. The cell pellets were washed with 1X cold PBS and resuspended in an ice-cold 1X assay buffer  containing 0.4 mM Phenylmethylsulfonyl fluoride (PMSF) and 1% Triton X-100. The cell suspension was incubated on ice and vortexed periodically for 30 min. The cell lysate was centrifuged at 10,000× g for 15 min at 4 °C, and the supernatant was collected and stored at −80 °C until the assay was performed. The protein concentration of the cleared cell lysate was determined with a nanodrop spectrophotometer. GPx activity in the supernatant was determined according to the protocol provided by the manufacturer. A cellular GPx assay kit  was used to measure GPx activity. Briefly, media in a flask were discarded after treatments and HUVECs were washed with 1X PBS. The cells were harvested by scrapping the cell monolayer in PBS. The cells were then centrifuged at 400× p-value of less than 0.05 (p < 0.05) was regarded as statistically significant.All data were presented as means ± SEM of three independent experiments carried out in triplicates. Statistical analysis was performed using one-way analysis of variance (ANOVA) Tukey’s post hoc test in IBM SPSS statistics version 22.0. A 2O2-induced cytotoxicity and apoptosis in HUVECs. In addition, omentin inhibits intracellular ROS formation and enhances the antioxidant defense system in HUVECs exposed to oxidative stress-induced injury. As oxidative stress-stimulated endothelial cell injury plays an important role in the development of atherosclerosis, the findings of the present study may shed light on the pharmacological basis and the clinical application of omentin in treatment of atherosclerosis.Taken together, our present work shows that omentin possesses a protective effect against H


The ubiquitin–proteasome system (UPS) as a major degradative system plays a crucial role in the maintenance of Ca2+ homeostasis. The E3 component N-recognin (UBR) subfamily is a part of the UPS; however, the role of UBR in regulating cardiac CICR is unknown. In the present study, we found that among the UBR family, single knockdown of UBR3 or UBR6 significantly elevated the amplitude of sarcoplasmic reticulum Ca2+ release without affecting Ca2+ transient decay time in neonatal rat ventricular myocytes. The protein expression of alpha 1 C subunit of L-type voltage-dependent Ca2+ channel (Cav1.2) was increased after UBR3/6 knockdown, whereas the protein levels of RyR2, SERCA2a, and PLB remained unchanged. In line with the increase in Cav1.2 proteins, the UBR3/6 knockdown enhanced the current of Cav1.2 channels. Furthermore, the increase in Cav1.2 proteins caused by UBR3/6 reduction was not counteracted by a protein biosynthesis inhibitor, cycloheximide, suggesting a degradative regulation of UBR3/6 on Cav1.2 channels. Our results indicate that UBR3/6 modulates cardiac CICR via targeting Cav1.2 protein degradation.Ca  Through the CICR process, calcium transients are generated. The increased cytoplasmic calcium binds to Troponin C, moving the tropomyosin complex off the actin binding site and allowing the myosin to bind the actin filament. Through ATP hydrolysis, the myosin head pulls the actin filament toward the center of the sarcomere. Then, a calcium ion pump sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), which is under the precise control of phospholamban (PLB), transfers most of the Ca2+ in the cytoplasm back into the SR, and the muscle fiber relaxes [The cardiac Ca2+ stores. This pr relaxes .The ubiquitin–proteasome system (UPS) is a cascade reaction consisting of ubiquitin (Ub), E1 (Ub-activating enzyme), E2 (Ub-conjugating enzyme), E3 (Ub-protein ligase) and proteasome. Ub is catalyzed by E1 in an ATP-dependent manner, and E2 accepts the activated Ub on cysteine. E3 mediates the transfer of the activated Ub from E2 to target protein. Finally2+ transients in UBR-knockdown rat cardiomyocytes and found that UBR3/6 changed CICR. Further analysis revealed that the knockdown of UBR3 and UBR6, significantly increased the level of Cav1.2 channel proteins, not other sarcolemmal or SR membrane proteins involved in CICR. The function of Cav1.2 channel was also enhanced in response to UBR3/6 knockdown. The employment of protein synthesis inhibitor cycloheximide showed that UBR3/6-knockdown-mediated increases of Cav1.2 proteins were not derived from de novo synthesis.The aim of this study is to elucidate the implication of the modulation of ubiquitin ligase UBR on cardiac calcium-handling proteins. We studied CICR by measuring CaAll of the animal experiments were approved by the Animal Experiments Committee of Tongji University and conformed to the Guide for the Care and Use of Laboratory Animals established the US National Institutes of Health.◦C. The same procedure was repeated five times. The isolated cells were collected, cultured in a CO2 incubator, and purified by differential adhesion for 2 h.Neonatal rat ventricular myocytes (NRVMs) from the ventricles of 1–2-day-old Sprague–Dawley neonatal rats were isolated as previously described. The heaThe NRVMs were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum  and 1% penicillin and streptomycin in a humidified incubator at 37°C with 5% CO2. 48 h later, the NRVMs were transfected with siRNAs for 48 h and collected for downstream assay.Rat UBR1-7 were knocked down by specific small interference RNAs (siRNAs) which were synthesized by Jima Biotechnology Co., Ltd. . A 21-mer scrambled double-stranded RNA was used as the negative control. All of the siRNA sequences are listed in Adherent cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and then blocked with 1% BSA for 1 hr. After incubation with anti-α-actin (Sigma) as primary antibody overnight at 4°C, FITC-conjugated goat anti rabbit IgG (Abcam) was used as secondary antibody. 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. The slides were photographed using fluorescence microscopy . The dilution concentration of the primary antibodies was 1:10 to 1:100, and the secondary antibodies at a dilution of 1:200.Intracellular calcium was measured using a dual-excitation fluorescence photomultiplier system (Ion Optix) as described . NRVMs wThe cells were lysed using RIPA lysis buffer , 1% sodium deoxycholate, 1% NP-40, 1 mM PMSF and 1 mM EDTA) at 4°C for 20 min. The proteins were separated by SDS-PAGE gel (Invitrogen), and transferred to polyvinylidene fluoride membranes (Invitrogen). The membranes were incubated overnight at 4°C with the appropriate primary antibodies followed by the peroxidase-conjugated secondary antibody for 1 hr at room temperature. Then the immunoblots were visualized using chemiluminescence reagents. The primary antibodies included anti-UBR3 antibodies (Santa Cruz Biotechnology), anti-UBR6 antibodies (Santa Cruz Biotechnology), anti-Cav1.2 antibodies (Alomone Labs), anti-RYR2 antibodies (Abcam), anti-SERCA2a antibodies (Abcam), anti-PLB antibodies , and anti-GAPDH antibodies . The dilution concentration of the primary antibodies was 1:200 to 1:1000, and the secondary antibodies at a dilution of 1:1000. Results are representative of at least three independent experiments.2+ current as previously described [2 2, MgCl2 0.8, dextrose 10, and HEPES 10. The intracellular solution  contained (in mM): CsCl 130, GTP 0.1, EGTA 10, Na− phosphocreatine 5, MgCl2 1, MgATP 5, and HEPES 10.Standard voltage clamp technique was used to record the cardiac L-type Caescribed . After 2v1.2 channel protein levels.This was accomplished by treating UBR3 or UBR6 knockdown cells with or without cycloheximide to inhibit protein translation. The NRVMs were treated with cycloheximide (100 µg/ml)  for 0, 4, 8, and 12 h, following UBR3 or UBR6 siRNAs treatment for 24 h. Total proteins were extracted from the cell samples, and Western blot was performed to analyze Ca2+ transient and voltage-clamp data were analyzed using Pulsefit (HEKA) and Origin 7.5 . Each experiment was repeated at least three times.All data were analyzed using SPSS 13.0 software  and presented as means ± SEM. In all experiments, unpaired Student’s t-test or one-way ANOVA was used to compare experimental groups with their appropriate controls. p < 0.05 was considered statistically significant. Ca2+ transient in cardiomyocytes, the UBR1-7 siRNAs were individually transfected into NRVMs. After 48 hours, the knockdown efficiency of UBR1-7 siRNA was tested by RT-PCR. The cross-inhibition and inhibition efficiency of UBR1-7 siRNAs are listed in 2+ transient amplitude and Ca2+ transient decay time was measured in Fura-2/AM loaded NRVMs under electric field stimulation (2+ transient amplitude in F340/F380 ratio from (0.141 ± 0.006) to (0.224 ± 0.031) and (0.192 ± 0.017) respectively. However, the Ca2+ transient decay time (T50) was not significantly changed by UBRs knockdown % and (166 ± 22)%, respectively. However, the expression levels of RyR2, SERCA2a, and PLB were unchanged. These results suggest that UBR3 and UBR6 modulate CICR through targeting Cav1.2 channel proteins.To further determine the CICR-associated proteins regulated by UBR3/6, the levels of Ca/6 siRNA . The resv1.2 channels. As shown in the current–voltage (I–V) curves (v1.2 channels during the depolarizing step from – 50 to +50 mV in UBR3 or UBR6 knockdown NRVMs was significantly increased (p < 0.01). The peak current density of the Ca2+ channels in UBR3 knockdown cells increased from (−5.21 ± 0.36) to (−6.46 ± 0.31) pA/pF . The peak current density generated by the Ca2+ channels in UBR6 knockdown cells increased to (−6.17 ± 0.3) pA/pF . These results suggest that the downregulation of UBR3 and UBR6 enhances the activity of Cav1.2 channels.Based on the above results, we further examined whether the knockdown of UBR3 or UBR6 could enhance the function of Ca) curves , the peav1.2 channels is derived from de novo synthesis in NRVMs treated with UBR3 and UBR6 siRNA, we employed CHX, a pan inhibitor of protein synthesis that inhibits ribosome translocation. CHX treatment resulted in time-dependent decreases of endogenous Cav1.2 levels in normal cells. In contrast, CHX treatment did not counteract the increases in Cav1.2 proteins caused by UBR3 and UBR6 knockdown , and Cavβ-free Cav1.2 channels can be ubiquitinated by E3 ubiquitin-protein ligase complex slx8-rfp subunit (RFP2) and degraded by the proteasome [2+ transient amplitude. We further found that in the Ca2+-handling proteins located on the sarcolemmal and SR membranes, the knockdown of UBR3/6 increased the protein level of Cav1.2 channels, but not RyR2, SERCA2a, or PLB proteins, suggesting that Cav1.2 channel is the key target in the modulation of UBR3 and UBR6 on cardiac CICR. Based on these results, we speculate that UBR3 and UBR6 are likely to be new modulators of CICR in cardiomyocytes.The balance of Caoteasome ,17. Calpoteasome . Overexpoteasome . The abooteasome . There aoteasome . The UBRoteasome . It consv1.2 channel is a major channel that mediates Ca2+ influx from extracellular space into the cytoplasm of cardiomyocytes, which triggers Ca2+ release from SR through RyR2 channels and generates CICR[v1.2 channel, as an important kind of ion channel in cardiomyocytes, is also crucial for the physiological function of heart[v1.2 channel are associated with a variety of cardiac disorders, for example, arrhythmia[v1.2 was reported to be ubiquitinated by RFP2 and degraded at the proteasome[v channels for degradation by both the proteasome and the lysosome[v1.2 channels can be degraded by different kinds of E3 ubiquitin-protein ligases. UBR3 and UBR6-mediated regulation could be a novel mechanism of the regulation of Cav1.2 channels. In the present studies, we observed that the knockdown of both UBR3 and UBR6 increased not only the level of Cav1.2 channel proteins, but also its peak current density. That is to say, UBR3 and UBR6 can modulate Cav1.2 channels, and affect the electrophysiological properties of the whole heart. The effect of UBR3/6 on Cav1.2 channels may be an essential precondition for normal cardiac physiology. Our results showed that only UBR3 and UBR6 facilitated the degradation of Cav1.2 channels among the UBR family. Although UBR3 and UBR6 belong to the UBR family, unlike other typical N-recognins, they are not bound to the known N-end rule substrates (N-degrons) of UBR1 and UBR2 [v1.2 channel via their distinctive functional domains in different physiological and pathologic conditions.Caates CICR. Cav1.2  of heart. Abnormarrhythmia, heart frrhythmia,27, Brugrrhythmia, and carrrhythmia. In receroteasome. Meanwhi lysosome. These cand UBR2 ,30, so tand UBR2 . UBR3 coand UBR2 . So, we v1.2 in NRVMs; nevertheless, the mechanism of CICR shows developmental changes in rat hearts. In NRVMs, the elevation of cytosolic calcium concentration upon depolarization is mainly mediated by sarcolemmal Cav1.2-mediated calcium influx, whereas, calcium release from the sarcoplasmic reticulum contributes to the majority of calcium elevation in adult cardiomyocytes upon membrane depolarization. This indicates an important functional transition of the L-type Ca2+ channels from Ca2+ entry path to a predominantly Ca2+ trigger [v1.2 protein homeostasis, which implicates the potency of UBR in controlling Cav1.2 protein expression in adult cardiomyocytes [v1.2 channels. This discovery has novel pathophysiological implication in heart diseases associated with CICR. UBR3 and UBR6 might become potential targets for therapeutic intervention in diseases associated with electrical and contractile dysfunction.In the present study, we found the regulation of UBR3/6 on Ca trigger ,33. It smyocytes ,35. Howe


Current study focused on the bioactivity of PH46 (2) and related novel indane dimers (6-10) to investigate the impact of changes in substitution and stereochemistry at the C-1 and C-2 positions of the PH46 (2) scaffold.PH46A (m using 5-lipoxygenase (LOX) and 15-LOX enzymes, and 5-LOX binding was evaluated in silico against NDGA, nitric oxide (NO) released from LPS-induced SW480 cells and cytokines in THP-1 macrophages  and in SW480 cells (IL-8).Cytotoxicity profiles of compounds were established using THP-1 macrophages and SW480 cells. Effects of the compounds were then evaluated at 10 µ2) and 7 cause reduction in NO, inhibition of 5-LOX with high binding energy and no cytotoxicity effects in THP-1 macrophages and SW480 cell lines (up to 50 µm). The cytokine profiling of the series demonstrated inhibition of IL-6 and TNF-α in THP-1 macrophages together with IL-8 in SW480 cells.PH46 (in vivo effects demonstrated by indane dimers and PH46A (1) in murine models of colitis.The observed profile of cytokine modulation  and inhibition of release of NO and 5-LOX may contribute to the  H-indene) ring system consists of a combination of aromatic benzene and aliphatic cyclopentane ring as shown in  This versatility has contributed to indane being identified as a privileged structure. The indane ‘scaffold’ also commonly occurs naturally or in medicinal chemistry as indanones, indandiones and indanols.The indane  or 6-(Methylamino)hexane-1,2,3,4,5-pentanol 4--1-hydroxy-2,3-dihydro-1H,1′H--2-yl)methyl)benzoate) has advanced to clinical trial (ISRCTN90725219) (singular). PH46A (1) is a first in class with a novel scaffold. In early studies, simple monomeric indanes, with a structure based on the natural pterosin scaffold, were designed and shown to demonstrate smooth muscle relaxant and mast cell stabilisation effects. Subsequent work led to the development of dimeric indane molecules which demonstrated mast cell stabilisation activities, with twice the order of magnitude than the monomeric indane series.,10 At that point we considered the potential of the series for their clinical use as new chemical entities in the prevention of asthma based on their combined mast cell stabilisation and muscle relaxant properties.The current study is based on molecules that have been developed in our group over the last 20 years. These studies centred on the synthesis and evaluation of the pharmacological activity of nature identical and synthetically modified indanes, indanones and dimeric indane. One of these molecules, PH46A  reduced colon cytokine levels of TNFα, IL-6 and IL1b in DSS colitis mice. These molecules also prevented histological damage to colons. From these results, a lead molecule PH46A (1) demonstrated efficacy in two murine models of inflammatory bowel disease (IBD): an acute dextran sulphate sodium (DSS) model of murine colitis and the more chronic spontaneous model of −/−IL-10 colitis mice. The scaffold of PH46A (1) incorporates the indane skeleton and is the N-glucamine salt of a single enantiomer PH46 (2) of this novel drug with 1S, 2S stereochemistry at the C-1 and C-2 positions. The success of PH46A (1) in ameliorating the effects of in vivo activity in the DSS and −/−IL-10 murine model of colitis has led to its potential as a therapeutic agent in IBD. It is interesting to note that of the four possible enantiomers forming the isomeric dimeric indane scaffold that with the 1S, 2S stereochemistry at C-1 and C-2 , the absence of understanding the molecular target has prompted further investigation into the activity of PH46A (1) and PH46 (2), and some analogues in order to build understanding of what may be a novel, complex mechanism of action or biological fingerprint. Within the series there is currently no clear understanding of the effect of substitution at the C-1 and C-2 positions on the pharmacological activity of analogous compounds, although our earlier work has identified what appears to be an optimal stereochemical configuration for the enantiomers of PH46 (2).Despite the impressive in vitro, in vivo and in silico models. This allows us to understand the relationship of stereochemistry and substitution at the C-1 and C-2 positions on indanol ring B on biological activity  and PH46 (2) were synthesised according to the experimental method previously reported. The key compounds of interest 6 and 7 were synthesised from 3, with intermediate compounds 4 and 5,12  for 48 h. The PMA-differentiated macrophages were cultured and left at resting period for 3 days (PMAr stage). For SW480, the cells were seeded in 96-well plates 24 h prior to drug incubation at 10 µm. After 24 h, the media were removed and each well was washed with 100 µl of PBS and the step was repeated twice. After washing, 100 µl of freshly prepared phosphatase substrate  was added into each well. The plates were incubated in the dark at 37°C and 5% CO2 for 1 h. The reaction was stopped by adding 20 µl of Na OH. Readings of the wells were recorded at 405 nm.THP-1 monocytes  and SW480 cells were seeded in 96-well plates at concentration 4 × 10m CaCl2 , 100 µl of 132 µg/ml L-α-phosphatidylcholine type II-S  and 100 µl of 1.1 mm ATP  at RT for 5 min in the dark. The reactions were started by adding 100 µl of 275 µm arachidonic acid  to the mixture in quartz microcuvettes. The mixture was gently mixed by pipetting three times and the increase in absorbance at 234 nm was measured using the Shimadzu UV-1602 spectrophotometer. The compounds were screened using one concentration point to determine the percentage inhibition by following the formation of the conjugated diene product 5-HpETE at 234 nm (ε = 25 000 M−1 cm−1).Briefly, 100 µl of 2.75-unit human recombinant 5-LOX   was pre-incubated with 50 µl of compounds/standard inhibitor/buffer, 100 µl of 2.2 mm) at 25°C for 5 min. The control well consisted of 50 µl of rabbit reticulocyte 15-LOX-1 and 50 µl of buffer in 0.2% DMSO. The reaction was initiated by adding 50 µl of arachidonic acid  into both the sample and control wells. The reaction mixture was incubated at 25°C for 15 min in the dark. The reaction was terminated by adding 100 µl of FOX reagent after reaction period of 15 min. The FOX reagent contained 30 mm sulphuric acid, 100 µm xylenol orange and 100 µm ferrous (II) sulphate in MeOH : water (9 : 1). The absorbance readings were measured at 584 nm after 30 min of colour development at RT.In the sample wells, 50 µl of rabbit reticulocyte 15-LOX-1  was pre-incubated with 50 µl of compounds/standard inhibitors . The treatment of cells with LPS was carried out for 48 h, at 37°C under 5% CO2. After treatment, the culture supernatant was collected and immediately tested for nitrite quantitation using GREISS reagent. Equal volume of fresh culture supernatant and sulphanilamide solution was loaded into each well of 96-well plates. The mixture was incubated for 10 min, in the dark at RT. Next, same volume of N-1-napthylethylenediamine dihydrochloride (NED) was added into each well and incubated for 20 min. A purple/magenta colour was formed in the wells and the absorbance was recorded at 540 nm. A standard curve (0–100 µm) of NO2− was developed using known standards as provided by the kit.SW480 cells were stimulated with 1 µg/ml of 5 cells per well in 500 µl. The differentiation process and resting period of the THP-1 macrophages were carried out and adapted. After 1 h of drug exposure, the cells were stimulated with 1 µg/ml of Escherichia coli 0111: B4 LPS (Sigma-Aldrich). At 24 h, the media were collected from each well, transferred into 2.0-ml microcentrifuge tubes and centrifuged at 17 950 g for 10 min at 4°C. Supernatant collected was analysed using MSD V-PLEX Proinflammatory Panel 1 Human (4-Plex) Cat. No K15052D and V-PLEX individual assay Cat. No. K151QUD kits .THP-1 monocytes were seeded in 24-well plates at density of 2.5 × 104 cells/well and left to grow until 80% confluency. The cells were stimulated with 1 µg/ml of E. coli 0111: B4 LPS after 1 h of drug treatment. Supernatant was collected from each well at 24 h. The supernatant was transferred into 2.0-ml microcentrifuge tubes and centrifuged at 13 000 rpm for 10 min at 4°C. Supernatant collected was analysed using MSD U-PLEX Biomarker Group 1 Human (10-Plex) Cat. No. K15067L-2  plates.SW480 cells were seeded into 96-well plates at density 4 × 102, 6, 7, 8, 9 and 10 and the reference standard nordihydroguaiaretic acid (NDGA) were prepared using LigPrep tool of the Schrödinger software to generate the 3D structures. During the process, input ionisation states were retained and chiralities were determined from 3D structures. Final structures were minimised using optimised potential liquid simulations (OPLS3e) force field. Structure of the target protein also needs to be prepared before going further into in silico calculations. Accordingly, crystal structure of human 5-LOX  in complexed with arachidonic acid was retrieved from RSCB protein data bank and subjected to Protein Preparation Wizard module of Schrödinger's.,22 Bond orders were assigned, missing hydrogens were added, chain B, all water molecules and heteroatoms except arachidonic acid were removed. In the next step, the structure was optimised and further minimised using OPLS3e force field to avoid steric clashes. Arachidonic acid was taken as a reference to define the binding site of 5-LOX. A grid representing the binding site was generated using default settings. Glide XP (extra precision) module was used to dock the compounds into the binding pocket.As a first step in molecular modelling compounds P < 0.05 considered as statistically significant. Dunnett's Multiple Comparison Test was chosen to compare between the control group and samples groups. Subsequently, Bonferroni's Multiple Comparison Test was used to compare the samples groups in the cytokines ‘profiling to evaluate the SARs’. The results were expressed as the mean ± standard error mean (n = 3 experiments performed on different days), unless otherwise stated. Asterisks indicate the value is significant .Statistical comparisons  were made using one-way ANOVA with m was selected as the tested concentration with addition of one higher and one lower concentrations: 50 and 10 µm, respectively. From m in either THP-1 macrophages or SW480. This study allowed us to establish a dose range for incubation of test compounds in a non-toxic dose range.In the current study, 30 µm, only PH46 (2) and methylene analogue 7 inhibited 5-LOX significantly at 45.30 ± 5.28% and 39.71 ± 4.95%, respectively, P < 0.05. Results were comparable to the degree of inhibition of zileuton at 45.22 ± 5.36%, P < 0.05. The % inhibition of control NDGA was observed at 51.92 ± 1.01%, P < 0.05, as shown in m inhibited 15-LOX activity at 72.43 ± 3.25%. The Z′-factor in this 96-well microplate ferrous oxidation-xylenol orange (FOX) assay was determined as 0.891 which demonstrated the suitability for mid-throughput to high-throughput screening. The IC50 value for NDGA was determined at 4.61 ± 0.93 µm, similar to the literature values..Of the compounds screened at 10 µ2 and 7, together with the reference compound NDGA, had the highest score. Besides a number of hydrophobic interactions, NDGA makes strong hydrogen bonds with Val671, HIS367, THR 364 and GLN363 in the binding pocket. In addition, a favourable T-shaped π- π interaction with HIS367 is also observed. Docking analysis of the active compound 2 indicates that the hydroxy group at C-1 position participates a hydrogen bonding with HIE550 of 5-LOX. No other hydrogen bond is observed but the carboxy group orients itself towards polar LYS409 suggesting a possible favourable interaction.As shown from 2 and reduce the energy of protein–ligand complex which in turn enhances activity. In compound 6 where hydroxy group at C-1 position of 2 is replaced by carbonyl group, a new hydrogen bond is observed between carboxy group and VAL671 which may compensate the absence of favourable interaction of -OH group. However, compound 6 was observed to adopt a different orientation in the 5-LOX binding pocket. Most noticeable, an unfavourable face-to-face π-π interaction between indene ring and PHE177 is observed (the angle between the ring planes is 20.5°). Hence, the favourable hydrophobic interaction of indene moiety is now replaced with unfavourable π-π stacking interaction  but did not show statistical significance due to a slight increase in SEM at n = 3. A second positive control, the natural product and nitric oxide (NO) inhibitor, α-mangostin gave an inhibition of 61.63 ± 7.50% with P < 0.01. Of the analogues examined, PH46 (2) exhibited the highest inhibition with 56.50 ± 3.09%. Para-carboxybenzyl derivatives 6, 7 and the para-unsubstituted benzyl 9 also inhibited NO production. IC50 values were determined for analogues which inhibited at greater than 50% at the selected 10 µm concentration. The IC50 value of α-mangostin was determined at 8.87 ± 1.18 µm and this value is close to the reported values of IC50: 12.2 µm. The IC50 value of hydrocortisone was 48.66 µm consistent with literature values. PH46 (2) displayed the lowest IC50 value of 19.70 ± 7.88 µm of all the analogues, followed by compound 9 (IC50: 20.49 ± 2.89 µm).Preliminary screening of the indane dimers  showed c The level of IL-6 in THP-1 macrophages stimulated with LPS without drug treatment was 1.666 ± 0.256 pg/µg ml in CTRL 1 as shown in P < 0.001. Infliximab did not inhibit IL-6 significantly from the value of 1.666 ± 0.256 pg/µg ml to 0.924 ± 0.180 pg/µg ml. Infliximab was used as the positive control as it inhibits the production of TNF-α and is used as anti-TNF-α therapy for IBD treatment clinically.,30 PH46 (2) and compound 8, its unsubstituted benzyl analogue, displayed potent IL-6 inhibition with levels of inhibition of 92.07% (0.132 ± 0.038 pg/µg ml) and 95.49% (0.075 ± 0.019 pg/µg ml), respectively, over CTRL 1.IL-6 plays a central role in the maintenance of chronic intestinal inflammation in IBD. In P < 0.001, at 1.784 ± 0.052 pg/µg ml. Infliximab did not have significant inhibitory effect on IL-1β, with production level at 0.329 ± 0.059 pg/µg ml. No significant inhibition was observed by analogues in the study. TNF-α has been shown to induce various inflammatory mediators in the inflamed mucosa in IBD. In the cytokine profiling for TNF-α, the level of TNF-α for CTRL 1 was measured at 22.305 ± 2.753 pg/µg ml, whereas CTRL 2 (without LPS stimulation) measured at 0.060 ± 0.019 pg/µg ml as shown in m effectively inhibited the production of TNF-α with % inhibition of 99.98%, measuring at 0.002 ± 0 pg/µg ml, p < 0.001. Cyclosporine A slightly inhibited the release of TNF-α at 17.57% (17.460 ± 2.249 pg/µg ml) at 10 µm, but the value was not statistically significant. The profiling of TNF-α was like IL-6 where PH46 (2) and 8 inhibited TNF-α at 45.18% (11.613 ± 0.162 pg/µg ml) and 32.99% (14.193 ± 1.685 pg/µg ml) respectively. These compounds both have a hydroxyl group with the S-configuration at C-1 position which may play a role in assisting TNF-α inhibitory activity.Cytokine IL-1β triggers intestinal inflammation by augmenting the recruitment of granulocytes and the activation of intestinal epithelial cells (IECs). In this study, the release of IFN-γ was low compared with the other three analytes under the same experimental conditions. The level of IFN-γ in LPS-stimulated THP-1 macrophages was 0.024 ± 0.007 pg/µg ml and 0.001 ± 0.000 pg/µg ml in THP-1 macrophages without LPS stimulated. Cyclosporine A inhibited IFN-γ with % inhibition of 68.05%  as shown in 6 and the activities are comparable with cyclosporine A. Some groups exhibited greater variability due to the low detection of IFN-γ.Studies have shown that the production of IFN-γ is increased in lamina propria and T cells in patients with IBD.,35 Overall the inhibition over CTRL 1 values range from 12.70 to 25.0% for the mentioned molecules as shown in P < 0.001. Cyclosporine A inhibited IL-8 with P < 0.05, measuring at 2.689 ± 0.293 pg/µg ml. The inhibitory effects observed with PH46 (2) and compound 8 on IL-6 and TNF-α release from THP-1 macrophages were also observed on the release of IL-8 from SW480 cells with P < 0.05, 23.92% (2.529 ± 0.058 pg/µg ml) and 31.17% (2.288 ± 0.059 pg/µg ml) respectively. In addition, compound 7 inhibited IL-8 measuring at 19.49% (2.676 ± 0.142 pg/µg ml), P < 0.05. The effect of this molecule on IL-6 release from THP-1 macrophages was less potent but statistically significant.IL-8 is a potent angiogenic factor in colon wound healing, colitis and cancer. In patients with IBD, IL-8 is overexpressed and generally localised to areas of active inflammation and tissue damage.2) and the indane dimers (6-10) on 5-LOX, NO and on the production of inflammatory mediators from THP-1 macrophages and SW480 cells were investigated. PH46 (2) was selected for these studies, to facilitate comparison with other close analogues for which there was no salt available or for which suitable salts could not be prepared. The selection of PH46 (2) over PH46A (1) is justified given that in vitro and clinical data has shown that PH46A (1) dissociates in solution to PH46 (2) and the N-glucamine salt.The effects of PH46  are independent of cyclooxygenase (COX) (Frankish & Sheridan unpublished results). The zileuton (12), is a bioisostere of indane, and is also an orally active inhibitor of the 5-LOX enzyme and was used in this study as a positive control. The ability of bioisosteres to elicit similar biological activity has been attributed to common physicochemical properties. The 5-LOX plays a unique role in the production of leukotrienes which makes it a potential therapeutic target for inflammatory conditions like asthma, rheumatoid arthritis, psoriasis and IBD. Due to the involvement of 5-LOX in IBD and the profound activity observed for PH 46A (1) and PH 46 (2) and its analogues in in vivo colitis models, the activity of PH46 (2), the indane dimers (6-10) and positive controls 12 and phenidone was evaluated on 5-LOX and 15-LOX enzymes, to see if LOX activity is part of the pharmacological fingerprint of the series. Furthermore, molecular docking calculations were performed to further support biological activity results towards 5-LOX. Docking calculations revealed that binding site of 5-LOX is composed of both hydrophilic and hydrophobic amino acid residues.Earlier studies have shown that the  NO overproduction via iNOS upregulation by intestinal epithelium has been consistently associated with IBD. The effects of compounds, PH46 (2) and the indane dimers (6-10) on NO inhibition were evaluated in this study, to elucidate if such activity may be a contributing factor to the observed beneficial in vivo effects of the novel scaffold in murine colitis models. Although this study does not probe the mechanism of NO inhibition, and will need to be expanded, it suggests that NO inhibition may be a significant activity for the lead molecule PH46 (2) and may be a contributing factor to the effects observed in in vivo models.Active inflammation of the colon lining during IBD is associated with increased production of cytokines released by the colonic cells, macrophages and neutrophils. These cytokines such as IFN-γ and TNF-α induce the release of inducible NO synthase (iNOS) via nuclear factor-κB (NF-κB) pathway which results in increased production of NO.2) and the related dimers (6-10) on the four primary cytokines released by the THP-1 macrophage cell line; TNF-α, IL-1β, IL-6 and IFN-γ, and on IL-8 released from SW480 cells. The indanes screened initially at 10 µm with drug incubation for 24 h. The total protein for the samples was determined. Measurement of each cytokine was represented by the concentration of each cytokine (pg/ml), expressed in relative to total protein quantified (µg) using BCA assay to give the final unit in pg/µg ml. Infliximab and cyclosporine A were used as positive controls similar to the previous study and they are known as standard medications used to treat IBD. The results obtained for the quantification of IL-8 in SW480 showed that there was a statistically significant inhibition of IL-8 release by some of the indane dimers with a methylene group at C-2 position.To understand the impact of the indane dimer scaffold on cytokine production, we evaluated the effects of PH46 (2) and related molecules that might explain activity observed in in vivo phenotypic models. The study of the analogous series also aimed at understanding how substitution and stereochemistry at the C-1 and C-2 position of the B ring in the indane dimer scaffold might contribute to the observed biological activity. Specifically, we investigated the impact of subtle modification at C-1 and C-2 positions of the B ring in the dimers. The indane dimers, PH46 (2) and its C-1 methylene derivative 7 inhibited 5-LOX significantly (p < 0.05). These compounds have the S-para-carboxybenzyl substituent at C-2. The molecules differ in substitution at C-1, where 7 has no OH group, but both molecules retain sp3 hybridisation at this position, which may impact on binding with the isozymes. The observed inhibition of 5-LOX was further supported during molecular docking analysis which has shown highest docking scores for indane dimers PH46 (2) and 7.The current study focused on probing activities that contribute to the pharmacological fingerprint of PH46  and the novel analogues (6-10) on the key mediators with significant values at p < 0.05 are summarised in The activity of 2) and the related novel indane dimers (6-10) has been evaluated in in vitro, in vivo and in silico models and these studies have contributed to our understanding of this novel class of molecule. The cytokine profiling of the series demonstrated inhibition of IL-6 and TNF-α in THP-1 macrophages and IL-8 in SW480 cells. PH46 (2) and 7 cause a statistically significant reduction in NO production and reduction in 5-LOX activity in the arachidonic acid pathway. These activities should be considered to form part of the indane dimer biological fingerprint and may contribute to the in vivo effects demonstrated by PH46 (2) in murine models of colitis. PH46 (2) reduced DAI in the acute 5% DSS and IL-10 -/- murine model of colitis and significantly reduced MPO activity, a measure of inflammatory cell infiltration and inflammatory colon cytokines . The observation of inhibition of NO and 5-LOX by PH46 (2) and other related indane molecules warrants further study and may help to elucidate a clearer understanding of the relationship between the reduction in inflammation in the in vivo models and the decreased level of cytokines observed. Further investigations will lead to a deeper understanding of the pharmacological fingerprint to identify potential development of anti-inflammatory therapeutic agents.The biological fingerprint of PH46 (The Author(s) declare(s) that they have no conflicts of interest to disclose.KC and TZ synthesised and characterised the compounds discussed in this manuscript. KC performed all biological assays with assistance from AC on the multiplex assays. AE performed the 5-LOX molecular docking analysis. HS, NF and JOS were involved in all aspects of the project design, supervision of chemical and biological experiments and data interpretation. SW-480 cell line was a gift from JOS. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.jphp13269-sup-0001-AppendixS1Appendix S1. Materials, chemicals and synthesis of indane dimers.Click here for additional data file.


Overall, this study explores the multiple prognostic values and oncologic functions of the S100A family in PC.S100 calcium‐binding protein A (S100A) family members regulate multiple biological functions related to pancreatic cancer (PC) progression and metastasis. However, the prognostic and oncologic values of S100A family have not been systematically investigated in PC. In the present study, the mRNA expression and potential functions of S100A family were investigated by bioinformatic analysis. Our results demonstrated that overexpression of S100A2, S100A6, S100A10, S100A11, S100A14 and S100A16 was significantly associated with higher T stage, advanced histologic grade and worse prognosis in PC. Besides, one CpG of S100A2, three CpG of S100A6, four CpG of S100A10, four CpG of S100A11, two CpG of S100A14 and five CpG of S100A16 were negatively associated with corresponding S100A family members expression and positively associated with overall survival (OS). The signature based on four CpGs showed good prediction ability of OS. Besides, S100A2 overexpression took part in the regulation of mitotic cell cycle, ECM‐receptor interaction and HIF‐1α transcription factor network. Overexpression of S100A6, S100A10, S100A11, S100A14 and S100A16 may impair the infiltration and cytolytic activity of CD8 We also investigated the potential biological roles of S100A family members in PC using functional enrichment analysis in ConsensuspathDB (22.1https://cancergenome.nih.gov/) database. The CpG methylation data of S100A family members in TCGA PC cohort were downloaded through MethSurv (https://biit.cs.ut.ee/methsurv/). Among the 177 PC cases in TCGA PC cohort, 171 were cases with OS >1 month. Besides, GSE28735 data set were used for paired differential expression analysis for S100A family members. TCGA PC data set and GSE28735 data set are both freely available as public resources. Therefore, local ethics approval was not needed.First, we downloaded the mRNA expression data recorded based on FPKM and corresponding clinical information for PC from the Cancer Genome Atlas  based on the TCGA and GTEx projects. Then, the differential expression of S100A family members between PC samples (n = 45) and the corresponding adjacent non‐tumour tissues (n = 45) was investigated using GSE28735 data set. In addition, the overlapped significantly differential expression S100A family members between GEPIA database and GSE28735 database were selected for further analysis.First, differential expression analysis of S100A family in PC was performed using the GEPIA database , a website database based on resources from TCGA database. The final prognostic KM plots were presented with a hazard ratio (HR), 95% confidence interval (CI) and log‐rank P value. A P value < .05 was considered statistically significant.The prognostic values of differentially expressed S100A family in PC were analysed using Kaplan‐Meier (KM) plotter database ; the best cut‐off points were determined by the X‐tile 3.6.1 .CpG methylation data of S100A family members were extracted from MethSurv. Correlation analysis between the mRNA expression of S100A family members and the corresponding CpG methylation in PC was conducted with the threshold of Pearson correlation coefficients <−0.4 and http://software.broadinstitude.org/gsea/) was performed to investigate the differences in potential biological functions between the high‐ and low‐risk groups.In addition, we performed Lasso regression and multivariate Cox analysis to develop a prognostic signature based on critical CpG. Risk score = ∑ the multivariable Cox regression coefficients × the methylation of critical CpG. High/low‐risk groups were determined according to the best cut‐off point determined by the X‐tile 3.6.1. The predictive ability of the CpG signature was assessed by C‐index, and the AUC of ROC curve. Moreover, gene set enrichment analysis (GSEA)  in the TCGA PC cohort.+ T cell between the high‐ and low‐risk groups. Besides, correlation analysis between S100A family and the CD8+ T infiltration and cytolytic activity in PC was performed with Pearson correlation coefficients.With the help of GSVA, single‐sample gene set enrichment analysis (ssGSEA) was performed to quantify the activity or enrichment levels of CD82.8www.r-project.org/), SPSS 25.0 software and GraphPad prism 8.0 software  were used for statistical analyses. Correlations were calculated using Pearson correlated coefficient. Group differences were analysed by Wilcoxon test or Kruskal‐wallis test. P values <.05 were considered as statistically significant.R software  in PC patients, we examined the correlation between mRNA expression and OS or RFS on log‐rank test using KM plotter database. KM curves for OS demonstrated that patients with lower expression of S100A2, S100A4, S100A6, S100A10, S100A11, S100A13, S100A14 and S100A16 had superior OS than those with higher expression of S100A2, S100A4, S100A6, S100A10, S100A11, S100A13, S100A14 and S100A16    Figure . The C‐i) Figure . Further) Figure .3.6The top 50 co‐expressed genes of S100A2, S100A6, S100A10, S100A11, S100A14 and S100A16 were respectively enrolled into ConsensuspathDB and subjected to pathway enrichment analysis. S100A2 was significantly involved in the regulation of mitotic cell cycle, ECM‐receptor interaction and HIF‐1α transcription factor network Figure . Interes3.7+ T cell between high‐ and low‐risk groups. Using ssGSEA, we found that the enrichment fraction of CD8+ T cell infiltration and cytolytic activity in low‐risk group were significantly higher than those in high‐risk group . These results further suggested the potential functions of S100A6, S100A10, S100A11, S100A14 and S100A16 in decreasing the infiltration and anti‐tumour activity of CD8+ T cells.Pathway enrichment analysis also revealed that S100A6, S100A10, S100A11, S100A14 and S100A16 may be associated with T cell receptor signalling pathway, indicating their association with TIL infiltration in the TME of PC. In addition, ssGSEA revealed that S100A6, S100A10, S100A11, S100A14 and S100A16 overexpression significantly correlated with low infiltration and cytolytic activity of CD8+ T cells.+ T cells.Focal adhesion signalling pathway is widely considered as one of the oncologic cell‐intrinsic pathway influencing tumour immunity.+ T cells infiltration and cytolytic activities through focal adhesion‐Ras‐stimulated signalling pathway. However, some limitations should be acknowledged in this study. The first limitation of this study is the lack of experimental validation and externally clinical cohort validation. Second, the study outcome was largely dependent on the quality of data from publicly available databases.To the best of our knowledge, we are the first to systematically describe the prognostic values of the mRNA expression and the DNA methylation of S100A family in PC. Moreover, for the first time, we demonstrated that S100A6, S100A10, S100A11, S100A14 and S100A16 may suppress CD85In the current study, we comprehensively described the prognostic value and potential biological functions of S100A family members in PC, providing insights for further investigation of S100A family members as potential targets in PC.The authors declare that they have no potential conflict of interest.Hongkai Zhuang: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Software (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Xinming Chen: Formal analysis ; Methodology ; Validation ; Visualization ; Writing‐original draft . Fengying Dong: Software ; Writing‐review & editing . Zedan Zhang: Methodology (supporting); Writing‐original draft (supporting). Zixuan Zhou: Methodology (supporting). Zuyi Ma: Methodology (supporting). Shanzhou Huang: Data curation (supporting); Methodology (supporting). Bo Chen: Conceptualization . Chuanzhao Zhang: Conceptualization ; Funding acquisition ; Writing‐original draft ; Writing‐review & editing . Baohua Hou: Conceptualization ; Funding acquisition ; Writing‐original draft ; Writing‐review & editing .