Expression and localization of absent in melanoma 2 in the injured spinal cord

In traumatic brain injury, absent in melanoma 2(AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day(post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+(neurons), GFAP+(astrocytes), CNPase+(oligodendrocytes) and CD11 b+(microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+(leukocytes) and CD68+(activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus:Implications for EG95 vaccine application

Objective:To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus(E. granulosus). Methods:We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1(DNASTAR Inc.,Madison,WI),and the phylogenetic analysis was performed by using MEGA5.1(CEMI,Tempe,AZ,USA). In addition,linear and conformational epitopes were analysed,including secondary structure,NXT/S glycosylation,fibronectin type ecoⅢ(Fnndary Ⅲ) domain and glycosylphosphatidylinositol anchor signal(GPIanchor). The s structure was predicted by PSIPRED method. Results:Our results indicated that most isolates overall shared 72.6-100% identity in EG95 gene sequence with the published standard EG95 sequence,X90928. However,EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates,respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine,X90928,and they belonged to Subgroup 1. However,in comparison to X90928,several amino acid mutations occurred in most isolates besides oncosphere,which potentially altered the immunodominant linear epitopes,glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions:This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates,and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Cloning identification and functional analysis of human IL-17A promoter

Objective: To conduct the cloning identification and characterization of the sequence of human IL-17 A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17. Methods: First of all, the potential promoter region of IL-17 A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system. Results: Two transcriptional start points of the upper region, 600 bp and 1000 bp, of IL-17 A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17 A activator STAT3, which could start the expression of the reported gene. Conclusions: Clone established the regulatory region of human IL-17 A promoter, which provided bases to the subsequent function research.

Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

Coronavirus MHV-A59 infects the lung and causes severe pneumonia in C57BL/6 mice

It remains challenging to develop animal models of lung infection and severe pneumonia by severe acute respiratory syndrome coronavirus(SARS-CoV) and Middle East respiratory syndrome cornavirus(MERS-Co V) without high level of containment. This inevitably hinders understanding of virushost interaction and development of appropriate countermeasures. Here we report that intranasal inoculation of sublethal doses of murine coronavirus mouse hepatitis virus A-59(MHV-A59), a hepatic and neuronal tropic coronavirus, can induce acute pneumonia and severe lung injuries in C57BL/6 mice. Inflammatory leukocyte infiltrations, hemorrhages and fibrosis of alveolar walls can be observed 2-11 days after MHV-A59 infection. This pathological manifestation is associated with dramatical elevation of tissue IP-10 and IFN-γ and moderate increase of TNF-α and IL-1β, but inability of anti-viral type I interferon response. These results suggest that intranasal infection of MHV-A59 would serve as a surrogate mouse model of acute respiratory distress syndrome by SARS-CoV and MERS-CoV infections.

Blocking postsynaptic density-93 binding to C-X3-C motif chemokine ligand 1 promotes microglial phenotypic transformation during acute ischemic stroke

We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.

Cilostazol inhibits plasmacytoid dendritic cell activation and antigen presentation

Background Cilostazol, an anti-platelet drug for treating coronary heart disease, has been reported to modulate immune cell functions Plasmacytoid dendritic cells （pDCs） have been found to participate in the progression of atherosclerosis mainly through interferon ct （IFN-ct） production. Whether cilostazol influences pDCs activation is still not clear. In this study, we aimed to investigate the effects of cilostazol on cell activation and antigen presentation ofpDCs in vitro in this study. Methods Peripheral blood mononuclear cells isolated by Ficoll cen- trifugation and pDCs sorted by flow cytometry were used in this study. After pretreated with cilostazol for 2 h, cells were stimulated with CpG-A, R848 or virus for 6 h or 20 h, or stimulated with CpG-B for 48 h and then co-cultured with naive T cell for five days. Cytokines in supernatant and intracellular cytokines were analyzed by ELISA or flow cytometry respectively. Results Our data indicated that cilostazol could inhibit IFN-α and tumor necrosis factor α （TNF-α） production from pDCs in a dose-dependent manner. In addition, the ability of priming na ve T cells of pDCs was also impaired by cilostazol. The inhibitory effect was not due to cell killing since the viability of pDCs did not change upon cilostazol treatment. Conclusion Cilostazol inhibits pDCs cell activation and antigen presentation in vitro, which may explain how cilostazol protects against atherosclerosis.

Safety and immunogenicity of a SARS-CoV-2 inactivated vaccine in patients with chronic hepatitis B virus infection

Coronavirus disease 2019(COVID-19),caused by SARS-CoV-2 infection,has become a major global public health threat.Although significant advances have been made in developing and applying different vaccines in clinical trials[1,2],data are limited on the safety and efficacy of the inactivated vaccine in patients with chronic liver disease[3].Recent studies have preliminarily described the safety and immunogenicity of SARS-CoV-2 vaccines in patients with nonalcoholic fatty liver disease and in liver transplant recipients[4,5].

Pyroptosis:Induction and inhibition strategies for immunotherapy of diseases

Cell death is a central process for organismal health.Pyroptosis,namely pyroptotic cell death,is recognized as a critical type that disrupts membrane and triggers pro-inflammatory cytokine secretion via gasdermins,providing a robust form of cytolysis.Meanwhile,along with the thorough research,a great deal of evidence has demonstrated the dual effects of pyroptosis in host defense and inflammatory diseases.More importantly,the recent identification of abundant gasdermin-like proteins in bacteria and fungi suggests an ancient origin of pyroptosis-based regulated cell death in the life evolution.In this review,we bring a general overview of pyroptosis pathways focusing on gasdermin structural biology,regulatory mechanisms,and recent progress in induction and inhibition strategies for disease treatment.We look forward to providing an insightful perspective for readers to comprehend the frame and challenges of the pyroptosis field,and to accelerating its clinical application.

An improved method for the detection of double-stranded RNA suitable for quality control of mRNA vaccines

In more than 200 years since Edward Jenner successfully developed the first vaccine for combating smallpox,vaccines have played pivotal roles in preventing outbreaks of several infectious diseases,and it could even be asserted that each one of us has benefited from them.The recent severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)outbreak has also demonstrated the importance of vaccines in interrupting the transmission of infectious diseases(Deng and Peng,2020).

VX-765 reduces neuroinflammation after spinal cord injury in mice

Inflammation is a major cause of neuronal injury after spinal cord injury. We hypothesized that inhibiting caspase-1 activation may reduce neuroinflammation after spinal cord injury, thus producing a protective effect in the injured spinal cord. A mouse model of T9 contusive spinal cord injury was established using an Infinite Horizon Impactor, and VX-765, a selective inhibitor of caspase-1, was administered for 7 successive days after spinal cord injury. The results showed that:(1) VX-765 inhibited spinal cord injury-induced caspase-1 activation and interleukin-1β and interleukin-18 secretion.(2) After spinal cord injury, an increase in M1 cells mainly came from local microglia rather than infiltrating macrophages.(3) Pro-inflammatory Th1 Th17 cells were predominant in the Th subsets. VX-765 suppressed total macrophage infiltration, M1 macrophages/microglia, Th1 and Th1 Th17 subset differentiation, and cytotoxic T cells activation;increased M2 microglia;and promoted Th2 and Treg differentiation.(4) VX-765 reduced the fibrotic area, promoted white matter myelination, alleviated motor neuron injury, and improved functional recovery. These findings suggest that VX-765 can reduce neuroinflammation and improve nerve function recovery after spinal cord injury by inhibiting caspase-1/interleukin-1β/interleukin-18. This may be a potential strategy for treating spinal cord injury. This study was approved by the Animal Care Ethics Committee of Bengbu Medical College(approval No. 2017-037) on February 23, 2017.

The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection

Entero virus 71 （EV71） causes hand, foot, and mouth disease （HFMD） and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 （SCARB2） is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody （mAb） that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving a-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.

The function and clinical application of extracellular vesicles in innate immune regulation

The innate immune system plays a crucial role in the host defense against viral and microbial infection.Exosomes constitute a subset of extracellular vesicles(EVs)that can be released by almost all cell types.Owing to their capacity to shield the payload from degradation and to evade recognition and subsequent removal by the immune system,exosomes efficiently transport functional components to recipient cells.Accumulating evidence has recently shown that exosomes derived from tumor cells,host cells and even bacteria and parasites mediate the communication between the invader and innate immune cells and thus play an irreplaceable function in the dissemination of pathogens and donor cell-derived molecules,modulating the innate immune responses of the host.In this review,we describe the current understanding of EVs(mainly focusing on exosomes)and summarize and discuss their crucial roles in determining innate immune responses.Additionally,we discuss the potential of using exosomes as biomarkers and cancer vaccines in diagnostic and therapeutic applications.

Role of tegument proteins in herpesvirus assembly and egress

Morphogenesis and maturation of viral particles is an essential step of viral replication.An infectious herpesviral particle has a multilayered architecture,and contains a large DNA genome,a capsid shell,a tegument and an envelope spiked with glycoproteins.Unique to herpesviruses,tegument is a structure that occupies the space between the nucleocapsid and the envelope and contains many virus encoded proteins called tegument proteins.Historically the tegument has been described as an amorphous structure,but increasing evidence supports the notion that there is an ordered addition of tegument during virion assembly,which is consistent with the important roles of tegument proteins in the assembly and egress of herpesviral particles.In this review we first give an overview of the herpesvirus assembly and egress process.We then discuss the roles of selected tegument proteins in each step of the process,i.e.,primary envelopment,deenvelopment,secondary envelopment and transport of viral particles.We also suggest key issues that should be addressed in the near future.

Efficacy and safety of the Qiguiyin formula in severe pneumonia: study protocol for a randomized, double-blind, placebo-controlled clinical trial

OBJECTIVE: To evaluate the clinical efficacy and safety of Qiguiyin(QGY) formula in patients with severe pneumonia in China compared with a placebo.METHODS: This is a multicenter double-blind, placebo-controlled, randomized clinical trial with two parallel arms. There will be 530 patients enrolled and randomized into either the experimental group(QGY formula) or the control group(placebo). Therapies for patients in the two groups above will be based on the conventional therapy. The primary outcome is 28-day mortality. Secondary outcomes include:(a) duration of hospital stay;(b) duration of time in the intensive care unit(ICU) stays;(c) duration of mechanical ventilation;(d) antibiotic DDD value(which means the doses of antibotics during the treatment period);(e) serum procalcitonin(PCT) level;(f) serum C-reactive protein(CRP)level;(g) Pneumonia severity index(PSI) score;(h)Sequential Organ Failure Assessment(SOFA) score;(i) sputum culture results;(j) blood routine examination results;(k) routine urine test results;(l) stool routine examination results;(m) electrocardiogram results;(n) alanine aminotransferase levels;(o) aspartate amino transferase levels;(p) total bilirubin;(q) creatinine levels;(r) urea nitrogen levels;and(s)adverse events.ETHICS AND DISSEMINATION: The protocol has been approved by the Research Ethics Committee of Beijing Hospital of Traditional Chinese Medicine,Affiliated with Capital Medical University(2018 BL-053-02). This trial aims to provide evidence for QGY formula combined with conventional therapy in treating patients with severe bacterial pneumonia,and to verify the clinical effectiveness and safety of QGY formula in China compared with placebo. Additionally, this trial will reveal the effect of QGY formula on delaying/reversing the characteristics of drug-resistant bacteria.Trial registration number: Chi CTR1800019785.

Architecture of the herpesvirus genomepackaging complex and implications for DNA translocation

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs.Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown.We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP·BeF3-bound states.Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins,pUL28 and pUL33-unlike bacteriophage terminases.Distal to the nuclease domains,six ATPase domains form a central channel with conserved basicpatches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation.Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode.Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

IL-17-triggered downregulation of miR-497 results in high HIF-1α expression and consequent IL-1β and IL-6 production by astrocytes in EAE mice

Interleukin 17(IL-17)is increasingly recognized as a key factor that contributes to the pathogenesis of multiple sclerosis(MS)and its experimental mouse autoimmune encephalomyelitis(EAE)model.However,the roles and regulatory mechanisms of IL-17-induced pro-inflammatory cytokine production in EAE mice remain largely unclear.In this study,the expression of IL-17,hypoxia inducible factor-1α(HIF-1α),IL-1β,IL-6 and microRNA-497(miR-497),as well as their intrinsic associations,was investigated using EAE model mice and cultured astrocytes exposed to IL-17 in vitro.We observed markedly increased production of IL-17,HIF-1α,IL-1βand IL-6 in the brain tissues of EAE mice,while the expression and secretion of HIF-1α,IL-1βand IL-6 were also significantly increased when cultured primary astrocytes from mice were stimulated with IL-17.Meanwhile,the expression of miR-497 was downregulated both in vivo and in vitro.Subsequent in vitro experiments revealed that IL-17 induced the production of IL-1βand IL-6 in astrocytes through the upregulation of HIF-1αas a transcriptional factor,indicating that IL-17-mediated downregulation of miR-497 enhanced HIF-1αexpression.Furthermore,astrocytespecific knockdown of IL-17RA and HIF-1αor astrocyte-specific overexpression of miR-497 by infection with different lentiviral vectors containing an astrocyte-specific promotor markedly decreased IL-1βand IL-6 production in brain tissues and alleviated the pathological changes and score of EAE mice.Collectively,these findings indicate that decreased miR-497 expression is responsible for IL-17-triggered high HIF-1αexpression and consequent IL-1βand IL-6 production by astrocytes in EAE mice.

OTUD5 promotes innate antiviral and antitumor immunity through deubiquitinating and stabilizing STING

Stimulator of interferon genes(STING)is an adaptor protein that is critical for effective innate antiviral and antitumor immunity.The activity of STING is heavily regulated by protein ubiquitination,which is fine-tuned by both E3 ubiquitin ligases and deubiquitinases.Here,we report that the deubiquitinase OTUD5 interacts with STING,cleaves its K48-linked polyubiquitin chains,and promotes its stability.Consistently,knockout of OTUD5 resulted in faster turnover of STING and subsequently impaired type I IFN signaling following cytosolic DNA stimulation.More importantly,Lyz2-Cre Otud5^(fl/Y) mice and CD11-Cre Otud5^(fl/Y) mice showed more susceptibility to herpes simplex virus type 1(HSV-1)infection and faster development of melanomas than their corresponding control littermates,indicating that OTUD5 is indispensable for STING-mediated antiviral and antitumor immunity.Our data suggest that OTUD5 is a novel checkpoint in the cGAS-STING cytosolic DNA sensing pathway.

Circular RNA circZbtb20 maintains ILC3 homeostasis and function via Alkbh5-dependent m6A demethylation of Nr4a1 mRNA

Group 3 innate lymphoid cells(ILC3s)play critical roles in innate immunity and gut homeostasis.However,how ILC3 homeostasis is regulated remains elusive.Here,we identified a novel circular RNA,circZbtb20,that is highly expressed in ILC3s and required for their maintenance and function.CircZbtb20 deletion causes reduced ILC3 numbers,increasing susceptibility to C.rodentium infection.Mechanistically,circZbtb20 enhances the interaction of Alkbh5 with Nr4a1 mRNA,leading to ablation of the m6A modification of Nr4a1 mRNA to promote its stability.Nr4a1 initiates Notch2 signaling activation,which contributes to the maintenance of ILC3 homeostasis.Deletion of Alkbh5 or Nr4a1 also impairs ILC3 homeostasis and increases susceptibilities to bacterial infection.Thus,our findings reveal an important role of circular RNA in the regulation of innate lymphoid cell homeostasis.

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1(TBK1)is an essential protein kinase for activation of interferon regulatory factor 3(IRF3)and induction of the type I interferons(IFN-I).Although the biochemical regulation of TBK1 activation has been studied,little is known about how enterovirus 71(EV71)employs the deubiquitinases(DUBs)to regulate TBK1 activation for viral immune evasion.Here,we found that EV71 infection upregulated the expression of ubiquitinspecific protease 24(USP24).Further studies revealed that USP24 physically interacted with TBK1,and can reduce K63-linked polyubiquitination of TBK1.Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination,promoted the phosphorylation and nuclear translocation of IRF3,and in turn improved IFN-I production during EV71 infection.As a consequence,USP24 knockdown dramatically inhibited EV71 infection.This study revealed USP24 as a novel regulator of TBK1 activation,which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.