Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease.Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both  Parkin, a RING-HECT hybrid E3 ubiquitin ligase , is muta5–Increasing evidence has shown that parkin functions as a tumor suppressor. Human parkin gene localizes at chromosome 6q25-q26 region, which is a common fragile site. It has been found that this region is often deleted in ovarian, lung, and breast cancers . Parkin Glycolysis is the essential metabolism pathway for cell growth and survival. Compared with normal cells, tumor cells often have an increased rate of glycolysis and utilize much more glucose to keep the balance among the production of ATP, biosynthesis of building blocks, and reducing equivalents for rapid proliferation , 15. The16–25–In this study, we identified parkin as a regulator of PKM2 through biochemical purification of protein complex. Parkin is a specific PKM2-interacting protein and catalyzes ubiquitin conjugation to PKM2 mainly on sites Lys-186 and Lys-206. Ubiquitylation of PKM2 decreases its enzymatic activity. In contrast, PKM2 enzymatic activity is enhanced after ablation of parkin, hence increasing the steady state metabolite levels of glycolysis in cells. This is the first direct evidence to support the concept that parkin suppresses tumor growth by inhibiting glycolysis through PKM2 ubiquitylation.The HA-parkin plasmid was obtained from Addgene. The full-length parkin was amplified by PCR from HA-parkin and subcloned into pCin4-FLAG-HA, pCin4-FLAG, or pCMV-myc expression vectors. Parkin was subcloned into pTRIPZ lentiviral inducible vector. The full-length PKM2 was amplified by RT-PCR from human cells. The cDNA sequences corresponding to full-length parkin and different fragments of parkin and those corresponding to full-length PKM2 were amplified by PCR and subcloned into pGEX (GST) vector for expression in bacteria. Antibodies used in Western blotting assay were β-actin (A15), FLAG M2 from Sigma; HA (3F10) from Roche Applied Science; PKM2 (3198), Parkin (2132) and COX IV from Cell Signaling; citrate synthase form Abcam; Myc (9E10) from Santa Cruz.H1299, 293T, U87 and IMR32 cells were cultured in DMEM medium (Cellgro), supplemented with 10% fetal bovine serum (Gibco). MCF10A cells were maintained in DMEM/F12 50/50 medium (Cellgro), supplemented with 5% horse serum, 20 ng/ml EGF, 10 μg/ml insulin, 100 ng/ml cholera toxin and 0.5 mg/ml hydrocortisone. The FLAG-HA-parkin/H1299 stable line or FLAG-HA-PKM2/H1299 stable line was established by transfecting H1299 cells with plasmid expressing pCin4-FLAG-HA-parkin or pCin4-FLAG-HA-PKM2, respectively. And cells were selected with 1 mg/ml G418 (EMD Biosciences). The inducible parkin/H1299 cell lines were established by transfecting H1299 cells with pTRIPZ lentiviral inducible parkin plasmid, and selected with puromycin.Transfections with plasmid DNA or siRNA oligos were performed by Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.m Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mm EDTA, 10% glycerol, and freshly supplemented proteinase inhibitor mixture).For Western blotting analysis, cells were lysed in cold radioimmune precipitation assay buffer , Dounce homogenizing (20 strokes with a Type A pestle), and pelleting nuclei (1000 × g for 10 min). The supernatant was kept as the cell cytoplasmic extracts. Cytoplasmic extracts were adjusted to a final concentration of 100 mm NaCl and 0.1% CHAPS.The immunoprecipitation assay was performed with cell cytoplasmic extracts. Cytoplasmic extracts were prepared by resuspending cell pellet in hypotonic buffer , the bound proteins were eluted by 1× SDS loading buffer with heat to denature proteins. Alternatively, cell cytoplasmic extracts were incubated with FLAG-agarose beads (Sigma) or HA-agarose beads (Roche Applied Science) at 4 °C overnight to analyze cells transfected with FLAG-tagged or HA-tagged plasmid. The beads were washed five times with BC100 buffer, and the bound proteins were eluted using FLAG peptide or HA peptide in BC100 buffer for 2 h at 4 °C.Cell cytoplasmic extracts were incubated with parkin-specific antibody or PKM2-specific antibody at 4 °C overnight followed by Protein A/G beads for 4 h to analyze endogenous parkin or PKM2. After washing five times with BC100 buffer . GST or GST-tagged proteins were incubated with 35S-labeled proteins at 4 °C overnight in BC100 buffer + 0.2% BSA and then incubated with GST resins (Novagen) for 4 h. The resins were washed five times with BC100 buffer. The bound proteins were eluted with 20 mm reduced glutathione (Sigma) in BC100 buffer for 2 h at 4 °C and resolved by SDS-PAGE. The pulled down 35S-labeled protein was detected by autoradiography.GST or GST-tagged fusion proteins were purified as described previously , 31. methionine-labeled PKM2. Following immobilization with GST resins and recovery of captured complexes using reduced glutathione, the eluted complexes were resolved by SDS-PAGE and analyzed by autoradiography. 35S-Labeled PKM2 bound immobilized GST-tagged full-length parkin but not the control GST (E). Parkin was divided into different fragments according to its four domains: 1–100 fragment containing the ubiquitin-like domain, 100–300 fragment containing RING 1 domain, and 300–465 fragment containing the IBR and RING 2 domains. Purified full-length GST-PKM2 fusion proteins or GST alone was incubated with in vitro translated [35S]methionine-labeled full-length parkin protein (F) or different parkin fragments (G). The immobilized complexes were resolved by SDS-PAGE and analyzed by autoradiography. GST-PKM2 interacts with 35S-labeled full-length parkin and 100–465 fragment that contains RING 1-IBR-RING 2 domains (F and G).To investigate the direct interaction between parkin and PKM2, we performed an trol GST E. Parkin protein F or diffragments G. The im domains , F and GH). Notably, although the level of PKM2 and parkin does not change, PKM2 binding by parkin was significantly enhanced upon glucose starvation. These data demonstrated that the interaction between parkin and PKM2 is regulated by the level of glucose.To investigate the dynamic process of the interaction between parkin and PKM2 under the condition of glucose starvation, we subjected the FLAG-HA-parkin/H1299 stable line to glucose starvation for 0, 1, and 2 h, respectively. The cytoplasmic extracts were immunoprecipitated with FLAG M2-agarose beads. Western blotting analysis revealed that parkin binds much more PKM2 protein under glucose starvation H. NotablA). The structure of parkin was determined recently (––A), and the constitutively active parkin in which the N-terminal autoinhibitory domain was removed (parkin/d79) (–B). We also transfected inducible parkin H1299 stable lines with HA-Ub plasmid and induced the stable lines to express a high level of parkin by treatment with doxycycline. Western blotting analysis revealed that monoubiquitin-conjugated PKM2 was easily detected in HA immunoprecipitates of cell extracts from the cells induced to express parkin .To investigate the relationship of parkin and PKM2, we examined whether parkin can ubiquitylate PKM2. 293 cells were transiently transfected with plasmids expressing His-ubiquitin (Ub), FLAG-PKM2, and parkin. Western blotting analysis showed that monoubiquitin-conjugated PKM2 bound Ni-NTA resin with wild type parkin but not with mutant parkin  Fig. 3AAFig. 3A.recently –35. We pecently ––35  –35 had sin/d79) –B. We alsC).To confirm that parkin ubiquitylates PKM2, we performed a FLAG M2 and HA double immunoprecipitation assay. 293 cells were transiently transfected with plasmids expressing FLAG-PKM2, HA-Ub, and parkin. Cell extracts were subjected to a FLAG M2 and HA tandem immunoprecipitation under stringent condition. Western blotting analysis showed that monoubiquitin-conjugated PKM2 was readily detected by antibodies against FLAG and HA C.C. The proteins were analyzed by LC-MS/MS, and Lys-62, Lys-66, Lys-186, Lys-206, Lys-305, and Lys-367 were identified as the candidate modified sites. We further mutated PKM2 on those sites to arginine and performed a ubiquitylation assay. The results showed that the major ubiquitylation sites on PKM2 are located at Lys-186 and Lys-206 .To identify the modification sites of PKM2, we purified ubiquitylated PKM2 proteins by FLAG M2 and HA double IP as in Fig. 3in vitro ubiquitylation assay. We purified PKM2, parkin, and the constitutively active parkin (parkin/d79) from transfected 293 cells under stringent condition. The results showed that the constitutively active parkin can catalyze PKM2 ubiquitylation with phosphorylated Ser-65 ubiquitin in vitro (E).To further confirm that parkin ubiquitylates PKM2, we performed an in vitro E.C). These data demonstrated that ubiquitin-modified PKM2 has decreased activity. To confirm this result, we purified PKM2 and PKM2-Ub fusion protein, which mimics the monoubiquitylated PKM2, and assessed their pyruvate kinase activity. The pyruvate kinase activity of PKM2-Ub was significantly reduced compared with wild-type PKM2 (D), although the pyruvate kinase activity of PKM2 K186R,K206R did not change compared with the wild-type PKM2 (E).To understand the role of PKM2 ubiquitylation, we examined whether ubiquitylation of PKM2 has an effect on its enzymatic activity. 293 cells were transiently transfected with plasmids expressing His-Ub, FLAG-PKM2, and parkin. All His-Ub-conjugated proteins from cell extracts were obtained by Ni-NTA pulldown, and His-Ub-conjugated PKM2 was further purified by FLAG M2 immunoprecipitation. The pyruvate kinase activity of PKM2 and Ub-conjugated PKM2 was assessed, and although the amount of proteins was almost equal, the activity of ubiquitylated PKM2 was significantly lower than that of PKM2 C. These ype PKM2 D, althouype PKM2 E.in vivo. Parkin was knocked down in H460 cells by transfection with the pool of four parkin-specific siRNA oligos (A) or different parkin-specific siRNA oligos (B). Although endogenous parkin was severely reduced, the level of PKM2 was unaffected. In MCF10A cells, parkin was ablated by transfection with the pool of four parkin-specific siRNA oligos (C) or by infection with different shRNA lentiviruses (D). The same as in H460 cells, the level of PKM2 remained the same. We further overexpressed parkin in cells to examine the levels of PKM2. MCF10A cells were transfected with an increasing amount of wild-type parkin or mutant parkin. Western blotting analysis showed that the level of endogenous PKM2 was stable regardless of the level of wild-type or mutant parkin overexpression (E). In an inducible parkin/H1299 cell lines, parkin was induced for overexpression, and the endogenous PKM2 remained stable (F). H1299 cells were transfected with FLAG-PKM2 and an increasing amount of myc-parkin. Western blotting analysis showed no change of the level of FLAG-PKM2 no matter how much parkin was expressed (G). H1299 cells also were transfected with FLAG-PKM2 and an increasing amount of FLAG-parkin or constitutively active FLAG-parkin (parkin/d79). The results showed that the level of FLAG-PKM2 did not change no matter how much parkin was expressed (H). These data demonstrated that parkin does not regulate PKM2 stability.As shown above, parkin catalyzes ubiquitin conjugation to PKM2, so we examined whether parkin degrades PKM2 A oligos A or diffA oligos B. AlthouA oligos C or by iiviruses D. The sapression E. In an d stable F. H1299 xpressed G. H1299 xpressed H. These F).To investigate the relationship of parkin and PKM1, we examined the interaction between these two proteins. 293 cells were transiently transfected with expression plasmids for FLAG-tagged PKM1 and HA-tagged parkin. Cell cytoplasmic extracts were subjected to immunoprecipitation with FLAG M2-agarose beads or HA-agarose beads. Western blotting analysis showed that parkin clearly associates with PKM1 F.G).We examined whether parkin can ubiquitylate PKM1. 293 cells were transiently transfected with plasmids expressing His-Ub, FLAG-PKM1, and parkin. Western blotting analysis showed that ubiquitin-conjugated PKM1 bound Ni-NTA resin with wild-type parkin G.I).To confirm that parkin ubiquitylates PKM1, we performed a FLAG M2 and HA double immunoprecipitation assay. 293 cells were transiently transfected with plasmids expressing FLAG-PKM1, HA-Ub, and parkin. Cell extracts were subjected to a FLAG M2 and HA tandem immunoprecipitation under stringent condition. Western blotting analysis showed that ubiquitin-conjugated PKM1 was readily detected by antibodies against FLAG and HA I.H). These data demonstrated that ubiquitin-modified PKM1 has decreased activity.To understand the role of PKM1 ubiquitylation, we examined whether ubiquitylation of PKM1 has an effect on its enzymatic activity. 293 cells were transiently transfected with plasmids expressing His-Ub, FLAG-PKM1, and parkin. All His-Ub-conjugated proteins from cell extracts were obtained by Ni-NTA pulldown. His-Ub-conjugated PKM1 was further purified by FLAG M2 immunoprecipitation. The pyruvate kinase activity of PKM1 and Ub-conjugated PKM1 was assessed. The activity of ubiquitylated PKM1 was significantly lower than that of PKM1 H. These A) or different parkin-specific siRNA oligos (B). Although endogenous parkin was severely reduced and the level of PKM2 was unaffected, the pyruvate kinase activity of cell extracts was strikingly increased (A and B). In MCF10A cells, parkin was ablated by transfection with the pool of four parkin-specific siRNA oligos (C) or by infection with different shRNA lentiviruses (D). The same as in H460 cells, the level of PKM2 was unchanged, and the pyruvate kinase activity of cell extracts was strikingly increased (C and D).To further elucidate the effect of parkin on pyruvate kinase activity under physiological condition, we performed the pyruvate kinase activity assay in cells after inactivation of endogenous parkin. Parkin was knocked down in H460 cells by transfection with the pool of four parkin-specific siRNA oligos A or diffA oligos B. Althouncreased , A and BA oligos C or by iiviruses D. The sancreased , C and DE). The inducible parkin/H1299 cells were treated with doxycycline to induce expression of a high level of parkin. The endogenous PKM2 remained stable, and the pyruvate kinase activity of cell extracts decreased with different amounts of overexpressed parkin (F). These data demonstrated that parkin regulates pyruvate kinase activity of cells.To further confirm the effect of parkin on pyruvate kinase activity, we performed the pyruvate kinase activity assay in parkin-overexpressing cells. MCF10A cells were transfected with increasing amounts of wild-type parkin and mutant parkin. Although Western blotting analysis showed that the level of endogenous PKM2 was unchanged, no matter how much wild-type or mutant parkin was overexpressed, the pyruvate kinase activity of cell extracts decreased with more wild-type parkin and increased with more mutant parkin E. The ind parkin F. These To investigate the physiological function of regulation of PKM2 by parkin, we examined whether inactivation of endogenous parkin has an effect on the cell metabolic pathway. U87 cells or FLAG-HA-parkin/U87 stable cell lines were transfected once with the pool of four siRNA oligos specific for parkin 3′-UTR region or control siRNA oligos. The cells were cultured in DMEM for 24 h. The metabolism intermediate products were extracted with 80% cold methanol and analyzed by mass spectrometry. The mass spectrometric peak strength of each compound was normalized to the amount of total proteins. As shown in Parkin plays a central role in mitochondrial homeostasis and mitophagy . Several40–in vivo (It has been shown that PKM2 is important for tumor cell survival and growth –24. PKM2–in vivo . PKM2 isin vivo . PKM2 isin vivo . PKM2 isin vivo . ERK1/2 in vivo . PKM2 isin vivo . Mitogenin vivo . Our finMany proteins can be regulated by monoubiquitylation including histones , DNA repPyruvate kinase M gene encodes two isoenzymes through mRNA differential splicing , 20. PyrK. L., F. L., and H. H. executed the experiments. Y. C. and Y. Z. executed the LC-MS/MS analysis. B. Z. executed cell metabolites analysis. W. G. and W. Z. designed, executed, analyzed, and supervised the experiments. Z. M. and J. L. analyzed the experiments. W. Z. wrote the manuscript, and J. L. helped to edit the manuscript. All authors reviewed the results and approved the final version of the manuscript.


Surgical-site infection is the most frequent health care-associated infection in the developing world, with a strikingly higher prevalence than in developed countries We studied the prevalence of resistance to antibiotics in Enterobacteriaceae isolates from surgical-site infections collected in three major tertiary care centres in Bangui, Central African Republic. We also studied the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae.blaCTX-M-15 and aac(6′)-Ib-cr genes were detected in all strains, usually associated with qnr genes (98.5%). Escherichia coli, the most commonly recovered species , occurred in six different sequence types, including the pandemic B2-O25b-ST131 group . Resistance transfer was studied in one representative strain of the resistance gene content in each repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence-PCR banding pattern. Plasmids were characterized by PCR-based replicon typing and sub-typing schemes. In most isolates , blaCTX-M-15 genes were found in incompatibility groups F/F31:A4:B1 and F/F36:A4:B1 conjugative plasmids. Horizontal transfer of both plasmids is probably an important mechanism for the spread of blaCTX-M-15 among Enterobacteriaceae species and hospitals. The presence of sets of antibiotic resistance genes in these two plasmids indicates their capacity for gene rearrangement and their evolution into new variants.Between April 2011 and April 2012, 195 patients with nosocomial surgical-site infections were consecutively recruited into the study at five surgical departments in three major tertiary care centres. Of the 165 bacterial isolates collected, most were Enterobacteriaceae . Of these, 65/102 (63.7%) were 3GC-R, which were characterized for resistance gene determinants and genetic background. The Diverse modes are involved in transmission of resistance, plasmid dissemination probably playing a major role. This situation is of great concern, as ESBL enzymes can hydrolyse almost all beta-lactams (except carbapenems and cephamycins) and are frequently associated with genes that confer resistance to several other classes of antibiotic. During the past decade, CTX-M enzymes have gradually replaced the classical TEM and SHV-type ESBLs in many countries [Escherichia coli clones, such as sequence type 131 (ST131) harbouring blaCTX-M-15 on incompatibility group (Inc) FII conjugative plasmids [Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat in both hospital and community settings worldwide . The resountries . Rapid iplasmids .Plasmid-mediated quinolone resistance has emerged in Enterobacteriaceae, with three mechanisms described: Qnr, which mediates target protection; AAC(6′)-Ib-cr, which mediates drug modification; and OqxAB and QepA, which mediate drug efflux . These mSurgical-site infection is the most frequent health care-associated infection in the developing world, with a strikingly higher prevalence than in developed countries . The alan = 33), the orthopaedics department of the Community Hospital (n = 64), and the general surgery (n = 41), gynaecology (n = 46) and urology (n = 11) departments of the Amitié Hospital. Most of the operations were class I  in the Altemeier classification, mainly with a pre-operative physical status score of I . The duration of most operations did not exceed 2 h (98.0%). Antibiotic prophylaxis was administered to 193 patients (99.0%), most frequently consisting of beta-lactams (92.3%), mainly ceftriaxon , penicillin G  and ampicillin .A total of 195 patients  with surgical-site infections were included during the study period in the general surgical departments of the Complexe Pédiatrique (E. coli (n = 47), Staphylococcus aureus (n = 44), Enterobacter cloacae (n = 18), Acinetobacter baumanii (n = 11), Klebsiella pneumoniae (n = 19), Pseudomonas aeruginosa (n = 8), Proteus mirabilis (n = 8), Morganella morganii (n = 3), Salmonella spp. (n = 2), Enterobacter aerogenes (n = 2), Enterobacter amnigenus (n = 1), Enterobacter sakazakii (n = 1) and K. oxytoca (n = 1).Of the 195 non-duplicate biological samples taken, 151 were culture positive (77.4%). As 14 cultured specimens had mixed growth, a total of 165 bacterial isolates were collected. The bacteria isolated were P. aeruginosa nor A. baumanii was resistant to imipenem, whereas 9% (4/44) of S. aureus isolates were methicillin-resistant, and 63.7% (65/102) of Enterobacteriaceae  were resistant to 3GC-R antibiotics. C3G-R Enterobacteriaceae strains were resistant to all the beta-lactams tested, except for cefoxitin , and all were susceptible to ertapenem . In addition, they were characterized by high rates of resistance to netilmicin , kanamycin , gentamicin , tetracycline , ciprofloxacin  and cotrimoxazole , and a high rate of susceptibility to amikacin . The double-disc synergy test was positive for all strains. Older patients were more susceptible to C3G-R Enterobacteriaceae infection than younger ones (p = 0.04) (Table Neither 63.7% 65/02 of EntblaCTX-M-15 and aac(6′)-Ib-cr genes were detected in all strains. Of the 64 (98.5%) qnr-positive strains, 62 were qnrB, 56 were qnrS and none were qnrA, with 55 strains each harbouring 2 qnr genes.The E. coli strains, 6 for Enterobacter cloacae, 5 for K. pneumoniae and 2 for P. mirabilis. Three clones were found in at least two surgical departments in the same hospital and six clones in at least two different tertiary care centres: two Enterobacter cloacae clones, three K. pneumoniae clones and three E. coli clones , ST10 , ST405 , ST156 , ST354  and ST146  (Table E. coli B2-O25b-ST131 group.Six multilocus STs were found in the 33 ESBL by conjugation with E. coli J53-2 was successful for 22 (64.7%) of the 34 ESBL clones, which consisted of 17 E. coli isolates, 4 Enterobacter cloacae and 1 Enterobacter amnigenus. ESBL transfer by plasmid DNA electroporation into E. coli DH10B was successful for 5 (41.7%) of the 12 remaining isolates: 2 K. pneumoniae isolates, 2 P. mirabilis and 1 Enterobacter cloacae. The presence of blaCTX-M-15 and aac(6′)-Ib-cr was confirmed by PCR in the 27 transconjugants and transformants -Ib-cr, in contrast to qnr genes, as no qnrS and 45.8% (11/24) of qnrB genes were co-transferred with blaCTX-M-15. Additional resistance genes  were probably transferred, as shown by antimicrobial susceptibility testing  and IncHI2 (one Enterobacter cloacae)  and F36:A4:B1 . These two conjugative plasmids were found in strains collected from different hospitals. The other combinations were F1:A1:B1 (one E. coli), F1:A1:B20 (one E. coli), Fnew:A-:B20 (one E. coli and one Enterobacter cloacae) and K4:-:B- (two K. pneumoniae)  were 3GC-R, as described previously in Africa [re units , but alsountries . FurtherE. coli was the most frequent species among C3G-R Enterobacteriaceae. The 33 isolates belonged to six STs, of which three corresponded to at least two REP- and ERIC-PCR patterns, namely ST131, ST405 and ST10. The pandemic E. coli B2-O25b-ST131 group has contributed extensively to global dissemination of CTX-M-15 [E. coli multi-drug resistant STs also occurs in African hospitals. The reasons for the wide dissemination and expansion of these clones are unknown but may include increased transmissibility, greater ability to colonize and/or persist in the intestinal tract, enhanced virulence and more extensive antimicrobial resistance than other E. coli [CTX-M-15 , as has CTX-M-15 -13, show E. coli . The thr E. coli : ST146 iblaCTX-M-15. The ESBL blaCTX-M-15 gene was carried in all strains on plasmids of IncF incompatibility groups, except in one Enterobacter cloacae. IncF plasmids were the most prevalent in E. coli and K. pneumoniae carrying ESBL genes and also genes that confer resistance to tetracyclines, aminoglycosides and fluoroquinolones. These plasmids can be considered pandemic, as they have been detected in different countries and in bacteria of different origins and sources. The occurrence of these plasmid types appears to be closely linked to positive selection exerted by antibiotic use, incrementing their prevalence over that in bacterial populations that are not preselected for antimicrobial resistance. Their occurrence is also linked to virulence factors that contribute to the fitness of their bacterial host [E. coli STs, blaCTX-M-15 genes were found within IncF conjugative plasmids, with identical replicon ST formulae (F31:A4:B1 and F36:A4:B1), two plasmids reported to be abundant in humans [blaCTX-M-15 spread, as described previously [K. pneumoniae, E. coli and Enterobacter cloacae isolates in at least two different hospitals.Plasmid typing showed that horizontal gene transfer by IncF plasmid exchange was probably also involved in the dissemination of ial host ,18. In tn humans . The F31eviously . The disESBL blaCTX-M-15 and aac(6′)-Ib-cr genes were inconsistently associated with qnrB on these plasmids and also with other resistance genes, as shown by antimicrobial susceptibility testing of transconjugants. This suggests the presence of a multidrug resistance region, as described for IncFII plasmids pEK499 and pEK516 isolated in the United Kingdom from the E. coli ST131 group and for the pC15-1a plasmid disseminated in E. coli STs in Canada [blaCTX-M-15, qnrB and aac(6′)-Ib-cr genes has been described in K. pneumoniae isolates in North, West, Central and East Africa, consistent with the hypothesis that these resistance-determinant genes are carried together on the same plasmid [Various sets of antibiotic resistance genes within F31:A4:B1 and F36:A4:B1 plasmids indicate their capacity for gene rearrangement and their evolution into new variants. n Canada . Further plasmid . Such ac plasmid  but was This study is of particular importance because of the difficulty of carrying out such studies in hospitals in countries with inadequate health care systems. Our data suggest that diverse modes of transmission of resistance are involved, probably with a major role of plasmid dissemination. All necessary measures should be taken in African hospitals to prevent nosocomial infections and the selection of resistant bacteria, with efficient nosocomial infection surveillance programmes. Standard hygiene and especially adherence to hand hygiene policies are the cornerstone for preventing transmission of multidrug-resistant bacteria.The study protocols were approved by the National Ethics Committee of Central African Republic. Written informed consent to participate in the study was obtained from all patients.Patients with a surgical-site infection were recruited consecutively into the study between April 2011 and April 2012 in five surgical departments in three major tertiary care centres in Bangui: the Complexe Pédiatrique, the Amitié Hospital and the Community Hospital. A surgical-site infection was defined as an infection that occurred at or near a surgical incision within 30 days of the procedure or within 1 year if an implant was left in place . Patienthttp://www.sfm-microbiologie.org). Production of ESBL in C3G-R Enterobacteriaceae was detected by the double-disc synergy test [After superficial cleaning of wounds with physiological saline, a specimen was collected by rotating a sterile swab across the surface of the lesion, specifically targeting moist and necrotic areas. The specimens were placed in sterile tubes without transport medium at 4°C immediately after sampling and were processed within 2 h at the Pasteur Institute medical laboratory in Bangui. Sheep blood, chocolate and bromocresol purple agar plates were inoculated, incubated aerobically at 37°C and examined after 24 h and 48 h. Obligate anaerobes were not isolated. Bacterial isolates were identified by standard microbiological methods. Antibacterial drug susceptibility was determined by the disc diffusion method on Mueller–Hinton agar , according to the guidelines of the French Society of Microbiology -Ib gene and the quinolone resistance qnrA/B/S and qepA genes [blaCTX-M and blaSHV were then characterized by direct DNA sequencing of the PCR products. All aac(6′)-Ib positive strains were further analysed by digestion with BtsCl  of the PCR product to identify aac(6′)-Ib-cr [Genomic DNA was extracted with the QIAmp™ kit . Previously described polymerase chain reaction (PCR) methods were used to screen for plasmid-encoded pA genes . blaCTX-′)-Ib-cr .In order to assess the closeness of clonal relations between isolates at the local level, repetitive extragenic palindromic-PCR (REP-PCR) and enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR) were performed with REP-1R, REP-2 T and ERIC-2, respectively, as described previously . StrainsE. coli strains was performed as recommended (http://mlst.ucc.ie/) on one strain randomly selected for each of the identical REP- and ERIC-PCR banding patterns. All isolates of ST131 were analysed by duplex PCR targeting the pabB and trpA genes to determine whether the isolate belonged to the O25b-ST131 group [Multilocus sequence typing of E. coli K12 J5 resistant to sodium azide as the recipient strain [E. coli DH10B  by electroporation according to the manufacturer’s instructions (Bio-Rad). Transformants were incubated at 37°C for 1.5 h and then selected on Drigalski agar (Bio-Rad) supplemented with 2.5 μg/mL cefotaxime. Transconjugants and transformants were tested for ESBL production followed by PCR amplification of the ESBL and plasmid-mediated quinolone resistance genes, as well as by plasmid replicon typing.Resistance transfer experiments were carried out on one randomly selected strain that was representative of the resistance gene content of each REP- and ERIC-PCR banding pattern. Conjugations were performed on solid media with t strain . TranscoPCR-based replicon-typing analysis was performed as described previously . Plasmidt test were used to compare categorical and continuous variables in univariate analysis, respectively. We considered p values < 0.05 to indicate significant associations.Microsoft Access 2003 was used for data entry, and Stata version 11 for statistical analysis. The chi-squared test and Student’s


Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity  on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.Hainantoxin-IV (HNTX-IV) from the venom of the spider  Selenocosmia hainana [2. The toxin can inhibit TTX-S voltage-gated sodium channels with an IC50 value of 34.0 nM in adult rat dorsal root ganglion (DRG) neurons. Because of its compact ICK motif and robust activity, HNTX-IV can act as a potential scaffold for drug design. HNTX-IV was traditionally obtained through natural sources [HNTX-IV is a 35 amino-acid neuronal toxin with an inhibitory cystine knot (ICK) motif isolated from the venom of the Chinese bird spider  hainana . The seqE. coli was established in this report. In our study, GST (glutathione S-transferase) combined with SUMO was utilized to promote the solubility and folding of rHNTX-IV. The GST fusion tag is widely used to promote the solubility and folding of recombinant proteins [To obtain adequate HNTX-IV for the further study of structure-activity relationships and exploration of its pharmaceutical applications by molecular engineering, an efficient, simple and cost-effective strategy for the functional production of HNTX-IV in proteins ,5. SUMO,proteins –9 for itproteins . The funFurthermore, an affinity-chromatography-independent purification strategy was developed by introducing TCA precipitation to purify rHNTX-IV peptides. Another improvement is that a ligation-independent RF (restriction free) cloning method  was adopAll animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Hunan Normal University.E. coli DH5α competent cells were purchased from TaKaRa . The BL21(DE3) strain, Rosetta strain, and pET-43a (+) vector were purchased from Novagen . The SHuffle strain was from NEB . The cDNA of HNTX-IV [ HNTX-IV  and GST-GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCCCCTATACTAGGTTATTG-3ʹ (forward primer); 5ʹ-GATGGTGATGGTGATGACCAGAACCACTAGTAGTCACGATGCG GCCGCTC-3ʹ (reverse primer). The underlined sequences of the forward primer and the reverse primer were complementary to the 5ʹ end and the 3ʹ end of the insertion or replacement point of the recipient vector pET-43a (+) with a Tm value of 80°C, respectively. The italicized portions were complementary to the 5ʹ end and the 3ʹ end of GST with Tm value of 60°C, respectively. The resultant construct was verified by sequencing and named pET-GST. Similarly, the SUMO sequence was cloned into the pET-GST vector downstream of GST by the primer pair: 5ʹ-TCCGGGAGCTCGTGGATCCGAATTCTCTGACTCTGAAGTTAACCAGGAAG-3ʹ (Sumo—forward); 5ʹ-CGATGGTACCGTCGACGTCCTGCAGACCACCGATCTGTTCACGGTGAG-3ʹ (Sumo-reverse). The construct was verified by sequencing and named pET-GS. Subsequently, the HNTX-IV gene with a stop codon was inserted behind the SUMO sequence by the primer pair: 5ʹ-TCGAAGCTCACCGTGAACAGATCGGTGGTGAGTGCTTAGGGTTTGG-3ʹ (HNTX-IV-forward); 5ʹ-CGATGGTACCGTCGACGTCCTGCAGTTATATTTCATATTTACACCACC-3ʹ (HNTX-IV-reverse). The resulting plasmid was named pET-GS-HNTX-IV. The map of the expression vector is shown in The RF cloning strategy was applied to construct the expression vector . The GSTE. coli BL21(DE3) competent cells. After incubating overnight, one colony from the LB plate was picked and inoculated into 5 ml LB medium with 100 μg/ml ampicillin sodium, which was incubated at 37°C in a shaker at 220 rpm until the OD600 reached 0.6. The 5 ml seed culture was transferred into 500 ml LB medium supplemented with 100 μg/ml ampicillin sodium, which was further incubated at 37°C in a shaker at 220 rpm until the OD600 reached 0.6. The culture was induced by 0.2 mM IPTG at 220 rpm and 37°C for 4 h. The induced bacteria were harvested by centrifugation at 4500 × g for 10 min. The pellet was suspended using 20 ml PBS buffer  complemented with 1 mM PMSF, 5% glycerol, 1 mM MgCl2, 10 mg/L RNaseA and 20 mg/L DNase I. Cells were lysed by sonication for 5 min at 20% amplitude using a Branson SONIFIER S-250D  in ice water. The lysates were centrifuged at 15000 × g and 4°C for 30 min. The supernatant was stored at 4°C for further purification. Induction at 16°C was carried out overnight to verify the optimal temperature for rHNTX-IV production.The pET-GS-HNTX-IV plasmid was chemically transformed into 2+- and Mg2+-free PBS. The GST-tagged protein was eluted by 2 resin-bed volumes of elution buffer .The supernatant was loaded onto a 5 ml pre-equilibrated glutathione resin column with a flow rate of 0.5 ml/min. Flow-through was applied to the column once again to ensure maximal binding. The resin was washed by 5 column volumes with CaE. coli, purified as described previously, and stored in 50% glycerol in -20°C [SUMO protease was produced in in -20°C ,10. 30 μTCA, acetonitrile and acetone were tested for the removal of tags and intact recombinant protein after SUMO protease cleavage individually. For TCA precipitation, 1/10 protein volume of 100% TCA was added into the digested protein solution, kept at 4°C for 10 min, and then centrifuged for 15 min at 4°C and 15000 × g. For acetone precipitation, 4 volumes of cold acetone were added and kept at -20°C for 10 min then centrifuged for 15 min at 4°C and 15000 × g. Acetonitrile precipitation was performed as previously described : the cleAfter sonication and centrifugation, instead of chromatography affinity, the supernatant of the induced cell lysates was subject to dialysis by 20 kDa cut-off dialysis tubing against Tris-HCl buffer  overnight. Subsequently, the dialyzed solution was digested by SUMO protease at 30°C for 1 h or 4°C overnight. Then, the cleaved peptides were extracted by TCA precipitation, acetonitrile precipitation or acetone precipitation. The supernatant was dialyzed against distilled water by 1 kDa dialysis tubing with three buffer changes and lyophilized for HPLC purification.2O) for 15 min to clear residual salts or small molecules. The eluted compounds were detected by UV absorption at 215 nm. The eluted peaks were collected and lyophilized then stored at -20°C.The dialyzed solution was lyophilized and dissolved in 500 μl distilled water and filtered through 0.45 μm micro-filter before loading. Then, the solution was applied to a Symmetry C18  column and eluted at a flow rate of 1 ml/min using a gradient of 5%–45% buffer B (0.1% TFA in acetonitrile) over 35 min after washing with buffer A . Ionization was achieved by irradiation with a nitrogen laser (337 nm), with a 20-kV acceleration voltage. α-Cyano-4-hydroxy-cinnamic acid was used as the matrix.Molecular mass identification of rHNTX-IV was performed on a Voyager-DE2 asphyxiation and decapitated. DRG neurons were harvested from rats and collected in Dulbecco’s modified Eagle’s medium. The DRGs were treated with 20 U/ml protease for 20 min followed by 0.28 U/ml collagenase for 40 min. Neurons were dissociated in Ham’s F12 medium supplemented with 10% horse serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 3.0 mM L-glutamine. Cells were plated on glass coverslips coated with poly-L-lysine and maintained at 37°C in a 95% O2, 5% CO2 incubator for 24 h before electrophysiological recordings [Sprague-Dawley rats (30 days old) of both sexes were euthanized by COcordings .Ionic currents were recorded under the whole cell patch-clamp mode using an EPC-10 amplifier and the Pulse program (HEKA Electronics). Patch pipettes fabricated from borosilicate glass tubes using a P-97 puller (Sutter Instruments) were pulled to a resistance of 2.0–2.9 MΩ after heat-polishing. The membrane currents were usually filtered at 5 kHz and sampled at 20 kHz. Voltage errors were minimized using 60%–80% series-resistance compensation, and the capacitance artifact was canceled using the amplifier circuitry. Because the leak currents were relatively small and could be a nonlinear function of the voltage, no electronic compensation for the voltage-dependent leak current was used .2, 10 mM HEPES, 10 mM EGTA, 10 mM glucose, and 2 mM ATP (pH 7.2); and the bath solution contained 145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). Tetrodotoxin  was added to the bath solution when recording the TTX-Resistant currents from the DRG neurons. For recording calcium currents, the internal solution contained 110 mM Cs-methane sulfonate, 14 mM phosphocreatine, 10 mM HEPES, 10 mM EGTA, and 5 mM ATP-Mg (pH 7.3); and the external solution contained 10 mM BaCl2, 125 mM tetraethylammonium-Cl, 0.3 mM TTX and 10 mM HEPES (pH 7.4). To eliminate any influence of differences in osmotic pressure, all of the internal and external solutions were adjusted to 280±5 mOsmol/L with sucrose.On DRG neurons, for recording TTX-sensitive sodium currents, the pipette solution contained 145 mM CsCl, 4 mM MgClp<0.05 and calculated with the two-tailed Student’s t test. The data are presented as the mean ± SEM.Electrophysiological data were analyzed and displayed by Pulsefit (HEKA Electronics). Curve fitting and statistical analysis were performed with OriginPro 9.0 (OriginLab) and GraphPad Prism 5.0 software. The statistical significance was determined at 600 reaching 0.6 in approximately 3 h; compared with Rosetta (DE3), approximately 4 h; and SHuffle, more than 5 h (data not shown). Parameters including temperature and IPTG concentration were optimized to improve the target protein solubility and yield. Inductions at 37°C for 4 h and 16°C overnight were compared. As shown in The BL21(DE3) strain was selected as the expression host because of its rapid growth rate, with the ODThe recombinant GST-SUMO-rHNTX was routinely purified by GST affinity chromatography. However, the recovery efficiency was less than 10%, even though excessive glutathione resin was used .http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10002940.pdf.With respect to the low affinity of GST-SUMO-rHNTX-IV for the glutathione beads, GST-SUMO-rHNTX-IV in the crude cell extract was digested directly by SUMO protease without affinity purification. Given the robustness of the recombinant enzyme prepared in-house in various buffers and volumes (data not shown), the dialyzed lysates (20 ml) were completely digested by 300 μg SUMO protease at 4°C overnight. Because there were dramatic differences in hydrophobicity between the GST-SUMO tagged protein and rHNTX-IV, TCA precipitation (10% TCA), acetonitrile precipitation and acetone precipitation were tested for the removal of the GST-SUMO tag and unspecific proteins. The results showed that TCA precipitation and acetone precipitation can retrieve target peptides with high purity, while acetonitrile is less efficient . After TThe peptide supernatant after TCA precipitation could be easily purified by RP-HPLC . The finINa, and 10 μM rHNTX-IV inhibited TTX-S INa currents by more than 90% , indicating that the inactivation of the sodium channel was enhanced in the presence of rHNTX-IV. On the other hand, 100 nM rHNTX-IV showed little influence on the activation curve (V0.5 from −26.02 ± 0.51 to −27.34 ± 0.22 mV) (n = 5) (50 = 34 nM). Beyond that, additional experiments were performed to support the results. It was found neither native HNTX-IV  . With th HNTX-IV  nor rHNT HNTX-IV  could caSpider venoms represent a rich source for biologically active compounds, such as ion channel modulators , bioinseE. coli because of its high growth rate, low cost and labor efficiency [E. coli is not easy [One plausible approach to solve the problem is recombinant expression in ficiency . Howevernot easy , 22. TheIn our study, we found that fusion tags could be essential for the functional expression of disulfide bond-rich toxins. Additionally, the temperature has an obvious impact on the solubility of recombinant proteins. Because toxin peptides tend to aggregate into inclusion bodies, fusion tags such as NusA, GST, thioredoxin and SUMO are useful for solubility. However, improved solubility might not guarantee the proper folding of CKTs even if combined with the folding-assisted tags  . The scrin vitro manner, although the mechanism is unclear.In our study, by a combination of GST and SUMO, rHNTX-IV, a potent antagonist that acts at site 1 on tetrodotoxin-sensitive (TTX-S) sodium channels with 3 pairs of disulfide bonds, was functionally expressed in BL21(DE3). As is traditionally known, the reducing environment of the cytoplasm of BL21(DE3) is not preferable for disulfide-bond formation. However, as suggested by the identical molecular weight of rHNTX-IV and its theoretical value, and a similar activity with the native toxin, the disulfide bonds of rHNTX-IV indeed form. It is speculated that rHNTX-IV folded naturally in an The stability of the GST-SUMO fused protein seems to be promoted by GST because, in our experience, the His-SUMO tagged rHNTX-IV was prone to aggregate after elution.Affinity purification as a classical method can yield relative pure products. However, it could be costly for the large-scale production of recombinant peptides. Taking advantage of the hydrophobicity differentiation between the toxin peptides and proteins, purification strategies based on TCA precipitation, or solvent agent (acetonitrile or acetone) precipitation, were economical choices. Our study further supported this fact and suggested that TCA precipitation seems to be more effective than precipitation by organic solvents. This result may be because the cysteine knot toxins, which usually have small sizes and compact structures, are relatively resistant to denaturation from heat, acid/alkali, detergents, and so on .In conclusion, an efficient, simple and cost-effective strategy for rHNTX-IV expression is described in the article and is amenable to large-scale of rHNTX-IV production for further study of activity-structure relationships and pharmaceutical applications. The strategy also provides references for the large-scale production of active peptides with disulfide bonds.


TNFRSF13B mutations and the clinical correlates in an Italian cohort of 189 CVID, 67 IgAD patients, and 330 healthy controls to substantiate the role of TACI genetic testing in diagnostic workup. We found that 11% of CVID and 13% of IgAD carried at least one mutated TNFRSF13B allele. Seven per cent of CVID had monoallelic-mutations and 4% had biallelic-mutations. The frequency of C104R monoallelic-mutations was not higher than that found in healthy controls. Biallelic-mutations were exclusively found in CVID. CVID patients carrying monoallelic-mutations had an increased prevalence of lymphadenopathy, granulomata, and autoimmune cytopenias. CVID carrying biallelic-mutations had a low prevalence of autoimmunity in comparison with TACI wild-type CVID. Moreover, biallelic-mutated CVID had higher frequency of switched memory B-cells and higher IgM and IgA antibodies to polysaccharide antigens than TACI wild-type and monoallelic-mutated CVID. TACI-mutated IgAD patients had only monoallelic-mutations and did not display clinical difference from IgAD wild-type patients. In conclusion, TNFRSF13B genetic screening of antibody deficiencies may allow the identification of mutational patterns. However, as with counseling for risk assessment, geneticists should be aware that the interpretation of genetic testing for TACI mutations is difficult and the potential impact on clinical management is still limited.We assessed the prevalence of In the absence of genetic mutation(s) these criteria are based on clinical and immunological abnormalities, allowing a “possible” and not a “definitive” diagnosis. The clinical spectrum of PADs varies between different entities and even within the same entity, ranging from severe disease with a reduced life expectancy, to very mild or even asymptomatic forms. For CVID the clinical phenotype ranges from a poorly symptomatic one to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies ;[R72H], [I87N];[Y164X], [I87N];[A181E], [C104R];[C193X], [C104R];[A181E], [Y164X];[A181E], and ;[Y102X] (Twenty-one CVID patients (11.1%) carried at least one;[Y102X] . TNFRSF13B variant: 3 patients (4.7%) were sibs and had the heterozygous C104R mutation and 6 patients (9.4%) carried non-C104R monoallelic mutations. None of the IgAD patients had biallelic mutations ( TNFRSF13B. Two of them (0.9%) were heterozygous for the C104R variant and one (0.4%) was heterozygous for the I87N allele. No other monoallelic or biallelic missense mutations were found in healthy controls.Nine IgAD patients (13.4%) carried a singleutations . A total TNFRSF13B gene mutations was significantly higher in CVID compared to healthy controls (p < 0.0001), with the exception of monoallelic C104R mutations (p = ns)  (The frequency of(p = ns) . The fre, resp.) .Among PAD patients carrying variants, 25 were unrelated and 5 belonged to two different families: three sibs with IgAD were heterozygous for C104R and two sibs with CVID were heterozygous for I87N. In both cases the mutation was inherited from one unaffected parent.Seventeen different genetic alterations were observed, one of which has not been previously described: a novel nonsynonymous G190A variant found in an IgAD patient. The polymorphisms V220A  and P251L  were observed in the cohort of antibody-deficient patients at frequencies that were not significantly different from those observed in healthy control populations.All the nonsynonymous variants, with the exception of G190A, were present in dbSNP with a MAF ranging from <0.0001 to 0.005 . The posClinical details of 21 TACI-mutated CVID patients are reported in (a) Monoallelic TNFRSF13B Mutations. As already demonstrated [ TNFRSF13B heterozygous mutations had a higher prevalence of autoimmunity compared with patients with wild-type TNFRSF13B  ; in particular, patients with TNFRSF13B C104R mutations had a higher prevalence of ITP , whereas patients with non-C104R TNFRSF13B mutations had a higher frequency of AHA . The overall prevalence of lymphoproliferative manifestations was similar among CVID groups . No difference was observed between TACI wild-type CVID and CVID carrying monoallelic TACI mutations on the frequencies of infective manifestations, including pneumonia, chronic diarrhea, and the presence of bronchiectasis.nstrated , 34 CVID= 0.005) . AutoimmD groups . However(b) Biallelic TNFRSF13B Mutations. No clinical differences were observed between CVID carrying TNFRSF13B biallelic mutations and wild-type. In particular, the prevalence of autoimmunity was similar between the groups  (ificant) . No diff(a) Monoallelic TNFRSF13B Mutations. A severe B-cell lymphopenia (<1%) was identified only in one TACI-mutated patient (C193X). The frequency of IgM−IgD−CD27+ switched memory B-cells was similar in CVID carrying monoallelic TACI mutations and in TACI wild-type (5.6%  ±  4.4 versus 3.4%  ± 5), while the frequency of IgD+IgM+CD27− naïve B-cells was lower . Among TACI-mutated patients, the group carrying a non-C104R heterozygous mutation had the lowest B naïve frequency compared to wild-type subjects (37.5%  ± 53). No differences were found in the frequency of CD21low cells.p = 0.04). In particular, the group carrying non-C104R heterozygous TACI mutations had the lowest Treg subset frequency . No difference was observed between CVID with TACI wild-type sequence and CVID carrying biallelic mutations on the frequency of other T cells subsets. Immunophenotype data are summarized in Overall, TACI-mutated patients had a lower frequency of Treg cells than CVID wild-type subjects .Serum immunoglobulin levels at diagnosis were similar in TACI-mutated and TACI wild-type CVID . The gro(b) Biallelic TNFRSF13B Mutations. Biallelic TACI-mutated CVID had higher frequency of switched memory B-cells compared to TACI wild-type CVID patients  but similar frequency of naïve B-cells. Biallelic TACI-mutated CVID had also higher frequency of switched memory B-cells in comparison with patients with monoallelic TACI mutations . No differences were found in the frequency of other B-cell subsets (data not shown). Moreover, patients carrying two mutated TNFRSF13B alleles had higher IgA levels than TACI wild-type CVID . TNFRSF13B mutations had higher IgM anti-23-valent polysaccharide antigens compared to wild-type CVID patients  and compared to CVID carrying monoallelic TNFRSF13B mutations . The same was observed for specific IgA: CVID patients carrying biallelic mutations had higher prevaccination IgA anti-23-valent polysaccharide antigens compared to wild-type CVID .Due to the fact that all patients were under IgG replacement at the time of the study, we analyzed IgM and IgA antibodies to 23-valent polysaccharide antigens. At baseline, CVID patients carrying biallelicp = 0.05; 15 mU/mL ± 14, p = 0.05, resp.) while CVID carrying monoallelic-mutated or wild-type TNFRSF13B sequences had a mild but not significant increase of specific IgM  and of specific IgA .As expected, all CVID patients had a low vaccination response. Only biallelic-mutated patients had a significant increase in postvaccination IgM and IgA titers . As shown in  TNFRSF13B mutations, such as C104R and A181E, in B-cell tolerance [Despite consolidated evidence for a pathogenic role of specificolerance  and impaolerance , further TNFRSF13B genetic screening of hypogammaglobulinemic patients may allow the identification of novel mutational patterns, offering insight into the biological mechanisms underlying the pathogenesis of PADs. However, as with counseling for risk assessment, geneticists should be aware of the fact that the interpretation of genetic testing for TACI mutations is difficult and the potential impact on clinical management is still limited.In conclusion,


Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge  Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes including cell growth, differentiation, survival, and repair . MultiplAutophagy is a catabolic process that digests cellular contents within lysosomes ,6. AutopXestospongia exigua (Kirkpatrick) [trans-2,9-disubstituted 1-oxaquinolizidine rings predominantly maintain a trans-decaline-like conformation, unlike the cis-disubstituted rings, which adapt a cis-decaline-like conformer [Marine sponges are rich sources of bioactive, unique, and chemically diverse natural products . Xestosppatrick) . Chemicaonformer . Conformonformer . Bioactionformer , cytotoxonformer , antifunonformer , antimalonformer , as wellonformer . Xestosponformer ,16,17. Xonformer ,16,17.in vitro and to characterize the mechanisms associated with the anticancer activity of araguspongine C in breast cancer cells.Some marine-derived xestospongins and araguspongines were patented for modest antitumor activities in 1997 but little is known about their anticancer properties . Therefotrans-2,9-disubstituted 1-oxaquinolizidines, with mono and dihydroxy substitutions , N-oxides (araguspongines K and L), and monohydroxy and C-3′ methyl substitution (xestospongin B). Two day treatment of BT-474 cells resulted in antiproliferative activity and inhibition of breast cancer cell growth. The effect of oxaquinolizidine alkaloids treatment on breast cancer cells is shown in 50 values of 9.3 and 15.2 μM, respectively -oleocanthal in this assay was 5.0 µM, which was consistent with the reported value (~4.8 µM) validating the results of this study [Given the critical role of RTKs in controlling cell survival and death in response to external stimuli, we investigated the potential that araguspongine C-induced autophagic death to be associated with suppression of c-Met and/or HER2 signaling in BT-474 cancer cells. The Z-LYTE Kinase Assay-Tyr6 Peptide kit (Invitrogen) was used to assess the ability of araguspongines A and C to inhibit c-Met phosphorylation (activation) . In thes 19.9 μM A. On theis study . The remBT-474 is a HER2-overexpressing breast cancer cell line. Thus, further docking studies were conducted for araguspongine C on the crystal structure of HER2. Molecular docking study of araguspongine C on HER2 crystal structure (PDB: 3RCD ) suggestPI3K/Akt/mTOR signaling cascade is an essential pathway that is activated in response to ligand binding and activation of RTKs. Western blot studies showed that araguspongine C treatment caused a dose-dependent reduction of the levels p-PDK, p-Akt and p-mTOR in BT-474 cells A. In addN-oxide-containing araguspongines K and L due to the loss of one oxaquinolizidine nitrogen lone pair of electrons to form the N-oxide bond. Xestospongin B also showed modest activity, possibly due to different oxaquinolizidine ring geometry and stereochemistry. Chemically, araguspongine C has C-9,9′-dihydroxylated groups, unlike the C-9 monohydroxylated araguspongines A and B. The additional C-9'-hydroxyl was virtually proved to be the main binding and anchoring pharmacophoric group at the c-Met and HER2 kinase domains  (Demospongiae: Haplosclerida) and identified by spectral analyses [1H NMR and TLC analyses. Primary antibodies were purchased from Cell Signaling Technology , except for IP3 receptor primary antibody which was purchased from Santa Cruz Biotechnology, Inc. . Secondary antibodies were purchased from Cell Signaling Technology .All chemicals and reagents were purchased from Sigma-Aldrich , unless otherwise stated. Araguspongines A, C, K, L and xestospongin B were isolated from the Red Sea sponge analyses . A purit2 at 37 °C. Araguspongines and xestospongin B were first dissolved in a volume of DMSO to provide final 10 mM stock solutions which were used to prepare various concentrations of treatment media. Final concentration of DMSO was maintained as the same in all treatment groups within a given experiment and never exceeded 0.1%.The human breast cancer cell lines MDA-MB-231, MCF-7, BT-474, T-47D, and SKBR3 cells were purchased from ATCC . Breast cancer cell lines were maintained in RPMI-1640 media supplemented with 10% FBS, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin. All cell lines were maintained in a humidified atmosphere of 5% COViable cell count was determined using the 3--2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay . The opt4 cells per well (6 wells/group) in 96-well culture plates and maintained in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were divided into different treatment groups and given various treatments in RPMI-1640 medium containing 40 ng/mL HGF. Viable cell number after 48 h treatment was determined using the MTT assay.Cells were plated at a density of 1 × 10in Vitro Tumor Sensitivity Assay (Soft Agar Colony Formation) Kit , according to manufacturer’s protocol. Briefly, 50 μL of Base Agar Matrix Layer was dispensed into each well of a 96-well tissue culture plate. Cells (5 × 103) in 75 μL of Cell Suspension/Agar Matrix Layer were dispensed into each well. The cells were treated with 50 μL of culture medium containing DMSO or 10 μM of araguspongine C. After 8 days’ incubation, visible colonies were photographed under an inverted phase-contrast microscope.The colony-forming capacity of BT-474 breast cancer cells was assessed using the CytoSelect 96-Well Cells were treated according to the methods described previously . At the 6 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or araguspongine C treated with defined serum-free medium containing 40 ng/mL of HGF for 48 h. At the end of the experiment, cells in each treatment group were isolated with trypsin and then washed twice with ice cold PBS. Cells were then resuspended in 96 μL of ice-cold 1× Annexin V Binding Buffer. Afterwards, 1 μL Annexin V-FITC Conjugate and 12.5 μL Propidium Iodide (PI) Solution were added to each 96 µL cell suspension. The cells were then incubated for 10 min on ice in the dark. The cell suspension was then diluted to a final volume of 250 μL per assay with ice-cold, 1× Annexin V Binding Buffer. Dot plots were generated using CellQuest software , and they were divided into 4 quadrants . The LL quadrant shows cells negative for both annexin V and PI . The LR quadrant shows cells positive for annexin V, but negative for PI . The UL quadrant shows cells positive for PI, but negative to annexin V (dead), whereas the UR quadrant shows cells positive for both annexin V and PI (late apoptotic). All experiments were repeated at least three times.Induction of apoptosis was assessed by the binding of annexin V to phosphatidylserine, which is externalized to the outer leaflet of plasma membrane early during induction of apoptosis. Analysis of annexin V was determined using Annexin V-FITC Early Apoptosis Detection Kit . BT-474 cells were plated at a density of 5 × 10Cyto-ID is a proprietary reagent specifically labels autophagic vacuoles and co-localizes with light chain 3 (LC3). Cyto-ID Autophagy detection kit  was used according to the manufacturer’s protocol. The fluorescence was measured by a plate reader . Cyto-ID Green reagent staining showed the relative fluorescence intensity of cells was increased in a dose-dependent manner, indicating the occurrence of autophagy. Rapamycin was used as a positive control .Z-LYTE Kinase Assay-Tyr6 Peptide kit  was used to assess the ability of araguspongines A and C to inhibit c-Met phosphorylation. Briefly, 20 μL/well reactions were set up in 96-well plates containing kinase buffer, 200 μM ATP, 4 μM Z-LYTE Tyr6 Peptide substrate, 2500 ng/mL c-Met kinase and compound of interest as an inhibitor. After 1 h of incubation at room temperature, 10 μL development solution containing site-specific protease was added to each well. Incubation was continued for 1 h. The reaction was then stopped, and the fluorescent signal ratio of 445 nm (coumarin)/520 nm (fluorescein) was determined on a plate reader  which reflects the peptide substrate cleavage status and/or the kinase inhibitory activity in the reaction.in-silico experiments were carried out using Schrödinger molecular modeling software package installed on an iMac 27-inch Z0PG workstation with a 3.5 GHz Quad-core Intel Core i7, Turbo Boost up to 3.9 GHz, processor and 16 GB RAM .The The X-ray crystal structure of the human c-Met kinase domain; residues 1048–1350,  was retThe structure of each araguspongine was sketched in the Maestro 9.3 panel . The Lig Prep 2.3 module  of the Schrödinger suite was utilized to generate the 3D structure and to search for different conformers. The Optimized Potentials for Liquid Simulation  force field was applied to geometrically optimize the ligand and to compute partial atomic charges. Finally, at most, 32 poses per ligand were generated with different steric features for the subsequent docking studies.The prepared X-ray crystal structure of c-Met kinase domain was employed to generate receptor energy grids using the default value of the protein atomic scale (1.0 Å) within the cubic box centered on the cocrystallized ligand. After receptor grid generation, the prepared ligands were docked using the Glide 5.8 module  in extra precision (XP) mode .® version 18. A difference of P < 0.05 was considered statistically significant as compared to the vehicle-treated control group. The IC50 values (concentrations that induce 50% cell growth inhibition) were determined using non-linear regression curve fit analysis using GraphPad Prism software version 6.The results are presented as means ± SEM of at least three independent experiments. Differences among various treatment groups were determined by the analysis of variance (ANOVA) followed by Dunnett’s test using PASW statisticsin-silico docking analysis. Suppression of RTK activity was also linked to PI3K/Akt/mTOR pathway inhibition (This is the first study to comprehensively characterize and evaluate the anticancer properties of the oxaquinolizidine alkaloids in breast cancer models. Results showed that araguspongine C-induced suppression of BT-474 cancer cell growth is mediated by induction of autophagic cell death. Autophagic activity of araguspongine C was associated with downregulation of c-Met and HER2 RTKs and suppression of receptor activation . Inhibithibition . In addi


Sphingosine-1-phosphate (S1P) is a versatile lipid signaling molecule and key regulator in vascular inflammation. S1P is secreted by platelets, monocytes, and vascular endothelial and smooth muscle cells. It binds specifically to a family of G-protein-coupled receptors, S1P receptors 1 to 5, resulting in downstream signaling and numerous cellular effects. S1P modulates cell proliferation and migration, and mediates proinflammatory responses and apoptosis. In the vascular barrier, S1P regulates permeability and endothelial reactions and recruitment of monocytes and may modulate atherosclerosis. Only recently has S1P emerged as a critical mediator which directly links the coagulation factor system to vascular inflammation. The multifunctional proteases thrombin and FXa regulate local S1P availability and interact with S1P signaling at multiple levels in various vascular cell types. Differential expression patterns and intracellular signaling pathways of each receptor enable S1P to exert its widespread functions. Although a vast amount of information is available about the functions of S1P and its receptors in the regulation of physiological and pathophysiological conditions, S1P-mediated mechanisms in the vasculature remain to be elucidated. This review summarizes recent findings regarding the role of S1P and its receptors in vascular wall and blood cells, which link the coagulation system to inflammatory responses in the vasculature. Sphingosine-1-phosphate (S1P), a highly active lipid mediator, exhibits a broad range of cellular activities including proliferation, survival, adhesion, and migration . S1P is  in vitro and in vivo . Th. Th81].  ex vivo . Express ex vivo , suggest ex vivo . This ob in vitro platelet function testing such as light transmission aggregometry, S1P does not appear to function as a potent direct platelet agonist [The biological effects of S1P released from activated platelets in the vasculature include inhibition of platelet aggregation , angioge agonist .Due to lack of S1P lyase activity in platelets , S1P abuSince S1P represents an amphiphilic anion, its translocation across the plasma membrane supposedly does require active transport proteins. As mentioned above, several studies in various cell types point to the involvement of a transporter of the ABC family , 95. HowDuring vascular inflammation, monocytes secrete several proinflammatory cytokines and adhesion molecules, a response mechanism which facilitates recruitment and adherence to the inflamed and activated endothelium . ThrombiS1P is a recently recognized novel regulator also of monocyte functions , 105. HuS1P induced PAR-1 and PAR-4 mRNA and total protein expression in human monocytes and U937 cells in a concentration- and time-dependent manner, respectively. However, only PAR-4 cell-surface expression was increased significantly by S1P, whereas cell-surface PAR-1 remained unaffected. This response was associated with activation of the Akt, ERK1/2, and p38 pathway and induction of COX-2 but not COX-1. PAR-4-mediated induction of COX-2 was prevented by pharmacological inhibition of the PI3 kinase pathway and incubation of human monocytes with S1P resulted in an enhanced PAR-4-dependent chemotaxis response to thrombin. Thus, S1P enhances monocyte responses to thrombin via upregulation of PAR-4 protein and cell-surface expression, which promotes migration and COX-2 abundance. These studies establish a direct link between S1P receptor activation and regulation of thrombin receptor expression in human monocyte and the subsequent cellular responses to thrombin. This mechanism may facilitate monocyte recruitment to sites of vessel injury and inflammation .Taken together, complex (patho)physiological interactions between blood coagulation factors and S1P and their respective signaling receptors are being increasingly recognized see . This in


Systemic lupus erythematosus (SLE) is a chronic inflammatory disease mainly characterized by B cell hyperactivity. Glucocorticoid (GC) is widely used in SLE for its potent anti-inflammatory and immunosuppressive effects. Despite its important clinical efficacy, high-dose or long-term use of GC can cause severe side effects, such as osteoporosis, osteonecrosis, cataracts, hyperglycemia, coronary heart disease and cognitive impairment. Our early clinical studies have shown that Jieduquyuzishen prescription (JP) can effectively reduce the adverse effects and improve the curative effect of GC in the treatment of SLE. The BAFF/BAFF-R signaling pathway plays an important role in the development of SLE and has been regarded as a potential target for the therapy of SLE. In this study, we attempt to investigate the effect of JP on the BAFF/BAFF-R signaling pathway to explore the mechanism of JP in reducing the toxicity and enhancing the efficacy of GC. YAC-1 cells, isolated rat peripheral blood lymphocytes, polymorphonuclear neutrophils and spleen lymphocytes were treated with drug-containing serum. The results of RT-PCR, Western blot and dual-luciferase reporter gene assays indicate that either JP or GC can inhibit the mBAFF-induced up-regulation of BAFF, BAFF-R, Bcl-2, IL-10 and NF-κB in YAC-1 cells and WEHI-231 cells. Furthermore, MTS, flow cytometry and CFSE results reveal that the proliferation and survival of lymphocytes activated by mBAFF are suppressed by JP, GC and their combination. Contrary to GC, JP can reduce the apoptosis and raise the survival of polymorphonuclear neutrophils and can’t increase the apoptosis of the peripheral blood lymphocytes and spleen lymphocytes. Therefore, it is possible that JP can down-regulate the BAFF/BAFF-R signaling pathway as effectively as GC, which may result in the dosage reduction of GC, thus decreasing the toxicity and improving the efficacy of GC-based treatment of SLE. Artemisia annua L., rhizome of Cimicifuga heracleifolia Kom., dried aerial parts of Hedyotis diffusa Willd, et al, is widely used for SLE treatment in China and has been shown to have therapeutic effects [Systemic lupus erythematosus (SLE) is a generalized autoimmune disease featured by immunological dysfunction, involving hyperactivated B cells, abnormally activated T cells, and defective clearance of apoptotic cells and immune complexes ,2. A ser effects . More im effects –12, but Abnormal B-cell activation and differentiation is associated with the development of SLE. B cell-activating factor (BAFF) has been shown to be a powerful regulator of B cell biology. BAFF has a stimulatory effect on the peripheral B-cell population, and in vitro it can promote the survival of B cells. Excessive BAFF could lead to mature B-cell hyperplasia, driving autoimmune diseases. Overexpression of BAFF has been regarded as an important factor in the pathology of SLE, and levels of BAFF in serum were increased in some SLE patients. Thus, the symptoms of SLE may be improved by down-regulating the BAFF/BAFF-R signaling pathway ,14.In this study, we set out to investigate the effect of JP-treated rat serum on the BAFF/BAFF-R signaling pathway to explore the mechanism of JP in reducing toxicity and enhancing the efficacy of GC in the treatment of SLE. The mRNA levels of some key components in the BAFF/BAFF-R signaling pathway, including BAFF, BAFF-R, NF-κB, Bcl-2 and IL-10, were detected by RT-PCR. The protein expression of BAFF-R, NF-κB and phospho-NF-κB were detected by Western blot. The transcriptional activity of NF-κB was measured via a dual-luciferase reporter gene assay. Proliferation was analyzed using the MTS and CFSE methods, and apoptosis of the peripheral blood lymphocytes, polymorphonuclear neutrophils and spleen lymphocytes was evaluated by flow cytometry. The experiments revealed that both JP, GC and their combination can suppress the mBAFF-activated BAFF/BAFF-R signaling pathway in YAC-1 cells and WEHI-231 cells, inhibiting the proliferation and survival of YAC-1 cells and lymphocytes. These data suggest that JP can inhibit the BAFF/BAFF-R signaling pathway, which may be associated with the mechanism of the JP-mediated reduction of GC dosage for the treatment of SLE.The experimental protocol was approved by the Animal Welfare Committee of Zhejiang Chinese Medical University, Hangzhou, China (SYXK2008–0115). The animal housing facility is a barrier housing facility, and it is maintained in accordance with the national standards . Sprague-Dawley (SD) rats  were obtained from the Laboratory Animal Research Center of Zhejiang Chinese Medical University. YAC-1 cells and WEHI-231 were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.The raw herbal medicines for JP preparation were purchased from the Chinese Herbal Medicine Co., Ltd. of Zhejiang Chinese Medical University . RPMI-1640 culture medium (No. 11875093), fetal bovine serum , Lipofectamine 2000 (No. 11668) and Trizol reagent (No. 15596–026) were purchased from Invitrogen. The PrimeScript RT-PCR kit (DRR014S), TaKaRa Taq (DR001A) and 100 bp DNA Ladder (3422A) were purchased from TaKaRa company, Japan. Recombinant mouse BAFF  was purchased from R&D Systems. Dexamethasone  was purchase from Sigma, USA. The Bradford Protein Assay Kit (No. P0006) was purchased from Beyotime Inst. Biotechnology, China. The Dual-Luciferase reporter assay system kit (No. E1910) and Cell Titer 96 AQueous One Solution Cell Proliferation Assay  were purchased from Promega, USA. Rat peripheral blood lymphocyte separation medium kit (No. LTS1083) and rat polymorphonuclear neutrophil separation medium kit (No. LZS1091) were purchased from Tianjin Haoyang Biological Manufacture Co., Ltd., China. Cell Trace CFSE cell proliferation Kit (Life technologies C34554).BAFF-R (No. SC-19887) and NF-κB antibodies (No. SC-372) were purchased from Santa Cruz, USA. The pS536-NF-κB antibody (No. 3033) and GAPDH antibody (No. 5174) were purchased from Cell Signaling Technology. The goat anti-rabbit RDye 680 secondary antibody (No. 926–32221) and goat anti-mouse RDye 800 secondary antibody (No. 926–32210) were purchased from LI-COR Biosciences, USA.Artemisia annua L., rhizome of Cimicifuga heracleifolia Kom., dried aerial parts of Hedyotis diffusa Willd, prepared root of Paeonia veitchii Lynch, carapace of Trionyx sinensis Wiegmann, dried aerial parts of Centella asiatica (Linn.) Urban, fruit of Citrus medica Linn. var. sarcodactylis (Noot.) Swingle, rhizome of Glycyrrhiza uralensis Fisch, seed of Coix lacryma-jobi L.var.mayuen (Roman.) Stapf, prepared root of Rehmannia glutinosa (Gaert.) Libosch.. Traditional Chinese medicine of the above-mentioned herbs were crushed and mixed together at a ratio of 5:4:4:9:5:4:5:5:3:2. After soaking in water  for 1 h, the mixed herbs were boiled for 2 h for extraction. The residue was extracted again for another 2 h. The filtrates were collected, combined and concentrated to 0.5 g, 1.0 g, 1.5 g crude drug/mL.The JP formula used in the experiment was composed of 10 herbs, including dried aerial parts of Male SD rats weighing 200±20 g were divided into JP groups  and control groups. JP groups were administrated with different doses of JP or normal physiological saline via gastrogavage respectively, 5 mL/kg, twice a day, for 3 days. One hour after the last administration, the blood of the JP group and control group were sterilely collected separately through the celiac vein. After settling for 3–4 h at room temperature, the JP-treated rat serum and the blank serum were separated by centrifugation at 3000 r/min at 4°C for 20 min, and then stored at -70°C after inactivating at 56°C for 30 min.YAC-1 cells and WEHI-231 cells were cultured in RPMI-1640 containing 10% fetal bovine serum in a 5% CO2 incubator  at 37°C and rinsed twice with minimum essential medium (MEM)  before testing.After culture for 24 to 48 h, the YAC-1 cells were divided into 4 groups: 10% blank-control serum (C), 10% blank-control serum plus mBAFF (R), 10% blank-control serum plus mBAFF and DEX (G) and 10% moderate-dose JP-treated rat serum plus mBAFF (J).WEHI-231, a murine B lymphocyte, was used for the evaluation of dose-dependent effect of JP. WEHI-231 cells were divided into 5 groups treated with 10% blank-control serum group (C), 10% blank-control serum plus mBAFF group (R), 10% low-dose JP-treated rat serum plus mBAFF group (L), 10% moderate-dose JP-treated rat serum plus mBAFF group (M) and 10% high-dose JP-treated rat serum plus mBAFF group (H). Furthermore, the time-dependent effect of JP was investigated with moderate-dose JP-treated rat serum. WEHI-231 cells were treated with moderate-dose JP-treated rat serum for 3 h, 6 h, 12 h and 24 h independently.After culture for 1 h, the cells in groups which were demanded plus mBAFF were treated with mBAFF 0.1 μg/mL, and cells in groups which were demanded plus DEX were treated with DEX 40 ng/mL. Twenty-four hours later, the cells, protein and RNA were collected without rinsing for further assays.Female SD rats weighing 200±20 g were paunched to get spleens under 1% chloral hydrate anesthesia, and cut spleens into pieces, washing it with RPMI-1640 culture medium, centrifuged for 5min at 1500r/min after filtering it with 100 mesh nylon mesh, the supernatant was discarded, and the spleen cells were suspended with RPMI-1640 culture medium, then rat spleen lymphocytes were isolated and collected by using a rat peripheral blood lymphocyte separation medium Kit. Cells were divided into C, R, G, J and 10% moderate-dose JP-treated rat serum plus mBAFF and DEX group(G+J).Total RNA was extracted from the cell layer of each group with Trizol reagent. The quantity and purity of RNA were determined by measuring the absorbance at 260 and 280 nm using a spectrophotometer . The total RNA was reverse transcribed into complementary DNA (cDNA) with a cDNA synthesis kit and amplified in a PCR machine . The specific primers for the target genes and β-actin (synthesized by Shanghai Shenggong Co.) were used as described in A two-step PCR procedure was recommended as follows: pre-denaturation for 10 s at 94°C, 1 cycle; 94°C for 30 s, 54°C for 30 s and 72°C, 10 min, 30 cycles. The final products were identified by electrophoresis on a 1% agarose gel and analyzed with an automatic image analyzer. The mRNA level of β-actin was used as an internal control, and gene-specific mRNA expression was normalized against β-actin expression. Data are presented as mean±SD of triplicate measurementsTotal protein was extracted using Beyotime Lysis Buffer  and analyzed with a bicinchoninic acid (BCA) protein assay kit. The sample proteins were separated by electrophoresis on a 10% SDS-PAGE separating gel with the Bio-Rad electrophoresis system, then transferred to a polyvinylidene difluoride (PVDF) membrane. The transferred membranes were incubated with primary antibodies , then exposed to the corresponding secondary antibody (1:10000 dilution). The protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences) and the bands were quantitated with Odyssey Infrared Scanning system software. A GAPDH protein antibody was used as an internal control. Target protein expression was normalized against GAPDH protein expression. Data are presented as mean±SD of triplicate measurements.6 cells/ml. One hundred microliters of the solution(1 x 105 cells)were transferred into a 5 ml culture tube, then 5 μl of Annexin V-FITC and 5 μl of Propidium Iodide (PI) were added. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. Next, 400 μl of 1x Binding Buffer were added to each tube. The prepared samples were analyzed by flow cytometry (BECKMAN CYTOMICS FC 500) within 1 hour.The isolated peripheral blood lymphocytes, polymorphonuclear neutrophils and spleen lymphocytes were divided into four groups , spleen lymphocytes were divided into 5 groups . The cells were washed twice with cold PBS and then resuspended in 1 x Binding Buffer at a concentration of 1 x 10A double-parameter map was made showing FITC and PI fluorescence intensity. The cells were divided into four subgroups in the two-dimensional FCM map: left lower quadrant (B3), Annexin V–FITC–/PI– for the living cell group (LC); right lower quadrant (B4), Annexin V–FITC+/PI– for the viable apoptotic cell group (VA); right upper quadrant (B2), Annexin V—FITC+/PI+ cells for the non-viable apoptotic cell group (NVA); upper left quadrant (B1), AnnexinV—FITC–/PI+ for dead cells and other debris (DC).5 cells per well 1 hour prior to transfection. The NF-κB expression plasmid (0.6 μg per well), NF-κB firefly luciferase reporter plasmid (0.6 μg per well) and the internal control vector pRL-SV40 containing the Renilla firefly reporter gene (0.06 μg per well) were co-transfected into all cells using Lipofectamine 2000 transfection reagent. Twelve hours post-transfection, the cells were divided into 4 groups  as described in the previous section. Twenty-four hours later, the transfected cells were lysed and the fluorescence intensity of firefly and Renilla luciferase in the cell lysates was measured by a dual-luciferase reporter assay system. The transcriptional activity of NF-κB in the above 4 groups was expressed as the ratio of firefly to Renilla luciferase abundance (Fluc/Rluc).Experimental constructs were transfected into YAC-1 cells using a liposome-mediated method and Lipofectamine 2000 . YAC-1 cells with 500 μL fresh culture medium were plated on 24-well plates at a density of 1 × 10YAC-1 cells were seeded at a density of 2,500 cells per well in 96-well plates and allowed to grow to 80% confluency. After 24 hours, the medium was replaced and the cells were divided into 4 groups. The cells were cultured with 10% blank-control serum , or 10% JP-treated rat serum (group J). After treatment with mBAFF (group R and J) or mBAFF plus DEX (group G) for 24 h, MTS (20 μL/well) was added.Peripheral blood lymphocytes and polymorphonuclear neutrophils from 6 female SD rats were isolated and collected by using a rat peripheral blood lymphocyte separation medium kit and a rat polymorphonuclear neutrophil separation medium kit. The cells were also divided into four groups  to observe proliferation. The cells were incubated at 37°C for 4 h after the addition of MTS (20 μL/well). The absorbance was subsequently measured at 490 nm using an automatic microplate reader (Thermo Fisher Varioskan Flash 3001).7 cells/ml. Then 5 μl CFSE (5 mmol/L) was added into cells and its final concentration is 5 μmol/L. The cells were gently vortexed and incubated for 15min at room temperature in the dark. Next, the same volume of calf serum was added into cells for 10min to terminate reaction and then washed twice with PBS. CFSE labeled cells were collected and divided into 5 groups  and cultured with RPMI-1640 culture medium. 72 hours later, the cells were collected and washed with PBS twice, the proliferation dynamics of CFSE labeled cells were analyzed by flow cytometry (BECKMAN CYTOMICS FC 500) within 1 hour. The B region of the cells was showed as groups of proliferation cells.Rat spleen lymphocytes cells were washed twice with cold PBS and then resuspended with RPMI-1640 culture medium without serum at a concentration of 10t-test or Mann—WhitneyU test. Differences were considered statistically significant if the p value was less than 0.05. Statistical analysis was carried out by SPSS 16.0.All values were expressed as the mean ± standard deviation. One-Way ANOVA was used for comparisons between multiple groups (>2). Comparisons between 2 groups was done with p<0.05). The mRNA expression of BAFF, BAFF-R, NF-κB, Bcl-2 and IL-10 decreased significantly in the G group under the intervention of GC (p<0.05). A similar phenomenon was also found in the J group, indicating that JP plays a similar role as GC in regulating the mRNA expression of BAFF, BAFF-R, NF-κB, Bcl-2 and IL-10 . The protein expression levels of BAFF-R and NF-κB were significantly lower in the G group than in the R group (p<0.05), but the phospho-NF-κB level did not change significantly (p>0.05). Compared with the R group, the protein expression of BAFF-R, NF-κB and phospho-NF-κB decreased significantly in the J group . Compared with the R group, mRNA expressions of NF-κB, Bcl-2 and IL-10 decreased (p<0.05), but the BAFF-R did not change significantly in L group (p>0.05); mRNA expressions of BAFF-R, NF-κB, Bcl-2 and IL-10 decreased significantly in M and H groups (p<0.05). NF-κB and Bcl-2 had no significant difference between the L, M and H groups (p>0.05). BAFF-R and IL-10 was significantly lower in M and H groups than in L group (p<0.05), but BAFF-R and IL-10 did not change significantly between the M and H groups . Compared with the R group, mRNA expressions of BAFF-R, NF-κB decreased significantly in 3h group(p<0.05), and BAFF-R, NF-κB, Bcl-2 and IL-10 decreased significantly in 6h, 12h, 24h groups (p<0.05), but Bcl-2 and IL-10 did not changed significantly in 3h group (p>0.05). Bcl-2 in 12h group and IL-10 in 12h and 24h groups decreased significantly compared to 3h group(p<0.05), but BAFF-R and NF-κB had no significant difference in 3h, 6h, 12h groups compared to the 24h group . The transcriptional activity of NF-κB was reduced significantly in the G group upon the addition of GC to the culture (p<0.05). The same phenomenon was also observed in the J group . Compared to the R group, the proliferation was inhibited in both the G and the J groups (p<0.05). The influence of moderate-dose JP-treated rat serum on the proliferation of rat spleen lymphocytes was assayed using the CFSE method  was extracted into water. Yet, saline was used as vehicle control for rat gavage, and sera purified from the animals were used for cell culture experiments, which may affect the results of the experiments to some extent.


To evaluate whether the extra-cellular volume fraction (ECV) measured using cardiac MRI can detect myocardial tissue changes in dilated cardiomyopathy (DCM) without late gadolinium enhancement (LGE).Forty-one DCM patients and 10 healthy volunteers underwent pre- and post- T1 mapping using a modified Look-Locker inversion recovery (MOLLI) sequence, LGE, and cine MRI on a 3-T cardiac magnetic resonance (CMR) system. LGE-MR findings were used to divide DCM patients into two groups: Group A had no apparent LGE, and Group B had LGE apparent in at least one segment. The ECV of left ventricle (LV) myocardium (16 segments) was calculated in short-axis view as follows: ECV = [(ΔR1 of myocardium/ΔR1 of LV blood pool)] × (1 - hematocrit), where R1=1/T1, ΔR1 = Post-contrast R1 - Pre-contrast R1. The LV ejection fraction (LVEF) was obtained from cine MRI images. The mean myocardial ECV in LGE (-) segments in Group A+B was compared to that of controls. The mean myocardial ECV in Group A was compared to that of LGE (-) segments in Group B. The correlation between LV systolic function and the mean myocardial ECV of the whole myocardium was evaluated in all groups.Among the 41 DCM patients, 22 were in Group A, and 19 were in Group B. The mean ECV of DCM patents  was significantly higher (p < 0.001) than that of the control group . The ECV was inversely related to LVEF in Group A , Group B , and Group A+B .The ECV measured by MRI could be a useful parameter in evaluating diffuse myocardial changes in DCM patients.