Hepatitis B virus X protein represses expression of tumor suppressor PTPN18 in hepatocellular carcinoma.

HBV-associated hepatocellular carcinoma (HCC) represents the prevalent form of HCC, with HBx protein being a crucial oncoprotein. Numerous members of the protein tyrosine phosphatase non-receptor (PTPN) family have been confirmed to be significantly associated with the occurrence and progression of malignant tumors. Our group has previously identified the involvement of PTPN13 in HCC. However, the roles of other PTPNs in HCC still requires further investigation. In this study, we found PTPN18 expression was significantly downregulated within HCC tissues compared to that in adjacent non-tumor tissues and normal liver tissues. Functionally, PTPN18 exerted inhibitory effects on the proliferation, migration, invasion, and sphere-forming capability of HCC cells, while concurrently promoting apoptotic processes. Through phospho-protein microarray screening followed by subsequent validation experiments, we identified that PTPN18 could activate the p53 signaling pathway and suppress the AKT/FOXO1 signaling cascade in HCC cells. Moreover, we found that the HBx protein mediated the repression of PTPN18 expression by upregulating miR-128-3p. Collectively, our study unveiled the role of PTPN18 as a tumor suppressor in HBV-related HCC. Implications: Our findings revealed PTPN18 might serve as a potential diagnostic and therapeutic target for HBV-related HCC.

Immunogenicity and protective efficacy of Ag85A and truncation of PstS1 fusion protein vaccines against tuberculosis.

Tuberculosis (TB) is an important public health problem, and the One Health approach is essential for controlling zoonotic tuberculosis. Therefore, a rationally designed and more effective TB vaccine is urgently needed. To enhance vaccine efficacy, it is important to design vaccine candidates that stimulate both cellular and humoral immunity against TB. In this study, we fused the secreted protein Ag85A as the T cell antigen with truncated forms of the mycobacterial cell wall protein PstS1 with B cell epitopes to generate vaccine candidates, Ag85A-tnPstS1 (AP1, AP2, and AP3), and tested their immunogenicity and protective efficacy in mice. The three vaccine candidates induced a significant increase in the levels of T cell-related cytokines such as IFN-γ and IL-17, and AP1 and AP2 can induce more balanced Th1/Th2 responses than AP3. Strong humoral immune responses were also observed in which the production of IgG antibodies including its subclasses IgG1, IgG2c, and IgG3 was tremendously stimulated. AP1 and AP2 induced early antibody responses and more IgG3 isotype antibodies than AP3. Importantly, the mice immunised with the subunit vaccine candidates, particularly AP1 and AP2, had lower bacterial burdens than the control mice. Moreover, the serum from immunised mice can enhance phagocytosis and phagosome-lysosome fusion in macrophages, which can help to eradicate intracellular bacteria. These results indicate that the subunit vaccines Ag85A-tnPstS1 can be promising vaccine candidates for tuberculosis prevention.

Integrated analysis reveals an aspartate metabolism-related gene signature for predicting the overall survival in patients with hepatocellular carcinoma.

BACKGROUND: Deregulating cellular metabolism is one of the prominent hallmarks of malignancy, with a critical role in tumor survival and growth. However, the role of reprogramming aspartate metabolism in hepatocellular carcinoma (HCC) are largely unknown. METHODS: The multi-omics data of HCC patients were downloaded from public databases. Univariate and multivariate stepwise Cox regression were used to establish an aspartate metabolism-related gene signature (AMGS) in HCC. The Kaplan-Meier and receiver operating characteristic curve analyses were performed to evaluate the predictive ability for overall survival (OS) in HCC patients. Gene set enrichment analysis and immune infiltration analysis were operated to determine the potential mechanisms underlying the AMGS. Single-cell RNA sequencing (scRNA-seq) data of liver cancer stem cells were visualized by t-SNE algorithm. In vivo and in vitro experiments were implemented to investigate the biological function of CAD in HCC. In addition, a nomogram based on the AMGS and clinicopathologic characteristics was constructed by univariate and multivariate Cox regression analyses. RESULTS: Patients in the high-AMGS subgroup exerted advanced tumor status and poor prognosis. Mechanistically, the high-AMGS subgroup patients had significantly enhanced proliferation and stemness-related pathways, increased infiltration of regulatory T cells and upregulated expression levels of suppressive immune checkpoints in the tumor immune microenvironment. Notably, scRNA-seq data revealed CAD, one of the aspartate metabolism-related gene, is significantly upregulated in liver cancer stem cells. Silencing CAD inhibited proliferative capacity and stemness properties of HCC cells in vitro and in vivo. Finally, a novel nomogram based on the AMGS showed an accurate prediction in HCC patients. CONCLUSIONS: The AMGS represents a promising prognostic value for HCC patients, providing a perspective for finding novel biomarkers and therapeutic targets for HCC.

Application and Resistance Mechanisms of Lenvatinib in Patients with Advanced Hepatocellular Carcinoma.

Lenvatinib, a multitargeted tyrosine kinase inhibitor (TKI), is one of the preferred targeted drugs for the treatment of advanced hepatocellular carcinoma (aHCC). Since the REFLECT study showed that lenvatinib was noninferior to sorafenib in overall survival (OS), lenvatinib monotherapy has been widely used for aHCC. Moreover, lenvatinib combination therapy, especially lenvatinib combined with immune checkpoint inhibitors (ICIs), has shown more encouraging clinical results. However, drug development and comprehensive treatment have not significantly improved the prognosis, and lenvatinib resistance is often encountered in treatment. The underlying molecular mechanism of lenvatinib resistance is still unclear, and studies to solve drug resistance are ongoing. The molecular mechanisms of lenvatinib resistance in patients with aHCC include the regulation of signaling pathways, the regulation of noncoding RNAs, the impact of the immune microenvironment, tumor stem cell activation and other mechanisms. This review aims to (1) summarize the progress of lenvatinib in treating aHCC, (2) delineate the known lenvatinib resistance mechanisms of current therapy, and (3) describe the development of therapeutic methods intended to overcome these resistance mechanisms.

Circular RNA circSFMBT2 downregulation by HBx promotes hepatocellular carcinoma metastasis via the miR-665/TIMP3 axis.

Hepatitis B virus X protein (HBx) is considered as an oncogene in tumorigenesis and progression of hepatocellular carcinoma (HCC). In recent years, the important role of circular RNAs (circRNAs) in HCC has been increasingly demonstrated. However, the regulatory mechanisms of HBx on circRNAs remains largely unknown. In this study, we identified that a novel circRNA, circSFMBT2, was markedly downregulated by HBx. Low expression of circSFMBT2 was correlated with poor prognosis and vascular invasion. Functionally, overexpression of circSFMBT2 significantly inhibited HCC metastasis both in vitro and in vivo. The mechanism of circSFMBT2 was to as a sponge of miR-665, which is a negative regulator of tissue inhibitor of metalloproteinases 3 (TIMP3). However, HBx downregulated circSFMBT2 via the interaction with DExH-box helicase 9 (DHX9), which binds to flanking circRNA-forming introns. In conclusion, circSFMBT2, which is downregulated by HBx, acts as a tumor suppressor to inhibit tumor metastasis through the miR-665/TIMP3 axis. Our study suggests that circSFMBT2 could be a potential prognostic biomarker and therapeutic target for HCC.

Characterization of Shiga toxin-producing Escherichia coli isolated from Cattle and Sheep in Xinjiang province, China, using whole-genome sequencing.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen capable of causing severe gastrointestinal diseases in humans. Cattle and sheep are the natural reservoir hosts of STEC strains. Previously, we isolated 56 STEC strains from anal and carcass swab samples of cattle and sheep in farms and slaughterhouses. In this study, we performed whole-genome sequencing of these isolates and determined their serotypes, virulence profiles, sequence types (STs) and genetic relationships. Our results showed that the 56 isolates belong to 20 different STs, 29 O:H serotypes and 8 stx subtype combinations. The highly prevalent serotypes for bovine and ovine isolates were O8:H25 and O87:H16, respectively. Five serotypes of cattle or sheep isolates are novel. The majority (63%) of cattle isolates contain stx1 + stx2, subtyped into stx1a, stx2a and stx2c. In contrast, most of the sheep isolates contain stx1 only, primarily subtyped into stx1a and stx1c. None of the isolates tested eae-positive, but virulence factors such as ehxA and espP were present with variable prevalence rates. The prevalence of saa (19.6%) and espP (12.5%) in cattle isolates is much higher than that in sheep isolates, whereas that of subA (34%), katP (14.3%) and ireA (28.6%) in sheep isolates is considerably higher than that in cattle isolates. Core-genome SNP analysis revealed that the majority of isolates could be clustered based on their serotypes or STs, whereas some clustering is associated with more than one ST or serotype. Five sheep isolates (4 belonging to ST675 and serotype O76:H19 and 1 belonging to ST25 and serotype O128:H2) share STs, serotypes and stx profiles with two hemolytic uremic syndrome-associated enterohemorrhagic E. coli (HUSEC) isolates; a cattle isolate belonging to the same ST as HUSEC isolate HUSEC001 contains all the nine virulence genes tested. These data suggest a potential of the six isolates for causing severe human infections. Collectively, we described the characteristics of cattle and sheep STEC isolates from Xinjiang, China, which may be utilized in comparative studies of other geographic regions and sources of isolation, and for surveillance as well.

Analysis of the potential association between ferroptosis and immune in hepatocellular carcinoma and their relationship with prognosis.

Background: The development of targeted therapy and immunotherapy has enriched the treatment of hepatocellular carcinoma (HCC), however, have had poor or no reponse, or even no response. Previous research suggested that ferroptosis and tumor immune microenvironment (TIME) may have a fundamental impact on efficacy during HCC immunotherapy and targeted therapy. Therefore, there is a clinical need to develop a signature that categorizes HCC patients in order to make more accurate clinical decisions. Methods: Clinical data and gene expression data of HCC patients were obtained from The Cancer Genome Atlas (TCGA) portal and International Cancer Genome Consortium (ICGC) portal. To identify ferroptosis-related immune-related genes (ferroptosis-related IRGs), Pearson correlation analysis was conducted. The ferroptosis-related IRGs prognostic signature (FIPS) was constructed using Univariate Cox and LASSO Cox algorithms. The predictive effectiveness of FIPS was evaluated using Receiver Operating Characteristic (ROC) curves and survivorship curve. The correlation ship between FIPS and TIME was evaluated using single-sample Gene Set Enrichment Analysis (ssGSEA) and CIBERSORT. The relationship between FIPS and immunotherapy responsiveness was evaluated using immunophenoscore. The expression level of 10 ferroptosis-related IRGs in normal liver tissues and HCC tissues was compared using immunohistochemistry. Finally, we established a nomogram (based on FIPS, TNM stage, and age) for clinical application. Results: The FIPS was established with ten ferroptosis-related IRGs. The high-FIPS subgroup showed a poor clinical prognosis and an obviously higher proportion of HCC patients with advanced TNM stage, high WHO grade and high alpha fetoprotein(AFP) value. Analysis of TIME indicated that patients in the high-FIPS subgroup may be in immunosuppressed state. Meanwhile, we found that ferroptosis may be inhibited in the high-FIPS subgroup and this subgroup may be impervious to immunotherapy and sorafenib. Conclusion: We constructed a novel potential prognostic signature for HCC patients that predicts overall survival, ferroptosis and immune status, sorafenib sensitivity, and immunotherapy responsiveness.

Whole genome sequencing analysis of avian pathogenic Escherichia coli from China.

Avian pathogenic Escherichia coli (APEC) can cause localized or systemic infection in poultry herds, i.e., colibacillosis, which is an economically devastating bacterial disease of the poultry industry worldwide. Additionally, some APEC may have zoonotic potential. In this study, we sequenced 125 APEC isolates from chickens and ducks with obvious clinical symptoms in poultry farms in China and performed genomic epidemiological analysis along with 16 APEC reference genomes downloaded from NCBI. The phylogenetic analysis indicated a great diversity of APEC isolates, and a total of 35 different O types, 22 H types, and 29 ST types were identified. Several virulence-associated genes (VAGs), such as ompT (96.45 %), iss (97.87 %), and hlyF (90.78 %), as well as four complete siderophore gene clusters, including the Sit transport system (86.52 %), aerobactin (89.36 %), salmochelin (79.43 %), and yersiniabactin (54.61 %), were detected in APEC isolates with high prevalence, which could serve as virulence markers of APEC. Several virulence-associated gene clusters, including the two T6SS systems and the K1 capsule biosynthesis gene clusters, were significantly associated with APEC of phylogroups B2, D, and F but very rarely encoded by the APEC from phylogroups C and E. In addition, several virulence-associated genes, which have been reported in other E. coli pathotypes but have not been reported in APEC, were identified in this study. Our findings in this study have implications for a better understanding of APEC evolution and pathogenesis and may lead to the development of new diagnostic tools for APEC.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates Hypochlorous Acid Resistance and Impacts Expression of the Type IV Secretion System in Brucella melitensis.

The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCEBrucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.

Construction and Validation of a Combined Ferroptosis and Hypoxia Prognostic Signature for Hepatocellular Carcinoma.

Background: Ferroptosis, as a unique programmed cell death modality, has been found to be closely related to the occurrence and development of hepatocellular carcinoma (HCC). Hypoxia signaling pathway has been found to be extensively involved in the transformation and growth of HCC and to inhibit anti-tumor therapy through various approaches. However, there is no high-throughput study to explore the potential link between ferroptosis and hypoxia, as well as their combined effect on the prognosis of HCC. Methods: We included 370 patients in The Cancer Genome Atlas (TCGA) database and 231 patients in the International Cancer Genome Consortium (ICGC) database. Univariate COX regression and Least Absolute Shrinkage and Selection Operator approach were used to construct ferroptosis-related genes (FRGs) and hypoxia-related genes (HRGs) prognostic signature (FHPS). Kaplan-Meier method and Receiver Operating Characteristic curves were analyzed to evaluate the predictive capability of FHPS. CIBERSOR and single-sample Gene Set Enrichment Analysis were used to explore the connection between FHPS and tumor immune microenvironment. Immunohistochemical staining was used to compare the protein expression of prognostic FRGs and HRGs between normal liver tissue and HCC tissue. In addition, the nomogram was established to facilitate the clinical application of FHPS. Results: Ten FRGs and HRGs were used to establish the FHPS. We found consistent results in the TCGA training cohort, as well as in the independent ICGC validation cohort, that patients in the high-FHPS subgroup had advanced tumor staging, shorter survival time, and higher mortality. Moreover, patients in the high-FHPS subgroup showed ferroptosis suppressive, high hypoxia, and immunosuppression status. Finally, the nomogram showed a strong prognostic capability to predict overall survival (OS) for HCC patients. Conclusion: We developed a novel prognostic signature combining ferroptosis and hypoxia to predict OS, ferroptosis, hypoxia, and immune status, which provides a new idea for individualized treatment of HCC patients.

The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis.

Brucella, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H2O2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence.

A concise total synthesis of amphidinolide T2.

RCM + AD = T2: In the presence of the C16-methylene group, regioselective ring-closing metathesis (RCM) formed the (12E)-endocyclic double bond, which underwent Os-catalyzed asymmetric dihydroxylation (AD) to give the desired 12,13-diol intermediate required for the total synthesis of amphidinolide T2 in 16 linear steps in 8.0% overall yield.