Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

The importance and patterns for humanities education of Chinese ophthalmology residency

At this point in time,the importance of medical humanities education has not been fully understood during the standardized training of residents in China.The study of medical humanities is an important facet of ophthalmology residency that needs to be strengthened.During the standardized training of ophthalmology residents,not only the medical sciences should be strengthened,but also the nature and practice of medical humanities knowledge should be enhanced in multiple aspects.Offering medically-relevant literature,history,philosophy and other courses,simulating real medical activities,being enthusiastic in popular medical science and increasing the contents of the examination in medical humanities would all be areas that would further advance the ophthalmology residency.Along with medical science education,residents will be led and trained on medical humanities as to build good medical humanistic spirit of patient care so that they may better serve patients.

Dynamic Changes of Phenotypes and Secretory Functions during the Differentiation of Pre-DCs to Mature DCs

The dynamic expression of cytokines and phenotypes during the differentiation process of dendritic cell precursors（pre-DCs） to mature dendritic cells（DCs） was investigated,and the effects of inflammatory stimulation with lipopolysaccharide（LPS） on DCs differentiation were understood.The differentiation of bone marrow cells isolated from Balb/c mice was induced to DCs in an 8-day cell culture system with RPMI-1640 complete culture medium containing 10% FBS,20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor（rm GM-CSF） and 10 ng/m L recombinant mouse interleukin-4（rm IL-4）.On the day 3,6 and 7 after culture,DCs were divided into non-LPS group and LPS group,given 500 ng/m L LPS for 24 h stimulation and no stimulation respectively.The expression levels of CD11c~＋,MHC-Ⅱ~＋,CD40~＋,CD80~＋ and CD86~＋ were detected by flow cytometry,and those of IL-2,IL-4,IL-10,IL-12 p70 and IFN-γ in the supernatant by ELISA.On the day 3 and 6 after culture,the expression of IL-2,IL-4,IL-10 and IFN-γ in DCs showed no significant differences between non-LPS group and LPS group,whereas the differences were significant at day 7.The expression levels of cytokines（for IL-2,IL-4,IL-10,IFN-γ and IL-12 p70：152.86±6.91,778.33±8.35,44.55±2.54,58.26±1.09 and 2423.00±57.21 pg/mL respectively） in LPS group were higher than those in non-LPS group,especially IL-12 p70 increased obviously at day 7.It was concluded that during the differentiation process of pre-DCs to mature DCs,LPS stimulates DCs producing large amounts of IL-12 p70 and Th1-type cytokines as compared with Th2-type cytokines,and day 7 may be a key time-point for DCs polarization.

Iron Aggravates the Depressive Phenotype of Stressed Mice by Compromising the Glymphatic System

Dear Editor,The pathophysiologic al mechanisms underlying mood disorders including major depressive disorder(MDD)remain to be fully characterized.Iron is a key component in the development of the central nervous system and iron deficiency has been linked to impairments of mood and cognition[1].

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD 112(PVRL2,nectin-2)and a membranebound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which stably expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFATTIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions between CD 112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability assays,screening,and development of novel TIGIT-targeted therapeutic antibodies.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment.To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine,the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared.The neutralization activities of Alpha,Beta and Gamma variants’spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant,which were constructed using pseudotyped viruses.Meanwhile,the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants.Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019.Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds,whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold.Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results.This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

New strategy for COVID-19 vaccination:targeting the receptor-binding domain of the SARS-CoV-2 spike protein

The unexpected outbreak of SARS-CoV-2 infection,with over 42.5 million confirmed cases and more than 1.1 million deaths globally,^(1)has posed a great threat to public health.Despite global efforts to control this viral infection,the pandemic is still surging.While facing such a serious,unprecedented situation,vaccines are likely the most promising method to control this newly identified coronavirus.A recent study from Yang et al.^(2)showed that a recombinant spike receptor-binding domain(RBD)protein of SARS-CoV-2 prepared from insect cells induced protective immunity in nonhuman primates,providing a feasible vaccine candidate for COVID-19 control(Fig.1).

A bivalent recombinant vaccine:a promising strategy against both SARS-CoV-2 variants and wild type of the virus

A recent study published in Medcomm by He et al.1 introduced protein subunit vaccines with wild-type and mutant S1 subunit(S1-WT and S1-Mut)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)to evaluate their immunoprotective efficacy.The team further analyzed the neutralization activity of bivalent vaccine composed of S1-WT and S1-Mut for SARS-CoV-2 and mutant strains to explore a universal protective vaccine.

Quality-control method for the determination of biological activity of engineered calcineurin subunit B

The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.

Optimized functional and structural design of dual-target LMRAP,a bifunctional fusion protein with a 25-amino-acid antitumor peptide and GnRH Fc fragment

To develop fusion protein of a GnRH Fc fragment and the integrin targeting AP25 antitumor peptide for GnRH receptor-expressing cancer therapy.The LMRAP fusion protein was constructed.A transwell invasion assay was performed.The gene mRNA and protein levels of GnRHR-I,α5β1,andαvβ3 in different cancer cell lines were assessed.Cell proliferation was measured using a cell counting kit-8.An antagonist assay was performed on GnRH receptors.Anti-tumor activity was evaluated with a mouse xenograft tumor model.Immunohistochemistry(IHC)was applied to detect CD31 and CD34 expressions.Pharmacokinetic characteristics were determined with an indirect competition ELISA.The developed bifunctional fusion protein LMRAP not only inhibited HUVEC invasion,but also inhibited proliferation of GnRHR-I,α5β1,and high expression cancer cells.The IC50 for LMRAP in the GnRH receptor was 6.235 x 10-4 mol/L.LMRAP significantly inhibited human prostate cancer cell line22 RV1 proliferation in vivo and in vitro.LMRAP significantly inhibited CD31 and CD34 expressions.The elimination half-life of the fusion protein LMRAP was 33 h in rats.The fusion protein made of a GnRH Fc fragment and the integrin targeting AP25 peptide retained the bifunctional biological activity of GnRHR blocking,angiogenesis inhibition,prolonged half-life and good tolerance.

Epstein-Barr virus negative primary hepatic leiomyoma: Case report and literature review

Primary hepatic leiomyoma is a neoplasm of mesen-chymal origin and occurs only rarely. Secondary to benign smooth muscle proliferation, it is usually found in adult women and is associated with Epstein-Barr virus (EBV) infection. Here, we report the 29 th case of primary hepatic leiomyoma with its unique features related to diagnosis, treatment and developmental biology. A 48-year-old man, with an immunocompromised status, complained of pain in the upper quadrant of the abdomen. Serological analysis indicated no presence of hepatitis virus, no human immunodeficiency virus, and no EBV infection. The levels of α-fetoproteinand carcinoembryonic antigen were normal. A mass was detected in segment Ⅲ of the hepatic lobe by ultrasonography and an abdominal computed tomography scan. Endoscopy had negative findings. Exploratory laparotomy found no existing extrahepatic tumor and left lateral lobectomy was performed. Pathological examination showed the mass to be a typical leiomyoma. The cells were positive for α-smooth muscle actin and desmin, and negative for the makers of gastrointestinal stromal tumor (GIST), including CD117, CD34 and DOG1 (discovered on GIST1). In situ hybridization revealed negative status for EBV-encoded small RNA. After left lateral lobectomy, the patient was not given chemotherapy or radiotherapy. During a 2-year follow- up, no sign of local recurrence or distant metastasis was observed. In conclusion, we report a rare case of primary hepatic leiomyoma in a male patient without EBV infection. Hepatic resection was curative. This case presents data to expand our knowledge concerning the complex and heterogeneous nature of primary liver leiomyoma, indicating that EBV infection is important but neither necessary nor sufficient for the development of primary liver leiomyoma.

PRELIMINARY DNA IDENTIFICATION FOR THE TSUNAMI VICTIMS IN THAI-LAND

The 2004 Southeast Asia Tsunami killed nearly 5,400 people in Southern Thailand, including foreign tourists and local residents. To recover DNA evidence as much as possible from the seriously decomposed bodies, we explored procedures of sample preparation from both bone and tooth samples as well as both mitochondrial and nuclear markers. Despite having failed to recover enough DNA for nuclear marker typing, we succeeded in obtaining fully informative results for mitochondrial markers （HV1 and HV2） from 258 tooth samples with a success rate of 51% （258/507）. Using an organic DNA extraction method coupled with an ultrafiltration step, we obtained 16 STR （including 13 CODIS loci, one sex discrimination locus, and two Identifiler loci） profiles for 834 samples with a success rate of 79% （834/1,062）. In addition, by comparing the allelic frequencies between the typed samples as a group and other index populations, we conclude that the Thai tsunami victims are a combined group of several populations. Our results provide valuable evidence and protocols for the future forensic practice.

SARS-CoV-2 spike-specific TFH cells exhibit unique responses in infected and vaccinated individuals

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb)and B cell differentiation are tightly regulated by T follicular help (T_(FH)) cells. However, the longevity and functional role of T_(FH) cellsubsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection andinactivated vaccine elicited both spike-specific CXCR3^(+) T_(FH) cell and CXCR3^(-) T_(FH) cell responses, which showed distinct responsepatterns. Spike- specific CXCR3^(+) T_(FH) cells exhibit a dominant and more durable response than CXCR3^(-) T_(FH) cells that positivelycorrelated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3^(+) T_(FH) cell subset inducedby two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3^(+) T_(FH) cellshave a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3^(-) T_(FH)cells. In conclusion, the persistent and functional role of spike-specific CXCR3^(+) T_(FH) cells following SARS-CoV-2 infection andvaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. Thefindings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immunememory.

Spirulina platensis aqueous extracts ameliorate colonic mucosal damage and modulate gut microbiota disorder in mice with ulcerative colitis by inhibiting inflammation and oxidative stress

Ulcerative colitis(UC)is a chronic and recurrent inflammatory bowel disease(IBD)that has become a major gastroenterologic problem during recent decades.Numerous complicating factors are involved in UC development such as oxidative stress,inflammation,and microbiota disorder.These factors exacerbate damage to the intestinal mucosal barrier.Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products.However,there have been few studies on the treatment of UC using S.platensis aqueous extracts(SP),and the underlying mechanism of action of SP against UC has not yet been elucidated.Herein,we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier.Dextran sulfate sodium(DSS)was used to establish a normal human colonic epithelial cell(NCM460)injury model and UC animal model.The mitochondrial membrane potential assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and staining with Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model.Moreover,hematoxylin and eosin(H&E)staining,transmission electron microscopy(TEM),enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qPCR),western blot,and 16S ribosomal DNA(rDNA)sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice.In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury.SP also significantly reduced the excessive generation of intracellular reactive oxygen species(ROS)and prevented mitochondrial membrane potential reduction after DSS challenge.In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group.Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins(TJs)post-SP treatment.SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon.Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction,inhibition of DSS-induced ROS production,and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.

Biohazard levels and biosafety protection for Mycobacterium tuberculosis strains with different virulence

Tuberculosis(TB)is a chronic infectious respiratory disease caused by Mycobacterium tuberculosis(MTB),which seriously endangers human health.TB is transmitted through the respiratory tract and poses great biosafety risks to laboratory technicians and staff,through direct or indirect contact with MTB.Therefore,the comprehensive application of standard laboratory operating procedures,safety equipment,contingency plans,and other laboratory biosafety measures not only ensures the safe handling of potentially infectious microorganisms by laboratory workers but also strives to prevent the accidental exposure of potentially harmful pathogens to larger communities.In this article,we intend to discuss topics as biosafety risk assessments suitable for handling the biohazard levels of MTB strains,and the development of corresponding biosafety protection measures necessary for the biosafety of MTB testing and related research that will be implemented at different levels of laboratory and epidemiological situation.

Structural and functional insights into MCR-2 mediated colistin resistance

Dear Editor,The spread of antibiotic resistance genes among bacteria is a serious and growing threat to global health (Peleg and Hoopet,2010).One emerging case is transferable genes found in bacteria-encoding enzymes resistant to colistin.Colistin is a cationic polypeptide antibiotic with broadspectrum activity against Gram-negative bacteria through interactions with the lipid A moiety of bacterial lipopoly-saccharide(LPS),subsequently destroying the cell envelope.During late 2015,a plasmid-mediated gene,mcr-1,was first reported from Escherichia coli(E.coli)in China to confer resistance to colistin(Liu et a1.,2016),and detected world-wide soon afterwards(Gao et a1.,2016;Hu et a1.,2016).