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Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the incidence and mortality rates are increasing. Given the limited treatments of HCC and promising application of immunotherapy for cancer, we aimed to identify an immune-related prognostic signature that can predict overall survival (OS) rates and immunotherapy response in HCC.The initial signature development was conducted using a training dataset from the Cancer Genome Atlas followed by independent internal and external validations from that resource and the Gene Expression Omnibus. A signature based nomogram was generated using multivariate Cox regression analysis. The associations of signature score with tumor immune phenotype and response to immunotherapy were analyzed using single-sample gene set enrichment analysis and tumor immune dysfunction and exclusion algorithm. A cohort from Zhongshan Hospital was employed to verify the predictive robustness of the signature regarding prognostic risk and immunotherapy response.The prognostic signature, IGSIGS

Supplementary Figure from IFNα Potentiates Anti–PD-1 Efficacy by Remodeling Glucose Metabolism in the Hepatocellular Carcinoma Microenvironment

Lactobacillus bulgaricus is an important starter culture in the dairy industry, cell lysis is negative to the high density of this strain. This work describes the response of peptidoglycan synthases and hydrolases in Lactobacillus bulgaricus sp1.1 when pH decreasing in batch culture. First, the cell lysis was investigated by measuring the cytosolic lactate dehydrogenase released to the fermentation broth, a continuous increase in extracellular lactate dehydrogenase was observed after the lag phase in batch culture. Then, the peptidoglycan hydrolases profile analyzed using the zymogram method showed that eight proteins have the ability of peptidoglycan hydrolysis, three of the eight proteins were considered to contribute lysis of L. bulgaricus sp1.1 according to the changes and extents of peptidoglycan hydrolysis. In silico analysis showed that three putative peptidoglycan hydrolases, including N-acetylmuramyl-L-Ala amidase (protein ID: ALT46642.1), amidase (protein ID: ALT46641.1), and N-acetylmuramidase (protein ID: WP_013439201.1) were compatible with these proteins. Finally, the transcription of the three putative peptidoglycan hydrolases was upregulated in batch culture, in contrast, the expression of four peptidoglycan synthases was downregulated. These observations suggested the imbalance between peptidoglycan synthases and hydrolases involved in the lysis of Lactobacillus bulgaricus sp1.1.

Animal interphase chromosomes are organized into topologically associating domains (TADs). How TADs are formed is not fully understood. Here, we combined high-throughput chromosome conformation capture and gene silencing to obtain insights into TAD dynamics in Xenopus tropicalis embryos. First, TAD establishment in X. tropicalis is similar to that in mice and flies and does not depend on zygotic genome transcriptional activation. This process is followed by further refinements in active and repressive chromatin compartments and the appearance of loops and stripes. Second, within TADs, higher self-interaction frequencies at one end of the boundary are associated with higher DNA occupancy of the architectural proteins CTCF and Rad21. Third, the chromatin remodeling factor ISWI is required for de novo TAD formation. Finally, TAD structures are variable in different tissues. Our work shows that X. tropicalis is a powerful model for chromosome architecture analysis and suggests that chromatin remodeling plays an essential role in de novo TAD establishment., Hi-C and single-molecule sequencing analysis provide an improved assembly of the Xenopus tropicalis genome and insights into three-dimensional genome dynamics throughout embryogenesis.

A rotary laser optoelectronic measurement system is used as part of a multistation network for large-scale metrology. The line-structured laser surface of the transmitter is regarded as an ideal plane for the intersecting measurement. This article presents how the rotary laser surface model can be optimized for large-scale optoelectronic measurement systems. Initially, the deformation of the laser surface caused by the assembly error in a cylindrical lens is analyzed by Zemax simulation and studied by a visual evaluation method based on a high-precision triaxis rotary table. Based on the analysis result, the folded linear surface model and the quadric surface model are applied to reestablish the laser surface model, respectively, to reduce the shape error. A high-precision 3-D coordinate field is designed to acquire an accurate surface parametric model and validate the fitness of the new models. The experimental results based on workshop measurement positioning system (wMPS) show that both optimization models are conducive to improving the fitting accuracy of laser surfaces, and the quadric surface model fits better when the surface shape is closer to the commonest two-folded linear model. Finally, optimizing the laser surface model plays an important role in improving the system measurement accuracy.

Lactic acid has been proved to inhibit the proliferation of lactic acid bacteria in the fermentation process. To shed light on the cell cycle alterations in acidic conditions, the cell division of Lactobacillus bulgaricus sp1.1 in batch culture was analyzed directly by implementing of the intracellular fluorescent tracking assay in different pH adjusted by lactic acid. Cell proliferation and cell division were investigated to be negatively controlled by the decrease of pH, and pH 4.1 was the critical condition of downregulating cell division but retains cell culturability. The cell area and cell length in pH 4.1 were examined by using fluorescent labeling, and they reduced to about 29.18–34.89% and 32.67–40% of cells cultured in the unacidified medium, respectively. The DNA replication initiation was undergoing prompted by the low extent of DNA condensation and higher expression of the dnaA gene in this critical pH. The results indicated that the cell cycle progressions of Lactobacillus bulgaricus sp1.1 in acidic conditions were arrested at intracellular biomass accumulation and cell division stage. These findings provide fundamental insight into cell cycle control of the acidic environment in Lactobacillus bulgaricus sp1.1.

Cumulative evidence suggests that microRNAs (miRNAs) promote gene expression in cancers. However, the pathophysiologic relevance of miRNA-mediated RNA activation in hepatocellular carcinoma (HCC) remains to be established. Our previous miRNA expression profiling in seven-paired HCC specimens revealed miR-93-5p as an HCC-related miRNA. In this study, miR-93-5p expression was assessed in HCC tissues and cell lines by quantitative real-time PCR and fluorescence in situ hybridization. The correlation of miR-93-5p expression with survival and clinicopathological features of HCC was determined by statistical analysis. The function and potential mechanism of miR-93-5p in HCC were further investigated by a series of gain- or loss-of-function experiments in vitro and in vivo. We identified that miR-93-5p, overexpressed in HCC specimens and cell lines, leads to poor outcomes in HCC cases and promotes proliferation, migration, and invasion in HCC cell lines. Mechanistically, rather than decreasing target mRNA levels as expected, miR-93-5p binds to the 3'-untranslated region (UTR) of mitogen-activated protein kinase kinase kinase 2 (MAP3K2) to directly upregulate its expression and downstream p38 and c-Jun N-terminal kinase (JNK) pathway, thereby leading to cell cycle progression in HCC. Notably, we also demonstrated that c-Jun, a downstream effector of the JNK pathway, enhances miR-93-5p transcription by targeting its promoter region. Besides, downregulation of miR-93-5p significantly retarded tumor growth, while overexpression of miR-93-5p accelerated tumor growth in the HCC xenograft mouse model. Altogether, we revealed a miR-93-5p/MAP3K2/c-Jun positive feedback loop to promote HCC progression in vivo and in vitro, representing an RNA-activating role of miR-93-5p in HCC development.

The mechanistic target of rapamycin (mTOR) pathway, containing mTOR complex 1 (mTORC1) and mTORC2, is dysregulated in multiple cancers, including hepatocellular carcinoma (HCC). Mammalian lethal with sec-13 protein 8 (mLST8) is a shared constituent of both mTORC1 and mTORC2, yet little is known regarding its role in HCC development. mLST8 expression was detected in a total of 186 pairs of HCC and adjacent non-tumor specimens. The correlation between mLST8 level and clinicopathological features or prognostic significance were analyzed. The role of mLST8 on biological functions was also preliminarily studied. The study revealed that the mLST8 level was dramatically higher in HCC specimens than in adjacent non-tumor specimens. mLST8 overexpression positively correlated with tumor size, differentiation, and vessel invasion. Cases with elevated mLST8 level had more unfavorable overall survival (OS) and disease-free survival (DFS) than those with downregulated mLST8 level. Multivariate analysis demonstrated that mLST8 upregulation was an independent predictive marker for OS and DFS. Calibration curves from nomogram models indicated an excellent coherence between nomogram prediction and actual situation. Decision curve analysis proved that mLST8-based nomograms presented much higher predictive accuracy when compared with conventional clinical staging systems. Mechanistically, mLST8 enhanced cell proliferation and invasion through the AKT (protein kinase B) pathway. Our study demonstrates that mLST8 exerts an oncogenic role in HCC and may become a promising prognostic biomarker and therapeutic target for HCC patients.

Hepatocellular carcinoma (HCC) remains one of the most refractory malignancies worldwide. Schlafen family member 11 (SLFN11) has been reported to play an important role in inhibiting the production of human immunodeficiency virus 1 (HIV-1). However, whether SLFN11 also inhibits hepatitis B virus (HBV), and affects HBV-induced HCC remain to be systematically investigated. Methods: qRT-PCR, western blot and immunohistochemical (IHC) staining were conducted to investigate the potential role and prognostic value of SLFN11 in HCC. Then SLFN11 was stably overexpressed or knocked down in HCC cell lines. To further explore the potential biological function of SLFN11 in HCC, cell counting kit-8 (CCK-8) assays, colony formation assays, wound healing assays and transwell cell migration and invasion assays were performed in vitro. Meanwhile, HCC subcutaneous xenograft tumor models were established for in vivo assays. Subsequently, immunoprecipitation (IP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses were applied to understand the molecular mechanisms of SLFN11 in HCC. Co-IP, immunofluorescence and IHC staining were used to analyze the relationship between ribosomal protein S4 X-linked (RPS4X) and SLFN11. Finally, the therapeutic potential of SLFN11 with mTOR pathway inhibitor INK128 on inhibiting HCC growth and metastasis was evaluated in vitro and in vivo orthotopic xenograft mouse models. Results: We demonstrate that SLFN11 expression is decreased in HCC, which is associated with shorter overall survival and higher recurrence rates in patients. In addition, we show that low SLFN11 expression is associated with aggressive clinicopathologic characteristics. Moreover, overexpression of SLFN11 inhibits HCC cell proliferation, migration, and invasion, facilitates apoptosis in vitro, and impedes HCC growth and metastasis in vivo, all of which are attenuated by SLFN11 knockdown. Mechanistically, SLFN11 physically associates with RPS4X and blocks the mTOR signaling pathway. In orthotopic mouse models, overexpression of SLFN11 or inhibition of mTOR pathway inhibitor by INK128 reverses HCC progression and metastasis. Conclusions: SLFN11 may serve as a powerful prognostic biomarker and putative tumor suppressor by suppressing the mTOR signaling pathway via RPS4X in HCC. Our study may therefore offer a novel therapeutic strategy for treating HCC patients with the mTOR pathway inhibitor INK128.

Upregulated ubiquitin-conjugating enzyme E2M (UBE2M) is associated with poor prognosis in malignancies; However, the phenotype and mechanism of action of UBE2M in hepatocellular carcinoma (HCC) remain elusive. Here, we report that UBE2M is overexpressed and correlated with poor prognosis in HCC patients. The UBE2M level is an independent prognostic factor for HCC patients. UBE2M knockdown inhibits HCC cell proliferation, migration, and invasion, whereas its overexpression has an opposite effect. Mechanistically, upregulated UBE2M exerts oncogenic effects by translocation of accumulated β-catenin from the cytoplasm to the nucleus, thus activating downstream β-catenin/cyclin D1 signaling. In summary, our study demonstrates a notable role of UBE2M in promoting the growth of HCC, providing a novel strategy for HCC prevention and treatment.

BackgroundTransarterial chemoembolization (TACE) stands for the most commonly utilized therapy for hepatocellular carcinoma (HCC) worldwide. This study was to explore the potential predictive and prognostic roles of LAG-3 and PD-L1 as serum biomarkers in HCC patients underwent TACE treatment.MethodsA total of 100 HCC patients receiving TACE as well as 30 healthy controls were enrolled in the study. Serum LAG-3 and PD-L1 levels were determined at baseline and 3 day after TACE using enzyme-linked immunosorbent assay (ELISA).ResultsWe found serum levels of LAG-3 and PD-L1 were significantly elevated in HCC patients compared with healthy controls. Interestingly, patients with low pre-TACE and post-TACE levels of LAG-3 but not PD-L1 had a high probability of achieving an objective response (OR) after TACE treatment. Additionally, high pre-TACE LAG-3 level was correlated with poor disease outcome, and the patients with both high serum LAG-3 and PD-L1 level had the shorter overall survival (OS) than patients who are either PD-L1 or LAG-3 high or both PD-L1 and LAG-3 low. High pre-TACE serum LAG-3 level was positively associated with more cirrhosis pattern, advanced BCLC stage, pre-TACE alanine aminotransferase (ALT) level, and pre-TACE aspartate aminotransferase (AST) level. Furthermore, in 50 patients who underwent TACE, the serum LAG-3 level was significantly decreased at 3 day after TACE.ConclusionBoth pre-TACE and post-TACE serum LAG-3 levels could serve as powerful predictors for tumor response of TACE, and high pre-TACE serum LAG-3 level was an indicator for poor prognosis in HCC.

The overall response rate for anti–PD-1 therapy remains modest in hepatocellular carcinoma (HCC). We found that a combination of IFNα and anti–PD-1–based immunotherapy resulted in enhanced antitumor activity in patients with unresectable HCC. In both immunocompetent orthotopic and spontaneous HCC models, IFNα therapy synergized with anti–PD-1 and the combination treatment led to significant enrichment of cytotoxic CD27+CD8+ T cells. Mechanistically, IFNα suppressed HIF1α signaling by inhibiting FosB transcription in HCC cells, resulting in reduced glucose consumption capacity and consequentially establishing a high-glucose microenvironment that fostered transcription of the T-cell costimulatory molecule Cd27 via mTOR–FOXM1 signaling in infiltrating CD8+ T cells. Together, these data reveal that IFNα reprograms glucose metabolism within the HCC tumor microenvironment, thereby liberating T-cell cytotoxic capacities and potentiating the PD-1 blockade–induced immune response. Our findings suggest that IFNα and anti–PD-1 cotreatment is an effective novel combination strategy for patients with HCC. Significance: Our study supports a role of tumor glucose metabolism in IFNα-mediated antitumor immunity in HCC, and tumor-infiltrating CD27+CD8+ T cells may be a promising biomarker for stratifying patients for anti–PD-1 therapy. See related commentary by Kao et al., p. 1615. This article is highlighted in the In This Issue feature, p. 1599

Spray drying is an efficient method for preparing powdered products; however, its application for sensitive biological material, such as live lactic acid bacterial agent, is limited. This study examines the relationship between survival rates of Lactobacillus bulgaricus sp1.1 and water content/activities and identifies the injury sequence in cellular structures and constituents during spray drying. The results revealed that 90% bacterial cells were killed when the water content was

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination., Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Background Tumor-associated macrophages (TAM) are immunosuppressive cells that contribute to impaired anti-cancer immunity. Iron plays a critical role in regulating macrophage function. However, it is still elusive whether it can drive the functional polarization of macrophages in the context of cancer and how tumor cells affect the iron-handing properties of TAM. In this study, using hepatocellular carcinoma (HCC) as a study model, we aimed to explore the effect and mechanism of reduced ferrous iron in TAM. Methods TAM from HCC patients and mouse HCC tissues were collected to analyze the level of ferrous iron. Quantitative real-time PCR was used to assess M1 or M2 signature genes of macrophages treated with iron chelators. A co-culture system was established to explore the iron competition between macrophages and HCC cells. Flow cytometry analysis was performed to determine the holo-transferrin uptake of macrophages. HCC samples from The Cancer Genome Atlas (TCGA) were enrolled to evaluate the prognostic value of transferrin receptor (TFRC) and its relevance to tumor-infiltrating M2 macrophages. Results We revealed that ferrous iron in M2-like TAM is lower than that in M1-like TAM. In vitro analysis showed that loss of iron-induced immunosuppressive M2 polarization of mouse macrophages. Further experiments showed that TFRC, the primary receptor for transferrin-mediated iron uptake, was overexpressed on HCC cells but not TAM. Mechanistically, HCC cells competed with macrophages for iron to upregulate the expression of M2-related genes via induction of HIF-1α, thus contributing to M2-like TAM polarization. We further clarified the oncogenic role of TFRC in HCC patients by TCGA. TFRC is significantly increased in varieties of malignancies, including HCC, and HCC patients with high TFRC levels have considerably shortened overall survival. Also, TFRC is shown to be positively related to tumor-infiltrating M2 macrophages. Conclusions Collectively, we identified iron starvation through TFRC-mediated iron competition drives functional immunosuppressive polarization of TAM, providing new insight into the interconnection between iron metabolism and tumor immunity.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

The outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.

STARR-seq assesses millions of fragments in parallel measuring enhancer activity quantitatively. Here we show that STARR-seq is critically flawed with a systematic error in the cells of Arabidopsis thaliana (A. thaliana). Large amount of self-transcripts (STs) is lost during reverse transcription because these STs are polyadenylated after alternative polyadenylation sites (APAS) inside the test sequences. We solved this problem by using specially designed primer and recovered self-transcribed sequences independent from the PAS usage. In A. thaliana, we identified active enhancers and also enhancers quiescent in their endogenous genomic loci. Different from traditional STARR-seq identified enhancers, enhancers identified by new method are highly enriched in sequences proximal to the 5’ and 3’ ends of genes, and their epigenetic states correlate with gene expression levels. Our solution applies to methods based on self-transcript quantification. In addition, our results provide an invaluable functional enhancer activity map and insights into the functional complexity of enhancers in A. thaliana.

Background: Recently, natural products, particularly Inonotus obliquus (I. obliquus), have attracted lots of attention due to their effective anticancer effects with relatively low toxicity. Results: I. obliquus polysaccharides (IOP) with different molecular weights were obtained by filtering the aqueous extract through fractional membrane. IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) with the highest yield and inhibition ratio of HT-29 cancer cells was chosen to evaluate the apoptotic effect on HT-29 cancer cells. After treated with different concentrations of IOP60b, morphological changes including cell shrinkage and nuclear condensation and DNA fragmentation of HT-29 cancer cells were observed. In addition, the proportions of cells in early apoptosis and late apoptosis were significantly (pConclusion: These data suggested that IOP60b could be a potential candidate for the clinical prevention and treatment of colorectal cancer.

Lactic acid has been proved to inhibit the proliferation of lactic acid bacteria in the fermentation process. To shed light on the cell cycle alterations in acidic conditions, the cell division of Lactobacillus bulgaricus sp1.1 in batch culture was analyzed directly by implementing of the intracellular fluorescent tracking assay in different pH adjusted by lactic acid. Cell proliferation and cell division were investigated to be negatively controlled by the decrease of pH, and pH 4.1 was the critical condition of downregulating cell division but retains cell culturability. The cell area and cell length in pH 4.1 were examined by using fluorescent labeling, and they reduced to about 29.18-34.89% and 32.67-40% of cells cultured in the unacidified medium, respectively. The DNA replication initiation was undergoing prompted by the low extent of DNA condensation and higher expression of the dnaA gene in this critical pH. The results indicated that the cell cycle progressions of Lactobacillus bulgaricus sp1.1 in acidic conditions were arrested at intracellular biomass accumulation and cell division stage. These findings provide fundamental insight into cell cycle control of the acidic environment in Lactobacillus bulgaricus sp1.1.

Lactobacillus bulgaricus is an important starter culture in the dairy industry, cell lysis is negative to the high density of this strain. This work describes the response of peptidoglycan synthases and hydrolases in Lactobacillus bulgaricus sp1.1 when pH decreasing in batch culture. First, the cell lysis was investigated by measuring the cytosolic lactate dehydrogenase released to the fermentation broth, a continuous increase in extracellular lactate dehydrogenase was observed after the lag phase in batch culture. Then, the peptidoglycan hydrolases profile analyzed using the zymogram method showed that eight proteins have the ability of peptidoglycan hydrolysis, three of the eight proteins were considered to contribute lysis of L. bulgaricus sp1.1 according to the changes and extents of peptidoglycan hydrolysis. In silico analysis showed that three putative peptidoglycan hydrolases, including N-acetylmuramyl-L-Ala amidase (protein ID: ALT46642.1), amidase (protein ID: ALT46641.1), and N-acetylmuramidase (protein ID: WP_013439201.1) were compatible with these proteins. Finally, the transcription of the three putative peptidoglycan hydrolases was upregulated in batch culture, in contrast, the expression of four peptidoglycan synthases was downregulated. These observations suggested the imbalance between peptidoglycan synthases and hydrolases involved in the lysis of Lactobacillus bulgaricus sp1.1.

Despite the use of immune checkpoint blockade (ICB) therapy for hepatocellular carcinoma (HCC), developing more effective immunotherapy and predicting HCC’s response to ICB therapy remain top priorities. Ribosomal protein S3A (RPS3A) is a multifunctional molecule, but its association with tumor immune cell infiltration and prognosis in HCC patients is unknown. Thus, we aimed to investigate the correlation of RPS3A with HCC immune cell infiltration and prognosis to explore novel therapeutic strategies and prognostic biomarkers for this disease. Here, we showed that RPS3A expression levels were higher in HCC cell lines and samples than in normal hepatocytes and adjacent tumor-free tissues, respectively, and patients with high RPS3A expression had worse overall and recurrence-free survival durations than did patients with low expression. Moreover, single-sample gene set enrichment analysis (ssGSEA) and immunohistochemistry demonstrated a strongly negative correlation between RPS3A expression and tumor immune cell infiltration. Meanwhile, RPS3A was revealed to be positively correlated with that of most examined immune checkpoint molecules. GSEA also suggested that high RPS3A expression promoted the biological processes related to tumorigenesis, metastasis, and immunosuppression. Finally, RPS3A-based nomograms were constructed and exhibited better predictive accuracy for HCC prognosis and more net clinical benefits when compared with traditional prognosis-prediction staging systems. In short, these findings suggest that high RPS3A expression correlates with low tumor immune cell infiltration and poor prognosis in HCC patients. Furthermore, RPS3A-based nomograms are robust HCC prognostic predictors. RPS3A therefore may serve as a therapeutic target in and predict the efficacy of ICB therapy for HCC.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust

Background Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. Methods The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. Results We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC−). Furthermore, PD-L1 TC− in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis. Conclusions LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

Identifying novel prognostic biomarkers for hepatocellular carcinoma (HCC) and then, develop an effective individualized treatment strategy remain extremely warranted. The prognostic role of sulfiredoxin‐1(SRXN1), an antioxidant enzyme, remains unknown in HCC. This study aimed to explore the prognostic implications of SRXN1 in HCC patients after partial hepatectomy. The expression of SRXN1 in HCC and normal tissue were analyzed using the patients from the public databases and Zhongshan Hospital. The Cox regression, Kaplan‐Meier survival analysis, and time‐dependent receiver operating characteristic curves were performed to identify the predictive role of SRXN1 expression on HCC patients. A prognostic nomogram based on SRXN1 expression was constructed and validated to further confirm the predictive power of SRXN1 as a prognostic biomarker. Finally, functional enrichment analysis and protein‐protein interaction network analysis of SRXN1 and its associated genes were conducted. The results showed that SRXN1 was upregulated in HCC samples compared with the normal liver tissues. Patients with SRXN1 upregulation had shorter survival time. SRXN1 overexpression was significantly correlated with advanced clinicopathological parameters. The prognostic nomogram based on SRXN1 expression was proved to be more accurate than routine staging systems for the prediction of overall survival. Protein‐protein interaction network analysis demonstrated the first neighbor genes of SRXN1 mainly participated in response to oxidative stress. In brief, SRXN1 could be a prognostic biomarker for the management of HCC., In this work, we evaluated the prognostic power of SRXN1 for hepatocellular carcinoma using clinical specimens from both the public databases and Zhongshan Hospital. We also established a prognostic nomogram based on SRXN1 expression, which was proved to be more accurate than the routine staging systems for the prediction of overall survival. Finally, functional and pathway enrichment analysis and PPI network analysis of SRXN1 and its related genes were conducted, respectively, which may shed light on the mechanism of SRXN1 as an oncogene.

Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine’s synergistic effects with interferon-β at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.

Glutamate-cysteine ligase catalytic subunit (GCLC) has been reported to overexpress in a variety types of cancer and be related with tumor progression and drug resistance. However, little has been known about GCLC's prognostic significance and biological roles in hepatocellular carcinoma (HCC). In the present study, we evaluated GCLC expression level using immunohistochemical staining (IHC) in tissue microarray (TMA) containing paired tumor and peritumoral liver tissues from 168 patients with HCC who received curative resection. GCLC levels in tumor tissues were significantly higher than in peritumoral liver tissues, and tumor GCLC level was associated with overall survival (OS) and disease-free survival (DFS). Five-year OS and DFS rates were 41.15% and 25.88% for the group with high tumor GCLC level, compared with 68.09% and 47.51% for the group with low tumor GCLC level (P