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.

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.

Research Progress on a SARS-CoV-2 Vaccine in China

Although many countries have controlled the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through strict management, there are still many countries with record-breaking numbers of new cases. Therefore, it is very important to develop a vaccine that can cause wide cross reactivity in clinical trials. At present, more than 90 vaccines are entering clinical trials and progressing smoothly, including inactivated vaccines, adenovirus-vectored vaccines and other types of vaccines. Here, we review and summarize the efficacy and potential threats of a SARS-CoV-2 vaccine. We reviewed whole-virus vaccines, adenovirus-subunit vaccines and recombinant protein vaccines and discussed the positive and negative consequences of a SARS-CoV-2 vaccine. However, there are still heated debates on the mechanism, effectiveness, and breadth of protection. In conclusion, this study can predict the risk of new coronavirus outbreaks in the future by discussing the research and development status of new coronavirus vaccines in China and other countries. Looking to the future, it is important to mine the large amount of data generated in clinical trials of universal new coronavirus vaccines to ensure that these vaccine programs are equally useful in the face of new coronavirus mutations.

人MD-2/GST融合蛋白在大肠杆菌的表达

构建人髓样分化蛋白 2 (humanmyeloiddifferentiatonprotein 2 ,hMD 2 )与谷胱甘肽巯基转移酶 ( glutathione S transferase,GST)的融合蛋白(hMD 2 /GST)表达载体PGEX 4T 1/hMD 2 ,在大肠杆菌中融合表达hMD 2 /GST。以真核表达载体PEF BOS/hMD 2为模板 ,用PCR法在MD 2编码序列上下游引入酶切位点和终止密码子 ;将hMD 2编码序列克隆入原核表达载体PGEX 4T 1的相应酶切位点 ;用PCR和酶切筛选、鉴定重组质粒PGEX 4T 1/hMD 2 ,并对插入基因片断测序 ;重组质粒PGEX 4T 1/hMD 2转化大肠杆菌 ,经IPTG诱导后用SDS PAGE分析表达产物。结果PCR、酶切鉴定和序列测定证实MD 2编码序列正向插入原核表达载体PGEX 4T 1的相应酶切位点 ,片断与PCR扩增产物大小相同、序列无误、阅读框架正确 ;SDS PAGE证实hMD 2 /GST在大肠杆菌中表达 ,表达量约占菌体总蛋白的 3 0 %。说明成功构建了PGEX 4T 1/hMD 2载体 ,hMD 2 /GST融合蛋白在大肠杆菌中得到初步表达 ,为MD

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Plant-produced recombinant SARS-CoV-2 receptor-binding domain;an economical,scalable biomaterial source for COVID-19 diagnosis

The outbreak of the novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),spread rapidly causing a severe global health burden.The standard COVID-19 diagnosis relies heavily on molecular tests to detect viral RNA in patient samples;however,this method is costly,requires highly-equipped laboratories,multiple reagents,skilled laboratory technicians,and takes 3-6 hours to complete.To overcome these limitations,we developed a plant-based production platform for the SARS-CoV-2 receptor-binding domain as an economical source of detection reagents for a lateral-flow immunoassay strip(LFIA)which is suitable for detection of IgM/IgG antibodies in human samples.Further,we validated the plant-produced SARS-CoV-2 receptor-binding domain-based LFIA as a useful diagnostic tool for COVID-19.A total of 51 confirmed COVID-19 serum samples were tested using the LFIA,and the obtained results were consistent with those from polymerase chain reaction assays,while providing sensitivity and specificity of 94.1%and 98%,respectively.The developed LFIA is rapid,scalable,user-friendly,and relatively inexpensive with a simple test procedure,making it useful for the routine monitoring of COVID-19 in clinical settings.This study was approved on March 19,2020 by the Ethics Committee of the Faculty of Medicine,Chulalongkorn University(COA No.354/2020 and IRB No.236/63).

Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8^＋ T cells from a HLA-A2^＋ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION： The procedure is simple and efficient for generating peptide-MHC tetramers.

Binding of the SARS-CoV-2 spike protein to glycans

The pandemic of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a high number of deaths in the world.To combat it,it is necessary to develop a better understanding of how the virus infects host cells.Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate(HS)and sialic acid-containing glycolipids/glycoproteins.In this study,we examined and compared the binding of the subunits and spike(S)proteins of SARS-CoV-2,SARS-Co V,and Middle East respiratory disease(MERS)-Co V to these glycans.Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner and no binding with sialic acid residues was detected.Overall,this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells,and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.

SARS-CoV-2 spike protein and RNA dependent RNA polymerase as targets for drug and vaccine development: A review

The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.

Future of the current anticoronaviral agents: A viewpoint on thevalidation for the next COVIDs and pandemics

Despite the global decline in the severity of the coronavirus disease 2019 (COVID-19) cases, the disease stillrepresents a major concern to the relevant scientific and medical communities. The primary concern of drug scientists,virologists, and other concerned specialists in this respect is to find ready-to-use suitable and potent anticoronaviraltherapies that are broadly effective against the different species/strains of the coronaviruses in general, not only againstthe current and previous coronaviruses (e.g., the recently-appeared severe acute respiratory syndrome coronavirus 2“SARS-CoV-2”), i.e., effective antiviral agents for treatment and/or prophylaxis of any coronaviral infections, includingthose of the coming ones from the next species and strains (if any). As an expert in this field, I tried, in this up-to-dateperspective “viewpoint” article, to evaluate the suitability and applicability of using the currently-availableanticoronaviral agents for the next coronavirus diseases (COVIDs) and coronaviral pandemics, highlighting the mostimportant general guidelines that should be considered in the next pandemics from the therapeutic points of view.

高产信号分子AI-2乳酸菌的筛选及其Pfs基因的克隆和表达

【目的】从锡盟地区酸马奶酒分离的乳酸菌中筛选出高产信号分子自体诱导物2(Autoinducer-2,AI-2)的乳酸菌,通过优化其重组蛋白Pfs的诱导条件体外合成信号分子AI-2。【方法】利用生物学发光法对不同乳酸菌产信号分子AI-2的产量进行比较,以高产信号分子AI-2乳酸菌基因组DNA为模板,扩增其S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs)基因,构建原核表达载体。利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行重组蛋白的诱导表达,通过优化培养基、诱导温度、诱导前菌体密度、IPTG浓度以及诱导时间得到高表达的Pfs蛋白,使其与底物作用最终体外合成信号分子AI-2。【结果】10株乳酸菌均可产信号分子AI-2,其中屎肠球菌8-3分泌信号分子AI-2的产量明显高于其他菌株;重组蛋白的最佳诱导条件为:选取SOC(Super optimal broth with catabolite repression)作为诱导表达培养基,菌液OD600为0.5–0.7时加入终浓度为0.1 mmol/L的IPTG,37°C诱导12 h;利用最优诱导条件获得了浓度为4.08 g/L的纯化Pfs蛋白,体外合成了信号分子AI-2。【结论】酸马奶酒中分离出的10株乳酸菌均可产生信号分子AI-2,且屎肠球菌8-3可通过Pfs基因的作用生成信号分子AI-2。

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus

OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P

Healing of chronic cutaneous wounds by topical treatment with basic fibroblast growth factor

OBJECTIVE: To evaluate the safety and efficacy of topical application of recombinant bovine basic fibroblast growth factor (rbFGF) on the healing of chronic cutaneous wounds. METHODS: Twenty-eight patients with thirty-three chronic cutaneous wounds resulting from trauma, diabetes mellitus, pressure sore and radiation injuries were enrolled in this prospective, open-label crossover trial. Prior to treatment with rbFGF, all wounds failed to heal with conventional therapies within 4 weeks. All wounds were locally treated with rbFGF at a dose of 150 AU/cm(2). Healing time and the quality of wounds were used to evaluate the efficacy of the treatment. RESULTS: Healing of all chronic wounds was expedited. During the study, eighteen wounds completely healed within 2 weeks, four healed within 3 weeks, and another eight completely healed within 4 weeks. Only three wounds failed to heal within 4 weeks, but healed at 30, 40 and 42 days after treatment with rbFGF. Thus, compared with conventional therapies, the effective rate of rbFGF treatment within 4 weeks was 90.9%. Histological assessment showed more abundant capillary sprouts or tubes and that fibroblasts were differentiated in wounds treated with rbFGF. No adverse side effects related to basic fibroblast growth factor were observed. CONCLUSIONS: Our results indicate that rbFGF could be used to accelerate healing in chronic wounds. It is our belief that this may be a more effective method of chronic wound management.