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Periodontitis is the top reason for tooth loss, and smoking significantly increases severe periodontitis risk. Defective autophagy has been reported to play a vital role in periodontitis. This study aimed to elucidate the relationship between autophagy and inflammation factors production in nicotine-treated periodontal ligament stem cells (PDLSCs) and the underlying mechanism.In this study, transmission electron microscopy, immunofluorescence, and the mCherry-GFP-LC3 plasmid were used to study autophagy flux. The gene levels of inflammation factors and long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) were detected by quantitative real-time PCR (qRT-PCR). Western blot was performed to assess the protein levels of autophagic markers and α7 nicotinic acetylcholine receptor (α7nAChR).We found that nicotine impaired autophagosome-lysosome fusion and lysosome functions to block autophagy flux, contributing to inflammatory factors production in nicotine-treated PDLSCs. Moreover, nicotine upregulated NEAT1 by activating α7nAChR. NEAT1 decreased autophagy flux by downregulating syntaxin 17 (STX17).Our data indicate that NEAT1-decreased autophagy flux is pivotal for inflammation factors production in nicotine-treated PDLSCs.

RationaleNlrx1, a pattern recognition receptor belonging to the NLR family, is highly expressed in the heart and is significantly increased after myocardial infarction in vivo, however, the role of Nlrx1 in post-infarction inflammation and healing is poorly understood. Although the effects of infiltrating macrophages on survival of cardiomyocytes and healing of injured hearts have been intensely studied, whether or not and how surviving cardiomyocytes might influence the phenotype and function of macrophages in the infarcted hearts was largely unexplored.ObjectiveTo investigate the role of Nlrx1 in post-infarction inflammation and healing and to dissect the underlying mechanism.Methods and resultsSingle cell RNA-sequencing of 23,805 cells isolated from infarcted hearts revealed 7 clusters of infarct macrophages including CCR2+, Isg+, Mki67+proliferative, and LYVE1+cardiac resident macrophages, and identified a novel subset of CD9+Il1rn+macrophages, which were characterized by high expression ofCd9, Il1rn, GpnmbandSpp1,and downregulation of proinflammatory cytokines and chemokines. CD9+Il1rn+macrophages were largely found in infarcted hearts of Nlrx1KO mice, and were transcriptionally unique with reparative phenotype. Loss of Nlrx1 attenuated ventricular dysfunction and adverse remodeling, and decreased post-infarction mortality. Flow cytometry analysis showed markedly increased number of CD9+macrophages and higher mean fluorescence intensity of CD9 in Nlrx1KO mice. RNA-seq analysis revealed that CD9+macrophages of Nlrx1KO infarcted hearts exhibited a reparative and anti-inflammatory phenotype as featured by upregulation of wound healing-associated genes includingCD9, Spp1andFn1, and downregulation of proinflammatory genes. Conditioned medium from Nlrx1 deficient cardiomyocytes upon hypoxia stimulation skewed macrophage polarization towards a reparative and inflammation-suppressed phenotype. Gain-and loss-of function of Nlrx1 combined with RNA-seq analysis identified CCN2, whose expression and secretion were significantly increased in Nlrx1 deficient cardiomyocytes upon hypoxia, as a cardiomyocytes-derived mediator responsible, at least partly, for reprograming macrophage towards a reparative phenotype. In vivo, CCN2 expression was markedly elevated in Nlrx1KO infarcted hearts. Moreover, exogenous CCN2 partially mediated macrophage transition towards a reparative phenotype.ConclusionsCD9+IL1rn+macrophages modulated by Nlrx1 deficient cardiomyocytes-derived CCN2, is a novel subset of reparative macrophages contributing to infarct healing. Inhibition of myocardial Nlrx1 and/or activation of CCN2 to boost CD9+IL1rn+reparative macrophages may represent new therapeutics for infarct healing following ischemic injury.

Introduction The experimental cerebral malaria (ECM) model in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) has revealed microglia are involved in the ECM immune microenvironment. However, the regulation of microglia in the ECM immune response is not clear, and there is no safe and efficient treatment clinically for the protection of the nerve cells. Aims To elucidate the negative regulation mechanism in the ECM brain mediated by microglia. Furthermore, to investigate protective effect of the appropriate enhancement of the PD‐1/PD‐L1 pathway in the brain against ECM through the intrathecal injection of the adenovirus expressing PDL1‐IgG1Fc fusion protein. Results The PD‐1/PD‐L1 pathway was induced in the ECM brain and showed an upregulation in the microglia. Deep single‐cell analysis of immune niches in the ECM brainstem indicated that the microglia showed obvious heterogeneity and activation characteristics. Intrathecal injection of recombinant adenovirus expressing PD‐L1 repressed the neuroinflammation and alleviated ECM symptoms. In addition, the synergistic effect of artemisinin and intracranial immunosuppression mediated by PD‐L1 was more efficacious than either treatment alone. Conclusion The appropriate enhancement of the PD‐1/PD‐L1 pathway in the early stage of ECM has an obvious protective effect on the maintenance of immune microenvironment homeostasis in the brain. Regulating microglia and the PD‐1/PD‐L1 pathway could be considered as a promising approach for protection against human cerebral malaria in the future., Microglia activated and expressed PD‐L1 and PD‐1 induced by PbA infection.The PD‐L1 could interacted with PD‐1 on the infiltrated CTL cells and inhibited the killing activity of CTL cells.The intrathecal injection of expression vector PDL1‐IgGFc could inhibit intracerebral inflammation and improved ECM.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies. Rongrong Liu et al. find that Hantaan virus, a common cause of hemorrhagic fever with renal syndrome (HFRS) in China, can infect CD8+ T cells and that these cells can support the full viral replication cycle. They also see an association between CD8+ T cells infection and disease progression in HFRS patients.

Oxidative phosphorylation (OXPHOS) is a vital pathway provides ATP for intracellular activities. Here, we found that Hantaan virus (HTNV) exploited mitochondria OXPHOS to assist its replication in host cells and Protein Kinase B/AKT played a major function in this process. Inhibiting AKT activation by BEZ treatment can inhibit HTNV replication and prevent the increase of OXPHOS level caused by HTNV infection. We also found that HTNV infection can promote AKT translocation to mitochondria, where AKT phosphorylates Polynucleotide phosphorylase (PNPT). Taken together, our research demonstrates that HTNV replication exploits OXPHOS in host cells and it increases OXPHOS function by AKT-PNPT interaction in mitochondria.IMPORTANCEVirus depends on metabolic pathways in host cells to favor its replication. This is a vital process which needs complicated host-virus interaction and targeting this process is a new strategy for antiviral drug development. Hantaan virus (HTNV) is the major pathogen which causes Hemorrhagic Fever with Renal Syndrome (HFRS) in China. However, there are neither effective therapeutic drugs nor FDA-licensed vaccine against HFRS, a deeper understanding of HTNV infection characteristics is of great significance for global public health and safety. This research means to elucidate the major metabolic pathway exploited by HTNV during its replication in host cells and its underlying molecular mechanism, which can enrich our understanding about HTNV biological characteristics and pathogenesis, also provide a new view on anti-HTNV drug development.

Hantavirus can cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in America, with high mortality and unknown mechanisms. Innate immunity is the host’s first-line defense to bridge the acquired immunity against viral infections. However, hantavirus has evolved various strategies in both molecular and cellular aspects to evade the host’s natural immune surveillance. The Interferon-I (IFN-I) signaling pathway, a central link of host defense, induces various antiviral proteins to control the infection. This paper summarizes the molecular mechanisms of hantavirus evasion mechanisms of the IFN signaling pathway and cellular processes such as regulated cell death and cell stress. Besides, hantavirus could also evade immune surveillance evasion through cellular mechanisms, such as upregulating immune checkpoint molecules interfering with viral infections. Understanding hantavirus’s antiviral immune evasion mechanisms will deepen our understanding of its pathogenesis and help us develop more effective methods to control and eliminate hantavirus.

Objective To investigate the effects of nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) on the proliferation, migration and invasion of human colon cancer HCT116 and LoVo cells. Methods NLRC3 was knocked down in HCT116 and LoVo cells by NLRC3-specific siRNA (si-NLRC3). NLRC3 mRNA and protein expression was detected by real-time quantitative PCR and Western blotting. The proliferation ability of cancer cells was detected by CCK-8 assay; the clone formation ability was detected by clone formation assay; the invasion ability was detected by Transwell

Hantaan virus (HTNV) infects humans and causes hemorrhagic fever with renal syndrome (HFRS). The development of well-characterized animal models of HFRS could accelerate the testing of vaccine candidates and therapeutic agents and provide a useful tool for studying the pathogenesis of HFRS. Because NLRC3 has multiple immunoregulatory roles, we investigated the susceptibility of Nlrc3−/− mice to HTNV infection in order to establish a new model of HFRS. Nlrc3−/− mice developed weight loss, renal hemorrhage, and tubule dilation after HTNV infection, recapitulating many clinical symptoms of human HFRS. Moreover, infected Nlrc3−/− mice showed higher viral loads in serum, spleen, and kidney than wild type C57BL/6 (WT) mice, and some of them manifested more hematological disorders and significant pathological changes within multiple organs than WT mice. Our results identify that HTNV infected Nlrc3−/− mice can develop clinical symptoms and pathological changes resembling patients with HFRS, suggesting a new model for studying the pathogenesis and testing of candidate vaccines and therapeutics.

Purpose Hantavirus infections cause severe haemorrhagic fever with renal syndrome (HFRS) in humans and are associated with high fatality rates. In 2017, numerous outbreaks were reported in China and Germany. This represents a significant public-healthcare issue with no effective HFRS vaccines that offer a long-term immune response. In this study, we investigated the long-term humoral and cellular immune responses and protective immunity of Hantaan virus (HTNV) granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40 ligand (CD40L) virus-like particles (VLPs) in mice. Methodology GM-CSF and CD40L VLPs were constructed via co-transfection of pCI-S and pCI-M-CD40L, and pCI-S and pCI-M-GM-CSF, into dihydrofolatereductase (dhfr)-deficient Chinese hamster ovary cells, respectively. Mice were immunized with HTNV VLPs 2 weeks apart. The animals were challenged 6 months after immunization. Specific and neutralizing antibodies were assessed by ELISA; IFN-γ was measured by enzyme-linked immunospot (ELISpot) assay and effectiveness by cytotoxic T lymphocyte (CTL) cytotoxicity assays. Nucleic acid loads of HTNV were tested by quantitative real-time PCR and viral antigen was detected via indirect ELISA. Pathological alterations were detected via haematoxylin-eosin staining. Results GM-CSF and CD40L VLPs provided stable, long-term protection with a high titre of neutralizing antibody in mice 6 months after immunization. Furthermore, VLPs increased HTNV-specific cellular immune responses via higher expression of IFN-γ and CTL responses. HTNV challenge assay results showed long-term protection against HFRS. No significant pathological alteration was observed in the organs of mice after immunization. Conclusion This is, to the best of our knowledge, the first report demonstrating the long-term potency of HTNV VLP vaccines against HTNV infection and offers new insights into HTNV vaccine development.

Single nucleotide polymorphisms (SNP) can be used as genetic markers and for risk assessment of allele-linked diseases, which can provide information for clinical diagnosis. Large-scale microarray and next-generation sequencing methods have made genome-wide SNP genotyping possible. However, in addition to their high cost, these techniques are dependent on having specialized equipment. Thus, there is a need for a simple genotyping method that can be implemented in a resource-limited environment.We developed a strategy for SNP genotyping based on T7 Endonuclease I cleavage and an enzyme-linked microparticle immune assay. Using this method, we genotyped two common SNP sites (rs11526468 and rs12979860). The quality of the genotyping process was validated.Although a 70% false-negative rate was observed, no false-positive reactions were found. Therefore, multiple parallel repeat reactions can offset the possibility of mutation detection failure.This method employs a duplicate reagent-dependent procedure, and therefore has the potential for integration into a portable kit for field utilization.

Coronary artery disease (CAD) is among the main causes of morbidities and mortalities globally. It is also considered to be an outcome of acute thrombotic events which entail activating platelets as well as coagulation proteins. In particular, rivaroxaban along with aspirin have been considered to reduce thrombotic events. However, they are yet to be evaluated by combining with or putting them against each other in patients experiencing CAD. This study intends to carry out an evaluation of whether combining rivaroxaben with aspirin will be effective and safe in treating patients experiencing chronic CAD.We intend to search information from the following databases: MEDLINE, EMBASE, Web of Science, Cochrane library, WanFang, and China National Knowledge Infrastructure. In the search, we intend to regard randomized control trials written in either English or Chinese and only those published until December 2021, as well as only those that have assessed the effectiveness and safety of combining rivaroxaban and aspirin in treating patients suffering from chronic CAD. We intend to accompany the study identification with searching or relevant reference lists as well as citations. We will also contact respective authors to provide additional information or data were needful. From the search, we will collate all citations identified and remove all duplicates. Similarly, 2 independent authors will screen all the titles and abstracts and assess them against the inclusion criteria for the study. Only selected studies will be included for critical appraisal, extraction of data, and synthesis. We will then conduct statistical analyses by utilizing a random-effect model.This study does not require ethical approval as the findings will be published in a peer-review journal.10.17605/OSF.IO/WBGE4.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies., Rongrong Liu et al. find that Hantaan virus, a common cause of hemorrhagic fever with renal syndrome (HFRS) in China, can infect CD8+ T cells and that these cells can support the full viral replication cycle. They also see an association between CD8+ T cells infection and disease progression in HFRS patients.

Objective To construct nucleotide-binding oligomerization domain-like receptors family member X1 (NLRX1) lentivirus plasmid and stable NLRX1-overexpressing A549 cell line. Methods Full-length cDNA encoding human NLRX1 was amplified from pCMV3-NLRX1 plasmid and then was inserted into pCDH-CMV-MCS-EF1-Puro vector to obtain NLRX1-overexpressing plasmid pCDH-NLRX1. pCDH-NLRX1 lentivirus particles were obtained by co-transfecting pCDH-NLRX1 with packaging plasmids (pMD2 and pAX2) into HEK293T cells. A549 cells were infected with concentrated pCDH-NLRX1 lentivirus particles and then were screened by puromycin to obtain stable NLRX1-overexpressed A549 cell line. The mRNA transcription and protein expression of NLRX1 were detected by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results The lentivirus plasmid pCDH-NLRX1 was successfully constructed. After pCDH-NLRX1 and packaging plasmids were co-transfected into HEK293T cells, we obtained NLRX1 lentivirus particles with the titer of 1×10

Introduction. During chronic hepatitis C virus (HCV) infections, HCV antigens establish cross-tolerance of endotoxins, but additional lipopolysaccharide (LPS) stimulation effects in this condition are poorly understood. Aim. This study aims to investigate the effects of the upregulated LPS on MMP and TIMP expression during chronic hepatitis C infection. Methodology. In the present study, we analysed the effect of HCV antigens and LPS stimulation on peripheral blood mononuclear cells (PBMCs) both in vivo and in vitro. Macrophages from HCV patients were isolated and their association with endotoxin tolerance was examined. MMP/TIMP1 expression and the related signalling pathways in macrophages were analysed. The macrophage and Huh7.5 cell co-culture model was used to analyse the effects of the cross-tolerance on collagen I deposition. Results. LPS levels were found to be significantly higher in HCV patients, particularly in those with HCV-induced liver fibrosis. In addition, although LPS serum level was occasionally upregulated in the patients, it did not induce intense immune response in PBMCs due to endotoxin cross-tolerance, and this was measured according to the changes in IL-6 and TNF-α levels. However, TIMP1 expression increased significantly during stimulation, exhibiting a tolerance/resistance phenotype, which was associated with TGF-β/Erk activation in macrophages. However, MMP levels did not increase due to endotoxin tolerance, which ultimately led to MMP/TIMP imbalance and influenced the deposition of collagen I. Conclusion. Increased LPS stimulation of macrophage during HCV antigen-induced endotoxin cross-tolerance contributes to MMP/TIMP1 imbalance and collagen I deposition.

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR‑Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR‑Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Tuberculosis is chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) that is prevalent worldwide. Several specific antigens, such as Antigen 85B (Ag85B) and 6 kDa early secretory antigenic target (ESAT-6) protein of M.tb, are listed as some of the candidate subunit vaccines against M.tb. ESAT-6, as a virulent factor and differential gene in M.tb, shows insufficient immunogenicity in animal model. In order to investigate the ways to improve the immunogenicity of ESAT-6, we immunized ESAT-6 by subcutaneous and intramuscular routes with different adjuvants. We found that ESAT-6 immunized alone did not induce significant humoral immunity in both immunization routes. However, subcutaneous immunization of ESAT-6 plus incomplete Freund's adjuvant can induce a significant humoral immune response, enhanced proliferation and elevated secretion of IFN-γ from splenocytes. Intramuscular immunization of ESAT-6 plus adjuvant aluminum salt or poly(I:C) did not enhance humoral and cellular immune responses. Therefore, it is concluded that immunization of ESAT-6 subcutaneously plus incomplete Freund's adjuvant induces stronger humoral and cellular immune responses, which can be considered of ESAT-6 as a subunit vaccine in further research against tuberculosis.

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3-1mg of pure and highly active rαBtx per liter of E. coli culture.

The emergence of Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern worldwide. PVL is associated with community-associated MRSA and is linked to skin and soft tissue infections (SSTIs). However, PVL genes have also been detected in health care-associated (HA) MRSA isolates. The diseases associated with PVL-positive HA-MRSA isolates and the distributions of PVL-encoding bacteriophages in HA-MRSA have not been determined. In this study, a total of 259 HA-MRSA strains isolated between 2009 and 2012 in China from inpatients with SSTIs, pneumonia, and bacteremia were selected for molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, and staphylococcal protein A gene typing. The PVL genes and PVL bacteriophages in the MRSA isolates were characterized by PCR. Among the tested MRSA isolates, 28.6% (74/259) were PVL positive. The high prevalence of PVL-carrying HA-MRSA was observed to be associated with SSTIs but not with pneumonia or bacteremia. The PVL-positive HA-MRSA isolates were colonized mainly by infective PVL phages, namely, Φ7247PVL, ΦSLT, and ΦSa2958. The distribution of PVL-carrying bacteriophages differed geographically. Our study highlights the potential risk of the emergence of multidrug-resistant HA-MRSA strains with increased virulence.

Japanese encephalitis virus (JEV), closely related to dengue, Zika, yellow fever and West Nile viruses, remains neglected and not well characterized1. JEV is the leading causative agent of encephalitis, and is responsible for thousands of deaths each year in Asia. Humoral immunity is essential for protecting against flavivirus infections and passive immunization has been demonstrated to be effective in curing disease2,3. Here, we demonstrate that JEV-specific monoclonal antibodies, 2F2 and 2H4, block attachment of the virus to its receptor and also prevent fusion of the virus. Neutralization of JEV by these antibodies is exceptionally potent and confers clear therapeutic benefit in mouse models. A single 20 μg dose of these antibodies resulted in 100% survival and complete clearance of JEV from the brains of mice. The 4.7 A and 4.6 A resolution cryo-electron microscopy structures of JEV–2F2-Fab and JEV–2H4-Fab complexes, together with the crystal structure of 2H4 Fab and our recent near-atomic structure of JEV4, unveil the nature and location of epitopes targeted by the antibodies. Both 2F2 and 2H4 Fabs bind quaternary epitopes that span across three adjacent envelope proteins. Our results provide a structural and molecular basis for the application of 2F2 and 2H4 to treat JEV infection. Two known anti-Japanese encephalitis virus antibodies are shown to bind quaternary epitopes spanning three envelope proteins, block viral attachment and prevent fusion. One low dose in mice is protective, underscoring their therapeutic potential.

Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-β) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

Consequences of primary dsysmenorrhea (PD) can be severe. Increased prostaglandin production leads to uterine contraction and insufficient blood flow to the endometrium causing ischemia and pain symptoms. Protein tyrosine kinase/phosphatase activities contribute to the modulation of uterine contraction. In our previous study, we found the synthetic β-methoxyacrylates compound Fluacrypyrim (FAPM), significantly increased protein tyrosine phosphatases (PTPs) activity, resulting in dephosphorylation of tyrosine kinases. In the present study, we found that FAPM near completely inhibited prostaglandin F2α (PGF2α)-, oxytocin-, acetylcholine-, and high K+-induced uterine contractions in rats in vitro, and decreased rat myometrial myosin light chain (MLC20) phosphorylation induced by PGF2α. A structure–activity relationship assay indicated that the β-methoxyacrylates structure of FAPM is crucial for the inhibition of PGF2α-induced uterine contractions. FAPM caused a concentration-dependent parallel rightward shift of the concentration–response curve induced by oxytocin, dose-dependently reduced the number of abdominal constrictions and increased the latency time in PGF2α- and acetic acid-induced writhing test in mice in vivo. Furthermore, FAPM considerably inhibited the development of Carr-induced rat paw edemas and thexylene-induced mouse ear edemas. Taken together, our results indicate that FAPM exerts antinociceptive and anti-inflammatory effects in vivo with considerable potential as a novel uterine relaxant.

Growth factor receptor bound protein 2 (Grb2) is a key adaptor performing a principal role in the oncogenic Ras signaling pathway. In the present study, we generated two fusion proteins. One contained an Src homology 2 (SH2) domain of Grb2, a signal peptide sequence, FLAG-tag sequence, PTD region and we named it FPTD-Grb2-SH2, while the other contained one mutant SH2 domain, added to a signal peptide sequence, FLAG-tag sequence, PTD region and we named it FPTD-Grb2-SH2M. Western blot analysis and immunofluorescence assay were used to investigate the expression and location of the fusion proteins in breast cancer cells. The proliferation and migration of the cells were estimated by MTT and Transwell cell migration assays, respectively. Flow cytometric analysis was performed to evaluate the apoptosis of the breast cancer cells. The recombinant proteins FPTD-Grb2-SH2 and FPTD-Grb2-SH2M were successfully expressed in the breast cancer cell lines regardless of HER2-phenotype, and they suppressed breast cancer cell growth and migration as expected from the lack of SH3 domain. Both FPTD-Grb2-SH2 and FPTD-Grb2-SH2M exhibited significant toxicity to breast cancer cells. The present study demonstrated that the recombinant proteins FPTD-Grb2-SH2 and FPTD-Grb2-SH2M may be used for anticancer drug development.

A safe and effective Hantaan virus (HTNV) vaccine is highly desirable because HTNV causes an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Since the immunity of the inactivated vaccine is weak and the safety is poor, HTNV virus-like particles (VLPs) offer an attractive and safe alternative. These particles lack the viral genome but are perceived by the immune system as virus particles. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this enhancement, we generated chimeric HTNV VLPs containing glycosylphosphatidylinositol (GPI)-anchored granulocyte macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity in vitro. The immunization of mice with chimeric HTNV VLPs containing GM-CSF or CD40L induced stronger humoral immune responses and cellular immune responses compared to the HTNV VLPs and Chinese commercial inactivated hantavirus vaccine. Chimeric HTNV VLPs containing GM-CSF or CD40L also protected mice from an HTNV challenge. Altogether, our results suggest that anchoring immunostimulatory molecules into HTNV VLPs can be a potential approach for the control and prevention of HFRS.

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in multiple cellular processes and its expression has been shown to play a critical role in tumorigenesis. However, the role of AhR in tumorigenesis of hepatocellular carcinoma remains unclear. In the current study, we investigated the role of AhR in hepatocellular carcinoma tumorigenesis and progression by (a) measuring the expression levels of AhR in liver lesions and (b) assessing the correlation between AhR expression and clinicopathologic parameters. The tissue microarray used in this study contained hepatocellular carcinoma tissues (n = 94), cancer adjacent normal hepatic tissues (n = 5) and normal hepatic tissues (n = 5), which were immunohistochemically assessed for AhR expression. Significantly stronger AhR staining was observed for hepatocellular carcinoma tissues than for cancer adjacent normal hepatic tissues (P = 0.003) and normal hepatic tissues (P = 0.004). In addition, AhR expression was associated with T stage (P = 0.03). The results from this study suggest that an increase in AhR expression is associated with hepatocellular carcinoma progression and may have a potential role in the treatment of hepatocellular carcinoma.

// Qikang Ying 1, * , Tiejun Ma 1, * , Linfeng Cheng 1, * , Xiaoxiao Zhang 1 , Agnieszka D. Truax 2 , Ruixue Ma 1 , Ziyu Liu 1 , Yingfeng Lei 1 , Liang Zhang 1 , Wei Ye 1 , Fanglin Zhang 1 , Zhikai Xu 1 , Lei Shang 3 , Rongrong Liu 1 , Fang Wang 1 , Xingan Wu 1 1 Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi’an, 710032, China 2 The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA 3 Department of Statistics, Fourth Military Medical University, Xi’an, 710032, China * These authors have contributed equally to this work Correspondence to: Xingan Wu, email: wuxingan@fmmu.edu.cn Fang Wang, email: wangf07@fmmu.edu.cn Rongrong Liu, email: rong4713@163.com Keywords: Hantaan virus, virus like particle, virus vaccine, CD40 ligand, granulocyte macrophage colony-stimulating factor Received: March 28, 2016 Accepted: July 10, 2016 Published: August 17, 2016 ABSTRACT Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate.

Myasthenia gravis (MG) is an autoimmune disease in which antibodies directed to the nicotinic acetylcholine receptor (nAChR) at the postsynaptic neuromuscular junction (NMJ) cause neuromuscular transmission defects, culminating in weakness and fatigue of skeletal muscles.1-3 Although the bivalent autoantibody that was assumed to be the main pathogenic factor can modulate AChRs by cross-linking and accelerating endocytosis and degradation, as well as directly blocking AChR function, overwhelming evidence has demonstrated that it is the activation of complement through successive stimulation of C3 and C5 convertase that finally results in focal lysis of the postsynaptic membrane.4-6 Complement deposition has been observed at myasthenic NMJs of human subjects with MG and in animals with experimental autoimmune myasthenia gravis (EAMG).7,8 Complement seems to be a critical mediator of anti-AChR antibody pathogenicity in MG. Complement-inhibitor therapy has been shown to be effective in EAMG. The severity of EAMG can be alleviated by cobra venom factor,4 a monoclonal antibody (MAb) against C5,9 or a soluble form of complement receptor 1.10 The main regulatory membrane inhibitors of complement activation located on cell surfaces include complement receptor 1 (CR1; CD35), decay-accelerating factor (DAF; CD55), and membrane cofactor protein (MCP; CD46). These membrane regulators shield cells from activation of autologous complement on their surfaces and subsequent complement-mediated destruction.11 Among these proteins, DAF seems to be the most important in protection of muscle against complement-mediated injury in EAMG.12 A previous study demonstrated that, compared with wild-type littermates, Daf1-deficient animals were highly sensitive to EAMG induction and underwent more severe complement-mediated muscle damage.13 Therefore, administration of DAF may be a potential therapeutic strategy in treating MG. Several approaches have been shown to facilitate attachment of complement-regulatory agents to a cellular surface by a targeting vehicle, which would enhance the efficacy of complement regulators compared with the unattached parent agents.14-16 Zhang and colleagues used a recombinant chimera comprising CD59 or DAF joined to an anti-5-dimethylaminonaphthalene-1-sulfonyl (anti-dansyl) antibody fragment, a targeting vehicle, which successfully targeted both CD59 and DAF to the hapten dansyl on the surface of Chinese hamster ovary (CHO) cells.17,18 In addition, using the same approach, human DAF has been tagged to a clinically relevant target cell type, a red blood cell (RBC), with an scFv specific for TER-119, which is an RBC-restricted surface antigen of the mouse.19 One study demonstrated that the effectiveness of soluble DAF at inhibiting complement-mediated lysis can be significantly enhanced when it is targeted to cell membranes via a glycophosphatidylinositol (GPI) anchor.14 However, DAF molecules containing their targeting vehicle are targeted at a non-specific cell type for certain diseases. Therefore, there remain some unresolved questions related to the clinical use of systemic anti-complement agents. The effect of systemic complement inhibition on the capacity to prevent host infections is not yet clear, as reflected in immunocompromised MG patients. The capacity for complement inhibition and safety of anti-complement agents may be enhanced by the presence of DAF that targets the molecule to pathologic sites where complement activation occurs in MG. Moreover, specific targeting may allow a much lower effective serum concentration of agents. The key challenge of this study is how to specifically deliver DAF to the pathologic target sites of MG. As shown by previous research, univalent antibody fragments, such as fragment antigen-binding (Fab) or scFv, are not pathogenic; in contrast, such fragments derived from anti–main immunogenic region (anti-MIR) MAbs can shield the MIR from the binding of pathogenically intact antibodies and MG autoantibodies without affecting ion channel function.20-22 The previously constructed univalent antibody fragment scFv195, which is derived from anti-MIR MAb195, has been shown to be a good candidate therapeutic molecule.23 The use of the scFv1956 fragment in this study is expected to allow the fusion protein to specifically target cells or tissues with the MIR of nAChR. In this study, we assessed the feasibility of targeting the novel hybrid molecule scFv-DAF to the site of complement activation triggered by pathologic antibodies to the AChR.

Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.

Hantaan virus (HTNV) is a rodent-borne virus that causes hemorrhagic fever with renal syndrome (HFRS) in Asia and can be transmitted to humans through bites or the inhalation of aerosolized urine, droppings, or saliva of infected rodents. Keratinocytes predominate in the epidermis and reportedly serve as a replication site for multiple vector-borne viruses, little is known about the susceptibility of human skin cells to HTNV infection. Therefore, we aimed to evaluate whether human keratinocytes support HTNV replication and elicit an immune response against HTNV infection. We found that a human keratinocyte cell line, HaCaT, supports HTNV replication. In addition, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (MDA5) play key roles in the detection of HTNV infection in HaCaT cells and in the up-regulation of interferon (IFN)-β expression, which subsequently leads to the production of a large amount of antiviral interferon-stimulated genes (ISGs) and other chemokines used for immune cell recruitment. Furthermore, we suggest that interferon regulatory factor (IRF)-3, as opposed to NF-κB/p65 or IRF-7, is translocated to the nucleus to induce IFN-β. However, the early induction of chemokine CXCL10 was a direct result of HaCaT cells counteracting HTNV infection and was not due to the induction of IFN. Overall, our data demonstrate, for the first time, the permissiveness of human keratinocytes to HTNV infection.

Hantavirus glycoprotein Gc is one of the main components that contribute to the generation of humoral immune responses, while the nucleocapsid protein (NP) is involved in cellular immune responses through the induction of antibody-dependent cytotoxic T cells. In this study, a chimeric gene, GcS0.7, which encodes a fusion protein containing Gc and truncated NP, was constructed as a candidate for Hantaan virus (HTNV) vaccine development. The chimeric gene was cloned into an adenoviral vector in conjunction with the powerful hybrid cytomegalovirus (CMV) enhancer/chicken β-actin (CAG) promoter or the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), or both. Both elements increased the expression level of the fusion protein. The rAd-GcS0.7-pCAG group demonstrated the highest fusion protein expression level, with a 2.3-fold increase compared with the unmodified adenoviral vector. To further evaluate the humoral and cellular immunity induced by the recombinant adenovirus, the antibody titers, interferon (IFN)-γ secretion level and cytotoxic T cell ratio were detected in immunized mice. The strongest HTNV‑specific humoral and cellular immune responses were detected in the rAd-GcS0.7‑pCAG group. The immunogenicity of these recombinant adenoviruses was compared with that of the inactivated vaccine through a series of immunological assays. In terms of the cellular immune responses, the rAd-GcS0.7-pCAG group even exceeded those induced by the vaccine control. The CAG hybrid promoter improved not only the expression level, but also the immunogenicity of the fusion protein, and may thus provide a promising strategy for HTNV vaccine research.

Autoantibody-induced complement activation, which causes disruption of the postsynaptic membrane, is recognized as a key pathogenic factor in myasthenia gravis (MG). Therefore, specific targeting of complement inhibitors to the site of complement activation is a potential therapeutic strategy for treatment of MG.We assessed expression of single-chain antibody fragment-decay accelerating factor (scFv-DAF), comprising a single-chain fragment scFv1956 based on the rat complement inhibitor DAF in prokaryotic systems, and studied its inhibitory effect on complement deposition in vitro.The recombinant conjugate scFv-DAF completely retained the wild-type binding activity of scFv1956 to AChR and inhibited complement activation of DAF in vitro.We found that scFv-DAF could bind specifically to TE671 cells, and it is significantly more potent at inhibiting complement deposition than the untargeted parent molecule DAF. scFv-DAF may be a candidate for in vivo protection of the AChR in MG.

Hantavirus glycoproteins (Gn and Gc) are significant components of vaccines for haemorrhagic fever with renal syndrome (HFRS); however, they are not effective due to weak immunogenicity and low levels of production in expression systems. To circumvent this problem, a 0.7-kb fragment of the S segment was fused to Gn, and a hybrid CAG promoter/enhancer in conjunction with (or without) the WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) was used to improve the expression of fusion protein GnS0.7 in the adenoviral expression system. The expression level of the fusion protein as well as the response of mice immunized with recombinant adenoviruses containing GnS0.7 was investigated. In addition, a series of immunological assays were conducted to determine the immunogenicity of the recombinant adenoviruses. The results showed that the recombinant adenovirus with the CAG promoter/enhancer (rAd-GnS0.7-pCAG) expressed approximately 2.1-fold more GnS0.7 than the unmodified recombinant adenovirus containing GnS0.7 (rAd-GnS0.7-pShuttle). This enhanced expression level was also higher than for other modified recombinant adenoviruses studied. Animal experiments showed that rAd-GnS0.7-pCAG induced a stronger Hantaan virus (HTNV)-specific humoral and cellular immune response in mice, with the cellular immune response to the GnS0.7 being stronger than the HFRS vaccine control. These results demonstrate that the CAG promoter/enhancer improved significantly the expression of the chimeric gene GnS0.7 in the adenovirus expression system. These findings may have significant implications for the development of genetically engineered vaccines for HFRS.

Decay accelerating factor (DAF) is a very potent complement regulatory protein which holds promise for clinical usage. Here we report on an improved procedure for refolding both rat and human DAF over-expressed in Escherichia coli. It was shown that 50–70% of the inclusion body could be refolded to soluble active protein. This method excludes the use of l -arginine, which is expensive, and can be used to prepare a large quantity of recombinant DAF for therapeutic studies.

Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359-610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1-274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.