Xuebijing improves intestinal microcirculation dysfunction in septic rats by regulating the VEGF-A/PI3K/Akt signaling pathway.

BACKGROUND: This study aims to explore whether Xuebijing (XBJ) can improve intestinal microcirculation dysfunction in sepsis and its mechanism. METHODS: A rat model of sepsis was established by cecal ligation and puncture (CLP). A total of 30 male SD rats were divided into four groups: sham group, CLP group, XBJ + axitinib group, and XBJ group. XBJ was intraperitoneally injected 2 h before CLP. Hemodynamic data (blood pressure and heart rate) were recorded. The intestinal microcirculation data of the rats were analyzed via microcirculation imaging. Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the serum levels of interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α) in the rats. Histological analysis and transmission electron microscopy were used to analyze the injury of small intestinal microvascular endothelial cells and small intestinal mucosa in rats. The expression of vascular endothelial growth factor A (VEGF-A), phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) in the small intestine was analyzed via Western blotting. RESULTS: XBJ improved intestinal microcirculation dysfunction in septic rats, alleviated the injury of small intestinal microvascular endothelial cells and small intestinal mucosa, and reduced the systemic inflammatory response. Moreover, XBJ upregulated the expression of VEGF-A, p-PI3K/total PI3K, and p-Akt/total Akt in the rat small intestine. CONCLUSION: XBJ may improve intestinal microcirculation dysfunction in septic rats possibly through the VEGF-A/PI3K/Akt signaling pathway.

A novel SLC25A1 inhibitor, parthenolide, suppresses the growth and stemness of liver cancer stem cells with metabolic vulnerability.

Liver cancer stem cells (LCSCs) are recognized as key contributors to hepatocarcinogenesis, progression, and recurrence. Consequently, eradicating LCSCs has a great chance of increasing long-term survival in patients with liver cancer. Parthenolide (PTL), a natural sesquiterpene lactone product, possesses robust antitumor activity. However, the effects of PTL on LCSCs and underlying mechanisms remain unknown. Here we show that administration of PTL stimulated cell cycle arrest at the G1 phase, induced apoptosis, and decreased the stemness of LCSCs. Further research indicates that PTL caused the production of ROS and the reduction of oxidative phosphorylation (OXPHOS) and mitochondrial membrane potential (MMP) levels of LCSCs. RNA sequencing (RNA-Seq) further shows that PTL decreased SLC25A1 expression at the mRNA level and that inhibition of SLC25A1 synergistically decreased the expression of IDH2 and several pivotal genes involved in mitochondrial respiratory chain complex, resulting in the production of ROS and mitochondrial dysfunction. In addition, the inhibitory effect of PTL on mitochondrial function and self-renewal capacity of LCSCs was abolished by the knockdown of SLC25A1 or treatment with SLC25A1 inhibitor CTPI-2. Importantly, PTL prevented liver cancer growth in vivo without clearly causing toxicity. Our research shows that PTL inhibits the growth and stemness of LCSCs through SLC25A1-mediated mitochondrial function. PTL may be a potential candidate natural agent for liver cancer treatment.

HSP90, as a functional target antigen of a mAb 11C9, promotes stemness and tumor progression in hepatocellular carcinoma.

BACKGROUND: Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs). METHODS: The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets. RESULTS: MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90+ or ESA+ cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/ß-catenin signaling. CONCLUSIONS: The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/ß-catenin signaling.

The past, present, and future of liver cancer research in China.

Liver cancer is among the leading causes of cancer-related death worldwide and China accounts for nearly half of the global burden of liver cancer. Effective interventions such as hepatitis vaccinations, new blood tests and imaging tests significantly decreased the incidence worldwide, especially in China. Unraveling the systemic and molecular mechanisms of liver cancer would contribute to develop more effective therapies to prolong the 5 year survival of the patients. The Chinese funding agencies have been paying high attention to the basic and translational research of liver cancer. Over the last decade, the National Natural Science Foundation of China (NSFC) initiated a panel of research programs which supported liver cancer research in multiple directions. Besides, great progress has been made in basic and clinical research, platform construction and drug development in the field of liver cancer. In this article, we summarized the funding landscape, research progress, cooperation among countries and institutions, and drug discovery in China, with an attempt to compare the status and outcome with our peers globally.

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-µFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-µFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-µFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-µFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-µFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-µFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

Palladin promotes cancer stem cell-like properties in lung cancer by activating Wnt/Β-Catenin signaling.

BACKGROUND: Cancer stem cells (CSCs) are responsible for drug resistance, cancer relapse, and metastasis. Here, we report the first analysis of Palladin expression and its impacts on stem cell-like properties in lung cancer. METHODS: Tissue microarrays were used to investigate Palladin expression and its association with prognosis. Immunofluorescence (IF), flow fluorescence assay, and Western blot were performed to detect Palladin expression in 6 NSCLC cell lines. Cell phenotypes and drug resistance were evaluated. Xenograft models were constructed to confirm the role of Palladin in vivo. RESULTS: By using the tissue microarrays, Palladin was identified to be highly expressed in the cytoplasm, specifically in the cytomembrane of NSCLC, and its high expression is associated with poor prognosis. Palladin is widely expressed and enriched in the sphere cells. The in vitro and in vivo studies showed that Palladin promoted stem cell-like properties, including cell viability, invasion, migration, self-renewal abilities, taxol resistance, and tumorigenicity. Western blot revealed that Palladin promoted the accumulation of ß-catenin and activated Wnt/ß-catenin signaling. Tissue microarrays analysis further confirmed the positive correlation between Palladin and ß-catenin. Wnt/ß-catenin pathway inhibitor blocked the Palladin-induced enhancement of sphere-forming. CONCLUSIONS: Palladin might act as an oncogene by promoting CSCs-like properties and tumorigenicity of NSCLC cells via the Wnt/ß-catenin signaling pathway. Besides, Palladin was identified to have the potential as a cell surface marker for LCSCs identification. These findings provide a possible target for developing putative agents targeted to LCSCs.

Annotation and evaluation of base editing outcomes in multiple cell types using CRISPRbase.

CRISPR-Cas base editing (BE) system is a powerful tool to expand the scope and efficiency of genome editing with single-nucleotide resolution. The editing efficiency, product purity, and off-target effect differ among various BE systems. Herein, we developed CRISPRbase (http://crisprbase.maolab.org), by integrating 1 252 935 records of base editing outcomes in more than 50 cell types from 17 species. CRISPRbase helps to evaluate the putative editing precision of different BE systems by integrating multiple annotations, functional predictions and a blasting system for single-guide RNA sequences. We systematically assessed the editing window, editing efficiency and product purity of various BE systems. Intensive efforts were focused on increasing the editing efficiency and product purity of base editors since the byproduct could be detrimental in certain applications. Remarkably, more than half of cancer-related off-target mutations were non-synonymous and extremely damaging to protein functions in most common tumor types. Luckily, most of these cancer-related mutations were passenger mutations (4840/5703, 84.87%) rather than cancer driver mutations (863/5703, 15.13%), indicating a weak effect of off-target mutations on carcinogenesis. In summary, CRISPRbase is a powerful and convenient tool to study the outcomes of different base editors and help researchers choose appropriate BE designs for functional studies.

Serum metabolic profiling of targeted bile acids reveals potentially novel biomarkers for primary biliary cholangitis and autoimmune hepatitis.

BACKGROUND: Primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH) are two unexplained immune diseases. The golden standard for diagnosis of these diseases requires a liver biopsy. Liver biopsy is not widely accepted by patients because of its invasive nature, and atypical liver histology can confuse diagnosis. In view of the lack of effective diagnostic markers for PBC and AIH, combined with the increasingly mature metabolomics technologies, including full-contour metabolomics and target. AIM: To determine non-invasive, reliable, and sensitive biochemical markers for the differential diagnosis of PBC and AIH. METHODS: Serum samples from 54 patients with PBC, 26 patients with AIH and 30 healthy controls were analyzed by Ultra-high performance liquid chromatography-tandem mass spectrometry serum metabolomics. The metabolites and metabolic pathways were identified, and the metabolic changes, metabolic pathways and inter-group differences between PBC and AIH were analyzed. Fifteen kinds of target metabolites of bile acids (BAs) were quantitatively analyzed by SRM, and the differential metabolites related to the diagnosis of PBC were screened by receiver operating characteristic curve analysis. RESULTS: We found the changes in the levels of amino acids, BAs, organic acids, phospholipids, choline, sugar, and sugar alcohols in patients with PBC and AIH. Furthermore, the SRM assay of BAs revealed the increased levels of chenodeoxycholic acid, lithocholic acid (LCA), taurolithocholic acid (TLCA), and LCA + TLCA in the PBC group compared with those in the AIH group. The levels of BAs may be used as biomarkers to differentiate PBC from AIH diseases. The levels of glycochenodeoxycholic acid, glycochenodeoxycholic sulfate, and taurodeoxycholic acid were gradually elevated with the increase of Child-Pugh class, which was correlated with the severity of disease. CONCLUSION: The results demonstrated that the levels of BAs could serve as potential biomarkers for the early diagnosis and assessment of the severity of PBC and AIH.

The DPY30-H3K4me3 Axis-Mediated PD-L1 Expression in Melanoma.

Background: DPY30 is a common subunit of the human SET1/MLL complex and is an essential protein required for the activity of SET1/MLL methyltransferase. DPY30 regulates the histone H3K4 modification, and dysfunction of DPY30 might contribute to the regulation of cancer immune evasion. However, the functions and regulation of DPY30 in the expression of programmed cell death ligand 1 (PD-L1) is still not completely explored. Methods: Various online databases were used for data processing and visualization, including UALCAN, Oncomine, cBioPortal, SangerBox, TISIDB, TIMER, and GEPIA databases. The expression of DPY30 and PD-L1 in melanoma tissues were evaluated by IHC. Chromatin Immunoprecipitation (ChIP), RT-PCR and flow cytometry were used to elucidate the underlying molecular mechanism of PD-L1 expression regulation and its function. Results: The mRNA level of DPY30 in melanoma was higher than in normal tissues. The expression of DPY30 was positively associated with TMB, neoantigens and PD-L1 expression. Furthermore, DPY30 expression showed significant positive correlations with immune suppressor cells and ICP genes involved in T-cell exhaustion. IHC showed that the positive rates of DPY30 and PD-L1 in melanoma tissues were 62% and 58%, respectively. Correlation analysis revealed that DPY30 over-expression was positively associated with PD-L1 expression. Silencing of DPY30 by specific siRNA significantly inhibited PD-L1 expression. ChIP analysis revealed that H3K4me3 levels were enriched in the proximal PD-L1 promoter region in tumor cells. Inhibition of DPY30 still suppressed the PD-L1 level in IFN-γ treated MMAC-SF cells. Furthermore, the apoptosis of PD1+ T-cells in co-culture with MMAC-SF cells by knockdown of DPY30 were markedly reduced. Conclusion: This study shows the roles of DPY30 in regulating the cancer immune evasion in melanoma. Targeting the DPY30-H3K4me3 axis might be an alternative approach to enhance the efficacy of checkpoint immunotherapy.

Identification of Tumor Antigens and Immune Subtypes of Malignant Mesothelioma for mRNA Vaccine Development.

BACKGROUND: mRNA-based cancer vaccines have been considered a promising anticancer therapeutic approach against various cancers, yet their efficacy for malignant mesothelioma (MESO) is still not clear. The present study is designed to identify MESO antigens that have the potential for mRNA vaccine development, and to determine the immune subtypes for the selection of suitable patients. METHODS: A total of 87 MESO datasets were used for the retrieval of RNA sequencing and clinical data from The Cancer Genome Atlas (TCGA) databases. The possible antigens were identified by a survival and a genome analysis. The samples were divided into two immune subtypes by the application of a consensus clustering algorithm. The functional annotation was also carried out by using the DAVID program. Furthermore, the characterization of each immune subtype related to the immune microenvironment was integrated by an immunogenomic analysis. A protein-protein interaction network was established to categorize the hub genes. RESULTS: The five tumor antigens were identified in MESO. FAM134B, ALDH3A2, SAV1, and RORC were correlated with superior prognoses and the infiltration of antigen-presenting cells (APCs), while FN1 was associated with poor survival and the infiltration of APCs. Two immune subtypes were identified; TM2 exhibited significantly improved survival and was more likely to benefit from vaccination compared with TM1. TM1 was associated with a relatively quiet microenvironment, high tumor mutation burden, and enriched DNA damage repair pathways. The immune checkpoints and immunogenic cell death modulators were also differentially expressed between two subtypes. Finally, FN1 was identified to be the hub gene. CONCLUSIONS: FAM134B, ALDH3A2, SAV1, RORC, and FN1 are considered as possible and effective mRNA anti-MESO antigens for the development of an mRNA vaccine, and TM2 patients are the most suitable for vaccination.

Genetic association and single-cell transcriptome analyses reveal distinct features connecting autoimmunity with cancers.

Autoimmune diseases (ADs) are at a significantly higher risk of cancers with unclear mechanism. By searching GWAS catalog database and Medline, susceptible genes for five common ADs, including systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren syndrome, systemic sclerosis, and idiopathic inflammatory myopathies, were collected and then were overlapped with cancer driver genes. Single-cell transcriptome analysis was performed in the comparation between SLE and related cancer. We identified 45 carcinogenic autoimmune disease risk (CAD) genes, which were mainly enriched in T cell signaling pathway and B cell signaling pathway. Integrated single-cell analysis revealed immune cell signaling was significantly downregulated in renal cancer compared with SLE, while stemness signature was significantly enriched in both renal cancer or lymphoma and SLE in specific subpopulations. Drugs targeting CAD genes were shared between ADs and cancer. Our study highlights the common and specific features between ADs and related cancers, and sheds light on a new discovery of treatments.

Trichostatin A improves the inflammatory response and liver injury in septic mice through the FoxO3a/autophagy signaling pathway.

BACKGROUND: Sepsis-induced liver injury is a fatal complication of sepsis. Trichostatin A (TSA) regulates inflammation and autophagy in some human diseases, and forkhead box O3a (FoxO3a) has been shown to regulate autophagy. The present study aims to investigate whether TSA exerts its effects on septic liver injury through the FoxO3a/autophagy signaling pathway. METHODS: A sepsis mouse model was constructed by the cecal ligation and puncture (CLP) method, and AML12 cells were pretreated with lipopolysaccharide (LPS) (1 µg/mL) to establish a sepsis cell model. Forty mice were divided into four groups, namely control group, TSA group, CLP group, and CLP+TSA group, with 10 mice in each group. Cells were divided into control group, TSA group, LPS group, and LPS+TSA group. Hematoxylin-eosin (H&E) staining and biochemical methods were used to evaluate liver tissue injury. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the expression of proinflammatory cytokines, and Western blotting and immunofluorescence were used to measure autophagy-related protein expression. RESULTS: Compared with the CLP group (mice), the proinflammatory cytokines (interleukin-ß [IL-ß] 2,665.27±324.90 pg/mL to 2,080.26±373.66 pg/mL; interleukin-6 [IL-6] 399.01±60.98 pg/mL to 221.90±46.89 pg/mL) and the hepatocyte injury markers (aspartate transaminase [AST] from 198.18±27.07 U/L to 128.42±20.55 U/L; alanine aminotransferase [ALT] from 634.98±74.10 U/L to 478.60±32.56 U/L) were notably decreased after TSA intervention. Moreover, LC3 II and FoxO3a showed an obvious increase and P62 showed an obvious decrease in the CLP+TSA group. Cell experiment results showed the similar trend. After FoxO3a gene was knocked down in AML12 cells, the promotion of autophagy and the improvement of liver enzyme index and inflammation by TSA were weakened. CONCLUSION: TSA may improve the inflammatory response and liver injury in septic mice through FoxO3a/autophagy.

Gasdermin D in Different Subcellular Locations Predicts Diverse Progression, Immune Microenvironment and Prognosis in Colorectal Cancer.

BACKGROUND: Pyroptosis is a type of cell death that causes an immune reaction. Gasdermin D (GSDMD), as an executor of pyroptosis, has become an attractive target in cancer research. However, the clinical significance of GSDMD expression in different subcellular locations remains unclear. METHODS: GSDMD was detected by immunohistochemistry in 178 cases of colorectal cancer with follow-up information. General data and information on systemic inflammatory indicators were collected from case records, and the clinicopathological parameters were reviewed by microscopy. CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, and CD68+ macrophages were detected by immunohistochemistry. Univariate survival analysis (Kaplan-Meier method, Log rank test) and a multivariate Cox proportional hazard model were used to analyze the impact of GSDMD on overall survival. RESULTS: Survival analysis showed that high expression of cytoplasmic GSDMD was an independent favorable indicator for prognosis (P=0.027) and improved the efficacy of chemotherapy (P=0.012). Positive cytoplasmic GSDMD expression indicated lower probability of distant metastasis (P=0.024), yet nuclear GSDMD expression predicted deeper infiltration depth (P=0.007). Membranous GSDMD expression positively correlated with CD68+ macrophages in tumor center (P=0.002) and CD8+ lymphocytes in tumor invasive front (P=0.007). However, nuclear GSDMD was negatively related to CD68+ macrophages in tumor invasive front (P<0.001) and CD8+ lymphocytes in tumor center (P=0.069). Cytoplasmic GSDMD was associated with more CD3+ lymphocytes both in tumor center (P=0.066) and tumor invasive front (P=0.008). Moreover, positive membranous GSDMD indicated a lower neutrophil-to-lymphocyte ratio (P=0.013). CONCLUSION: GSDMD subcellular localization patterns are related to CRC progression and immune reaction, and should be investigated in future studies.

MYH9 is crucial for stem cell-like properties in non-small cell lung cancer by activating mTOR signaling.

The fatality rate of non-small cell lung cancer (NSCLC) has been high due to the existence of cancer stem cells (CSCs). Non-muscle myosin heavy chain 9 (MYH9) can promote the progression of various tumors, but its effect on the stem cell-like characteristics of lung cancer cells (LCCs) has not been clarified. Our research found that the stemness characteristics of LCCs were significantly enhanced by the overexpression of MYH9, and the knockout of MYH9 had the opposite effects. The in vivo with inhibitor blebbistatin further confirmed the effect of MYH9 on the stem cell-like behavior of LCCs. Furthermore, western blotting showed that the expression level of CSCs markers (CD44, SOX2, Nanog, CD133, and OCT4) was also regulated by MYH9. Mechanistic studies have shown that MYH9 regulates stem cell-like features of LCCs by regulating the mTOR signaling pathway, which was supported by sphere formation experiments after LCCs were treated with inhibitors Rapamycin and CHIR-99021. Importantly, high expression of MYH9 in lung cancer is positively correlated with poor clinical prognosis and is an independent risk factor for patients with NSCLC.

p53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.

p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice.

[Value of procalcitonin and critical illness score in etiological diagnosis and prognosis of sepsis caused by intra-abdominal infections].

OBJECTIVE: To compare the early and late predictive values of critical illness score (CIS) and procalcitonin (PCT) in septic patients with blood stream infection (BSI) induced by intra-abdominal infection (IAI), and to identify the value of PCT in etiological diagnosis. METHODS: The clinical data of patients with at least one positive blood culture within 24 hours admission to the emergency department of China-Japan Friendship Hospital from January 2014 to December 2019 and with final diagnosis of IAI induced sepsis were enrolled. Sequential organ failure assessment (SOFA), mortality in emergency department sepsis (MEDS), Logistic organ dysfunction system (LODS), and acute physiology and chronic health evaluation II (APACHE II) scores were calculated based on the parameters on the day of admission. Differences in various indicators among different Gram-stained bacterial infections and among patients with different prognosis at 28 days or 60 days were compared. Receiver operator characteristic curve (ROC curve) was used to analyze the value of PCT in differential etiological diagnosis of IAI induced sepsis caused by single bacterial infection, and the predictive value of CIS and PCT on 28-day and 60-day death of septic patients with BSI induced by IAI. RESULTS: A total of 221 septic patients with IAI caused by single bacterial infection were enrolled. The 28-day mortality was 19.9% (44/221), and the 60-day mortality was 25.8% (57/221). Mortality caused by Gram-positive (G+) bacterial infection of patients was significantly higher than that caused by Gram-negative (G-) bacterial infection (28 days: 34.6% vs. 11.4%, 60 days: 42.0% vs. 16.4%, both P < 0.01). Compared with patients with G+ bacterial infection, the PCT value of patients with G- bacterial infection was higher [µg/L: 4.31 (0.71, 25.71) vs. 1.29 (0.32, 10.83), P < 0.05]. Compared with survival group, the values of CIS and PCT in death group were higher, either in 28 days or in 60 days [death group vs. survival group in 28 days: SOFA score was 6.0 (4.0, 10.0) vs. 3.0 (2.0, 5.0), MEDS score: 11 (9, 14) vs. 6 (6, 9), LODS score: 4.0 (2.0, 6.0) vs. 1.0 (0, 2.0), APACHE II score: 17.0 (15.0, 24.0) vs. 12.0 (8.0, 15.0), PCT (µg/L): 3.48 (1.01, 26.70) vs. 2.45 (0.32, 15.65); death group vs. survival group in 60 days: SOFA score: 6.0 (4.0, 10.0) vs. 3.0 (2.0, 5.0), MEDS score: 9 (6, 14) vs. 6 (6, 9), LODS score: 4.0 (1.0, 5.0) vs. 1.0 (0, 2.0), APACHE II score: 16.5 (12.0, 20.0) vs. 12.0 (8.0, 15.0), PCT (µg/L): 2.67 (0.98, 17.73) vs. 2.22 (0.31, 16.75); all P < 0.05]. ROC curve showed that: (1) the area under ROC curve (AUC) of PCT in the diagnosis of IAI induced sepsis with single bacterial infection was 0.740 [95% confidence interval (95%CI) was 0.648-0.833]. When the optimal cut-off value of PCT was 1.82 µg/L, the sensitivity of diagnosis of G- bacterial infection was 74.0%, and the specificity was 68.2%. When PCT value was higher than 10.92 µg/L, the specificity of diagnosis of G- bacterial infection could reach 81.8%. (2) In the prediction of 28-day and 60-day mortality for septic patients with BSI induced by IAI, the APACHE II score achieved the highest AUC [28 days: 0.791 (95%CI was 0.680-0.902), 60 days: 0.748 (95%CI was 0.645-0.851)]. APACHE II score higher than 14.5 could help to predict 28-day and 60-day mortality for IAI patients with negative predictive values of 94.9% and 88.5%. However, the predictive value of PCT for septic patients with BSI induced by IAI was relatively lower [28-day AUC: 0.610 (95%CI was 0.495-0.725), 60-day AUC: 0.558 (95%CI was 0.450-0.667)]. CONCLUSIONS: PCT is more reliable in the identification of pathogen type among IAI induced sepsis with BSI, while APACHE II score may perform better in predicting early and late mortality.

BCAT1 Activates PI3K/AKT/mTOR Pathway and Contributes to the Angiogenesis and Tumorigenicity of Gastric Cancer.

BACKGROUND: Focusing on antiangiogenesis may provide promising choices for treatment of gastric cancer (GC). This study aimed to investigate the mechanistic role of BCAT1 in the pathogenesis of GC, particularly in angiogenesis. METHODS: Bioinformatics and clinical samples analysis were used to investigate the expression and potential mechanism of BCAT1 in GC. BGC823 cells with BCAT1 overexpression or silencing were induced by lentiviral transduction. Cell phenotypes and angiogenesis were evaluated. The relevant proteins were quantized by Western blotting, immunohistochemistry, or immunofluorescence. Xenograft models were constructed to confirm the role of BCAT1 in vivo. RESULTS: BCAT1 was overexpressed in GC patients and associated with lower survival. BCAT1 expression was correlated with proliferation-, invasion-, or angiogenesis-related markers expression and pathways. Silencing BCAT1 expression suppressed cell viability, colony formation, cycle progression, invasion, and angiogenesis of BGC823 cells, as well as the tumor growth of xenograft models, whereas overexpressing BCAT1 had the opposite results both in vitro and in vivo. Bioinformatics analysis and Western blotting demonstrated that BCAT1 activated the PI3K/AKT/mTOR pathway. The addition of LY294002 reversed the tumor growth induced by BCAT1 overexpression, further verifying this mechanism. CONCLUSION: BCAT1 might act as an oncogene by facilitating proliferation, invasion, and angiogenesis through activation of the PI3K/AKT/mTOR pathway. This finding could aid the optimization of antiangiogenesis strategies.

Lipopolysaccharide promotes metastasis via acceleration of glycolysis by the nuclear factor-κB/snail/hexokinase3 signaling axis in colorectal cancer.

BACKGROUND: Cancer cell is generally characterized by enhanced glycolysis. Inflammasome activation is interaction with glycolysis. The concentration of lipopolysaccharide (LPS), a classic inflammasome activator, is significantly higher in colorectal cancer tissue than in normal intestinal mucosa. However, the mechanism of LPS on glycolysis and metastasis has not been fully elucidated. This study aimed to investigate the roles of LPS on inflammasome activation, glycolysis, and metastasis, and unravel metformin's potential in treatment of CRC. METHODS: We detected inflammasome activation and cell motility following LPS exposure in CRC cell lines. Glycolysis analysis was performed, and the key glycolytic rate-limiting enzymes were detected. Dual-luciferase reporter gene assay, co-immunoprecipitation, chromatin immunoprecipitation (ChIP) analysis, and ChIP-reChIP assay were performed to identify the specific mechanisms of LPS on glycolysis. Mouse metastasis models were used to determine the effects of LPS and metformin on metastasis. Correlation analysis of the expression of various molecules was performed in 635 CRC samples from The Cancer Genome Atlas and 83 CRC samples from our lab. RESULTS: LPS activates caspase-1 through NF-κB and upregulates the expression of Snail and HK3 depending on caspase-1 activation. LPS potentiates migration and invasion depending on accelerated glycolysis, which could be reversed by knockdown of glycolytic rate-limiting enzyme HK3. Nuclear Snail is upregulated by NF-κB under LPS treatment and then forms a complex with NF-κB, then directly binds to the HK3 promoter region to upregulate the expression of HK3. Metformin suppresses the NF-κB/Snail/HK3 signaling axis that is activated by LPS and then inhibits LPS-induced metastasis. In vivo, LPS-treated cells form more metastasis in the lungs of mice, and metformin completely reverses this effect of LPS. CONCLUSION: LPS activates inflammasomes in cancer cells through NF-κB and promotes metastasis through glycolysis enhanced by the NF-κB/Snail/HK3 signaling pathway in CRC. Metformin could prevent this effect of LPS.

miR-98-5p inhibits gastric cancer cell stemness and chemoresistance by targeting branched-chain aminotransferases 1.

AIMS: Gastric cancer stem cells (GCSCs) have been used as a therapeutic target. This study aims to estimate the role of miR-98-5p (termed miR-98) in the development of GCSCs. MAIN METHODS: The expression of miR-98 in CD44+ GCSCs was verified by RT-PCR. The miR-98 was overexpressed in CD44+ GCSCs by Lentivirus. The ability of self-renewal, invasion, chemoresistance and tumorigenicity was detected in vitro or in vivo after overexpression of miR-98. The target genes of miR-98 were predicted and verified by luciferase reporter assays. The effects miR-98/BCAT1 signaling on the chemoresistance and tumorigenicity of CD44+ GCSCs were investigated in a xenograft model by rescue experiments. KEY FINDINGS: We have shown that miR-98 was decreased in CD44+ GCSCs. The overexpression of miR-98 could inhibit the expression of stem-related genes and the ability of self-renewal, invasion, and tumorigenicity of GCSCs. Also, we found that miR-98 overexpression enhances the sensitivity to cisplatin treatment in vitro. Using a xenograft model, we showed that miR-98 overexpression reversed paclitaxel resistance to CD44+ GCSCs. Finally, we found that branched-chain aminotransferases 1 (BCAT1) is a target gene of miR-98. Overexpressed BCAT1 reversed xenograft tumor formation ability and attenuated the paclitaxel chemosensitivity induced by miR-98 downregulation. Furthermore, BCAT1 restoration affected the expression of invasion and drug resistance-related genes. SIGNIFICANCE: This study revealed miR-98 inhibits gastric cancer cell stemness and chemoresistance by targeting BCAT1, suggesting that this miR-98/BCAT1 axis represents a potential therapeutic target in gastric cancer.