TACI signals activate B cell proliferation, isotype switch and antibody production in both normal immunity and autoimmune states. In contrast to murine TACI, the human TACI gene undergoes alternative splicing to produce short and long isoforms (TACI-S and TACI-L). In previous studies, we showed that transduction of the short, but not long isoform, into murine B cells or human pre-B cells lacking TACI, caused them to become transcriptional and morphologically identical to plasma cells. These data suggest that the expression of different isoforms in humans provides unique controls on B cell maturation. In these studies we show that TACI-S and TACI-L form complexes in a ligand-independent manner, not dependent on a single extracellular domain. Both TACI isoforms are detectable in the endosomal cellular compartment where they co-localize with MyD88, TRAF6, and the activated 65 kDa form of TLR9, depending on a conserved intracellular TACI sequence. In contrast to TACI-L expressing cells, or cells bearing both isoforms, TACI-S binds ligands BAFF and APRIL with substantially greater affinity and promotes enhanced NF-kB activation. Using isoform-specific monoclonal antibodies, we show that while TACI-L is predominant as a surface receptor surface on human B cells, significantly more TACI-S is noted in the intracellular compartment and also in marginal zone, isotype switched and plasmablast in resting B cells. TACI-S is increased in tonsillar B cells and also in the intracellular compartment of activated peripheral B cells. These data shows that alternative splicing of the human TACI gene leads to two isoforms both of which intersect with MyD88 and TRAF6 and form complexes with TLR9, but the two isoforms have different ligand binding capacities, subcellular locations and activation capabilities. Actioduction . While Tntiation , 5. In hecretion , 7. Reinecretion –11. Whil defects , 10.Tnfrsf13b gene with two ligand-binding domains, the human TNFRSF13B gene has an additional 5′ exon which encodes a short terminal sequence. This permits skipping of exon 2 containing the first cysteine rich domain (CRD1), leading to the production of a second, shorter isoform lacking the first ligand-binding domain  or rhBAFF  (R&D Systems) were added and samples were analyzed at different time-points . For validation of complexes found in FRET studies by immunoprecipitation, we then generated the constructs FLAG-TACI-L and hemagglutinin labeled (HA) TACI-L, TACI-S, and the selected mutants C104R, A181E, or S194X , along with 0.8 μg of NF-kB luc reporter and 0.5 μg control pRL-null plasmids. Cells were cultured for 48 h after transfection to allow full TACI expression, and examined with and without activation for 6 h with 100 ng/ml rhAPRIL or rhBAFF. Equal amounts of DNA (0.8 μg) were used for each transfection, using empty vector DNA as needed. Reporter gene activity was determined after 48 h and NF-kB luciferase induction was normalized to Renilla luciferase intensity. As both FRET and immune precipitation studies suggested that TACI-S and TACI-L could form complexes with each other, we then tested NF-kB luciferase induction in HEK-293 cells transfected with both isoforms, validating the presence of each by the different fluorescent probes. For these studies, the values reported represent Relative Luciferase Activity (RLA) and are the mean ± SD from 5 to 7 independent experiments.As TACI-S lacks the CRD1 but is clearly functionally active, this domain does not appear to be needed for TACI assembly, as previously suggested , 16. ForTo examine TACI isoforms in human B cells, the HA tagged human TACI-L and TACI-S GFP-constructs were stably transduced into the human B cell line BJAB, which lacks the endogenous expression of TACI . GFP posAs we had found that human TACI-S exhibits greatly enhanced activation in both transduced murine and human B cells , we testKd values of TACI-L and TACI-S for BAFF or APRIL, 100 nM of labeled FLAG-tagged TACI-L and TACI-S proteins were incubated with increasing concentrations (from 0 to 104 nM) of BAFF or Mega-APRIL (Adipogen), for 30 min at room temperature in binding buffer (PBS)  using the Monolith NT Protein Labeling Kit RED-NHS . Briefly, proteins at concentrations of 20 μM were incubated with 4X dye at a ratio of 1:1 in labeling buffer. To determine the er (PBS) . The samTACI is best known as a surface receptor, but it is assembled in the ER where it intersects with MyD88 along with other adaptors such as TNFR-associated factor 6 (TRAF6), important for NF-kB activation , 19, 20.Our previous experiments demonstrated functional synergies between TACI and TLR9, including the shared requirement for MyD88  and the Figure 7A). The specificity of these mAbs was further validated by MST as described above, in this case incubating from 0 to 104 nM concentrations mAb with 100 nM fluorescently labeled recombinant FLAG-tagged TACI-L or TACI-S proteins for 30 min at room temperature in binding buffer (PBS) , on isoform-bearing BJAB cells. Clone 2H12 was selected and the IgG2a antibody was purified from hybridoma cell supernatants by FPLC . The commercial mAb  was found to bind only TACI-L bearing BJAB cells (er (PBS) . The samlo/−CD38hi). To examine the effect of activation in TACI isoforms expression, mononuclear cells (PBMCs) were cultured for 4 days in the presence of ODN (3 μg/ml), CD40L/IL21 (200 and 20 ng/ml) (Invivogen) (R&D Systems) or anti IgM (10 μg/ml) (SouthernBiotech). Flow cytometry data analysis was performed using FlowJo data analysis software . As permeabilized cell staining results also include contributions from surface staining, we calculated the percentage of TACI surface and intracellular expression as previously described . Human tonsillar mononuclear cells were obtained from fresh samples by mechanical disruption followed by separation on a Ficoll-Hypaque gradient; buffy coats from normal blood donors were also obtained from the New York Blood Center. Mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque . To examine the expression of TACI isoforms on B cell subpopulations, B cells isolated by negative selection (CD19+) were stained with isoform specific mAbs in PBS with 1% BSA for 30 min at 4°C. Cells were subsequently washed and fixed with a 2% paraformaldehyde/PBS solution before analysis on BD LSRFortessa. For intracellular staining, cells were permeabilized after fixation for 20 min with BD FACS Permeabilizing solution 2 (BD), washed and stained with mAb TACI-L or mAb TACI-S. For indirect staining, cells were incubated with anti-mouse Alexa Fluor 488 (Invitrogen) using an IgG2a or IgG2b isotype control to validate the specificity of the staining. Living cells were identified by forward scatter and side scatter gating, and/or exclusion of 7-aminoactinomycin-D (eBioscience). B cell populations were discriminated by using CD19 APC-Cy7 (eBiosciences), CD27 PE (BD), CD38 Brilliant Blue, (eBiosciences), IgM APC (BD), IgD Pacific Blue (eBiosciences), CD20 FITC (BD), to designate naïve (IgD+CD27−), transitional (IgM+CD38+), marginal zone (IgD+CD27+), switched memory (IgD-CD27+), and plasmablasts . Values were expressed as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). Statistical significance was assessed by a two-tailed paired Student's Previous work on the murine TACI receptor suggested that the CRD1 region was essential for receptor assembly . HoweverTNFRSF13B mutations, we then examined complexes in HEK-293T cells transfected with either mCherry-labeled TACI-S or TACI-L, along with either isoform containing missense mutations found in CVID  or YFP-CD40L as control. As the FRET signal was preserved for each combination , the 79 extracellular amino acids linking the CRD2 to the transmembrane domain (Δ105-164), or both CRD1 and CRD2 (Δ21-104) Figure . In eachKd for both) than TACI-S , as it possesses specificity only for the longer isoform Figure . The spep < 0.05)  and plasmablast B cells (p < 0.01).While the surface expression of TACI on human B cells is well known, based on our confocal studies and the signaling requirements of this receptor, substantial intracellular TACI was also expected. Thus we used the above monoclonal antibodies to examine any differences in expression of isoforms on cell surface and in the intracellular compartment. As staining of permeabilized cells includes fluorescent contributions from cell surface staining, we determined the separate surface and intracellular expression for TACI-S and TACI-L, using a previously described method . While T) Figure . To diss) Figure . While TTo analyze the effect of B cell activation on the expression of TACI isoforms, peripheral blood mononuclear cells (PBMCs) of normal donors were stimulated with selected B cell activators: ODN, CD40L/IL21, or anti-IgM. After 4 days of incubation, while IgM stimulation had a somewhat decreased expression of both isoforms in non-permeabilized B cells Figure , cells aTNFRSF13B mutations are more likely to have lymphoid hyperplasia and autoimmunity (TACI is a complex B cell receptor that promotes T cell dependent and independent antibody secretion and plasma cell differentiation , 30. In immunity , 11, 33.immunity , 35. We immunity .TNFRSF13B gene contains an additional 5′ exon as compared to its murine counterpart. This allows differential RNA processing, leading to the production of two human isoforms (Many studies analyzing TACI function have been performed using murine models. However, the human isoforms , the rolisoforms –39. Our isoforms .We demonstrate here that the short TACI isoform displays clearly greater affinity for ligands BAFF and APRIL as compared to the long isoform. TACI-S also mediates markedly enhanced NF-kB activation, with or even without the addition of ligands BAFF or APRIL, a feature previously noted for this receptor , 15, 20.Interestingly, our FRET analysis revealed that cells could display hybrid TACI receptors, containing both isoforms. Further examining this possibility, we found that cells transfected or transduced with both TACI-S and TACI-L demonstrated both intermediate ligand binding and luciferase induction, as compared to cells expressing either isoform alone. As this may be true of human B cells, we suggest that the differential expression of either or both TACI isoforms could provide fine-tuning or tolerogenic differentiation responses for these cells. Since TACI-S more strongly mediates plasma cell differentiation, we postulate that B cells may express predominantly TACI-L during early B cell development and proliferation, while TACI-S would be more characteristic of terminal B cell differentiation. In line with this, additional controls for the same TACI/BAFFR/BCMA receptor system are provided by alternative splicing of genes encoding the ligands BAFF and APRIL, which also leads to structural variants with either activating or inhibiting properties , 41.Following ligand binding, TACI directly interacts with MyD88 leading to NF-kB activation, dependent on the intracellular adaptors IRAK1, IRAK4, and TRAF6 . Since TFor peripheral human B cells, which bear both TACI-S and TACI-L , we usedIn summary, alternative splicing of the human TACI gene leads to the production of two isoforms with different ligand binding and activation capabilities in human cells. TACI ligands BAFF and APRIL preferentially bind the short isoform, which is also more efficient at inducing NF-kB activation, driving B cell maturation to plasma cell development. While both isoforms are found in human B cells and co-localize in the cytoplasm with MyD88, TRAF6, and TLR9, there is differential surface and intracellular expression of these isoforms in human B cell populations.This study was carried out in accordance with the recommendations of Mount Sinai Medical Center. The protocol was approved by the Mount Sinai Medical Center. All subjects gave written informed consent in accordance with the Declaration of Helsinki.YG-C and CC-R contributed conception and design of the study and performed the statistical analysis. YG-C performed experiments. SA performed MST assay. LR performed some experiments. JC acquired some data. CC-R wrote the first draft of the manuscript. AT, EM, and AC contributed with the discussion of the data. All authors contributed to manuscript revision, read, and approved the submitted version. YG-C and CC-R conceived the studies. CC-R obtained funding, provided study oversight, and drafted this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Additionally, MEM reduced CRS-induced upregulation of NMDA receptor subunit levels  in the frontal cortex. However, MEM markedly enhanced tau phosphorylation in the frontal cortex and other cerebral cortical regions following 28 days of CRS. The stimulatory effect of MEM on CRS-induced tau phosphorylation was correlated with increased activities of AKT, JNK, and GSK3β, inactivation of PP2A, and downregulation of Pin1 and HSP70. Moreover, MEM did not effectively reverse the NMDA receptor upregulation induced by 28-day CRS and even increased GluN2B subunit levels. In contrast to the duration-dependent effects of MEM on P-tau levels, MEM produced an anxiolytic effect in both regimens as revealed by elevated plus maze testing. However, MEM did not affect the body weight reduction induced by CRS. Thus, MEM exerts distinctive effects on CRS-induced tau phosphorylation, which might be related to the expression of GluN2B. The differential effects of MEM on P-tau levels have crucial implications for its clinical application.Exposure to chronic psychiatric stress has been linked to Alzheimer's disease-related tau hyperphosphorylation and abnormalities in glutamate neurotransmission. However, the pathological relationship between glutamatergic dysfunction and tau phosphorylation in the cerebral cortex under chronic psychiatric stress is not fully understood. The present study investigated the effects of memantine , an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, on chronic restraint stress- (CRS-) induced tau phosphorylation in mice. CRS administered for 16 or 28 consecutive days (1 h daily) induced significant tau phosphorylation in the brain. MEM treatment suppressed the elevation of phosphorylated tau (P-tau) levels induced by 16-day CRS in a dose-dependent manner. P-tau reduction was accompanied by the attenuation of the upregulation of GSK3 However, the causes of AD remain controversial, and no effective treatments are available [Alzheimer's disease (AD) is a neurodegenerative brain disease and the most common cause of dementia. AD is pathologically characterized by the extracellular deposition of ated tau . Since tvailable .Multiple factors are involved in the pathogenesis of AD, including aging, sex, endocrine levels, social environment, lifestyle, and stress factors . Among tTau proteins are widely expressed in the central nervous system and play a crucial role in neuronal physiology . In pathChronic glutamate excitotoxicity has been hypothesized to play a role in AD , 24. Strβ toxicity and alleviated tau hyperphosphorylation in an AD animal model [MEM has been used to treat moderate to severe AD . MEM wasal model , 31. Theal model . Therefoβ and p-GSK3β (Ser9))  protein kinase B (AKT and p-AKT) , c-Jun N-terminal kinase (JNK and p-JNK)  extracellular signal-regulated kinases (ERK and p-ERK) , and P38 mitogen-activated protein kinases (P38MAPK and p-P38MAPK)  were used. Also, antibodies against peptidyl-prolyl cis-trans isomerase NIMA-interacting-1 (Pin1) , heat shock proteins 90 (HSP90) , HSP70 , and HSC70 , as well as against protein phosphatases pY307-PP2Ac and PP2Ac , demethylated protein phosphatase PP2A catalytic subunit (DM-PP2Ac) , β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  were used. MEM hydrochloride was purchased from Sigma-Aldrich  and was dissolved in 0.9% saline.The following primary antibodies were used to visualize tau proteins: AT8 , PS396 , anti-Tau 3-repeat isoform RD3 , anti-Tau 4-repeat isoform RD4 , and TAU5 . To visualize NMDARs, antibodies against GluN2A  and GluN2B  were used. To visualize kinases, primary antibodies against cyclin-dependent kinase 5 (CDK5) , glycogen synthase kinase 3 beta , weighing 24-27 g, were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Science . Mice were maintained at a constant temperature (22–24°C) and humidity (40–60%) under a 12-h light/dark cycle with food and water ad libitum. All procedures were performed strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the School of Basic Medical Sciences of Fudan University. Every effort was made to minimize animal suffering or discomfort and to reduce the number of animals used.Experimental animals were randomly divided into 4 groups (10 per group): (1) control  injection of isotonic saline), (2) CRS , (3) CRS + an i.p. injection of 5 mg/kg MEM, and (4) CRS + an i.p. injection of 10 mg/kg MEM. Groups 1 and 2 received identical volume of vehicle . Groups 3 and 4 received the same volume of MEM containing two different doses (5 mg/kg or 10 mg/kg) 30 min before exposure to stress. The MEM dosages used in this study were based on the previous report . To evalThe chronic restraint stress (CRS) method was modified according to procedures described previously . RepeateBody weight was measured every 7 days. The protocol for the EPM test was described previously . The EPM2 (1.5 mM), Na3VO4 (1 mM), NaF (10 mM), sodium deoxycholate (0.5 mM), EDTA (1 mM), EGTA (0.5 mM), NP40 (1%), and protease and phosphatase inhibitor cocktails (Roche). For the detection of NMDAR subunits , the samples were homogenized in ice-cold lysis buffer containing SDS (2%), Tris-HCl , Na3VO4 (0.1 mM), NaF (1 mM), and protease and phosphatase inhibitor cocktails [μg) of each sample was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to nitrocellulose membranes . The membrane was blocked with 5% nonfat skim milk in TBST  for 1 hour at room temperature and then incubated with the primary antibody in blocking buffer overnight at 4°C. After washing with TBST for 3 times, infrared fluorescent dye- (IRDye-) labeled secondary antibodies  were applied for 1 h at room temperature. After 3 additional washes with TBST buffer, blots were scanned and analyzed by the Odyssey IR Imaging System . Normalization of the results was accomplished by parallel probing for GAPDH or β-actin in blots.Mice (4 per group) were anesthetized with sodium pentobarbital (40 mg/kg), which has been demonstrated not to influence tau phosphorylation over the time frame used in this study . After socktails . HomogenIn general, frozen samples (brain sections) were washed 3 times with phosphate-buffered saline (PBS) and blocked for 1 hour in PBS containing 0.2% Triton X-100 and 10% normal serum at 37°C. After blocking, sections were washed 3 times with PBS. Immunodetection was performed by incubation with an AT8-specific antibody at the manufacturer-suggested dilution (1 : 200) at 37°C for 1 h, followed by overnight incubation at 4°C. After washing 3 times in PBS, the sections were incubated for 1 h at 37°C with an anti-mouse Alexa Fluor 488-conjugated secondary antibody . For nuclear labeling, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C. The sections were washed again 3 times in PBS and then mounted on glass slides with ProLong® Gold Antifade Reagent . Images were captured using a Nikon Eclipse Ni-E/Ni-U microscope (Japan).p < 0.05.Statistical analysis of data was carried out by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. Two-way ANOVA followed by Bonferroni's multiple comparison test was used to analyze the change in body weight during the CRS procedure. All statistical analyses were performed using the GraphPad Prism version 6.0 software . The data are presented as the means ± standard error (SEM). Statistical significance was set at A previous study showed that MEM has anxiolytic effects . We explF = 69.93, p < 0.001) also had a significant effect on body weight. In contrast to the gradual increase in body weight in the control group, CRS mice showed a significant weight loss on day 8, partially recovered on day 16, and tended to lose weight again on day 24  = 0.09118, p = 0.7661; 10 mg/kg, F = 0.09682, p = 0.7593) had a significant effect on body weight at any of the three time points as compared to the CRS group (Results showed that the time effect on body weight was significant during the period of CRS. CRS treatment (n day 24 . NeitherRS group .p < 0.001) and 33.3%  less time in the open arms compared to the control group. In contrast, the closed-arm residence time in the CRS group was increased by 22.7% (p < 0.05) and 28.9% (p < 0.05) at the respective time points compared with that of the control mice. Moreover, CRS significantly reduced the number of open-arm entries on day 14 (p < 0.001) and day 27 (p < 0.01), by 37.2% and 32.9%, respectively, relative to the control group. In contrast, no significant difference was found between the control and CRS groups on the number of closed-arm entries. These results indicated increasing severity of anxiety behavior in mice subjected to CRS commencement.In the EPM test Figures , CRS micp < 0.05; 10 mg/kg, p < 0.05). Moreover, mice treated with MEM also showed a trend of CRS-induced abnormality reversal in the number of open- and closed-arm entries and in closed-arm residence time. The differences in these indexes were not significant not only between the CRS-only and CRS + MEM groups (P > 0.05) but also between the control and CRS + MEM groups  in the cerebral cortex induced by daily 30-min restraint stress for 18 consecutive days , immunobβ , which is the major tau phosphatase, accounting for approximately 70% of tau dephosphorylation in the human brain . PP2A acNMDAR subunits are encoded by three gene families, GluN1, GluN2 (A-D), and GluN3 (A/B) . The mosp < 0.001, Figures p < 0.001, Figures Western blot analysis showed that CRS significantly increased the expression of NMDARs containing GluN2A  treatment elevated the total tau protein level  and pY307-PP2Ac (p < 0.05). MEM (10 mg/kg) treatment could significantly further upregulate the levels of DM-PP2Ac (p < 0.01) and pY307-PP2Ac (p < 0.05). Neither CRS nor CRS with MEM treatment had a statistically significant impact on the expression levels of PP2Ac. These data suggest that the inactivation of PP2A is involved in the increase of P-tau and the exacerbating effect of MEM treatment on tau phosphorylation during the 28 days of CRS.In addition, we found the increased of DM-PP2Ac (p < 0.05) and HSP70 (p < 0.05) in 28-day CRS mice  significantly downregulated the expression of Pin1  but not GluN2B expression. However, treatment with 10 mg/kg MEM did not significantly affect the GluN2A level in CRS-only mice, whereas GluN2B expression was further enhanced (p < 0.05). Because the overactivation of GluN2B-containing NMDARs was reported to result in the accumulation of hyperphosphorylated tau in the hippocampus [Western blot analysis showed a significant increase in GluN2A  and PP2A demethylation at Leu309 relative to CRS-only mice. Therefore, treatment with MEM for mice subjected to 28-day CRS may activate kinases, GSK3β in particular, and inactivate phosphatase PP2A, contributing to MEM enhanced CRS-induced tau phosphorylation. In addition, treating two doses of MEM without CRS caused an increase in AT8-reactive P-tau levels in the frontal cortex of mice but without statistical significance.We found that MEM enhanced tau phosphorylation in the frontal cortex and other brain regions, including the hippocampus, following 28-day CRS. In this condition, MEM treatment upregulated the p-JNK, p-AKT, and p-ERK levels compared to those in CRS-only mice. MEM treatment also significantly suppressed the phosphorylation of GSK3Moreover, the effects of MEM treatment on the expression of chaperone proteins might be also involved in the increased tau phosphorylation. Thus, we found the downregulation of Pin1 and HSP70 expression to correlate with increased tau phosphorylation. Our results are consistent with reports showing that Pin1 deficiency promotes tau phosphorylation, pathological changes, and neurodegeneration –69 and tin vitro studies of the effects of glutamate on tau phosphorylation have yielded contradictory results. Some studies found NMDAR activation to induce tau phosphorylation [Excitotoxicity-induced neuronal death or injury through NMDAR channels is known to be involved in both acute neuronal damage and chronic neurodegenerative diseases . Howeverrylation , while orylation –77; NMDArylation .Recent studies also have shown that the overactivation of GluN2B-containing NMDARs leads to the accumulation of hyperphosphorylated tau , 46. AnoμM and 0.82 ± 0.12 μM, respectively [The analysis of the effects of MEM on recombinant rat NMDA receptors expressed in HEK 293 cells showed that MEM blocked glutamate-mediated currents in a concentration-dependent manner in GluN1a/GluN2A- and GluN1a/GluN2B-transfected cells with IC50 values (at −70 mV) of 0.93 ± 0.15 ectively . The IC5ectively ; moreoveectively . Hence, ectively , while Mectively , which iTo our knowledge, the relationship between NMDAR signaling and tau phosphorylation after CRS has not been examined. In the present study, we demonstrated that MEM significantly attenuated aberrant tau phosphorylation in the frontal cortex following CRS exposure for 16 consecutive days, while enhancing the tau phosphorylation abnormality after 28-day CRS. The CRS duration-dependent effects of MEM on tau phosphorylation were correlated with the changes in tau kinases and phosphatase. The underlying mechanism of the stimulatory effect of MEM on CRS-induced tau hyperphosphorylation might be related to the upregulation of GluN2B expression. The differential effects of MEM on pathological tau changes have important implications for its clinical applications, including the need for refining the dosage and regimen of any potential MEM-based therapy. Future studies are also required to determine whether MEM may also deregulate the expression of GluN2B and promote tau phosphorylation when used for the treatment of other neurological disorders.


Cystatin C has been proposed as a novel marker of renal function and predictor of cardiovascular risk. The aim of this study was to investigate the role of cystatin C level as a predictor of cardiovascular events in patients with coronary artery disease (CAD).Three hundred and five coronary artery patients were included in this study. Serum cystatin C levels, high‐sensitive C‐reactive protein (hs‐CRP), and oxidative stress were measured. Estimated glomerular filtration rate (eGFR) and the CAD severity score were calculated.r = 0.631, P < 0.0001) and was significantly elevated in the CAD severity score >50. Every 0.1 mg/l increase in cystatin C, 2 mg/l increase in hs‐CRP, 0.2 mmol/l decrease in high‐density lipoprotein cholesterol, 13.7 ml/min decrease in eGFR, and 1.51 μmol/l increase in homocysteine caused a 34, 12, 5, and 22% increase in the risk of having CAD, respectively.Cystatin C was correlated with the CAD severity score (Cystatin C could be a useful laboratory biochemical marker in predicting the severity of CAD. Cystatin C is associated with biochemical atherosclerosis markers such as CRP and homocysteine. Renal function has been identified as a risk factor for the onset and prognosis of coronary artery disease CAD, . ConsequGlomerular filtration rate (GFR) is an important indicator of renal function. However, in practice, since GFR is usually not directly measured in routine clinical practice, markers such as creatinine are used to estimate GFR. Estimating equations such as the Cochroft‐Gault equation In recent years, cystatin C has been proposed as a more reliable marker of renal function than serum creatinine, in particular for the detection of small reductions in GFR It is more sensitive and specific for the estimation of GFR and less influenced by age, gender, race, muscle mass, and medication as compared to serum creatinine However, in the recent years, cystatin C has emerged as a potential marker for cardiovascular risk. In fact, previous studies have shown that a high level of cystatin C has been related to suspected or confirmed acute coronary syndrome. The results of studies investigating the value of cystatin C in suspected stable CAD patients are controversial Therefore, the clinical utility of cystatin C as a new predictive marker of CAD merits further investigations. The aim of this study was to investigate the role of cystatin C level as a predictor of cardiovascular events and the association between this protein, oxidative stress markers, and other biochemical risk factors for atherosclerosis in patients with CAD.We studied 305 consecutive coronary patients (234 men and 71 women) for coronary angiography recruited from Department of Cardiology, CHU–Fattouma Bourguiba, Monastir, Tunisia. The mean age of this group is 61.15 ± 10.64 years.All patients admitted to our study had a history of stable angina defined by the presence of chest pain or unstable angina and were found to have CAD at angiography. Two experienced cardiologists, unaware of the patients clinical history and biochemical results, visually assessed all angiograms to assess the extent of the CAD.Patients with severe renal dysfunction, life‐threatening arrhythmias, acute and chronic liver disease, infectious and inflammatory disease, and symptomatic heart failure were excluded. The detailed histories of patients included demographics data, and cardiovascular risk factors were recorded. All participants were interviewed, and data on dyslipidemia, diabetes mellitus, hypertension, and smoking habits were recorded. For coronary risk factors, the following definitions were used: individuals were defined as hypertensive if their blood pressure was >140/90 mmHg or if they were receiving any antihypertensive treatment; individuals with a history of diabetes mellitus or those receiving any antidiabetic medication were considered to be diabetic; individuals were deemed dyslipidemic if their total cholesterol (TC) concentration was ≥5.68 mmol/l, or their triglyceride (TG) concentration was ≥2.28 mmol/l, or they were receiving lipid‐lowering drugs. Smoking history was coded as never and current smoker.Levels of TC, TG, high‐density lipoprotein cholesterol (HDLc), and serum creatinine were measured using standardized enzymatic methods . GFR was estimated by the MDRD formula. Serum high‐sensitive C‐reactive protein (hs‐CRP), fibrinogen, and cystatin C were quantified according to the instructions of the manufacturer using particle‐enhanced immunonephelometric assay .Superoxide dismutase (SOD), glutathione peroxidase (GR), glutathione reductase (GR), glutathione reductase (GPX) activities, and total antioxidant status (TAS) concentration were checked using commercials tests manufactured by Randox Laboratories  in a Daytona analyser. The catalase (CAT) activity was determined spectrophotometrically as the rate of substrate decomposition per unit time All patients underwent coronary angiography, performed using standard Judkins techniques before the start of the study. Blood samples were collected before coronary artery angiography from the antecubital vein of the patients who were resting in the supine position. The CAD severity was determined by two experienced independent observers, blinded to both the arterial waveform and clinical patient data, according to the Gensini scoring system All analyses were performed using SPSS 15.0 . Categorical variables of cystatin C groups were compared by the Chi‐square test. Continuous variables were expressed as mean ± SD and compared by one‐way analysis of covariance.Correlations between continuous variables were assessed using Spearman's correlation analysis. A linear regression analysis was applied for CAD severity score, oxidative markers levels, homocysteine, and hs‐CRP.P‐value <0.05 was considered as statistically significant.In order to analyze the separate effect of CAD severity score on serum cystatin C levels and the possible interaction between the severity score and renal impairment, an analysis of covariance (ANCOVA) was used. CAD severity score and estimated GFR (eGFR) were entered as categorical independent variables, and log hs‐CRP and homocystein were entered as covariates in this analysis. A We studied 305 patients grouped according to the CAD severity score. Cystatin C concentration was significantly elevated in the patients with a CAD severity score >50% compared to the normal coronary artery patients (2.9 ± 2.2 mg/l vs. 0.7 ± 0.2 mg/l).P < 0.01). Creatinine and cystatin C were positively correlated to the increase in the CAD severity score using ANCOVA analysis (P < 0.01), as was the decrease in eGFR (<0.01).The variables measured in relation with the CAD severity score are shown in Table The concentration of lipid peroxides measured as malondialdehyde is significantly correlated to the increase in the CAD severity score. On the other hand, our results showed a decrease in the activities of antioxidant enzyme in the patients with CAD severity score >50 Table .P < 0.001). Overall, subjects with higher serum cystatin C were older. Also, body mass index (BMI), hs‐CRP, and fibrinogen were higher at higher serum cystatin C quartile. Similarly, the concentration of lipid peroxides measured as malondialdehyde, homocytein, and CAD severity score were higher at higher serum cystatin C quartile. But HDLc and antioxidant markers  decreased progressively with higher serum cystatin C quartile. Cigarette smoking, diabetes, HTA , and obesity are not associated with serum cystatin C.Subject characteristics and clinical laboratory variables of cystatin C quartile groups are presented in Table Independent predictors of incident CAD, determined by logistic regression, were as follows: cystatin C, hs‐CRP, eGFR, HDLc after adjustments for age, creatinine, eGFR, homocysteine, gender, presence of hypertension, and diabetes Table . Every 0A backward stepwise multiple regression analysis was performed in the combined population of normal CAD (score 0) subjects to severe CAD patients (score >50) with cystatin C as a dependent variable Table . In multr = 0.631, r = 0.023, r = 0.37, r = 0.136, and r = 0.039; P < 0.05, respectively).We found that the increased level of cystatin C persisted where either hs‐CRP or homocystein levels were added as covariates in the multivariate model. Linear regression analysis showed that cystatin C, fibrinogen, hs‐CRP, TAS, and homocysteine levels were the most significant predictors of CAD severity , homocyteine , and TAS . It was negatively correlated with eGFR , HDLc , and lipid peroxides as malondialdehyde .Our results showed a correlation between cystatin C and the levels of oxidative parameters. We observed an increase in the concentration of lipid peroxides as malondialdehyde and a decreased activity of antioxidant serum proteins associated with high concentration of cystatin C. However, cystatin C was positively correlated with hs‐CRP (Cystatin C is a protein that belongs to a group of cysteine proteinase inhibitors, one of the four types of proteinases in mammalian cells. These types of proteins are encoded by the so‐called housekeeping genes that regulate the factors necessary for global cell function, and all nucleated cells produce them at a stable production rate The study of Koc et al. In this study, cystatin C was correlated with CAD severity score, BMI, HDLc, creatinine, homocyteine, lipid peroxides as malondialdehyde, and GPX. Thus, cystatin C appears to be a marker of cardiovascular risk, and high correlations of circulating cystatin C have been shown to be consistently and strongly associated with the occurrence and the severity of CVD. Our results agree with the previous idea that demonstrates the positive correlation between cystatin C and the increase in the CAD severity score.Moreover, cystatin C seems to offer more complete prognostic information than other markers of renal disease. Above all, cystatin C appears to be useful for identifying individuals at a higher risk for cardiovascular events among patients classified as belonging to a low category according to both creatinine and eGFR value For secondary cardiovascular events, cystatin C is one of the strongest risk predictors. The contribution of cystatin C in a multivariate model remains significant, which indicates its added value to established risk factors. Because of the association of renal dysfunction with CVD, it is unclear whether cystatin C is a direct marker of CVD or merely a marker for renal failure, which has implications for therapeutic intervention Recently, Zethelius et al. This suggested that the predictive value of cystatin C is not dependent, although, in another study, cystatin C, independently, predicted smooth CAD lesions but not complex lesions or CAD severity Some studies found no relationship between cystatin C levels and CRP, and the association between cystatin C and cardiovascular mortality was independent of the CRP. Thus, our result is in agreement with some studies Among all predictors evaluated, cystatin C was the strongest correlate of fibrinogen and the second strongest correlate of CRP. Although it has been suggested that high cystatin C concentrations are directly related to both inflammation and atherosclerosis Our results are in agreement with the previous idea that demonstrates the correlation between cystatin C and the levels of oxidative parameters, although various studies have demonstrated an association between oxidative stress markers and CKD and CAD events Previous studies have demonstrated that high concentrations of cystatin C have been also associated with a hypermethabolic status There is evidence that both elastolytic cysteine proteases and their inhibitors, an important one being cystatin C, are involved in the pathogenesis of atherosclerosis Increased cystatin C is emerging as a marker of both CKD and cardiovascular risk. In patients with CAD, an increase in the plasma concentration of cystatin C indicates severity of coronary lesions. As the presence of CAD is one of the major determinants of the prognosis in patients with reduced renal function, the concentration of serum cystatin C is expected to be useful in detecting patients at risk of CAD.The results of our study indicate that serum cystatin C is as relevant as CRP. There is a considerable association between cystatin C and biochemical cardiovascular risk factors such as homocysteine, low HDL, oxidative stress markers, and CRP. With these results, we suggested that cystatin C could be used as a marker in clinical practice to predict the presence of severity of atherosclerosis in suspected CAD patients.


HssAChE), revealing that the metabolites can also bind in the PAS in the same way as AFB1. Here, we investigated the binding modes of these compounds on the catalytic anionic site (CAS) of HssAChE to compare the affinity of the metabolites for both binding sites as well as verify which is the preferential one. Our results corroborated with experimental studies pointing to AFB1 and its metabolites as mixed-type inhibitors, and pointed to the residues relevant for the stabilization of these compounds on the CAS of HssAChE.The most common type of aflatoxin (AFT) found in nature is aflatoxin B1 (AFB1). This micotoxin is extremely hepatotoxic and carcinogenic to mammals, with acute and chronic effects. It is believed that this could be related to the capacity of AFB1 and its metabolites in inhibiting the enzyme acetylcholinesterase (AChE). In a previous work, we performed an inedited theoretical investigation on the binding modes of these molecules on the peripheral anionic site (PAS) of human AChE ( Aspergillus (A. flavus and A. parasiticus) when they grow over grains in general, and dairy products, representing a serious problem for the storage and commercialization of agricultural commodities worldwide [In the beginning of the 1960s, contaminations of animal food with aflatoxins (AFT) were responsible for the death of turkeys, poults and pheasants in Great Britain, as well as adverse effects on ducks in South Africa, and trouts in the United States [orldwide .7-guaninine adduct, and triggering cancer and other damages to the liver [AFT B1 (AFB1) is the most common type of AFT and figures amongst the most carcinogenic chemicals found in nature . It can he liver ,6,7.Literature reports that AFB1 is capable of inhibiting the enzyme acetylcholinesterase (AChE) , and actHssAChE) were theoretically investigated by our research group in a former work [HssAChE, and elucidate which residues are responsible for their stabilization. In addition, a comparison between the results obtained in both sites (PAS and CAS) was done to indicate the preferential one. We believe that this work will contribute to obtain more information about the preferential type of inhibition of AFB1, and its metabolites on HssAChE, while helping to collect valuable information that will guide the design of new antidotes against AFT intoxication.It is known that AFB1, and probably also its metabolites, can bind to the peripheral anionic site (PAS) or the catalytic anionic site (CAS) of AChE . The potmer work . Here, wHssAChE is located close to residues Asp74, Trp86, Tyr337 and Tyr341, while the PAS is close to residues Tyr72, Trp286 and Arg296. Most of CAS presents a low electrostatic potential and a hydrophilic solvent accessible surface, as shown in As shown in The negative values of interacting energy observed for AFB1 and its metabolites  suggest HssAChE: on the PAS, as shown before [It is also important to notice that, for all metabolites, the binding energies on the CAS were lower than the values reported before for the PAS . This sun before , and on n before ,11.HssAChE/AFB1, are shown in HssAChE/metabolite complexes are shown in 5 kcal·mol−1.The poses selected in docking studies were further submitted to MD simulations to check if they present stabilization on the chosen site during the simulation time. The resulting plots of energy variation through the simulated time, for the complex Temporal RMSD plots shown in HssAChE presented higher flexibility when complexed with AFBO and AFQ1, near Asp74 and Trp86, respectively. It is important to mention that these are the same residues observed in interactions with those metabolites in the docking studies.RMSF plots shown in Plots of H-bond interactions observed for the complexes during the simulated time are shown in HssAChE/AFP1, while Superposition of MD frames during the MD simulations corroborate the stability of residues and ligands for all systems. The MMPBSA results shown in HssAChE on this site.From the prediction of the solvent accessible surface area, we can infer that the CAS presents both hydrophilic and hydrophobic regions. It is expected that AFB1 and its metabolites would bind close to these hydrophobic parts, as observed in the molecular docking results. The energies observed were lower than in the PAS for all metabolites, suggesting that they may preferentially bind to the CAS and come closer to the active site. One example is the interaction of AFL with His447, which is part of the catalytic triad. Asp74, Trp86, Ser125 and Tyr337 were the residues contributing to stabilization in most systems. Regarding AFB1, our results suggest that it may present two binding modes: on PAS, as a non-competitive inhibitor, and on CAS, coming close to active site, and acting as a competitive inhibitor. According to the energy and RMSD plots obtained from the MD studies, it is possible to observe that all complexes tend to stabilization. The metabolites AFP1 and AFQ1 presented similar results and the highest affinities for the CAS, according to the docking and MD studies. As in docking studies, H-bonds with residues Asp74 and Tyr337 mainly contributed for the stabilization of the systems. MM-PBSA calculations presented very similar results for all metabolites, although AFB2a and AFB1 were slightly better than the others with no unfavorable interaction with any residue. Nevertheless, AFQ1 and AFP1 also presented good results in MM-PBSA studies. Van Der Waals/electrostatic and apolar contributions to the energy were negative and favored the systems. On the other hand, the polar solvation energy was positive and contributed to disfavor the complexes. This is expected, since AFB1 and its metabolites are highly hydrophobic compounds. In summary, our results suggest that the CAS is the preferential binding site for all the metabolites, being AFP1 and AFQ1 the best inhibitors of HssAChE/AFB1 used in this work was obtained and validated as described before [®, Wavefunction, Irvine, CA, USA, 1999) [The model of the complex d before , throughd before  and 4EY4d before  and the A, 1999)  and RM1 A, 1999) . Figure HssAChE were performed with the software Molegro Virtual Docker (MVD)® [Docking studies for the ligands on CAS of r (MVD)®  (Versionr (MVD)® , with a r (MVD)® .HssAChE/ligand were simulated through GROMACS 5.1.4 [http://plasma-gate.weizmann.ac.il/Grace/)  and PyMOL [Poses from the docking studies were prepared for the forcefield OPLS/AA  from theCS 5.1.4 ,20 accorCS 5.1.4 ,24, and CS 5.1.4  (Versionnd PyMOL  (VersionAs before , the MM-


This work employed genomic substitutions blocking or mimicking phosphorylation at three proposed phosphorylation sites on Arp2 to demonstrate that they do not regulate Arp2/3 complex activity in fission yeast. Drosophila cells. Replacing both T233 and T234 with aspartic acid was lethal, suggesting that phosphorylation might be inhibitory. Nevertheless, blocking phosphorylation at these sites had the same effect as mimicking it: slowing assembly of endocytic actin patches. Mass spectrometry revealed phosphorylation at a fourth conserved Arp2 residue, Y218, but both blocking and mimicking phosphorylation of Y218 only slowed actin patch assembly slightly. Therefore, phosphorylation of Y198, T233, T234, and Y218 is not required for the activity of fission yeast Arp2/3 complex.LeClaire et al presented evidence that phosphorylation of three sites on the Arp2 subunit activates the Arp2/3 complex to nucleate actin filaments. We mutated the homologous residues of Arp2  in the fission yeast genome to amino acids that preclude or mimic phosphorylation. Arp2/3 complex is essential for the viability of fission yeast, yet strains unable to phosphorylate these sites grew normally. Y198F/T233A/T234A Arp2 was only nonfunctional if GFP-tagged, as observed by LeClaire et al in  Assembly of branched actin filament networks drives cellular processes including cell motility and clathrin-mediated endocytosis . The sevAcanthamoeba Arp2/3 complex lost its ability to nucleate actin filaments when treated with serine/threonine and tyrosine phosphatases. Furthermore, antibodies to phosphothreonine and phosphotyrosine interacted with the Arp2 and Arp3 subunits of the Arp2/3 complex from Acanthamoeba, Bos taurus, and humans. Mass spectrometry was used to identify phosphorylation of the highly conserved T237 and T238 residues of amoeba Arp2. The location of Y202 near these threonines suggested that it might also be phosphorylated. Drosophila Arp2/3 complex. Depletion of Arp2 compromised the formation of lamellipodia in Drosophila S2 cells. This defect was rescued by the expression of wild-type Arp2-GFP, T237A/T238A Arp2-GFP, or Y202A Arp2-GFP, but not by Y202A/T237A/T238A Arp2-GFP, indicating that phosphorylation of either the two threonines or the tyrosine is essential for Arp2/3 complex activity. A Drosophila kinase that phosphorylates these residues has been identified: In 2015, Dictyostelium Arp2 slowed development but not pseudopod extension in a chemotaxis assay  and restored the ability of phosphatase-treated Acanthamoeba Arp2/3 complex to nucleate actin filaments in vitro , at which point a further 70 g/l YE5S powder was added and cells were allowed to resume growing. Cultures were pelleted and frozen when the OD595 of a 1:10 dilution was 0.55–0.65 (5.5–6.5 × 107 cells/ml). The cells were thawed, lysed using a microfluidizer, and centrifuged, and the Arp2/3 complex was purified from the soluble fraction by chromatography on a GST-VCA affinity column , a MonoQ 5/50 ion exchange column , and a HiLoad Superdex 200 16/60 gel purification column  (−1·cm−1).Fission yeast was grown in 4–8 liters of YE5S culture medium until the ODciences) . Roche c4HCO3 buffer. Digested samples were analyzed using liquid chromatography–tandem mass spectrometry on a Waters/Micromass AB QSTAR Elite mass spectrometer (Purity was confirmed by SDS–PAGE and staining the seven bands corresponding to the Arp2/3 complex subunits with Coomassie blue. The Arp2 (44 kD) band was cut from the gel and digested with 6.67 μg/ml trypsin for 18 h at 37°C in 10 mM NHtrometer  by the Ytrometer  through trometer . Mass sptrometer  to ensurarp2 of diploid S. pombe strains because arp2 is an essential gene. Haploid S. pombe strains with complementary adenine auxotrophy mutations ade6-M210 and ade6-M216  for 36 h. The cells were harvested at OD595 0.4–0.7 (4–7 × 106 cells/ml), washed twice with EMM5S media, and mounted on EMM5S 2% agarose pads. Images were acquired at room temperature with an Olympus IX-71 microscope with a 100×/NA 1.4 Plan Apo lens (Olympus) and an Andor CSU-X1 spinning disk confocal system with an iXON-EMCCD camera (Andor Technology). Fluorescence was excited using a Coherent OBIS 488-nm LS 20-mW laser with adjustable power and an internal power meter. Time-lapse movies of endocytic actin patches were acquired with Andor iQ2 software (Andor Technology); other images were acquired with the μManager 1.4 plugin (4 plugin  for Imag4 plugin .21 Z-sections with 0.6 μm spacing were taken of eight yeast strains: one nonfluorescent strain (FY528) and seven endogenously expressing one of the following GFP-tagged proteins: Myo2p, Ain1p, Acp2p, ARPC5, Arp2p, Arp3p, and Fim1p . Sum projections of fluorescence image Z-stacks were corrected for camera noise and uneven illumination and segmented to isolate individual S. pombe cells. The total fluorescence intensity per cell in each strain was measured and plotted against the number of fluorescent molecules per cell, which was previously obtained by quantitative immunoblotting   plugin fWe used the resulting calibration curve  and the We took 3–4 time-lapse movies with 1-s intervals for 60 s of each haploid yeast strain expressing endogenous Fim1-GFP. Each image consisted of six Z-sections separated by 0.6 μm collected with 5 mW laser power . An original MATLAB (MathWorks) script corrected the images for uneven illumination, camera noise, and photobleaching . EndocytAll accepted patch tracks within each time-lapse movie were aligned and their total fluorescence intensities (integrated density) were averaged using temporal super-resolution realignment . The calhttps://github.com/tdpollardlab/findLinearRegions) to fit straight lines to the most linear 3.7 s regions of the assembly and disassembly phases . Shaded regions indicating SD were generated using the shadedErrorBar MATLAB script .To measure the rate at which actin patches assembled, we created an original MATLAB script (y phases . The meay phases . Patch ahttps://github.com/tdpollardlab/PhotoBleachingCorrector). Time-lapse movies were first corrected for camera noise and uneven illumination. Poorly illuminated areas were then identified and cropped using the best-fitting two-dimensional Gaussian function to the uneven illumination correction image. To identify intracellular regions, each of the first 10 frames of each time-lapse movie was thresholded by brightness. Pixels brighter than the threshold in eight of the 10 frames were assumed to be intracellular  was used to calculate the growth rate for each strain (Cells were grown in YE5S with shaking for 36 h in the log phase at 25°C. Cultures were diluted to ODand 26 h . The logh strain .595 of 0.4–0.6 (35–65 × 105 cells/ml) and diluted to OD595 0.1. After 1:10 serial dilutions, 10 μl samples were plated on YE5S and incubated for 24–48 h at 32°C or 36°C (Cells were grown in YE5S for 36 h in the log phase with shaking. Cultures were harvested at an OD or 36°C .http://www.ebi.ac.uk/biostudies) under the accession number S-BSST205.Source data for actin patch time-lapse movies (raw data and photobleaching-corrected sum projections), along with image stacks used to generate the calibration curve, are available in the BioStudies database (


Hydrogen sulfide is a toxic and corrosive gas, produced by the activity of sulfate-reducing microorganisms (SRM). Owing to the environmental, economic and human-health consequences of sulfide, there is interest in developing specific inhibitors of SRM. Recent studies have identified perchlorate as a promising emerging inhibitor. The aim of this work is to quantitatively dissect the inhibitory dynamics of perchlorate. Sulfidogenic mixed continuous-flow systems were treated with perchlorate. SRM number, sulfide production and community structure were monitored pre-, during and post-treatment. The data generated was compared to a simple mathematical model, where SRM growth slows as a result of inhibition. The experimental data supports the interpretation that perchlorate largely acts to suppress SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Surface-attachment was identified as an important parameter preventing SRM wash-out and thus governing inhibitory dynamics. Our study confirmed the lesser depletion of surface-attached SRM as compared to planktonic SRM during perchlorate treatment. Indirect effects of perchlorate  were also assayed by amending reactors with DPRB. Indeed, low concentrations of perchlorate coupled with DRPB amendment can drive sulfide concentrations to zero. Further, inhibition in a complex community was compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Finally, we quantified susceptibility to perchlorate across SRM in various culture conditions, showing that prediction of complex behavior in continuous systems from batch results is possible. This study thus provides an overview of the sensitivity of sulfidogenic communities to perchlorate, as well as mechanisms underlying these patterns. Microbial sulfate reduction (SR) by sulfate-reducing microorganisms (SRM) is one of the primary pathways of anaerobic terminal organic matter mineralization, and as such is a determinant of carbon and sulfur cycling in many environments . SR resuDesulfovibrio desulfuricans strain Hildenborough, and identified perchlorate, among other inhibitors  pothibitors . Due to hibitors .Much of the current insight on specific inhibition of SR has been gained via pure culture studies . AlthougWe characterize the effects of perchlorate in terms of the rate and level of inhibition of SR, as well as the rate and level of post-treatment rebound of SR. Empirical data is compared to a simple model, elucidating two factors key to inhibitory dynamics, namely: (1) the dose-response/inhibition curve of SRM against perchlorate and (2) surface-attachment of microbial cells. Surface-attachment, as well as the indirect effects of perchlorate (mediated by DPRB) are explored further, via direct manipulation. Finally, inhibition in a complex community is compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Together, the experiments performed provide a comprehensive illustration of the inhibitory dynamics of perchlorate, and factors that affect it, both in pure culture and in a community setting.2x6H2O, 3.04 g/100 mL CaCl2x2H2O; 50x: 4 g/100 mL NaCl, 5 g/100 mL NH4Cl, 0.5 g/100 mL KCl, 0.5 g/100 mL KH2PO4, 1 g/100 mL MgSO4x7H2O, 0.1 g/100 mL CaCl2x2H2O). Yeast extract (2 g/L) or sodium lactate (60 mM) served as the electron donor and carbon source and 20 mM sodium sulfate served as the electron acceptor. Media was flushed with N2 to render it anaerobic, and was pumped in from a reservoir bottle  at a set flow rate of approximately 2.6 ml hr−1, resulting in a residence time of 48 h. This flow rate was chosen to accommodate slow-growing microorganisms and maintain diversity within the community. Chemostats were kept on a stir plate at 30°C and effluent drained passively such that a constant volume of V = 125 ml was maintained. Replicate chemostats were sterilized, rendered anaerobic (by degassing with N2) and inoculated at 10% with a marine sulfidogenic community collected from San Francisco Bay sediment and pre-enriched on yeast extract and sulfate. Post inoculation, chemostats were allowed to reach steady-state (defined as stable sulfide production over approximately 7 days) prior to treatment with perchlorate. Control chemostats were left untreated. Once inhibition stabilized, treatment was ceased and rebound of sulfide was monitored  . Inflow onitored . Sodium onitored  and readArcobacter sp. CAB, Sedimenticola selenatireduces CUZ, Marinobacter vinifirmus UCB, and Denitromonas halophilus SFB-3  as ferrous iron reacts with sulfide and renders SRM colonies black. Single colonies were picked and cultured in APM. The purity of the isolate, Desulfovibrio sp. BMSR (IMG ID 2687453694), was confirmed via 16s rRNA sequencing, using primers 27F and 1525R  and the resulting pellet was immediately stored at −80°C. DNA was extracted using a DNeasy Powersoil HTP 96 kit (Qiagen), following manufacturer’s instructions. For surface-attached communities, 15–20 glass beads were collected and DNA was extracted immediately using a DNeasy PowerBiofilm Kit (Qiagen). DNA concentrations were quantified via a Quant-iT HS Kit , on an Infinite M1000 Pro microplate reader (Tecan), following manufacturer’s instructions.P liquid handling robot, and pooled. Sequencing was performed at the UC Davis Genome Center  on a Miseq (Illumina Miseq Reagent Kit V2). Demultiplexed samples were analyzed according to the Mothur Miseq SOP  . Librariiseq SOP . Brieflyiseq SOP  and remaPrimer 7  was usedPrimer 7 . SignifidsrA gene (a specific marker of dissimilatory SRM). The dsrA gene was PCR amplified with primers DSR1-F+ and DSR-R . Perchlorate leaves the chemostat proportional to its concentration in the chemostat .Where IC50 denotes the concentration inhibiting 50% of SRM, and HillSlope describes the steepness of the slope of the dose-response/inhibition curve . Thus, y is a fraction representing the effect of a specific concentration of P. As the model assumes that the only effect of perchlorate is a change in the growth rate, y multiplies by μp ) and surface-attached SRM (NA), with each population being subject to its own carrying capacity, Kp and KA respectively:Finally, growth was modeled additively as a combination of planktonic SRM (Nmax × y.and from Equation (3), μ = μtN, was converted to sulfide concentration, [sulfide], for easier comparison to experimental data, using the regression of measured sulfide and measured tN.The initial model output, Desulfovibrio sp. BMSR cultures were incubated in APM with 20 mM sulfate and with one of: (i) no lactate and no perchlorate; (ii) no lactate and 50 mM perchlorate; or (iii) 60 mM lactate and 50 mM perchlorate. Samples were taken at 24 h and mixed with LMT agarose (1%), SYTO 9  and propidium iodide . Cells were imaged using a Zeiss AxioImager M2 at the Berkeley Imaging Facility. ImageJ was used to merge images from different filters and cells were counted manually. Statistical analysis was performed in Graphpad Prism.Our current understanding of perchlorate inhibition implies a non-biocidal effect of perchlorate on SRM. To confirm this, triplicate P liquid handling robot. Data generated was normalized to a control and used to construct dose-response curves in GraphPad Prism 7  using the “Dose-Response – Inhibition, log(inhibitor) vs. normalized response – variable slope” function. Various donors for growth were used: sodium lactate (40 mM), sodium pyruvate (20 mM), sodium fumarate (20 mM), sodium succinate (20 mM), glucose (30 mM) and yeast extract (2 g/L). Dose-response/inhibition curves were also generated from growth-curve data. Briefly, triplicate anaerobic tubes were inoculated under various perchlorate concentrations and either/and OD600 (measured on a Spectronic 20D+) or sulfide (measured via cline assay) were monitored. Data generated was used to construct growth curves and calculate growth rates. Growth rates were normalized to the control growth rate and used to construct dose-response/inhibition curves, as described above. IC50 and Hillslope values determined were used in the model described above.Perchlorate inhibition of SR was quantified via dose-response/inhibition curves, generated as previously described . BrieflydsrA gene as well as by the percentage of 16S rRNA amplicons assigned to known SRM  to the difference between treated and control communities during treatment. Several non-SR organisms also decrease significantly with treatment: Spirochaeta, Acetoanaerobium, and Petrimonas . Conversely, during treatment, there is a relative enrichment of other, likely fermentative organisms, including Fusobacterium, the unclassified Bacteroidetes, and other unclassified Bacteria. Interestingly, Desulfocurvus, the only other SRM present in any significant proportion, as well as Dethiosulfovibrio were unaffected by perchlorate treatment , with treated communities undergoing the same temporal effect as control communities, and rebounding to the same end-point. Control and treated communities group together before and after the treatment phase (at > 70% similarity), and separate, at 35% dissimilarity, during treatment . SIMPER reatment .p = 0.0012, t-test for 20 mM 660–852 h vs. all control at all time-points), but then rebounding to control levels within 250 h . qPCR of dsrA shows a trend of slight inhibition of SRM throughout treatment, although this trend is non-significant . In subsequent runs, there was no significant effect of 20 mM perchlorate on sulfide production.A subsequent chemostat experiment was performed using 20, 50, and 80 mM perchlorate . At 80 mD. alaskensis G20 cells are uninhibited by >100 mM perchlorate . The proportion of dead:live cells was compared to that in control cultures with no lactate and no perchlorate, via live-dead staining. All cultures exhibited a similar proportion of dead cells post-incubation .A theoretical model of inhibition was constructed based on the assumption that perchlorate affects the growth rate of SRM. In support of this, it has been shown that SRM can overcome perchlorate inhibition via de-repression of the SR pathway , and additionally that pyruvate-fermenting chlorate ,b. To comax = 0.13h−1) for the model was determined from batch growth curves of community sulfide production. The dose-response/inhibition curve of perchlorate against SRM was also determined empirically. The final function for quantifying the perchlorate effect (y) on μ is based on growth rates  to give the actual growth rate, μ. In the final model, the population of SRM then follows Equations 4a – 4c.From here, tN was set to 40% based on the average % SRM in control chemostats over time (∼ steady state SRM population). Several surface-attached fractions were simulated before settling onAN = 0.5%. The model output was converted from % SRM to [sulfide], according to the regression of log(sulfide) against log(%reads) (R2 = 0.79):The model was calibrated using data from the initial chemostat run. max is critical in establishing rebound: if it is slower than the residence time, SRM do not rebound  As  rebound . (3) Fin rebound : in the p = 0.3). However, rebound in the biofilm culture chemostats was not as obvious as previously observed , increasing surface area for attachment, and treated with 50 mM perchlorate . Normaliobserved .Desulfovibrio species as contributing 11% to the difference between control and treated planktonic communities, and 8% for biofilm communities. A nMDS plot shows control samples and pre-treatment samples group together, and the treated samples clustered independently. The same trend was true for biofilm communities. However, planktonic communities showed a greater separation than biofilm communities upon treatment (36% vs. 32% dissimilarity between control/pre-treatment and treatment groups) . Planktonic SRM dropped to 3.9 ± 1.4% of the community while surface-attached SRM dropped to 12 ± 6.5% . Interestingly, Desulfobacterium and Desulforhopalus were enriched in the surface attached SRM fraction as compared to the planktonic  . Examini2 ± 6.5% . The proanktonic . DesulfoWhile our experimental data and model confirm the direct inhibitory effect of perchlorate on SRM, particularly at concentrations greater than or equal to media sulfate concentrations (20 mM), perchlorate also has indirect effects on sulfate-reduction. Dissimilatory perchlorate reducing bacteria (DPRB) can reduce perchlorate coupled to the oxidation of organic acids, an energetically favorable metabolism compared to sulfate-reduction. Thus, DPRB can outcompete SRM for organic donors. Further, DPRB can metabolize sulfide, enzymatically oxidizing it to elemental insoluble sulfur . PreviouShewanella. Nitrate treatment was suspended and perchlorate (20 mM) or perchlorate and a large inoculum (30%) of DPRB was added (phase 4). This eliminated sulfide in the perchlorate and DRPB-amended replicates, briefly. However, subsequent to and likely as a result of issues with flow, 20 mM and 20 mM + DPRB treatments became indistinguishable again in two out of the three replicates, starting at hour 1700. One replicate (replicate 3) did maintain complete sulfide inhibition for the duration of perchlorate treatment, and concurrent perchlorate reduction was seen (Amendment with a mix of DPRB (1:10 inoculum) was initially tested in batch communities treated with 20 mM perchlorate. This perchlorate concentration was chosen as it is minimally directly inhibitory to sulfidogenesis in our system, and thus allows us to maximally detect indirect inhibitory effects. Treatment with 20 mM perchlorate led to complete sulfide removal coupled to the reduction of 7.7 ± 2.6 mM perchlorate. Indirect effects of perchlorate were subsequently tested in chemostats . The conwas seen . InteresSedimenticola in group I, as well as a greater enrichment of Marinobacter , was isolated and tested in an additional chemostat run with 20 mM or 50 mM perchlorate . However, at 50 mM treatment, sulfide concentrations demonstrated a pronounced decline in two out of three replicates  and 20 mM sulfate. Interestingly, perchlorate sensitivity was donor dependent, particularly in the case of pyruvate   had the same dose-response curve against perchlorate as the pure-culture G20  . Furtherture G20 . Finally the SRM . Thus, sFusobacterium genus are known fermenters, producing butyrate and acetate from various sugars, amino acids and small peptides and the enrichment of Fusobacterium under perchlorate treatment has been previously characterized   . At low sat gene  as perchlorate concentrations followed the uniformly mixed model, which would not be expected in a non-uniform, diffusion-limited system . It is asat gene , which ssat gene . As notesat gene , possiblShewanella, Denitrovibrio, Petrimonas) are capable of nitrate reduction and nitrite accumulation. Nitrite is a known potent inhibitor of SRM and a current mechanism for reversing sulfide production in commercial oil reservoirs, which has been applied with varying degrees of success in various systems , DPRM can eliminate sulfide completely. However, in our chemostats, the establishment of DPRM and extent of inhibition was dependent on the community context and the existing sulfide concentrations. Perchlorate treatment in the field would enrich for native organisms capable of perchlorate reduction, likely more competitive in their given environment than exogenously introduced DPRM. Previous work in our lab has successfully enriched for perchlorate reduction in marine systems, concomitant with total suppression of sulfide . These w systems . Our res systems .Desulfovibrio sp. BMSR grown on pyruvate-sulfate is significantly more resistant than when grown on lactate-sulfate. The energetics and electron flow of pyruvate-sulfate grown D. alaskensis G20 and lactate-sulfate grown G20 are known to differ: electrons from lactate can flow through H2 as an electron carrier, whereas electrons from pyruvate can flow directly and rapidly to SR through the menaquinone pool  into cells is partially donor dependent. How these various factors translate to donor-dependent differences in sensitivity remains to be addressed. Further, sensitivity of BMSR did not depend on whether it was grown in pure culture or in co-culture with other fermenters. As such, at least in this system, inhibition of SRM is independent of other community members. The Desulfovibrio sp. BMSR chemostat run with 20 and 50 mM perchlorate mimics community results, and can be modeled by adjusting μ, the interconversion factor between tN and [sulfide] and the dose-response/inhibition curve of perchlorate. Hence, remarkably, whether in a complex community or in a pure culture, the batch determination of a few parameters can be used to predict inhibition. Finally, this pure-culture chemostat study uncovered the relative ease with which pure cultures of SRM adapt to perchlorate. Adaptation of SRM in the community was not seen over the course of this study, although it was specifically assayed for after each chemostat run. Adaptation has also not been seen in other long-term community studies of perchlorate inhibition in this lab, making our observation of adaption in pure-cultures all the more interesting. Future work will aim to describe mechanisms of adaptation to perchlorate, both in pure and mixed culture.Inhibition may also be modulated by donor used for growth. In our system, one pool . Currentsor, Rex . It is pnclusive 1. It is In summary, the addition of perchlorate leads to slower growth of SRM populations, which renders them susceptible to washout. This effect can be partially mitigated by increased surface-attachment. This principal governs inhibition in a pure culture as well as in a mixed community. The data presented here highlight how simple mathematical models constrained by a few experimentally determined parameters in batch can predict the behavior of complex continuous systems. Isolation, combined with the creation of synthetic co-cultures and mixed communities, allows us to parse out indirect effects of perchlorate as well as to define controls on inhibition when multiple organisms are present. In this way, this work provides both a framework for testing community-relevant ecological and physiological hypotheses, as well as practical insights for controlling sulfidogenesis across environments.The datasets generated for this study can be found in NCBI, PRJNA521657.MS and JC designed the study. MS performed the experiments with the help of GN and NG. YC helped with modeling. MS wrote manuscript, with edits from AE, HC, and JC.JC holds IP on the application of perchlorate to oil reservoir souring. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the original article, there was an error. Tyrosine-phosphatases were incorrectly referred to as tyrosine-kinases.A correction has been made to the following:Section: Signal transduction, page 2Subsection: Direct signaling, paragraph 1Corrected paragraph:Direct signaling by CD32 is mediated by immunoreceptor tyrosine-based activation motif (ITAM/CD32A and CD32C) (17) and by immunoreceptor tyrosine-based inhibitory motif (ITIM/CD32B) (18). These motifs determine whether the receptors are activating or inhibitory. It is important to emphasize that signaling starts by cross-linking of the receptor leading to activation of phosphatases such as SHP and SHIP, and members of the src-family of tyrosine kinases (19–21). This leads to phosphorylation of the important tyrosine residues in the ITAM/ITIM motifs from where various signaling cascades are initiated. Phosphorylation of ITAMs lead to activation of the cells (22), whereas phosphorylation of ITIMs lead to cell inhibition (23). The mechanisms involved in the control of CD32B have been excellently reviewed by Getahun and Cambier (24).The author apologizes for this error and states that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Esenbeckia alata (Rutaceae), a plant used in traditional medicine, led to the isolation of two alkaloids, kokusaginine 1 and flindersiamine 2, as main cytotoxic agents. Primary ethanolic extract and raw fractions exhibited cell inhibition against five cancer cell lines at different levels (25–97% inhibition at 50 µg/mL) as well as isolated alkaloids 1–2 (30–90% inhibition at 20 µM). Although alkaloid 2 generally was the most active compound, both alkaloids showed a selective effect on K562, a human chronic myelogenous leukemia cell line. The E1-like ubiquitin-activating enzymes  have been recently described as important targets for future treatment of cancer progression, such as leukemia, among others. Therefore, as a rationale to the observed cytotoxic selectivity, an in-silico evaluation by molecular docking and molecular dynamics was also explored. Compounds 1–2 exhibited good performance on the interaction within the active site of UBA5.Bio-guided fractionation performed on the leaves-derived ethanol extract of  Esenbeckia is one of the most abundant taxonomic group within this family, contributing a total of 30 species  were then depurated by CC using dichloromethane/acetone 9:1 (1.5 L) as eluent. Subfraction EA-EEa3 afforded 2 (135 mg) and EA-EEa6 yielded 1 (95 mg) as main compounds retaining the cytotoxic activity exhibited by extracts, fractions, and subfractions. These compounds were structurally elucidated by 1H and 13C-NMR  was evaluated using a previously reported protocol [E. alata leaves, fractions, subfractions, and the alkaloids 1–2, dissolved in dimethylsulfoxide (DMSO) and diluted in the medium, without exceeding the maximal amount of DMSO (<0.5%). Doxorubicin (10 µM)  was used as a positive control. 100 μL of a solution of the ethanolic extract (50 μg/mL), fractions (30 μg/mL), and subfractions (10 μg/mL) and alkaloids 1–2 (20 μM) were added to each cell culture (100 μL). The mixture was kept under exposure for 48 h. After the incubation period, the cells were fixed to the plastic bottom layer by the addition of 50 μL of cold 50% aqueous trichloroacetic acid. The plates were incubated at 4 °C for 1 h, washed with H2O, and air dried. The cells with trichloroacetic acid were stained by addition of sulforhodamine B (SRB)  at 0.4% (w/v) dissolved in 1% acetic acid for 30 min and then washed with 1% aqueous acetic acid. The plates were air dried and the dye was solubilized by adding 100 μL of Tris, pH 10. The plates were shaken for 5 min and the absorbance was measured by an ELISA plate reader  at 515 nm. These values were expressed as an inhibition percentage, relative to the incubations treated with negative control.The cytotoxicity of test substances , without geometric restrictions, included in SPARTAN software (Spartan 14v114 (2013), Wave function, Inc., Irvine, CA, USA) was performed with a 500-conformers limit. Energetically most-stable conformers were MMFF-optimized to be used into the docking protocol. Docking calculations were performed with the Autodock/Vina 1.1.2  plug-in for PyMOL 1.3r2  under a Python 2.5.2  environment for Windows . The strMolecular dynamics (MD) simulations were run in Gromacs 5.0.5  on an Ubuntu 12.04 server . The ligand best pose from docking and the crystal structure of enzyme were employed as input for dynamics simulations of complexes and stand-alone proteins, respectively. Ligands were prepared by adding hydrogen atoms and the corresponding charges using the AM1-BCC charge scheme in UCSF Chimera . Subsequently, ligand topologies were generated automatically with ACPYPE script. Protein topologies were obtained in Gromacs using the Amber 99SB force field and TIP3P water model was implemented. Solvation was performed in a triclinic box using a margin distance of 1.0 nm. The addition of 0.1 M NaCl to complexes and proteins was carried out by replacing randomly water molecules until neutrality was achieved. The systems were energy-minimized by 2000 steps of the steepest descent method. NVT equilibration at 310 K for 50 ps, followed by NPT equilibration during 500 ps using the Parrinello–Rahman method at 1 bar as reference were conducted on systems using position restraints. Finally, solute position restraints were released and a production run for 150 ns was performed. The temperature and pressure were kept constant at 310 K and 1 bar, respectively. Coordinates were recorded in a 1 fs time step. Electrostatic forces were calculated using the particle-mesh Ewald (PME) method. Periodic boundary conditions were used in all simulations and covalent bond lengths were constrained by the LINCS algorithm.1–2 and TAK-243 compounds using the molecular mechanic/Poisson-Boltzmann surface area (MM/PBSA) method [binding was calculated using the Lennard-Jones and Coulomb potential. The binding energy calculations were performed for 200 snapshots taken at an interval of 5 ns during the last stable 50 ns period of MD trajectory, using the g_mmpbsa tool of GROMACS, based on MM/PBSA technique [Binding free energy was calculated for test alkaloids ) method . Snapshoechnique . Thus, t1–2 against the cancer cell lines U251 , PC-3 (prostate), K562 (leukemia), HCT-15 (colon), MCF-7 (breast), and SKLU-1 (lung) expressed as cell growth inhibition percentages. Test substances showed effects at different levels on the cancer cell lines evaluated at the test doses, but they also exhibited lower cytotoxicity (4–21%) against the cell line MCR5 . A clear selectivity towards K562 cell line was observed. Thus, the ethanolic extract  showed values above 60% at 50 μg/mL against the lines K562, PC-3, MCF-7, and SKLU-1, although the inhibition percentage of K562 was particularly found to be the highest (~97%), so a clear selectivity on this cell line was observed. Such percentages were noticed to be similar (and even better) to that of positive control, whose inhibition values were located into the 82–99% range. In contrast, the ethanolic extract showed a low inhibition percentage for lines U251 and HCT-15 (~27–43%).n-hexane, EtOAC, MeOH, and water) were also evaluated for their cytotoxic activity on the same panel of cell lines. Three of these fractions  resulted in inhibition values below 30% (data not shown), so they were discarded for further depurating processing. However, AcOEt fraction  exhibited values between 27 and 97%, involving a similar profile to that of EEa, which indicated retention of the initial cytotoxic activity  exhibiting moderate cytotoxic activity (IC50 = 87.4 μg/mL) [2, kokusaginine B, also exhibited no cytotoxic on the same panel [1–2 exhibited weak cytotoxicity against A2780 human ovarian cancer cell line [2 displayed potent inhibitory effect on MCF-7 (breast cancer) by the inhibition of tubulin assembly [1–2 exhibited selectivity against K562, being the first report of the cytotoxicity assessment of these alkaloids against such a cell line.In the case of pure alkaloids ractions . Such co control . Alkaloi4 μg/mL)  as well 4 μg/mL) . Recentlme panel . In addiell line . Howeverassembly . These rassembly ,39. In tC-terminal carboxylate. Therefore, the inhibition of UBA5, through this ATP pocket, can result in low abundance of ubiquitinated proteins in leukemia and myeloma cells, inducing cell death [1–2 within the active site containing the ATP pocket of UBA5, in order to evaluate their plausible competing ability for this site acting as putative inhibitors. As mentioned above, K562 cancer cell line exhibits sensitivity to UPS perturbations, since this system is crucial for regulating protein degradation, subcellular localization, and cell cycle progression. The Ub-fold modifier 1 (UFM1) is particularly associated with several important biological functions . The firll death . Owing tll death . The disll death . Therefo1–2 and UBA5 was initially preformed. The resulting docking calculations resulted in good Vina scores  in comparison to that of TAK-243 , a known inhibitor for different E1-like Ub-activating enzymes [1–2 might exhibit a reasonable interaction with the binding site of UBA5 , but their best-docked poses were found to be located in a different position within this site to that of TAK-243  appeared to be non-significant for UBA5 inhibition, since the two-dimensional residual interaction diagrams  simulations as the next step to expand the information about the binding mode of these test alkaloids. Thus, the ligand:enzyme trajectories for those complexes between UBA5 and 1–2 and TAK-243 were monitored by means of geometric properties over the time. In this regard, the root mean square deviations (RMSD) were used to evaluate the structural stability of the receptor frame by measuring the time-dependent distance between different positions (in Å) of the set of atoms . The behavior of UBA5–ligand complexes was found to be different to that of the UBA5 apoenzyme, since the structural stabilization seems to be a two-step process. The UBA5–TAK-243 complex reached an initial stabilization step between 5 and 42 ns (0.15–0.20 nm) and, from this point, the complex changed its geometric properties until RMSD values of 0.30–0.35 nm. This complex stabilized after 100 ns (0.25–0.30 nm). In the case of UBA5–1 and UBA5–2 complexes, an early stabilization was also achieved (0.15–0.25 nm) between 10 and 65 ns, and a fluctuation was experienced for both complexes until RMSD values of 0.35–0.40 nm at 75 ns. This second stabilization was then maintained over the remaining simulation. Although both alkaloids exhibited a similar RMSD profile, compound 2 promoted fewer perturbations to those of 1. The analysis of such fluctuations of the atomic positions for each enzyme residue was extended by measurements of the root mean square fluctuation (RMSF), in order to scrutinize the flexibility and secondary structure of the UBA5 enzyme under interaction with the test ligands. The restricted mobility of some residues was then evidenced in apoenzyme and when each ligand was separately introduced to UBA5, while mobility for other residues was slightly higher. All examined complexes showed different behavior , so its inhibition mode may be achieved by system stabilization through a different manner to that of TAK-243. These results indicated a reasonable in-silico interaction performance of 1–2 with the UBA5 enzyme over the time.Hence, separately, 150-ns MD simulations for the UBA5 alone (apoenzyme) and distinctly docked with behavior b, with e2 showed the lower binding energy (−114.5 ± 4.1 kJ/mol) to that of 1  and TAK-243 (−71.6 ± 7.8 kJ/mol). These results might rationalize the observed best docking performance and the best experimental cytotoxic activity of 2 on K562 line. The main contribution to the binding energy of 1–2 was due to vdW energies (> −145 kJ/mol). The binding free energy for test ligands during interaction with UBA5 for the last 50 ns of MD trajectory was estimated by MM/PBSA approach, after the MD simulations . All thr1–2 or TAK-243), as mentioned above. For UBA5–TAK-243 complex, the most binding energy contributing residues were found to be SER208 (−4.9 kJ/mol), ARG188 (−3.8 kJ/mol), VAL207 (−3.3 kJ/mol), and PRO232 (−3.1 kJ/mol), whereas for UBA5–1 and –2, complexes were GLY82, GLY83, ASP104, and LYS127 , involving weak polar electrostatic interactions for the recognition of the acidic site of UBA5 proximal to the ATP pocket by these alkaloids due to basic nature, so they could be considered potential leads for the future development of UBA5 inhibitors based on furoquinolines. As computational hypotheses related to the insights into the interaction, these alkaloids showed a particularly different binding mode to that of a known UAE E1 inhibitor . Such a mode of action through UBA5 inhibition will be further explored by in-vitro enzymatic inhibition over purified enzyme. Thus, after data analyses, some important hits and structural requirements were established, indicating the potential of furoquinoline alkaloids for disrupting cancer progression by ubiquitin–proteasome pathway perturbation or acting synergistically with kinase inhibitors to induce apoptosis of K562 cells. Hence, this information can be preserved for further studies focused on the development of antitumor agents based on furoquinoline alkaloids.After a bio-guided fractionation was performed on the ethanol extract from leaves of