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Engineered T cell receptor-T cell (TCR-T) therapy and chimeric antigen receptor-T cell (CAR-T) therapy are currently the two most effective ways of adoptive T cell therapy. Because CAR can only recognize antigens on the surface of tumors, CAR-T therapy has not yet had satisfactory results in the treatment of solid tumors. TCR can not only recognize tumor surface antigens, but also intracellular antigens. Thus TCR-T therapy has shown unprecedented promise in the treatment of solid tumors, and has become an extremely attractive treatment modality. This review described the differences between TCR-T therapy and CAR-T therapy in recognizing cancer antigens, the clinical targets and different types of tumor antigens targeted by current TCR-T therapy, the clinical development status of TCR-T antitumor therapy, and discussed the criteria for preclinical evaluation of TCR titer and the advantages, limitations and possible effective countermeasures of current TCR-T therapy. Finally, we reviewed the current status of TCR-T therapy and some of the challenges, emphasized the importance of targeting tumor-specific antigens, and outlined neoantigen-specific TCR-T treatment strategies combining checkpoint blockage therapy and oncolytic viruses, which we expect will significantly improve cancer immunotherapy and provide some clues for future TCR-T therapy to eradicate multiple types of cancer.

The coronavirus disease 2019 (COVID-19) global pandemic, caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been marked by severe cases demonstrating a "cytokine storm", an upsurge of pro-inflammatory cytokines in the bloodstream. NLRP3 inflammasomes, integral to the innate immune system, are speculated to be activated by SARS-CoV-2 within host cells. This review investigates the potential correlation between NLRP3 inflammasomes and COVID-19, exploring the cellular and molecular mechanisms through which SARS-CoV-2 triggers their activation. Furthermore, promising strategies targeting NLRP3 inflammasomes are proposed to mitigate the excessive inflammatory response provoked by SARS-CoV-2 infection. By synthesizing existing studies, this paper offers insights into NLRP3 as a therapeutic target, elucidating the interplay between COVID-19 and its pathophysiology. It serves as a valuable reference for future clinical approaches in addressing COVID-19 by targeting NLRP3, thus providing potential avenues for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as "co-receptors" because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71's early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a complex disorder characterized by insufficient insulin production or insulin resistance, which results in a lifelong dependence on glucose-lowering drugs for almost all patients. During the fight with diabetes, researchers are always thinking about what characteristics the ideal hypoglycemic drugs should have. From the point of view of the drugs, they should maintain effective control of blood sugar, have a very low risk of hypoglycemia, not increase or decrease body weight, improve β-cell function, and delay disease progression. Recently, the advent of oral peptide drugs, such as semaglutide, brings exciting hope to patients with chronic diabetes. Legumes, as an excellent source of protein, peptides, and phytochemicals, have played significant roles in human health throughout human history. Some legume-derived peptides with encouraging anti-diabetic potential have been gradually reported over the last two decades. Their hypoglycemic mechanisms have also been clarified at some classic diabetes treatment targets, such as the insulin receptor signaling pathway or other related pathways involved in the progress of diabetes, and key enzymes including α-amylase, α-glucosidase, and dipeptidyl peptidase-IV (DPP-4). This review summarizes the anti-diabetic activities and mechanisms of peptides from legumes and discusses the prospects of these peptide-based drugs in type 2 diabetes (T2D) management. [ABSTRACT FROM AUTHOR]

Background: Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD) and sometimes causes several neurological complications. However, the underlying mechanism of the host response to the virus infection remains unclear. Objective: To reveal the cell-specific transcriptional response of cultured RD cells following infection with EV71, and better understand the molecular mechanisms of virus-host interactions. Methods: The RD cells were infected with or without EV71 for 24 h, and then transcriptome sequencing and qRT-PCR were performed to analyze the transcriptome difference of functional genes. Results: More than 15000 genes were identified in transcriptome sequencing. In comparison with uninfected RD cells, 329 DEGs were identified in cells infected with EV71. GO and KEGG pathway enrichment analysis showed that most of the DEGs were related to DNA binding, transcriptional regulation, immune response and inflammatory response, apoptosis inducing factors and enriched in JAK-STAT and MAPK signaling pathways. TXNIP (thioredoxin-interacting protein) gene was further demonstrated to play an important role participating in cellular apoptosis induced by EV71, and the apoptosis and death mediated by TXNIP during EV71 infection was triggered by viral 2A protease (2Apro), not 3C protease (3Cpro). Conclusion: Our study demonstrated that RD cells have a significant response to EV71 infection, including immune response and apoptosis. 2Apro might be a key inducer relative to the cellular apoptosis and death mediated by TXNIP during EV71 infection. These data would contribute to preferably understand the process at the molecular level and provide theoretical foundation for diagnosis and treatment of EV71-related diseases. [ABSTRACT FROM AUTHOR]

A synthetic peptide octaarginine which mimics human immunodeficiency virus‐1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental pH‐sensitive fluorophores in a same nanoparticle. The authors believe that this triple fluorescent pH sensor provides a new tool to pH measurements that can have application in cellular uptake mechanism study and new nanomedicine design. [ABSTRACT FROM AUTHOR]

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer. [ABSTRACT FROM AUTHOR]

Replication of hepatitis B virus (HBV) via protein-primed reverse transcription is initiated by binding of the viral P protein to the conserved ε stem-loop on the pregenomic (pg) RNA. This triggers encapsidation of the complex and the ε-templated synthesis of a short P protein-linked DNA oligonucleotide (priming) for subsequent minus-strand DNA extension. ε consists of a lower and upper stem, a bulge containing the priming template, and an apical loop. The nonhelical subelements are considered important for DNA synthesis and pgRNA packaging whereas the role of the upper stem is not well characterized. Priming itself could until recently not be addressed because in vitro generated HBV P - ε complexes showed no activity. Focussing on the four A residues at the base and tip of the upper ε stem and the two U residues in the loop we first investigated the impact of 24 mutations on viral DNA accumulation in transfected cells. While surprisingly many mutations were tolerated, further analyzing the negatively acting mutations, including in a new cell-free priming system, revealed divergent position-related impacts on pgRNA packaging, priming activity and possibly initiation site selection. This genetic separability implies that the ε RNA undergoes multiple distinct interactions with P protein as pgRNA encapsidation and replication initiation progress, and that the strict conservation of ε in nature may reflect its optimal adaptation to comply with all of them. The data further define the most attractive mutants for future studies, including as decoys for interference with HBV replication. [ABSTRACT FROM AUTHOR]

Introduction: The Hepatitis B Virus (HBV) genome contains four ORFs, S (surface), P (polymerase), C (core) and X. S is completely overlapped by P and as a consequence the overlapping region is subject to distinctive evolutionary constraints compared to the remainder of the genome. Specifically, a non-synonymous substitution in one coding frame may produce a synonymous substitution in the alternative frame, suggesting a possible conflict between requirements for diversifying and purifying forces. To examine how these contrasting requirements are balanced within this region, we investigated the relationship amongst positive selection sites, conserved regions, epitopes and elements of protein structure to consider how HBV balances the contrasting evolutionary pressures. Methodology/Results: 323 HBV genotype D genome sequences were collected and analyzed to identify sites under positive selection and highly conserved regions. Epitopes sequences were retrieved from previously published experimental studies stored in the Immune Epitope Database. Predicted secondary structures were used to investigate the association between structure and conservation. Entropy was used as a measure of conservation and bivariate logistic regression was used to investigate the relationship between positive selection/conserved sites and epitope/secondary structure regions. Our results indicate: (i) conservation in S is primarily dictated by α-helix elements in the protein structure, (ii) variable residues are mainly located in PreS, the major hydrophilic region (MHR) and the C-terminus, (iii) epitopes in S, which are directly targeted by the host immune system, are significantly associated with sites under positive selection. Conclusions: The highly variable spacer domain in P, which corresponds to PreS in S, appears to act as a harbor for the accumulation of mutations that can provide flexibility for conformational changes and responding to immune pressure. [ABSTRACT FROM AUTHOR]

Little is known about the extra- and intracellular stimuli inducing renal stem/progenitor cells to develop into three-dimensionally structured tubules. To study this specific development in a controlled environment, we used an advanced culture technique. Embryonic tissue derived from neonatal rabbit kidney was placed in a perfusion culture container at the interface of an artificial interstitium made of a polyester fleece. Culture was carried out in chemically defined Iscove’s Modified Dulbecco’s Medium (IMDM) for 13 days. Development of tubules was histochemically detected on cryosections labeled with Soybean Agglutinin (SBA). The experiments showed that aldosterone exerts a specific tubulogenic effect. Application of aldosterone (1 × 10−7 M) raised numerous SBA-labeled tubules, while in the absence of the steroid hormone the development of tubules was lacking. Specificity of hormone action was analyzed by the use of aldosterone antagonists. Administration of spironolactone (1 × 10−4 M) and canrenoate (1 × 10−5 M) completely inhibited the development of tubules. Finally, disrupting the intracellular molecular complex of the mineralocorticoid receptor (MR) and heat shock proteins by geldanamycin (2 μg/ml) prevented the development of tubules. Our results suggest that the tubulogenic effect induced by aldosterone is attributed to both hormone binding and an undisturbed intracellular response of the MR. [ABSTRACT FROM AUTHOR]

Abstract: Activation of hepatic stellate cells (HSCs) is the central event in the development of liver fibrosis and cirrhosis. The transdifferentiation process of quiescent into activated HSCs requires a complete reprogramming in gene expression, which is governed by modulation of transcriptional activators or repressors. Using microarray analysis to identify genes differentially expressed during the activation process of human HSCs, zinc finger protein 267 (ZNF267) mRNA was up-regulated in activated HSCs and in cirrhotic human liver. ZNF267 belongs to the family of Kruppel-like zinc fingers and contains a conserved KRAB (Kruppel associated box) A and B domain in the N-terminal part outside the C-terminal region of zinc fingers. ZNF267 constructs containing enhanced cyan fluorescence protein were constitutively localized in the nucleus. When fused to GAL4 DNA binding domain, full-length ZNF267 and all constructs encompassing KRAB A domain showed transcriptional repressor activity. Microarray analysis and RNase protection assays showed that ZNF267 represses MMP-10 gene expression, which was confirmed by reporter gene assays. Furthermore, ZNF267 binds to the MMP-10 promoter region as demonstrated by chromatin immunoprecipitation assays. In conclusion, our results suggest that ZNF267 as a negative transcriptional regulator of MMP-10 might promote liver fibrogenesis through alteration of matrix degradation in vivo. [Copyright &y& Elsevier]

Abstract: Liver fibrosis results from an excessive deposition of extracellular matrix proteins secreted by activated hepatic stellate cells (HSCs). The activation process is accompanied by an increased activity of various transcription factors, including zinc finger protein 267 (ZNF267). Recently, ZNF267 has been shown to modulate gene expression and to function as a transcriptional repressor. MMP-10 was identified as a target gene; its gene expression and promoter activity are inhibited by ZNF267, which might promote liver fibrogenesis through diminished matrix degradation. However, the transcriptional regulation of the ZNF267 gene is unknown. In the present study, we have cloned and characterized the human ZNF267 promoter containing a 1.5 kb fragment of the 5′-flanking region (−1414/+173). The ZNF267 gene has a TATA-less promoter with multiple transcription initiation sites. Analysis of serial 5′-deletions of luciferase reporter constructs revealed a minimal promoter between −72 and +173 bp. Mutational analysis of putative regulatory elements indicated that a CCAAT box within this region was essential for ZNF267 promoter activity. Electrophoretic mobility shift assays demonstrated that transcription factor nuclear factor Y (NF-Y) bound to the CCAAT box. In co-transfection experiments, NF-YA increased the promoter activity of ZNF267. In conclusion, our results suggest that the binding site for NF-Y is critical for ZNF267 gene regulation and, herewith, the activation of this transcriptional factor may play an important role in the activation process of HSCs and in liver fibrosis. [Copyright &y& Elsevier]

Coxsackievirus B3 (CVB3) is the most common cause of acute and chronic viral myocarditis, primarily in children, while human adenovirus infections represent a significant cause of morbidity and mortality worldwide, in people of all ages. A series of novel 2-benzoxyl-phenylpyridine derivatives were evaluated for their potential antiviral activities against CVB3 and adenovirus type 7 (ADV7). Preliminary assays indicated that some of these compounds exhibited excellent antiviral effects on both CVB3 and ADV7 viruses; they could effectively inhibit virus-induced cytopathic effects, reduce viral progeny yields, and had similar or superior antiviral activities compared with the control drug, ribavirin. Further, these compounds targeted the early stages of CVB3 replication in cells, including viral RNA replication and protein synthesis, rather than inactivating the virus directly, inhibiting virus adsorption/entry, or affecting viral release from cells. Our data demonstrate that the tested 2-benzoxyl-phenylpyridine derivatives are effective inhibitors of CVB3 and ADV7, raising the possibility that these compounds might be feasible candidates for anti-viral agents. [ABSTRACT FROM AUTHOR]

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI), which is widely used for anti-HIV-1. Evidences revealed that several central nervous system side effects could be observed in mice and patients with administration of EFV. However, the detailed mechanisms are still unknown. In this study, we investigated the effects of long-term EFV treatment on cognitive functions and the potential underlying mechanisms in mice. We maintained C57BL/6 mice aged 2 months with treatment containing 40 or 80 mg/kg/day EFV for 5 months, while control group treated with saline. The cognitive functions were evaluated by novel object recognition test, Barnes maze test and Morris water maze. The results showed significant short-term memory impairment in 40 and 80 mg/kg groups, and notable spatial learning and memory impairments in 80 mg/kg group, without any spontaneous activity alteration. Moreover, EFV induced impairments in dendritic integrity and synaptic plasticity in hippocampus. Furthermore, Significant increases were observed in the expression levels of pro-IL-1β, a similar tendency of TNF-α and phosphorylation of p65 of the 80 mg/kg group compared with control group. These results imply that long-term EFV treatment causes synaptic dysfunction resulting in cognitive deficits, which might be induced by the enhanced pro-inflammatory cytokines IL-1β and TNF-α via activating NF-κB pathway. • Long-term efavirenz administration causes cognitive deficits in C57BL/6 mice. • Severity of behavioral side effect in mice is dose-dependent. • The CNS is likely to be affected by efavirenz through the activation of NF-κB pathway. • Hippocampal impairments are strongly associated with cognitive deficits induced by long-term efavirenz administration. • Neither spontaneous activity alterations nor cortex impairments were detected. [ABSTRACT FROM AUTHOR]

Graphical abstract A decavanadate complex incorporating triethanolamine and sodium cations was synthesized which form a rare wavelike 2D network in the crystal structure and also showed potent inhibitory activities against human tumor cells. Abstract A decavanadate salt Na 4 {(HOCH 2 CH 2) 3 NH} 2 [V 10 O 28 ]·6H 2 O was synthesized via the reaction of sodium metavanadate and triethanolamine hydrochlorides in water solution, which exhibits an interesting wavelike 2D network in the crystal structure. The product also showed potent inhibitory activities against human laryngeal carcinoma epithelial cell line (Hep-2), human breast cancer cell line (MDA-MB-231) and hepatocellular liver carcinoma cell line (HepG 2), which was better than the positive control drug 5-fluorouracil (5-FU), implying that newly synthesized compound may be a potential candidate for the development of anti-cancer drugs. [ABSTRACT FROM AUTHOR]

A series of 2-Benzoxyl-Phenylpyridine derivatives were evaluated for their potential antiviral activities against EV71. The preliminary assays indicated that some of these compounds exhibited excellent antiviral effects on EV71, they could effectively inhibit virus-induced cytopathic effects (CPEs), reduce progeny viral yields, and present similar or better antiviral activities compared to the positive control drug ribavirin. Among these derivatives, compounds WY7, WY13 and WY14 showed the most potency against EV71. Investigation of the underlying mechanism of action revealed that these compounds target EV71 replication in cells post infection, they could profoundly inhibit viral RNA replication and protein synthesis, and inhibit virus-induced cell apoptosis. Further experiments demonstrated that compound WY7 potently inhibited the activity of the EV71 3C protease (3Cpro), and to some extent, it affected the activity of 3D polymerase (3Dpol), thus blocking viral replication, but not the activity of the 2A proteinase (2Apro). Modeling of the molecular binding of the 3Cpro-WY7 complex revealed that compound WY7 was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. These results indicate that these compounds might be feasible therapeutic agents against EV71 infection and that these compounds may provide promising lead scaffolds for the further design and synthesis of potential antiviral agents. [Display omitted] [ABSTRACT FROM AUTHOR]

Corona Virus Disease 2019 (COVID-19) continues to be a burden for human health since its outbreak in Wuhan, China in December 2019. Recently, the emergence of new variants of concerns (VOCs) is challenging for vaccines and drugs efficiency. In severe cases, SARS-CoV-2 provokes inappropriate hyperinflammatory immune responses leading to acute respiratory distress syndrome (ARDS) and even death. This process is regulated by inflammasomes which are activated after binding of the viral spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2) receptor and triggers innate immune responses. Therefore, the formation of "cytokines storm" leads to tissue damage and organ failure. NOD-like receptor family pyrin domain containing 3 (NLRP3) is the best studied inflammasome known to be activated during SARS-CoV-2 infection. However, some studies suggest that SARS-CoV-2 infection is associated with other inflammasomes as well; such as NLRP1, absent in melanoma-2 (AIM-2), caspase-4 and -8 which were mostly found during dsRNA virus or bacteria infection. Multiple inflammasome inhibitors that exist for other non-infectious diseases have the potential to be used to treat severe SARS-CoV-2 complications. Some of them have showed quite encouraging results during pre- and clinical trials. Nevertheless, further studies are in need for the understanding and targeting of SARS-Cov-2-induced inflammasomes; mostly an update of its role during the new VOCs infection is necessary. Hence, this review highlights all reported inflammasomes involved in SARS-CoV-2 infection and their potential inhibitors including NLRP3- and Gasdermin D (GSDMD)-inhibitors. Further strategies such as immunomodulators and siRNA are also discussed. As highly related to COVID-19 severe cases, developing inflammasome inhibitors holds a promise to treat severe COVID-19 syndrome effectively and reduce mortality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Diarimalala, Wei, Hu and Hu.)

Background: Hepatocellular carcinoma (HCC) is one of the most leading causes of cancer death worldwide. The 5-year survival rate of HCC patients remains low due to the lack of early-stage symptoms. Human complement factor H-related protein 4 (CFHR4) is a critical gene that belongs to the factor H family of plasma glycoproteins, which has not been linked to HCC development. The correlations between CFHR4 and prognosis and tumor-infiltrating lymphocytes in HCC are yet unknown. The present study demonstrated the involvement of CFHR4 in HCC via data mining approaches., Results: A total of 18 upregulated and 67 down-regulated differentially expressed genes (DEGs) were identified. Importantly, CFHR4, which was screened from DEGs, was shown to express at a lower level in HCC tumor tissue than normal tissues. Western blotting (WB), immunohistochemical (IHC) and quantitative reverse transcription PCR (qRT-PCR) experiments of clinical samples further validated CFHR4 was aberrantly expressed in HCC patients; Data from TCGA showed that CFHR4 was inversely correlated with a cancer family history, histological grade, tumor node metastasis (TNM) stage, and serum AFP level of HCC patients; Univariate and multivariate analyses revealed that low expression of CFHR4 was an independent predictive marker in patients with HCC; Kaplan-Meier analysis showed that the lower expression of CFHR4 was significantly associated with the progression of HCC and poor prognosis rates. Furthermore, TIMER analysis indicated that CFHR4 expression levels had correlations with infiltrating levels of immune cells in HCC., Conclusion: CFHR4 expression was low in HCC and was significantly related to the poor prognosis of HCC and the level of immune infiltration. CFHR4 played important roles in regulating the initiation and progression of HCC and could be a potential biomarker for the diagnosis and prognosis of HCC., Methods: The expression of CFHR4 was analyzed by GEO and TCGA-LIHC database and verified by WB and IHC assay. The biological function of CFHR4 was performed by GO and KEGG enrichment analysis, and the genomic alteration of CFHR4 was investigated by cBioPortal database.The correlation between CFHR4 expression and clinical relevance was evaluated through Cox proportional hazards model, and the correlation between CFHR4 expression and tumor immune infiltrates were studied by TIMER database., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ding, Li, Xu, Hu and Ye.)

During replication of the hepatitis B virus (HBV) in liver cells, the reverse transcription of pre-genomic RNA (pgRNA) is initiated by protein priming at an RNA packaging signal ε located near the 5' end of pgRNA. Heat-shock proteins (Hsps) such as Hsc70, Hsp40, and Hsp90 have been reported to be involved in the reconstitution of HBV polymerase (P protein) and E. The P - E complex initiates the reverse transcription and assembly of nucleocapsids. Hence, blockade of P - ε interactions is an attractive target for drug intervention. We explored the influence of the Hsp inhibitor KNK437 on replication and transcription of the HBV. Three working models were applied: HepG2. 2. 15 cell line; Huh7 cells transfected transiently with the 1. 05 X HBV (pCH9-3091) plasmid; Huh7 cells transfected transiently with the 1. 3 X HBV (pGEM-1. 3 X HBV) plasmid. Cytotoxic effects of KNK437 were detected by the CCK-8 method. Levels of hepatitis B surface antigen (HBsAg) and hepatitis B viral protein (HBeAg) in the media secreted from cells were measured using an ELISA. Intracellular HBV DNAs within nucleocapsids were measured by quantitative polymerase chain reaction (qPCR), and intracellular HBV RNAs by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Transcription of Hsps in cells was determined by qRT-PCR. Data suggested that KNK437 reduced the extracellular secretion of HBsAg and HBeAg in most cases; it downregulated expression of intracellular HBV DNAs within nucleocapsids and RNA transcripts. The lowest rate of viral DNAs in KNK437-treated hepatocytes for all experimental groups was ~1. 5%o (control, 100%), whereas that for RNAs was ~30%. Western blotting revealed KNK437 to inhibit intracellular core expression in HepG2. 2. 15. As a general inhibitor, KNK437 suppressed transcription of hsp70, hsp90b, and hsp4o. These data suggest that KNK437 may be a potent anti-HBV inhibitor for future therapy against chronic hepatitis.

The ultimate goal of chronic hepatitis B virus(HBV)therapy is full eradication of the virus from the liver. However, this is rarely achieved with the clinically available first-line agents (entecavir and tenofovir disoproxil fumarate) due to the inability to eliminate covalently closed circular DNA(cccDNA), which persists in the nucleus of infected hepatocyte cells,and failure of the host to induce an adequate specific immune response to control the infection. Currently, the clinical treatment for chronic HBV infection mainly includes nucleos(t)ide analogues (NAs), non-NAs and immune modulatory agents; however, each agent has individual advantages and drawbacks. It is, therefore, extremely urgent to identify novel targets involved in viral replication and develop novel anti-HBV drugs. In light of the breakthroughs in cccDNA research and host immune treatments, this review aims to summarize the state of the recent HBV drug research and development to highlight future therapeutic strategies to target the virus and host immune response.

The Ebola virus belongs to the Filovirus family, which causes Ebola hemorrhagic fever (mortality, 25%-90%). An outbreak of infection by the Ebola virus is sweeping across West Africa, leading to high mortality and worldwide panic. The Ebola virus has caused a serious threat to public health, so intensive scientific studies have been carried out. Several vaccines (e.g., rVSV-ZEBOV, ChAd3-ZEBOV) have been put into clinical trials and antiviral drugs (e.g., TKM-Ebola, ZMAPP) have been administered in the emergency setting to patients infected by the Ebola virus. Here, recent advances in vaccines and drugs against the Ebola virus are reviewed.

Background: Hepatitis B virus (HBV) transcription and replication are essentially restricted to hepatocytes. Based on the HBV enhancer and promoter complex that links hepatic glucose metabolism to its transcription and replication, HBV adopts a regulatory system that is unique to the hepatic gluconeogenic genes. CRTC2, the CREB-regulated transcription coactivator 2, is a critical switch modulating the gluconeogenic program in response to both hormonal and intracellular signals. However, the relationship between CRTC2 and HBV transcription and replication remains unclear., Methods: To analyze the influence of CRTC2 on HBV transcription and replication, CRTC2 expression construct or siRNA was cotransfected with plasmids containing enhancer II/core promoter complex-controlled luciferase or 1.3× wtHBV genome in Huh-7 cells. Luciferase activity, HBV core protein expression, HBV transcripts, and DNA replication intermediates were measured by luciferase assays, western blots, real-time polymerase chain reaction (PCR), and Southern blots, respectively. Forskolin (FSK) or phosphorylation-defective CRTC2 mutants were further utilized to elucidate the potential mechanism. siRNA against peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) was also used to examine the mediator involved in CRTC2-regulated HBV biosynthesis in Huh-7 cells., Results: CRTC2 overexpression increased HBV transcription and replication in Huh-7 cells, including levels of core protein expression, mRNA, and DNA replication intermediates. Correspondingly, CRTC2 knock down by siRNA reduced HBV biosynthesis. FSK treatment strongly enhanced the effect of CRTC2 through triggering the dephosphorylation and nuclear entry of CRTC2. The phosphorylation-defective mutant (S171A/S275A) of CRTC2 localized in the nucleus and was constitutively active, which dramatically promoted HBV transcription and replication similar to FSK-treated wild-type CRTC2. Knock down of PGC1α, whose expression was induced by CRTC2, greatly compromised the enhancing effect of CRTC2 on HBV transcription and replication., Conclusions: Our results clearly indicate that non-phosphorylated CRTC2 strongly enhances HBV biosynthesis through inducing PGC1α expression. Further study of the mechanisms will elucidate the importance of metabolic signals on HBV transcription and replication, and offer insight into potential targets for developing anti-HBV agents.

Introduction: The Hepatitis B Virus (HBV) genome contains four ORFs, S (surface), P (polymerase), C (core) and X. S is completely overlapped by P and as a consequence the overlapping region is subject to distinctive evolutionary constraints compared to the remainder of the genome. Specifically, a non-synonymous substitution in one coding frame may produce a synonymous substitution in the alternative frame, suggesting a possible conflict between requirements for diversifying and purifying forces. To examine how these contrasting requirements are balanced within this region, we investigated the relationship amongst positive selection sites, conserved regions, epitopes and elements of protein structure to consider how HBV balances the contrasting evolutionary pressures., Methodology/results: 323 HBV genotype D genome sequences were collected and analyzed to identify sites under positive selection and highly conserved regions. Epitopes sequences were retrieved from previously published experimental studies stored in the Immune Epitope Database. Predicted secondary structures were used to investigate the association between structure and conservation. Entropy was used as a measure of conservation and bivariate logistic regression was used to investigate the relationship between positive selection/conserved sites and epitope/secondary structure regions. Our results indicate: (i) conservation in S is primarily dictated by α-helix elements in the protein structure, (ii) variable residues are mainly located in PreS, the major hydrophilic region (MHR) and the C-terminus, (iii) epitopes in S, which are directly targeted by the host immune system, are significantly associated with sites under positive selection., Conclusions: The highly variable spacer domain in P, which corresponds to PreS in S, appears to act as a harbor for the accumulation of mutations that can provide flexibility for conformational changes and responding to immune pressure.

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.

Little information on the structural growth of renal tubules is available. A major problem is the technical limitation of culturing intact differentiated tubules over prolonged periods of time. Consequently, we developed an advanced culture method to follow tubule development. Isolated tissue containing renal progenitor cells was placed in a perfusion culture container at the interphase of an artificial polyester interstitium. Iscove's modified Dulbecco's medium without serum or protein supplementation was used for culture, and the culture period was 13 days. Tissue growth was not supported by addition of extracellular matrix proteins. The development of tubules was registered on cryosections labeled with soybean agglutinin (SBA) and tissue-specific antibodies. Multiple SBA-labeled tubules were found when aldosterone was added to the culture medium. In contrast, culture without aldosterone supplementation displayed completely disintegrated tissue. The development of tubules depended on the applied aldosterone concentration. The use of 1 x 10(-6) M and 1 x 10(-7) M aldosterone produced numerous tubules, while application of 1 x 10(-8) M to 1 x 10(-10) M led to a continuous decrease and finally a loss of tubule formation. The development of labeled tubules in aldosterone-treated specimens took an unexpectedly long period of at least 8 days. The morphogenic effect of aldosterone appeared to be mineralocorticoid hormone-specific since spironolactone and canrenoate abolished the development. Finally, dexamethasone induced widely distributed cell clusters instead of tubules.