A Single-chain Antibody for One-pot Fabrication of Luminescent Gold Nanoclusters and Rabies Virus Imaging in Cells

The fragile antibody leads to a great challenge as a scaffold to fabricate the luminescent metal nanoclusters using one-pot method.This study presents a stable single-chain anti-body(scFv57R-ATS)for the fabrication of luminescent gold nanoclusters(AuNCs@scFv57R-ATS)and a quick,sensitive rabies virus detection in living cells.In this paper,AuNCs@scFv57R-ATS was designed to specifically recognize antigen RV in modified HeLa cells,which promoted the demonstration of metal nanocluster fluorescent probes for antigen targeting and therapy.

Replication Kinetics of Coxsackievirus A16 in Human Rhabdomyosarcoma Cells

Coxsackievirus A16(CVA16),together with enterovirus type 71(EV71),is responsible for most cases of hand,foot and mouth disease(HFMD) worldwide.Recent findings suggest that the recombination between CVA16 and EV71,and the co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years.It is therefore important to further understand the virology,epidemiology,virus-host interactions and host pathogenesis of CVA16.In this study,we describe the viral kinetics of CVA16 in human rhabdomyosarcoma(RD) cells by analyzing the cytopathic effect(CPE),viral RNA replication,viral protein expression,viral RNA package and viral particle secretion in RD cells.We show that CVA16 appears to first attach,uncoat and enter into the host cell after adsorption for 1 h.Later on,CVA16 undergoes rapid replication from 3 to 6 h at MOI 1 and until 9 h at MOI 0.1.At MOI 0.1,CVA16 initiates a secondary infection as the virions were secreted before 9 h p.i.CPE was observed after 12 h p.i.,and viral antigen was first detected at 6 h p.i.at MOI 1 and at 9 h p.i.at MOI 0.1.Thus,our study provides important information for further investigation of CVA16 in order to better understand and ultimately control infections with this virus.

Detection of anti-aquaporin-4 autoantibodies in the sera of Chinese neuromyelitis optica patients

In this study, we recruited 10 neuromyelitis optica patients, two multiple sclerosis patients and two myelitis patients. Chinese hamster lung fibroblast （V79） cells transfected with a human aquaporin-4-mCherry fusion protein gene were used to detect anti-aquaporin-4 antibody in neuromyelitis optica patient sera by immunofluorescence. Anti-aquaporin-4 autoantibody was stably detected by immunofluorescence in neuromyelitis optica patient sera exclusively. The sensitivity of the assay for neuromyelitis optica was 90% and the specificity for neuromyelitis optica was 100%. The anti-aquaporin-4 antibody titers in sera were tested with serial dilutions until the signal disappeared. A positive correlation was detected between Expanded Disability Status Scale scores and serum anti-aquaporin-4 antibody titers. The anti-aquaporin-4 antibody assay is highly sensitive and specific in the sera of Chinese neuromyelitis optica patients. Detection of aquaporin-4 autoantibody is important for the diagnosis and treatment of neuromyelitis optica.

Self-assembled multiepitope nanovaccine based on NoV P particles induces effective and lasting protection against H3N2 influenza virus

Current seasonal influenza vaccines confer only limited coverage of virus strains due to the frequent genetic and antigenic variability of influenza virus(IV).Epitope vaccines that accurately target conserved domains provide a promising approach to increase the breadth of protection;however,poor immunogenicity greatly hinders their application.The protruding(P)domain of the norovirus(NoV),which can self-assemble into a 24-mer particle called the NoV P particle,offers an ideal antigen presentation platform.In this study,a multiepitope nanovaccine displaying influenza epitopes(HMN-PP)was constructed based on the NoV P particle nanoplatform.Large amounts of HMN-PP were easily expressed in Escherichia coli in soluble form.Animal experiments showed that the adjuvanted HMN-PP nanovaccine induced epitope-specific antibodies and haemagglutinin(HA)-specific neutralizing antibodies,the antibodies could persist for at least three months after the last immunization.Furthermore,HMN-PP induced matrix protein 2 extracellular domain(M2e)-specific antibody-dependent cell-mediated cytotoxicity,CD4^(+)and CD8^(+)T-cell responses,a nucleoprotein(NP)-specific cytotoxic T lymphocyte(CTL)response.These results indicated that the combination of a multiepitope vaccine and self-assembled NoV P particles may be an ideal and effective vaccine strategy for highly variable viruses such as IV and SARS-CoV-2.

Design and immunogenicity assessment of HIV-1 virus-like particles as a candidate vaccine

The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine.Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to safety concerns,HIV virus like particles(VLPs) offer an attractive alternative because they are considered safer since they lack viral genome.We got a stable eukaryotic cell line by G418 resistance selection,engineered to express the HIV-1 structure protein Gag and Env efficiently and stably.We confirmed the presence of Gag and Env proteins in the cell culture supernatant and that they could self-assemble into VLPs.These VLPs were found to be able to elicit specific humoral and cellular immune response after immunization without any adjuvant.

Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure

The Enterovirus 71(EV71)VP4 is co-translationally linked to myristic acid at its amino-terminal glycine residue.However,the role of this myristoylation in the EV71 life cycle remains largely unknown.To investigate this issue,we developed a myristoylation-deficient virus and reporter(luciferase)pseudovirus with a Gly-to-Ala mutation(G2A)on EV71 VP4.When transfecting the EV71-G2 A genome encoding plasmid in cells,the loss of myristoylation on VP4 did not affect the expression of viral proteins and the virus morphology,however,it did significantly influence viral infectivity.Further,in myristoylation-deficient reporter pseudovirus-infected cells,the luciferase activity and viral genome RNA decreased significantly as compared to that of wild type virus;however,cytopathic effect and viral capsid proteins were not detected in myristoylation-deficient virus-infected cells.Also,although myristoylation-deficient viral RNA and proteins were detected in the second blind passage of infection,they were much fewer in number compared to that of the wild type virus.The replication of genomic RNA and negative-strand viral RNA were both blocked in myristoylation-deficient viruses,suggesting that myristoylation affects viral genome RNA release from capsid to cytoplasm.Besides,loss of myristoylation on VP4 altered the distribution of VP4-green fluorescent protein protein,which disappeared from the membrane structure fraction.Finally,a liposome leakage assay showed that EV71 myristoylation mediates the permeability of the model membrane.Hence,the amino-terminal myristoylation of VP4 is pivotal to EV71 infection and capsidmembrane structure interaction.This study provides novel molecular mechanisms regarding EV71 infection and potential molecular targets for antiviral drug design.

Norovirus P particle-based tau vaccine-generated phosphorylated tau antibodies markedly ameliorate tau pathology and improve behavioral deficits in mouse model of Alzheimer’s disease

Dear Editor,Currently,there are no FDA-approved disease-modifying therapies that can prevent,halt,or reverse Alzheimer's disease(AD).As the unsatisfactory of amyloid-p-targeted treatment in recent years,development of Tau-targeted active immunotherapy takes much concern.1 Tau protein,a major microtubule-associated protein in the nervous system,was found to be abnormally hyperphosphorylated at six epitopes:Ser396/404,Ser202,Thr205,Ser238,and Ser262 in AD patients.2 Hence,immunotherapy targeting more highly-expressed phosphorylated Tau(pTau)species may induce a sufficient pool of pTau antibodies to eliminate pathological tau and elicit cognitive improvement.

A biepitope,adjuvant-free,self-assembled influenza nanovaccine provides cross-protection against H3N2 and H1N1 viruses in mice

Currently,the incorporation of multiple epitopes into vaccines is more desirable than the incorporation of a single antigen for universal influenza vaccine development.However,epitopes induce poor immune responses.Although the use of adjuvants can overcome this obstacle,it may raise new problems.Effective antigen delivery vehicles that can function as both antigen carriers and intrinsic adjuvants are highly desired for vaccine development.Here,we report a biepitope nanovaccine that provides complete protection in mice against H3N2 virus as well as partial protection against H1N1 virus.This vaccine(3MCD-f)consists of two conserved epitopes(matrix protein 2 ectodomain(M2e)and CDhelix),and these epitopes were presented on the surface of ferritin in a sequential tandem format.Subcutaneous immunization with 3MCD-f in the absence of adjuvant induces robust humoral and cellular immune responses.These results provide a proof of concept for the 3MCD-f nanovaccine that might be an ideal candidate for future influenza pandemics.

Immunological and virological characteristics of human immunodeficiency virus type i superinfection： implications in vaccine design

Superinfection is frequently detected among individuals infected by human immunodeficiency virus type I （HIV-1）. Superinfection occurs at similar frequencies at acute and chronic infection stages but less frequently than primary infection. This observation indicates that the immune responses elicited by natural HIV-1 infection may play a role in curb of superinfection; however, these responses are not sufficiently strong to completely prevent superinfection. Thus, a successful HIV-1 vaccine likely needs to induce more potent and broader immune responses than those elicited by primary infection. On the other hand, potent and broad neutralization responses are more often detected after superinfection than during monoinfection. This suggests that broadly neutralizing antibodies are more likely induced by sequential immunization of multiple different immunogens than with only one form of envelope glycoprotein immunogens. Understanding why the protection from superinfection by immunity induced by primary infection is insufficient and if superinfection can lead to cross-reactive immune responses will be highly informative for HIV-1 vaccine design.

Enhancing the antitumor activity of an engineered TRAIL-coated oncolytic adenovirus for treating acute myeloid leukemia

The use of oncolytic viruses has emerged as a promising therapeutic approach due to the features of these viruses,which selectively replicate and destroy tumor cells while sparing normal cells.Although numerous oncolytic viruses have been developed for testing in solid tumors,only a few have been reported to target acute myeloid leukemia(AML)and overall patient survival has remained low.We previously developed the oncolytic adenovirus rAd5pz-zTRAIL-RFP-SΔ24E1a(A4),which carries the viral capsid protein IX linked to tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and results in increased infection of cancer cells and improved tumor targeting.To further improve the therapeutic potential of A4 by enhancing the engagement of virus and leukemia cells,we generated a new version of A4,zA4,by coating A4 with additional soluble TRAIL that is fused with a leucine zipper-like dimerization domain(zipper).ZA4 resulted in enhanced infectivity and significant inhibition of the proliferation of AML cells from cell lines and primary patient samples that expressed moderate levels of TRAIL-related receptors.ZA4 also elicited enhanced anti-AML activity in vivo compared with A4 and an unmodified oncolytic adenoviral vector.In addition,we found that the ginsenoside Rh2 upregulated the expression of TRAIL receptors and consequently enhanced the antitumor activity of zA4.Our results indicate that the oncolytic virus zA4 might be a promising new agent for treating hematopoietic malignancies such as AML.

Functionalization of Magnetic Titanium Dioxide for Targeted Drug Delivery and UV-induced Release

Magnetic titanium dioxide core-shell nanoparticles（Fe3O4@TiO2 NPs） were synthesized as particles withappropriate size for intravenous injection. They subsequently were functionalized with p-aminothiophenol（PATP） tofacilitate the covalent attachment of target drugs through azo coupling reaction, which can be universally observed ina variety of drugs containing phenols, anilines, and imidazoles. The proposed drug nanocarrier was designed to po-ssess the ability to release drugs upon UV irradiation because of the photocatalytic property of the TiO2 shells. All theresults indicate the potential application of the Fe3O4@TiO2 NPs in targeted drug delivery with greater chemical sta-bility and universality.

Potential coordination role between O-GIcNAcylation and epigenetics

Dynamic changes of the post-translational O-GIcNAc modification （O-GIcNAcylation） are controlled by O-linked β-N-acetylglucosamine （O-GIcNAc） transferase （OGT） and the glycoside hydrolase O-GIcNAcase （OGA） in cells. O-GIcNAcylation often occurs on serine （Ser） and threonine （Thr） residues of the specific substrate proteins via the addition of O-GIcNAc group by OGT. It has been known that O-GIcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GIcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GIcNAcylation as ＇protein code＇ or ＇histone code＇ may provide recognition platforms or executive instructions for subse- quent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GIcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O- GIcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GIcNAcylation with epigenetic changes in intracellular biological processes.

Exosomes Released from Rabies Virus-Infected Cells May be Involved in the Infection Process

Exosomes are cell-derived vesicles that are secreted by many eukaryotic cells. It has recently attracted attention as vehicles of intercellular communication. Virus-infected cells release exosomes, which contain viral proteins, RNA, and pathogenic molecules. However, the role of exosomes in virus infection process remains unclear and needs to be further investigated.In this study, we aimed to evaluate the effects of exosomes on rabies virus infection. OptiPrep^(TM) density gradient centrifugation was used to isolate exosomes from rabies virus-infected cell culture supernatants. A rabies virus G protein enzyme-linked immunosorbent assay and acetylcholinesterase activity assays were performed to verify the centrifugation fractions. Exosomes were then characterized using transmission electron microscopy and Western blotting. Our results showed that rabies virus infection increased the release of exosomes. Treatment with GW4869 and si-Rab27 a, two exosomal secretion inhibitors, inhibited exosome release. Furthermore, the inhibitors reduced the levels of extracellular and intracellular viral RNA. These data indicated that exosomes may participate in the viral infection process. Moreover, our results establish a basis for future research into the roles of exosomes in rabies virus infection and as potential targets for developing new antiviral strategies.

Role of helicity of a-helical antimicrobia peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic a-helical antimicrobial peptide A12L/A20L （Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide） was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activi- ties of o-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitu- tions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Gram- positive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial pep- tides with enhanced specificity.

Inhibition of Mcl-1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells

Acute myeloid leukemia(AML)is a serious disease.The 5-year survival rates remain frustratingly low(65%for children and 26%for adults).Resistance to frontline chemotherapy(usually cytarabine)often develops;therefore a new treatment modality is needed.Bcl-2 family proteins play an important role in balancing cell survival and apoptosis.The antiapoptotic Bcl-2 family proteins have been found to be dysregulated in AML.ABT-199,a BH3 mimetic,was developed to target antiapoptotic protein Bcl-2.Although ABT-199 has demonstrated promising results,resistance occurs.Previous studies in AML show that ABT-199 alone decreases the association of proapoptotic protein Bim with Bcl-2,but this is compensated by increased association of Bim with prosurvival protein Mcl-1,stabilizing Mcl-1,resulting in resistance to ABT-199.In this study,we investigated the antileukemic activity of the Mcl-1-selective inhibitor A-1210477 in combination with ABT-199 in AML cells.We found that A-1210477 synergistically induced apoptosis with ABT-199 in AML cell lines and primary patient samples.The synergistic induction of apoptosis was decreased upon Bak,Bax and Bim knockdown.While A-1210477 treatment alone also increased Mcl-1 protein levels,combination with ABT-199 reduced binding of Bim to Mcl-1.Our results demonstrate that sequestration of Bim by Mcl-1,a mechanism of ABT-199 resistance,can be abrogated by combined treatment with the Mcl-1 inhibitor A-1201477.

Human INO80/YY1 chromatin remodeling complex transcriptionally regulates the BRCA2-and CDKNIA-interacting protein （BCCIP） in cells

The BCCIP （BRCA2. and CDKNIA-interacting protein） is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCClP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and col- orectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 sub- units catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCClP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation （CHIP） experiments clarify the enrichment of INO80 and YY1 at ＋0.17 kb downstream of the BCClP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCClP promoter region. Our findings strongly indicate that BCClP is a potential target gene of the INO80/YY1 complex.

Effects of the Molecular Weight of PLGA on Degradation and Drug Release In vitro from an mPEG-PLGA Nanocarrier

We successfully synthesized four kinds of copolymers with varying molecular weights of poly（lactide- co-glycolide）（PLGA） to yield methoxy-poly（ethylene glycol）-block-poly（lactide-co-glycolide）（mPEG-PLGA） nano- carriers： mPEG-PLGA（3k）, mPEG-PLGA（9k）, mPEG-PLGA（llk） and mPEG-PLGA（16k）. An antitumor drug, 10-hydroxycamptothecin（HCPT）, was encapsulated into the mPEG-PLGA nanocarrier cores by self-assembly in dialysis. The lower molecular weight nanocarriers degraded more quickly, resulting in mass loss, pH decline, and a rapid HCPT release rate in vitro. The degradation and drug release of the nanocarriers were dependent on the PLGA molecular weight. However, the larger molecular weight nanocarriers could not increase the loading content and encapsulation efficiency. Considering the antitumor effect of these nanocarriers, the mPEG-PLGA（9k） nanocarrier, which had the highest drug loading content[（7.72±0.57）%] and a relatively high encapsulation efficiency [（22.71±5.53）%], is an optimum agent for drug delivery.

HIV-1 Vif suppresses antiviral immunity by targeting STING

HIV-1 infection-induced cGAS–STING–TBK1–IRF3 signaling activates innate immunity to produce type I interferon(IFN).The HIV-1 nonstructural protein viral infectivity factor(Vif)is essential in HIV-1 replication,as it degrades the host restriction factor APOBEC3G.However,whether and how it regulates the host immune response remains to be determined.In this study,we found that Vif inhibited the production of type I IFN to promote immune evasion.HIV-1 infection induced the activation of the host tyrosine kinase FRK,which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif(ITIM)of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN.Mechanistically,the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162.However,the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection.These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING.These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.

Detection and Comparison of Structure and Function of Wild-type Pneumolysin and Its Novel Mutant PIyM2

Here is reported a novel pneumolysin（Ply） mutant（PlyM2） that addresses a long-standing problem for vaccine development in this field： detoxification of Ply in the premise of retaining antigenic integrity. Structure and function of wild-type Ply（PIyWT） and PIyM2 mutants were detected and compared. Their structures were not signi- ficantly different according to the analysis by thermal-dependent fluorescence spectroscopy and circular dichroism spectroscopy. PlyM2 was confirmed to have lost hemolytic activity and yet could induce neutralizing antibodies to prevent in vitro hemolysis by PIyWT and Streptococcus pneumoniae（S, pneumoniae）. These results give support to PIyM2 to be a new protein antigen for inclusion in the development of an effective pneumocoecal multiprotein vaccine.

Identification of Cinobufagin and Resibufogenin as Inhibitors of Enterovirus 71 Infection

In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71（EV71） infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations（lCs0） of einobufagin and resibufoge- nin were （10.94±2.36） and （218±31） nmol/L, respectively, the 50% cytotoxic concentrations（CCs0） of them were （1277±223） and （1385±254） nmol/L, respectively, and the anti-EV71 selectivity index（SI50） of cinobufagin was 116.7, which are promisingly developed into drug. Using a VP1 detection assay and a constructed reporter luciferase, we found that cinobufagin and resibufogenin disrupted the synthesis of EV71 protein. However, neither of them inhibited EV71 RNA replication. Our study suggests that cinobufagin and resibufogenin are the promising candidates that should he fllrther investigated for the treatment of EV71 caused disease.