Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).

Differential expression and regulation of integral membrane protein 2b in rat male reproductive tissues

Aim： To examine the expression and regulation of integral membrane protein 2b （Itm2b） in rat male reproductive tissues during sexual maturation and under different treatments by in situ hybridization. Methods： Testis, epididymis, and vas deferens were collected on days 1-70 to examine Itm2b expression during sexual maturation. To further examine the regulation of Itm2b, adult rats underwent surgical castration and cryptorchidism. Ethylene dimethane sulfonate and busulfan treatments were carried out to test the regulation of Itm2b after destruction of Leydig cells and germ cells. Results： In testis, Itm2b expression was moderately detected in the adluminal area of seminiferous cords on days 1-10, and detected at a low level in the spermatogonia on days 20 and 30. The Itm2b level was markedly increased in Leydig cells from day 20 to day 70. In epididymis and vas deferens, Itm2b was detected from neonate to adults, and the signal gradually increased in accordance with sexual maturation. Itm2b expression was significantly downregulated in epididymis and vas deferens of castrated rats, and strongly stimulated when castrated rats were treated with testosterone. Cryptorchidism led to a significant decline of Itm2b expression in testis and caput epididymis. Itm2b expression in epididymis and vas deferens was significantly decreased after the Leydig ceils were destroyed by ethylene dimethane sulfonate. Busulfan treatment produced no obvious change in Itm2b expression in epididymis or vas deferens. Conelusion： Our data suggested that Itm2b expression is upregulated by testosterone and might play a role in rat male reproduction.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2

Coronavirus disease 2019(COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2), has spread rapidly worldwide with high rates of transmission and substantial mortality. To date, however, no effective treatments or enough vaccines for COVID-19 are available. The roles of angiotensin converting enzyme 2(ACE2) and spike protein in the treatment of COVID-19 are major areas of research. In this study, we explored the potential of ACE2 and spike protein as targets for the development of antiviral agents against SARS-Co V-2. We analyzed clinical data, genetic data, and receptor binding capability.Clinical data revealed that COVID-19 patients with comorbidities related to an abnormal reninangiotensin system exhibited more early symptoms and poorer prognoses. However, the relationship between ACE2 expression and COVID-19progression is still not clear. Furthermore, if ACE2 is not a good targetable protein, it would not be applicable across a wide range of populations. The spike-S1 receptor-binding domain that interacts with ACE2 showed various amino acid mutations based on sequence analysis. We identified two spike-S1 point mutations(V354 F and V470 A) by receptorligand docking and binding enzyme-linked immunosorbent assays. These variants enhanced the binding of the spike protein to ACE2 receptors and were potentially associated with increased infectivity. Importantly, the number of patients infected with the V354 F and V470 A mutants has increased with the development of the SARS-Co V-2 pandemic. These results suggest that ACE2 and spike-S1 are likely not ideal targets for the design of peptide drugs to treat COVID-19 in different populations.

Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

Highly Sensitive Poly-N-isopropylacrylamide Microgel-based Electrochemical Biosensor for the Detection of SARS-COV-2 Spike Protein

Objective Late 2019 witnessed the outbreak and widespread transmission of coronavirus disease 2019(COVID-19),a new,highly contagious disease caused by novel severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Consequently,considerable attention has been paid to the development of new diagnostic tools for the early detection of SARS-CoV-2.Methods In this study,a new poly-N-isopropylacrylamide microgel-based electrochemical sensor was explored to detect the SARS-CoV-2 spike protein(S protein)in human saliva.The microgel was composed of a copolymer of N-isopropylacrylamide and acrylic acid,and gold nanoparticles were encapsulated within the microgel through facile and economical fabrication.The electrochemical performance of the sensor was evaluated through differential pulse voltammetry.Results Under optimal experimental conditions,the linear range of the sensor was 10-13-10-9 mg/m L,whereas the detection limit was 9.55 fg/mL.Furthermore,the S protein was instilled in artificial saliva as the infected human saliva model,and the sensing platform showed satisfactory detection capability.Conclusion The sensing platform exhibited excellent specificity and sensitivity in detecting spike protein,indicating its potential application for the time-saving and inexpensive detection of SARS-CoV-2.

Bioabsorbable Barrier Membrane Combined with rhBMP-2 Improved Bone Formation in an Experimental Model of Compromised Healing But Was Not Superior to rhBMP-2 Alone

Objective: Bioabsorbable barrier membranes placed over alveolar ridge bone defects are routinely used in dental surgery to promote bone formation. Combining these osteoconductive membranes with osteoinductive Bone Morphogenetic Proteins could prove useful in long bone fracture treatment. The hypothesis was tested in a clinically relevant model of compromised healing. Methods: Four groups of 8 rabbits underwent unilateral mid-tibial osteotomy, excision of periosteum and endosteum, and plate fixation. One group had rhBMP-2 deposited between the bone ends and Membrane wrapped around the osteotomy, the second group had Membrane wrapped around the osteotomy, the third group had rhBMP-2 placed between the bone ends, and the fourth group received no additional treatment. Results: After 7 weeks, callus size and blood flow were significantly higher in the Membrane+rhBMP-2 group than in the rhBMP-2 treated group, but torsion to failure test showed no significant difference. Membrane treatment and no treatment led to non-union. Conclusion: Absorbable barrier membrane combined with rhBMP-2 enhances bone formation, but has no advantage to rhBMP-2 alone. Membrane alone wrapped around the osteotomy was unable to prevent non-union formation.

Extracorporeal membrane oxygenation for coronavirus disease 2019-associated acute respiratory distress syndrome:Report of two cases and review of the literature

BACKGROUND Coronavirus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus-2,is a worldwide pandemic.Some COVID-19 patients develop severe acute respiratory distress syndrome and progress to respiratory failure.In such cases,extracorporeal membrane oxygenation(ECMO)treatment is a necessary life-saving procedure.CASE SUMMARY Two special COVID-19 cases—one full-term pregnant woman and one elderly(72-year-old)man—were treated by veno-venous(VV)-ECMO in the Second People’s Hospital of Zhongshan,Zhongshan City,Guangdong Province,China.Both patients had developed refractory hypoxemia shortly after hospital admission,despite conventional support,and were therefore managed by VV-ECMO.Although both experienced multiple ECMO-related complications on top of the COVID-19 disease,their conditions improved gradually.Both patients were weaned successfully from the ECMO therapy.At the time of writing of this report,the woman has recovered completely and been discharged from hospital to home;the man remains on mechanical ventilation,due to respiratory muscle weakness and suspected lung fibrosis.As ECMO itself is associated with various complications,it is very important to understand and treat these complications to achieve optimal outcome.CONCLUSION VV-ECMO can provide sufficient gas exchange for COVID-19 patients with acute respiratory distress syndrome.However,it is crucial to understand and treat ECMO-related complications.

Membrane Proteins as Potential Colon Cancer Biomarkers: Verification of 4 Candidates from a Secretome Dataset

Colorectal cancer (CRC) is an important health issue in Taiwan. There were over ten thousand newly diagnosed CRC patients each year. The outcome of late stage CRC still remains to be improved, and tumor markers are expected to improve CRC detection and management. From a colorectal cancer cell secretome database, we chose four proteins as candidates for clinical verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), and tetraspanin-6 (TSPAN6), and tumor necrosis factor receptor superfamily member 16 (NGFR). Different groups of 30 CRC patients’ tissue samples collected from Chang Gung Memorial Hospital were analyzed by immunohistochemistry (IHC) for the four proteins, and the results were scored by pathologist. For all the four candidate proteins, marked differences of IHC score existed between tumor and adjacent non-tumor counterpart. However, there were only trends between higher protein expression levels and worse outcome. Three proteins (TROP2, TM9SF2 and NGFR) had trends between higher tissue expression and tumor stage or lymph node metastasis. Our study revealed that tissue expression of four proteins (TROP2, TM9SF2, TSPAN6, and NGFR) was markedly different between tumor and adjacent non-tumor counterparts. Overexpression of all these four proteins showed some trends with poorer survival.

<i>Morinda citrifolia</i>(Noni) Fruit Juice Inhibits SARS-CoV-2 Spike Protein Binding of Angiotensin-Converting Enzyme 2 (ACE2)

COVID-19 is a global pandemic that has claimed millions of lives. This disease is caused by a coronavirus, SARS-CoV-2, which requires the binding of its spike protein to angiotensin-converting enzyme 2 (ACE2) for infection of the host cell. Morinda citrifolia (noni) fruit juice has antiviral activity that involves enhancement of immune system function. SARS-CoV-2 spike-ACE2 interaction experiments were carried out to further investigate the antiviral properties of noni juice and its major iridoids. Noni juice inhibited binding by approximately 69%. Scandoside was the most active of the three iridoids evaluated, reducing average spike protein-ACE2 interaction by 79.25%. The iridoids worked synergistically towards inhibiting spike protein binding when assayed together, improving activity by more than 22% above the expected level. But the modest activity of the most abundant iridoid, deacetylasperulosidic acid, indicates that other phytochemicals (i.e. scopoletin, quercetin, rutin and kaempferol) are also involved. Our results suggest that the presence of several biological active phytochemicals in noni juice enhances resistance to SARS-CoV-2 by interfering with its ability to bind ACE2. This is a new and significant anti-viral mechanism of noni juice that does not directly involve its immunomodulatory properties.

Universal COVID-19 Vaccine Targeting SARS-CoV-2 Envelope Protein

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused over 382 million cases and over 2.7 million deaths globally as of 23 March 2021. By that date, at least 10 SARS-CoV-2 variants had emerged. The transmissibility and lethality of the variants are higher than those of the Wuhan reference strain. Therefore, a universal vaccine for the reference strain and all variants (present and future) is indispensable. The coronavirus envelope (E) protein is an integral membrane protein crucial to the viral lifecycle and the pathogenesis of coronaviruses. The SARS-CoV-2 E protein has a postsynaptic density protein 95/Drosophila disc large tumor suppressor/zonula occludens-1 (PDZ) binding motif (PBM), and its interaction with PDZ-domain-2 of the human tight junction protein may interrupt the integrity of lung epithelium. Furthermore, the SARS-CoV-2 E protein itself is a homopentameric cation channel viroporin, which may be involved in viral release. This protein is thus a potential target for the development of a universal COVID-19 vaccine, because of its highly conserved amino acid sequence. The variant mutations occur mainly in the spike protein, and conservation of E protein remained in most Variants of Concern (VOC). Only one of the extant VOC have mutations in the E protein that P71L mutation occurs in the South African variant 501Y.V2 (B.1.351). If a vaccine is designed to target E protein, two scenarios are possible: 1) SARS-CoV-2 maintains a highly conserved E protein amino acid sequence, rendering the virus consistently or permanently susceptible to the vaccine;or 2) the E protein mutates and new variants evolve accordingly. In scenario 2, the tertiary structure and function of the E protein homopentameric cation channel viroporin, PBM, or other aspects affecting pathogenicity would be attenuated. Either scenario would thus ameliorate the pandemic. I therefore propose that a vaccine targeting the SARS-CoV-2 E protein would be effective against the Wuhan reference strain and all current and future SARS-CoV-2 variants. Efforts to create E protein-based vaccines are ongoing. Further research and clinical trials are needed to realize this universal COVID-19 vaccine.

血清IL-2、sST2表达与特发性膜性肾病免疫抑制剂治疗反应性的相关性

目的探讨特发性膜性肾病患者血清白介素-2(IL-2)、可溶性生长刺激表达基因2蛋白(sST2)表达水平与免疫抑制剂治疗反应性的相关性。方法选取2020年1月至2022年10月于医院接受免疫抑制剂治疗的135例特发性膜性肾病患者,于入院时检测患者血清IL-2、sST2,并于治疗完成后测定24 h尿蛋白定量,依据患者治疗反应性分为缓解组与未缓解组。对比两组患者一般资料及入院时血清IL-2、sST2水平,采用点二列相关性分析血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性的关系,并绘制受试者工作特征(ROC)曲线评估血清IL-2、sST2水平预测特发性膜性肾病免疫抑制剂治疗反应性的价值。结果135例特发性膜性肾病患者中共有132例完成规律治疗,经免疫抑制剂治疗6个月后,101例患者疾病缓解,纳入缓解组,其余31例患者纳入未缓解组。未缓解组年龄、入院时肾功能分级、疾病分期、血清IL-2、sST2水平均高于缓解组,差异有统计学意义(P<0.05);点二列相关性分析显示,血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性不良风险呈正相关(r 1=0.428,P 1<0.001;r 2=0.344,P 2<0.001);绘制ROC曲线,结果显示,血清IL-2、sST2预测特发性膜性肾病免疫抑制剂治疗反应性不良的曲线下面积均>0.7,具有一定预测价值,且联合预测价值更高。结论血清IL-2、sST2表达水平与特发性膜性肾病患者免疫抑制剂治疗反应性密切相关,二者表达水平越高,治疗反应性越差,且联合检测可作为预测特发性膜性肾病患者免疫抑制剂治疗反应性的敏感指标。

溶酶体相关膜蛋白-2对系统性血管炎的诊断及治疗转归的临床价值

目的探讨溶酶体相关膜蛋白-2(Lysosomal associated membrane protein-2,LAMP2)在系统性血管炎(Systemic vasculitis,SV)的诊断和治疗转归中的临床价值。方法纳入2019年1月至2020年12月期间我科住院的SV患者58例、其他免疫性疾病58例、原发性高血压101例。收集三组患者的临床资料,使用ELISA方法检测三组患者的血清LAMP2水平。比较SV组与其他免疫性疾病组、原发性高血压组之间血清LAMP2水平的差异。对SV患者进行随访,比较活动期的SV患者治疗前后血清LAMP2水平变化。结果SV组中的血清LAMP2水平显著高于原发性高血压组[0.37(0.17-1.05)ug/mL vs.0.20(0.09-0.34)ug/mL,P<0.001]和其他免疫疾病组[0.37(0.17-1.05)ug/mL vs.0.23(0.08-0.43)ug/mL,P=0.002]。SV活动期患者血清LAMP2水平显著高于非活动期患者[活动期vs非活动期:0.78(0.25-2.50)vs 0.23(0.11-0.35)ug/mL,P=0.010]。活动期SV患者治疗后与治疗前相比血清LAMP2水平明显下降[0.53(0.30-2.34)ug/dl vs.0.22(0.17-0.42)ug/dl,P=0.019]。结论SV患者的血清LAMP2水平明显高于其他免疫疾病和原发性高血压患者,且血清LAMP2水平可以反映SV活动期的严重程度及治疗反应情况。血清LAMP2水平在SV的诊断、鉴别诊断、疾病评估及治疗等方面有很好的评估价值。

Structural basis of SARS-CoV-2 3CL^pro and anti-COVID-19 drug discovery from medicinal plants

The recent pandemic of coronavirus disease 2019(COVID-19)caused by SARS-CoV-2 has raised global health concerns.The viral 3-chymotrypsin-like cysteine protease(3CL^pro)enzyme controls coronavirus replication and is essential for its life cycle.3CL^pro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV).Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV.Therefore,herein,we analysed the 3CL^pro sequence,constructed its 3D homology model,and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds.Our analyses revealed that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19.

穿山龙总皂苷对膜性肾病大鼠肾组织M型PLA2R和IgG4表达的影响及其机制

目的 分析穿山龙总皂苷对膜性肾病大鼠肾组织M型磷脂酶A2受体(Phospholipase A2 receptor, PLA2R)和免疫球蛋白G亚型4(Immunoglobulin G4,IgG4)表达影响及可能机制。方法 将40只SPF级雄性SD大鼠按随机数字表法分为对照组、模型组、贝那普利组(10 mg/kg)、低和高剂量穿山龙总皂苷组(80 mg/kg、160 mg/kg),每组各8只。除对照组,其余4组采用Border法制备膜性肾病模型,造模成功后,贝那普利组灌胃给予贝那普利10 mg/(kg·d),低和高剂量穿山龙总皂苷组分别灌胃给予穿山龙总皂苷80 mg/(kg·d)、160 mg/(kg·d),对照组、模型组灌胃给予10 ml/(kg·d)生理盐水。连续给药4周后,检测24 h尿蛋白、白蛋白、血肌酐、血尿素氮、尿酸水平,HE染色观察肾脏病理改变,蛋白免疫印迹法检测肾脏中M型PLA2R、IgG4、磷酸化磷脂酰肌醇3-激酶(Phosphorylated phosphoinositide 3-kinase, p-PI3K)、磷酸化蛋白激酶B(Phosphorylated protein kinase B,p-AKT)、核因子E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)、血红素加氧酶(Heme oxygenase-1,HO-1)表达水平。结果 与模型组比较,贝那普利组、高剂量穿山龙总皂苷组白蛋白水平明显升高,血肌酐、血尿素氮、尿酸水平明显降低,差异均有统计学意义(P>0.05)。与模型组比较,贝那普利组、低剂量和高剂量穿山龙总皂苷组肾脏病理改变明显改善,24 h尿蛋白水平及肾脏中M型PLA2R、IgG4、p-PI3K、p-AKT表达水平明显降低,肾脏中Nrf2、HO-1表达水平明显增加,差异均有统计学意义(P<0.05)。结论 穿山龙总皂苷对膜性肾病大鼠的肾脏具有保护作用,其机制可能与降低PLA2R、IgG4表达,抑制PI3K/AKT通路,激活Nrf2/HO-1通路相关。

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Charge‑Transfer Resonance and Electromagnetic Enhancement Synergistically Enabling MXenes with Excellent SERS Sensitivity for SARS‑CoV‑2 S Protein Detection

The outbreak of coronavirus disease 2019 has seriously threatened human health.Rapidly and sensitively detecting SARSCoV-2 viruses can help control the spread of viruses.However,it is an arduous challenge to apply semiconductor-based substrates for virus SERS detection due to their poor sensitivity.Therefore,it is worthwhile to search novel semiconductor-based substrates with excellent SERS sensitivity.Herein we report,for the first time,Nb2C and Ta2C MXenes exhibit a remarkable SERS enhancement,which is synergistically enabled by the charge transfer resonance enhancement and electromagnetic enhancement.Their SERS sensitivity is optimized to 3.0×10^6 and 1.4×10^6 under the optimal resonance excitation wavelength of 532 nm.Additionally,remarkable SERS sensitivity endows Ta2C MXenes with capability to sensitively detect and accurately identify the SARS-CoV-2 spike protein.Moreover,its detection limit is as low as 5×10^−9 M,which is beneficial to achieve real-time monitoring and early warning of novel coronavirus.This research not only provides helpful theoretical guidance for exploring other novel SERS-active semiconductor-based materials but also provides a potential candidate for the practical applications of SERS technology.