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Tissue inhibitor matrix metalloproteinase 1 (TIMP1) has been reported to act as a tumor oncogene in colon cancer. However, little is known about the biological role of TIMP1 in gastric cancer. In this study, we found that the expression of TIMP1 in GC tissues was upregulated compared with the normal gastric tissues. TIMP1 was confirmed as a direct target of miR-6745 and silencing TIMP1 mimicked the effects of miR-6745 in GC cells. Further mechanism studies have shown that miR-6745 inhibits the Wnt/β-catenin pathway by targeting TIMP1, thereby inhibiting cell proliferation, migration and invasion. In addition, through the analysis of GC tissues, a negative correlation between miR-6745 and TIMP1 was found in 42 GC tissues. Our findings indicate that the miR-6745-TIMP1 axis regulates Wnt/βcatenin signaling and participates in GC tumorigenesis and provide a potential therapeutic target for preventing GC progression.

CAR T cell therapy has shown dramatic clinical success in relapsed or refractory (r/r) B-ALL and other haematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal bone marrow cells. T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide, maintained their cytotoxic function and mediated powerful antitumour effects. Subsequently, we conducted a pilot clinical study to evaluate the safety and feasibility of therapy with anti-CD99 CAR T cells in 4 patients with r/r T-LBL (n=1), AML (n=2) or myeloid sarcoma (MS) (n=1). The clinical overall response rate (ORR) was 50% (2/4 patients), and 1 patients (25%) achieved complete remission (CR) for 2 month. Mild cytokine release syndrome (CRS) occurred in 2 patients and the CRS no more than grade 2. Together, our results demonstrate that anti-CD99 CAR T cells specifically recognize and efficiently eliminate CD99+ leukemia cells.

The novel duck reovirus (NDRV) is an emerging pathogen that causes disease in various waterfowl species. Since the outbreak, it has caused huge economic losses to the duck industry in China. A rapid, reliable, and high-throughput method is required for epidemiological investigation and evaluation of vaccine immunogenicity. A good first step would be establishing an enzyme-linked immunosorbent assay (ELISA) that could detect NDRV antibodies in different breeds of ducks and geese from the serum and egg yolk. This study used a recombinant NDRV σB protein and a corresponding horseradish peroxidase (HRP)-labeled monoclonal antibody to develop a blocking ELISA (B-ELISA). The cutoff value of the B-ELISA was 37.01%. A total of 212 serum samples were tested by the B-ELISA, and the virus neutralization test (VNT) was the gold standard test. The sensitivity and specificity of the B-ELISA were 92.17% (106/115) and 97.94% (95/97), respectively. The agreement rates between the B-ELISA and VNT were 94.81% (kappa value, 0.896). The B-ELISA could specifically recognize anti-NDRV sera without cross-reacting with other positive serums for other major diseases in ducks and geese. The inter- and intra-assay coefficients of variation (CVs) of the B-ELISA and VNT assays were acceptable. In conclusion, the novel B-ELISA could be a rapid, simple, safe, and economically attractive alternative to the VNT in assessing duck flocks' immunity status and in epidemiological surveillance in multiple waterfowl species.

Matching material degradation with host remodeling, including endothelialization and muscular remodeling, is important to vascular regeneration. We fabricated 3D PGS-PCL vascular grafts, which presented tunable polymer components, porosity, mechanical strength, and degrading rate. Furthermore, highly porous structures enabled 3D patterning of conjugated heparin-binding peptide, dimeric thymosin β4 (DTβ4), which played key roles in antiplatelets, fibrinogenesis inhibition, and recruiting circulating progenitor cells, thereafter contributed to high patency rate, and unprecedentedly acquired carotid arterial regeneration in rabbit model. Through single-cell RNA sequencing analysis and cell tracing studies, a subset of endothelial progenitor cells, myeloid-derived CD93

Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3’UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.

Liver cancer is a malignant cancer phenotype for which there currently remains a lack of reliable biomarkers and therapeutic targets for disease management. Tryptophan 2,3‑dioxygenase (TDO2), a heme‑containing polyoxygenase enzyme, is primarily expressed in cells of the liver and nervous systems. In the present study, through the combination of cancer bioinformatics and analysis of clinical patient samples, it was shown that TDO2 expression in liver cancer tissue samples was significantly higher than that in normal tissues, and liver cancer patients with high TDO2 expression had a poor prognosis. Mechanistic studies on liver cancer cells showed that TDO2 promoted cancer cell migration and invasion via signal transduction through the Wnt5a pathway. Such regulation impacted the expression of cancer‑associated biomarkers, such as matrix metalloprotease 7 (MMP7) and the cell adhesion receptor CD44. Treatment with a calcium channel blocker (azelnidipine) reduced TDO2 levels and inhibited liver cancer cell migration and invasion. A mouse xenograft cancer model showed that TDO2 promoted tumorigenesis. Furthermore, azelnidipine treatment to downregulate TDO2 also decreased liver cancer development in this mouse cancer model. TDO2 is thus not only a useful liver cancer biomarker but a potential drug target for management of liver cancer.

Liraglutide is an analogue of human glucagon-like peptide-1 (GLP-1), an endogenous intestinal hormone which play essential roles in the regulation of glycolipid metabolism. To investigate the role of lactic acid bacteria (LAB) in the lipid-lowering effect of liraglutide, forty mice were divided into normal saline-treated basal diet (NFD), normal saline-treated high-fat food (HFD), 10.0 mg/kg/d simvastatin-treated HFD (SIM + HFD), 200 and 400 μg/kg/d liraglutide-treated HFD (LL + HFD and HL + HFD) groups for 5 weeks. 16S rDNA sequencing, real-time quantitative PCR and western blot were used to detected changes of intestinal flora, cholesterol 7α-hydroxylase (CYP7A1), LDL-receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Results showed that liraglutide could up-regulate CYP7A1 and LDLR, whereas down-regulate HMGCR. Besides, liraglutide enhance the abundance of lactobacillaceae in gut of hyperlipidemic mice and increase the bile tolerance ability of LAB by up-regulating bile salt hydrolases, and the lysate of liraglutide-sensitive LAB could also directly down-regulate HMGCR, the key enzyme in cholesterol synthesis, and inhibit hepatocyte steatosis. These findings might provide new theoretical guidance for clinical application of liraglutide and threw a light on research and development of anti-obesity, hypolipidemic and cholesterol-lowering drugs or functional foods.

Myocardial remodelling is a common phenomenon in cardiovascular diseases, which threaten human health and the quality of life. Due to the lack of effective early diagnosis and treatment methods, the molecular mechanism of myocardial remodelling should be explored in depth. In this study, we observed the high expression of MBNL1 in cardiac tissue and peripheral blood of an isoproterenol (ISO)‐induced cardiac hypertrophy mouse model. MBNL1 promoted ISO‐induced cardiac hypertrophy and fibrosis by stabilizing Myocardin mRNA in vivo and in vitro. Meanwhile, an increase in MBNL1 may induce the apoptosis of cardiomyocytes treated with ISO via TNF‐α signalling. Interestingly, MBNL1 can be activated by p300 in cardiomyocytes treated with ISO. At last, Myocardin can reverse activate the expression of MBNL1. These results suggest that MBNL1 may be a potential target for the early diagnosis and clinical treatment of myocardial remodelling.

Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.

Goose astrovirus (GAstV) is an emerging pathogen with a wide distribution in China that causes visceral gout and leads to significant economic losses in the goose industry. Here, 10 GAstV strains were isolated from different farms in southeast China. We performed an integrated analysis of the full-genome sequences of these new strains alongside comprehensive epidemiological surveillance information from the database. Interestingly, the results showed two distinct genotypes of GAstV, which were evolutionarily distant from each other. Group I GAstVs were closely related to DAstV IV, and group II strains were classified with duck astrovirus (DAstV) II and turkey astrovirus (TAstV) II. Further investigation showed that among the GAstV I strains, ZJC14 and AHDY differed from FLX. Comparative analysis of 58 available genomes clustered the GAstV II strains into two subgroups. We identified two major mutation sites, 456 (E/D) and 540 (L/Q), in the capsid protein, which were related to distinct subgroups according to evolution. GAstV II subgroup 1a strains are the predominant strains in the current prevalent epidemiology. Phylogeographic analysis based on 90 reported cases from 13 provinces revealed the complexity and severity of GAstV epidemics in China, within which Henan, Anhui and Jiangsu provinces have suffered great impacts. According to these phylogeographic investigations, following the initial introduction of GAstV from Hunan Province, the dispersal of GAstV with different subgenotypes on a nationwide scale may be explained by the live gosling trade. Our findings have important implications for the evolution and dispersal of GAstV and will contribute to understanding the potential risk of GAstV.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit. STING ER exit is the rate-limiting step in STING signaling, but the mechanism that drives this process is not understood. Paludan and colleagues identify CxORF56, called STEEP here, as a positive regulator of STING signaling.

Cardiac hypertrophy is an early milestone of many heart diseases. LncRNAs often play a leading role in this process. However, its mechanism of action in cardiac hypertrophy has not been fully explained. In a previous study, we showed a new mode by which lncRNA-Mhrt inhibited cardiac hypertrophy by inhibiting myocardin. However, the underlying molecular mechanism remains unclear. This study aims to explore potential action modes of Mhrt in regulating the expression of myocardin in the process of cardiac hypertrophy. Here, we find that Mhrt reduces myocardin expression through KLF4 in vivo and in vitro. Meanwhile, Mhrt promotes the expression of KLF4 through direct binding to miR-145a-5p or inhibiting phosphorylation of KLF4 by forming a complex with KLF4 to prevent the binding of ERK and KLF4, thereby inhibiting myocardin expression and the development of myocardial hypertrophy. Taken together, our findings reveal a new pathway, Mhrt-KLF4-myocardin, that regulates cardiac hypertrophy and revealed additional possible action modes of Mhrt in the occurrence and development of cardiac hypertrophy. The new regulatory pathway serves as a potential therapeutic avenue for cardiac hypertrophy.

Thyroid cancer remains the most common endocrine malignancy worldwide, and its incidence has steadily increased over the past four years. Papillary Thyroid Cancer (PTC) is the most common differentiated thyroid cancer, accounting for 80–85% of all thyroid cancers. Mitochondrial proteins (MRPs) are an important part of the structural and functional integrity of the mitochondrial ribosomal complex. It has been reported that MRPL9 is highly expressed in liver cancer and promotes cell proliferation and migration, but it has not been reported in PTC. In the present study we found that MRPL9 was highly expressed in PTC tissues and cell lines, and lentivirus-mediated overexpression of MRPL9 promoted the proliferation and migration ability of PTC cells, whereas knockdown of MRPL9 had the opposite effect. The interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) was found by immunofluorescence and co-immunoprecipitation experiments (Co-IP). In addition, GGCT is highly expressed in PTC tissues and cell lines, and knockdown of GGCT/MRPL9 in vivo inhibited the growth of subcutaneous xenografts in nude mice and inhibited the formation of lung metastases. Mechanistically, we found that knockdown of GGCT/MRPL9 inhibited the MAPK/ERK signaling pathway. In conclusion, our study found that the interaction of GGCT and MRPL9 modulates the MAPK/ERK pathway, affecting the proliferation and migration of PTC cells. Therefore, GGCT/MRPL9 may serve as a potential biomarker for PTC monitoring and PTC treatment.

BackgroundA better understanding of the molecular mechanisms that manifest in the immunosuppressive tumor microenvironment (TME) is crucial for developing more efficacious immunotherapies for hepatocellular carcinoma (HCC), which has a poor response to current immunotherapies. Regulatory T (Treg) cells are key mediators of HCC-associated immunosuppression. We investigated the selective mechanism exploited by HCC that lead to Treg cells expansion and to find more efficacious immunotherapies.MethodsWe used matched tumor tissues and blood samples from 150 patients with HCC to identify key factors of Treg cells expansion. We used mass cytometry (CyTOF) and orthotopic cancer mouse models to analyze overall immunological changes after growth differentiation factor 15 (GDF15) gene ablation in HCC. We used flow cytometry, coimmunoprecipitation, RNA sequencing, mass spectrum, chromatin immunoprecipitation and Gdf15–/–, OT-I and GFP transgenic mice to demonstrate the effects of GDF15 on Treg cells and related molecular mechanism. We used hybridoma technology to generate monoclonal antibody to block GDF15 and evaluate its effects on HCC-associated immunosuppression.ResultsGDF15 is positively associated with the elevation of Treg cell frequencies in patients wih HCC. Gene ablation of GDF15 in HCC can convert an immunosuppressive TME to an inflammatory state. GDF15 promotes the generation of peripherally derived inducible Treg (iTreg) cells and enhances the suppressive function of natural Treg (nTreg) cells by interacting with a previously unrecognized receptor CD48 on T cells and thus downregulates STUB1, an E3 ligase that mediates forkhead box P3 (FOXP3) protein degradation. GDF15 neutralizing antibody effectively eradicates HCC and augments the antitumor immunity in mouse.ConclusionsOur results reveal the generation and function enhancement of Treg cells induced by GDF15 is a new mechanism for HCC-related immunosuppression. CD48 is the first discovered receptor of GDF15 in the immune system which provide the possibility to solve the molecular mechanism of the immunomodulatory function of GDF15. The therapeutic GDF15 blockade achieves HCC clearance without obvious adverse events.

Since 2010, Tembusu virus (TMUV) has spread widely in China, causing huge economic losses to the poultry industry. Due to the infectious and zoonotic nature of flaviviruses, their potential threat to public health is of great concern. Cellular immune responses usually play a critical role in combating viral infections. To study the molecular basis of cell immunity induced by TMUV, 14 cytotoxic T lymphocyte (CTL) epitope peptides of TMUV antigen E, NS1 and NS3 were predicted by bioinformatics tools. Their abilities to induce cellular immune responses were determined by IFN-γ ELISpot assay, and 4 peptides were found to exhibit highly significant responses upon stimulation. In addition, the cytotoxic activity induced by the epitope peptides was assessed by lactate dehydrogenase (LDH) release assay. Finally, among these peptides, we identified two murine TMUV NS3-derived H-2d-restricted CTL epitopes in BALB/c mice, which could be used to further study of epitope vaccines against TMUV infection.

To compare the effects of three mannans, Konjac glucomannan (KGM), guar gum (GG) and locust bean gum (LBG), on obesity and obesity-related metabolic disorders in mice fed with high-fat diet (HFD), and to investigate the potential modulation of gut microbiota, we performed a 14 week study on C57BL/6J mice fed a HFD with/without mannan supplementation. The results showed that supplementing 8% KGM, GG, and LBG to a HFD dramatically reduced the body weight gain and adipose accumulation, attenuated liver injury, and antagonized glycolipid metabolism and inflammation-related parameters of HFD-fed mice in different degrees. However, only LBG had such roles when the supplement dose was reduced to 2%. In addition, it was found that LBG required more time to exert its impacts on weight control and lipid metabolism. Furthermore, 16S rRNA gene sequencing of gut microbiota indicated that mannans with different structures and supplement doses affected the overall structure of the gut microbiota to a varying extent and specifically changed the abundance of some OTUs. Moreover, several OTUs belonging to the genera Muribaculum, Staphylococcus, [Eubacterium] fissicatena group, and Christensenella had a high correlation with obesity and obesity-related metabolic disorders of the host. In summary, all the three mannans had the potential to be used as alternative dietary supplements or functional foods to prevent obesity and obesity-related metabolic disorders induced by a HFD, but the effects of the dose and time varied, and the functions of the mannans were associated with their ability to regulate the gut microbiota.

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken’s codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck’s codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

A number of studies microRNAs (miRNA) play an important role in cardiac physiological and pathological processes. In these studies, miRNA regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we reveal a new regulatory mechanism in the process of cardiac hypertrophy which miRNA may also regulate the promoter activity of target genes. In the course of the study, we find that miR-466h-5p regulates the occurrence of myocardial hypertrophy via myocardin and it can upregulate the expression of myocardin through directly binding to the promoter region of myocardin. Meanwhile, we also find that myocardin can reverse-activate miR-466h-5p expression through binding to the CarG box. Thus, miR-466h-5p and myocardin form a positive regulation loop in the process of cardiac hypertrophy. These findings reveal a new mode by which miRNAs may regulate gene expression and may play a positive role in revealing the complete mechanisms of cardiac hypertrophy.

WDR1 is a major cofactor of the actin depolymerizing factor (ADF)/cofilin, accelerating ADF/cofilin-mediated actin disassembly. We had previously showed that WDR1-mediated actin dynamics is required for postnatal myocardial growth and adult myocardial maintenance in mice, in which the detailed phenotypes of adult cardiomyocyte-specific Wdr1 deletion mice had not been analyzed. In this study, we systematically analyzed the role of Wdr1 in adult mouse heart. Adult cardiomyocyte-specific Wdr1 deletion mice (cKO) exhibited cardiac hypertrophy and myocardial fibrosis. Echocardiographic study and electrocardiography revealed impaired contractile function, prolonged QT interval and Tpeak-Tend interval, and abnormal T-wave amplitude in cKO mice. Increased levels of sarcomeric proteins, adherens junction proteins and cofilin, and severe actin filament (F-actin) accumulations were observed in cKO mice heart. Taken together, this finding demonstrates that WDR1 is a critical factor for normal structure and function of adult mouse heart.

Tumor targeting agents are being developed for early tumor detection and therapeutics. We previously identified the peptide SNFYMPL (SNF*) and demonstrated its specific binding to human esophageal specimens of high-grade dysplasia (HGD) and adenocarcinoma with imaging ex vivo. Here, we aim to identify the target for this peptide and investigate its potential applications in imaging and drug delivery. With SNF* conjugated affinity chromatography, mass spectrum, Western blot, enzyme-linked immunosorbent assay (ELISA), and molecular docking, we found that the epithelial cell adhesion molecule (EpCAM) was the potential target of SNF*. Next, we showed that FITC-labeled SNF* (SNF*-FITC) colocalized with EpCAM antibody on the surface of esophageal adenocarcinoma cells OE33, and SNF*-FITC binding patterns significantly changed after EpCAM knockdown or exogenous EpCAM transfection. With the data from TCGA, we demonstrated that EpCAM was overexpressed in 17 types of cancers. Using colon and gastric adenocarcinoma cells and tissues as examples, we found that SNF*-FITC bound in a pattern was colocalized with EpCAM antibody, and the SNF* binding did not upregulate the EpCAM downstream Wnt signals. Subsequently, we conjugated SNF* with our previously constructed poly(histidine)-PEG/DSPE copolymer micelles. SNF* labeling significantly improved the micelle binding with colon and gastric adenocarcinoma cells in vitro, and enhanced the antitumor effects and decreased the toxicities of the micelles in vivo. In conclusion, we identified and validated SNF* as a specific peptide for EpCAM. The future potential use of SNF* peptide in multiple tumor surveillance and tumor-targeted therapeutics was demonstrated.

MicroRNAs (miRNAs) are a type of small non-coding RNAs that participate in diverse cellular processes including microbial invasion and immune defense. In a previous study, we identified a large amount of Japanese flounder (Paralichthys olivaceus) miRNAs responsive to megalocytivirus infection. In the present study, we examined the function of one of these miRNAs, pol-miR-194a, in association with the infectivity of Edwardsiella tarda, an intracellular bacterial pathogen to many fish species including flounder. We found that pol-miR-194a was induced in expression to a significant extent in the spleen, liver, and gill of Japanese flounder infected by E. tarda. Transfection of flounder cells with pol-miR-194a mimic significantly enhanced the intracellular replication of E. tarda. pol-miR-194a was able to interact specifically with the 3′UTR of IRF7 in a negative manner, resulting in inhibition of IRF7 expression. Consistently, pol-miR-194a significantly blocked the promoter activity of type Ⅰ interferon. Taken together, these results indicate that pol-miR-194a plays an important role in the regulation of flounder immune response as well as microbial infection, and that pol-miR-194a probably serves as a target for E. tarda to manipulate and escape host immune defense.

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development in the liver. However, its extremely short half-life in vivo greatly compromises its potential applications. Here, we report an Ac-SDKP analog peptide with d-amino acid replacement (Ac-SDD KD P). The stability of Ac-SDD KD P and its prevention of liver fibrosis were investigated in vitro and in vivo. The stabilities of Ac-SDKP and Ac-SDD KD P exposed to angiotensin-1-converting enzyme (ACE) and their half-lives in rats and human sera were determined by high-performance liquid chromatography. The inhibitory effects of Ac-SDKP and Ac-SDD KD P on the proliferation and activation of hepatic stellate cells (HSC-T6) were evaluated using the Cell Counting Kit-8, Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assays. Finally, the protective effects of Ac-SDKP and Ac-SDD KD P on carbon tetrachloride (CCl4 )-induced liver fibrosis in rats were compared. d-Amino acid replacement significantly enhanced the stability of the peptide to ACE and prolonged the half-life of Ac-SDKP in rats and human sera. The Ac-SDKP-mediated inhibition of HSC-T6 cell proliferation was well preserved, and Ac-SDD KD P exerted inhibitory effects comparable to Ac-SDKP on α-smooth muscle actin (α-SMA), collagen I and III expression, and phosphorylated-Smad-2 expression. After intraperitoneal (i.p.) administration, Ac-SDD KD P exhibited significantly greater protection than Ac-SDKP against CCl4 -induced liver fibrosis in rats. The serum alanine aminotransferase, aspartate aminotransferase, albumin, and total protein levels of the Ac-SDD KD P-treated rats were significantly lower than those of the Ac-SDKP-treated rats. α-SMA, CD45, and collagen I and III expression, as well as Smad-2 phosphorylation were significantly attenuated in the livers of the Ac-SDD KD P-treated rats compared to those of the Ac-SDKP-treated rats. Furthermore, we showed that the Ac-SDD KD P concentration in the rat liver increased to a physiological level of 60 min after i.p. administration, although i.p. administration of Ac-SDKP failed to enhance the peptide concentration in the rat liver. Our findings indicate that d-amino acid replacement is a simple and effective method to enhance the stability of Ac-SDKP. Ac-SDD KD P represents potential application of Ac-SDKP in fibrosis treatment and provides a new potential treatment strategy for liver fibrosis. © 2019 IUBMB Life, 71(9):1302-1312, 2019.

Extracellular vesicles (EVs) are shed from most mammalian cells by different processes. EVs possess several distinct advantages, including excellent biocompatibility, good bio-stability and low immunogenicity. Moreover, they play significant roles in physiological and pathological processes. Challenges in EV research mainly concern highly efficient isolation, specific membrane surface engineering and further development of EV applications in biomedical fields. In this review, we summarize the recent and representative research regarding isolation, engineering and biomedical applications of EVs, which represent important research focus areas. These three aspects have not ever been systematically classified and summarized in previous reviews. Finally, we give our insights into the key issues concerning EVs and their future development for biomedical applications.

Multiple articles have confirmed that an imbalance of the intestinal microbiota is closely related to aberrant immune responses of the intestines and to the pathogenesis of inflammatory bowel diseases (IBDs). Probiotic strains have been identified for the treatment and prevention of IBDs. The aim of this study was to screen a new probiotic strain with anti-inflammatory activity and investigate the potential mechanisms underlying its activity. We identified a new probiotic strain, L. lactis ML2018, that has anti-inflammatory properties and was isolated from traditional fermented food. In an in vitro experiment, L. lactis ML2018 prevented the release of nitric oxide (NO) and the production of inflammatory factors induced by lipopolysaccharides (LPS) in RAW264.7 cells. The in vivo anti-inflammatory effects of L. lactis ML2018 were evaluated using a dextran sulfate sodium (DSS)-induced animal model of colitis. Oral administration of L. lactis ML2018 significantly ameliorated colitis induced by DSS, which included preventing a decrease in body weight, shortening of the colon length and apoptosis of epithelial cells. L. lactis ML2018 could inhibit DSS-induced intestinal inflammation by preventing the overproduction of proinflammatory factors, suppressing the infiltration of macrophages, controlling the fibrosis, improving the integrity of the intestinal epithelial barrier and upregulating the concentrations of short-chain fatty acids (SCFAs). Moreover, L. lactis ML2018 could prevent inflammation by inhibiting the activation of the NF-κB and MAPK signaling pathways. These data suggest that L. lactis ML2018 could have therapeutic potential for treating IBDs.

Myocardin is an important factor that regulates cardiac hypertrophy, and its activity can be regulated by GATA4. However, the molecular mechanism of the above process remains unclear. This paper presents three kinds of possible molecular mechanisms of GATA4 inhibiting myocardin activity in the process of cardiac hypertrophy. First, a competitive combination of GATA4 and SRF with myocardin could reduce the formation of the myocardin-SRF-CarG box complex when GATA4 was overexpressed. Second, overexpression of GATA4 could inhibit the combination of myocardin and p300 and downregulate acetylated myocardin levels. Finally, GATA4 could upregulate the phosphorylation of myocardin protein upon activation of the ERK pathway. These findings may provide insight into the function of GATA4 and myocardin in the occurrence and development of cardiac hypertrophy.

Streptococcussuis (S.suis) is an important zoonotic pathogen that causes septicaemia, meningitis and streptococcal toxic shock-like syndrome in its host, and recent studies have shown that S.suis could be competent for natural genetic transformation. Transformation is an important mechanism for the horizontal transfer of DNA, but some elements that affect the transformation process need to be further explored. Upon entering the competent state, Streptococcus species stimulate the transcription of competence-related genes that are responsible for exogenous DNA binding, uptake and processing. In this study, we performed conserved promoter motif and qRT-PCR analyses and identified CrfP as a novel murein hydrolase that is widespread in S.suis and stimulated with a peptide pheromone in the competent state through a process controlled by ComX. A bioinformatics analysis revealed that CrfP consists of a CHAP hydrolase domain and two bacterial Src homology 3-binding (SH3b) domains. Further characterization showed that CrfP could be exported to extracellular bacterial cells and lytic S.suis strains of different serotypes, and this finding was verified by TEM and a turbidity assay. To investigate the potential effect of CrfP in vivo, a gene-deletion mutant (ΔcrfP) was constructed. Instead of stopping the natural transformation process, the inactivation of CrfP clearly reduced the effective transformation rate. Overall, these findings provide evidence showing that CrfP is important for S.suis serovar 2 competence.

Crocins, as a kind of water-soluble carotenoid pigment, are a series of ester compounds formed from crocetin and gentibiose or glucose, and mainly distributed among Crocus sativus L. (CSL), Gardenia jasminoides Ellis. (GJE). Crocins exhibit a wide range of pharmacological effects on neurodegeneration, cardiovascular disease, cerebrovascular disease, depression, liver disease, arthritis, tumor, diabetes, etc. This review systematically discussed the pharmacologic study of crocins in the aspect of structural characteristic and pharmacokinetics, and summarized the mechanism of treating disease. It summarized the abundant research of crocins from 1984 to 2020 based on the above aspects, which provide a reference for the deeply development and application of crocins.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi.METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes.FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels.INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus.FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

Duck reovirus (DRV) is a fatal member of the genus Orthoreovirus in the family Reoviridae. The disease caused by DRV leads to huge economic losses to the duck industry. Post-translational modification is an efficient strategy to enhance the immune responses to virus infection. However, the roles of protein phosphorylation in the responses of ducklings to Classic/Novel DRV (C/NDRV) infections are largely unknown. Using a high-resolution LC–MS/MS integrated to highly sensitive immune-affinity antibody method, phosphoproteomes of Cairna moschata spleen tissues under the C/NDRV infections were analyzed, producing a total of 8,504 phosphorylation sites on 2,853 proteins. After normalization with proteomic data, 392 sites on 288 proteins and 484 sites on 342 proteins were significantly changed under the C/NDRV infections, respectively. To characterize the differentially phosphorylated proteins (DPPs), a systematic bioinformatics analyses including Gene Ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation were performed. Two important serine protease system-related proteins, coagulation factor X and fibrinogen α-chain, were identified as phosphorylated proteins, suggesting an involvement of blood coagulation under the C/NDRV infections. Furthermore, 16 proteins involving the intracellular signaling pathways of pattern-recognition receptors were identified as phosphorylated proteins. Changes in the phosphorylation levels of MyD88, NF-κB, RIP1, MDA5 and IRF7 suggested a crucial role of protein phosphorylation in host immune responses of C. moschata. Our study provides new insights into the responses of ducklings to the C/NDRV infections at PTM level.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease with a poor prognosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development. However, it's extremely short half-life greatly limits its applications. Previously, we reported an Ac-SDKP analog peptide in which Asp and Lys residues were replaced with D-amino acids (Ac-SD

To explore the effect of Tangshen Formula (, TSF), a Chinese herbal medicine, on interstitial cells of Cajal (ICC) in the colon of diabetic rats.Fifty-four male Wistar rats were randomly divided into normal control (NC, n=14) and high-fat diet (HFD) groups (n=40). After 6 weeks, the rats in the HFD group were injected intraperitoneally streptozotocin once (30 mg/kg). Thirty rats with fasting blood glucose higher than 11.7 mmol/L were randomly divided into diabetes (DM) and TSF groups, 15 rats in each group. Rats in the NC and DM groups were intragastrically administered with saline, and those in the TSF group were given with TSF (2.4 g/kg) once daily for 20 weeks. Expression levels of Bax, Bcl-2, and caspase-3 in colonic smooth muscle layer were measured by Western blotting and immunohistochemical staining. The number of ICC was determined by immunohistochemical staining. Immunofluorescence was used for analyzing the ratio of classically activated macrophages (M1) and alternatively activated macrophages (M2) to total macrophages. Electron microscopy was used to observe the epithelial ultrastructure and junctions.TSF appeared to partially prevented loss of ICC in DM rats (P0.05). Compared with the NC group, expression levels of Bcl-2, Bax, caspase-3, and TNF-α as well as the ratio of M1 to total macrophages increased in DM rats (all P0.05), and the ratio of M2 to total macrophages decreased (P0.05 or P0.01). Compared with the DM group, TSF decreased the expression levels of abovementioned proteins and restore M2 to total macrophages ratio (P0.05 or P0.01). TSF appeared to attenuate the ultrastructural changes of epithelia and improve the tight and desmosome junctions between epithelia reduced in the DM rats.Reduced number of ICC in DM rats may be associated with damage of the intestinal barrier. The protective effects of TSF on ICC may be through repair of the epithelial junctions, which attenuates inflammation and inflammation-initiated apoptosis in colon of DM rats.

Endothelial cell proliferation disorder caused by vascular injury seems to be one of the causes of atherosclerosis, which is the pathological basis of coronary heart disease. The role of STAT3 in the regulation of microRNAs and endothelial dysfunction in atherosclerosis is unclear. STAT3 can be activated by cytokine IL-6 and up regulate the expression of CX3CL1. In addition, microRNA-15a-5p (miR-15a-5p) inhibited the transcription of CX3CL1, the proliferation of vascular endothelial cells and the proliferation of STAT3 regulated vascular endothelial cells. STAT3 positively regulates the expression of CX3CL1, and then down-regulates the inhibition of CX3CL1 by over-expression of miR-15a-5p, thus forming an elimination feedback loop to control the proliferation of HUVECs and affect the progression of atherosclerosis. In conclusion, miR-15a-5p may be the therapeutic target of the pathological basis of coronary atherosclerosis.

Probiotics are thought to have immunomodulatory functions, improve inflammatory disorders and treat inflammatory bowel disease (IBD). Here, we screened a new probiotic strain with anti-inflammatory activity and investigated its effect on the immune cell response and histone acetylation. Lactobacillus casei (L. casei) LH23 inhibited the production of nitric oxide and inflammatory factors induced by lipopolysaccharides in RAW264.7 cells, which was associated with inhibiting the over-activation of the JNK/p38 signaling pathway. Furthermore, L. casei LH23 can significantly ameliorate dextran sulfate sodium (DSS)-induced mouse colitis in vivo by reducing numbers of macrophages (CD11b+F4/80+) and their secreted inflammatory cytokines. Myeloperoxidase activity was also decreased in mice treated with LH23. The administration of L. casei LH23 induced the increase of CD3+CD4+CD25+ regulatory T cells among mesenteric lymph nodes. Meanwhile, LH23 treatment could augment short chain fatty acid contents. Importantly, we reported here for the first time that DSS treatment significantly decreased the level of histone H3K9 acetylation, while supplementation of L. casei LH23 restored the level of histone H3K9 acetylation in colon tissues. These data suggest that L. casei LH23 may have been beneficial for preventing and treating IBD.

Fumonisins have posed hazardous threat to human and animal health worldwide. Enzymatic degradation is a desirable detoxification approach but is severely hindered by serious shortage of detoxification enzymes. After mining enzymes by bioinformatics analysis, a novel carboxylesterase FumDSB from Sphingomonadales bacterium was expressed in Escherichia coli, and confirmed to catalyze fumonisin B1 to produce hydrolyzed fumonisin B1 by liquid chromatography mass spectrometry for the first time. FumDSB showed high sequence novelty, sharing only ~34% sequence identity with three reported fumonisin detoxification carboxylesterases. Besides, FumDSB displayed its high degrading activity at 30–40 °C within a broad pH range from 6.0 to 9.0, which is perfectly suitable to be used in animal physiological condition. It also exhibited excellent pH stability and moderate thermostability. This study provides a FB1 detoxification carboxylesterase which could be further used as a potential food and feed additive.

Purpose: miR-5100 participates in the proliferation of lung cancer and pancreatic cancer cells, and participates in the differentiation of osteoblasts. However, the regulation of gastric cancer cells in gastric cancer cells remains unclear. Experimental design: The blood of patients was collected to detect the expression level of miR-5100, and the apoptosis and autophagy levels of cells were detected using western blot, flow cytometry, and confocal. At the same time, in vitro tumor formation experiments in nude mice were used to verify the results of in vitro experiments. Results: The expression of miR-5100 is related to the prognosis of gastric cancer, miR-5100 can enhance the apoptosis level of gastric cancer cells and inhibit the occurrence of autophagy by targeting CAAP1. MKL1 can inhibit the apoptosis of gastric cancer cells and promote the occurrence of autophagy by targeting CAAP1. At the same time, MKL1 can also increase the expression of miR-5100. Conclusions: Our research reveals the mechanism by which the MKL1/miR-5100/CAAP1 loop regulates apoptosis and autophagy levels in gastric cancer cells, and miR-5100 is expected to become a new potential target for gastric cancer treatment. Keywords: MKL1, miR-5100, CAAP1, Autophagy, Apoptosis, Gastric cancer

Intestinal mucosal barriers help the body resist many intestinal inflammatory diseases, such as inflammatory bowel disease (IBD). In this study, we identified a novel bacterium promoting the repair of intestinal mucosa and investigated the potential mechanisms underlying its activity. Culture supernatant of Bacillus subtilis RZ001 upregulated the expression of mucin 2 (MUC2) and tight junction (TJ) proteins in HT-29 cells in vitro. Oral administration of B. subtilis RZ001 may have significantly reduced symptoms such as the dextran sulfate sodium (DSS)-induced decrease in body weight, shortening of colon length and overproduction of proinflammatory factors. The number of goblet cells and levels of MUC2 and TJ proteins were significantly increased in adult mice fed with B. subtilis RZ001. B. subtilis RZ001 cells upregulated the levels of MUC2 in the intestinal organoids. Furthermore, culture supernatant of B. subtilis RZ001 could suppress the Notch signalling pathway and activate the expression of atonal homolog 1 (Atoh1). The transcription factor Atoh1 is required for intestinal secretory cell differentiation and activates transcription of MUC2 via binding to E-boxes on the MUC2 promoter. Taken together, B. subtilis strain RZ001 has the potential for treating IBD. The present study is helpful to elucidate the mechanisms of B. subtilis action.

MicroRNAs (miRNAs) are post-transcriptional regulators that play vital roles in diverse physiological processes including immunity. In this study, we investigated the regulatory mechanism and function of a novel Japanese flounder (Paralichthys olivaceus) miRNA, pol-miR-3p-2. pol-miR-3p-2 was responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. pol-miR-3p-2 negatively regulated the expression of p53 through interaction with the 3′UTR of p53. Overexpression of pol-miR-3p-2 promoted autophagy, resulting in augmented production of LC3-II, while knockdown of p53 increased the level of beclin, a key factor of autophagy. In vivo and in vitro studies showed that E. tarda infection induced autophagy in flounder, and pol-miR-3p-2 inhibited the infectivity of E. tarda. Together these results indicate that pol-miR-3p-2 regulates autophagy through the target gene p53, thus revealing a regulatory link between p53 and autophagy in teleost, and that pol-miR-3p-2 plays an important role in the immune defense against E. tarda.

Chitooligosaccharides (COS) have a variety of biological activities due to their positively charged amino groups. Studies have shown that COS have antidiabetic effects, but their molecular mechanism has not been fully elucidated. The present study confirmed that COS can reduce hyperglycemia and hyperlipidemia, prevent obesity, and enhance histological changes in the livers of mice with type 2 diabetes mellitus (T2DM). Additionally, treatment with COS can modulate the composition of the gut microbiota in the colon by altering the abundance of Firmicutes, Bacteroidetes, and Proteobacteria. Furthermore, in T2DM mice, treatment with COS can upregulate the cholesterol-degrading enzymes cholesterol 7-alpha-hydroxylase (CYP7A1) and incretin glucagon-like peptide 1 (GLP-1) while specifically inhibiting the transcription and expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the key enzyme in cholesterol synthesis. Furthermore, using an oleic acid-induced hepatocyte steatosis model, we found that HMGCR can be directly transactivated by SET and MYND domain containing 3 (SMYD3), a transcriptional regulator, via 5&prime, CCCTCC-3&prime, element in the promoter. Overexpression of SMYD3 can suppress the inhibitory effect of COS on HMGCR, and COS might regulate HMGCR by inhibiting SMYD3, thereby exerting hypolipidemic functions. To the best of our knowledge, this study is the first to illustrate that COS mediate glucose and lipid metabolism disorders by regulating gut microbiota and SMYD3-mediated signaling pathways.

Inflammatory bowel disease (IBD) is a chronic, recurrent, and remitting inflammatory disease resulting from immune dysregulation in the gut. As a clinically frequent disease, it can affect individuals throughout their lives, with multiple complications. Glucagon-like peptide 2 (GLP-2) is a potent epithelium-specific intestinal growth factor. However, native GLP-2 has a relatively short half-life in human circulation because of extensive renal clearance and rapid degradation by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV). Previously, We prepared a recombinant GLP-2 variant (GLP-2②), which has increased half-life and activity as compared to the [Gly2]GLP-2 monomer. The aim of the present study was to investigate the protective potential of GLP-2② in IBD models. LPS-induced in vitro model and dextran sulfate sodium (DSS)-induced in vivo model were used to study the anti-inflammatory and therapeutic effect of GLP-2②. We found that treated with GLP-2② showed a significantly reduction in the secretion of inflammatory cytokines. Furthermore, GLP-2② alleviated symptoms of DSS-induced colitis. GLP-2② treated mice displayed an increase in body weight, lower colitis scores, and fewer mucosal damage compared with GLP-2 treated mice. MPO activities, protein expression of NLRP3 and COX2 in the colon tissues were significantly reduced in GLP-2② groups. Importantly, the ameliorative effect of GLP-2② was related to anti-apoptosis effect in colon tissues. These findings demonstrated that GLP-2② may offer a superior therapeutic benefit over [Gly2]GLP-2 monomer for treatment of IBD.

The aim of this paper was to investigate the hemostatic effect and mechanism of carbonized Scutellariae Radix on uterine bleeding in the rats caused by early pregnancy termination. Eight unpregnant female rats were selected as normal group. Forty female rats conceived on the same day received mifepristone(11. 4 mg·kg-1) and misoprostol(125 μg·kg-1) to induce model of incomplete abortion in early pregnancy. Abortion models were randomly divided into model group,carbonized Scutellariae Radix water extract low dose group(0. 55 g·kg-1),medium group(1. 10 g·kg-1),high dose group(2. 20 g·kg-1) and positive control group(0. 45 g·kg-1).The uterine bleeding volume was detected by ultraviolet spectrophotometry. The pathological changes of endometrium were detected by HE(hematixylin-eosin) staining. The levels of interleukin(IL-1β),IL-6 and tumor necrosis factor(TNF-α) in the plasma of rats were determined by ELISA. The expression levels of IL-1β,IL-6 and TNF-α mRNA in the uterus of rats were determined by RT-PCR.The protein expression levels of VEGF,MMP-2 and MMP-9 were determined by Western blot. As compared with the normal group,the uterine bleeding volume and histopathological score were increased significantly; microvessel density of endometrial tissues was decreased significantly; the contents of TNF-α,IL-1β and the levels of TNF-α mRNA and IL-1β mRNA in the plasma were increased,while the content of IL-6 and level of IL-6 mRNA were decreased significantly. The protein expression levels of VEGF,MMP-2 and MMP-9 in the uterine tissues were also decreased. As compared with the model group,the uterine bleeding volume was decreased significantly in the carbonized Scutellariae Radix medium dose and high dose groups; endometrial repair was promoted,and the microvessel density of endometrial tissues was increased significantly; the contents of IL-1β and TNF-α in the plasma of rats were decreased significantly,while the content of IL-6 in the plasma of rats was increased significantly; the expression levels of IL-1β and TNF-α mRNA in the uterus of rats were decreased and the expression level of IL-6 mRNA showed an increase; the protein expressions of VEGF,MMP-2 and MMP-9 were decreased significantly in carbonized Scutellariae Radix medium and high dose groups. In conclusion,carbonized Scutellariae Radix showed good hemostatic effect,and its mechanism may be related to the repair of endometrium and inhibition of inflammatory reaction.

To establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture. Bcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα). A CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars.

Background Thymosin beta 4 (Tβ4) is a 43-amino-acid peptide with protective properties in myocardium injury. Previously, we produced a recombinant human dimeric Tβ4 (DTβ4). Here, the cardioprotective effects of DTβ4 and the molecular mechanisms underlying its enhanced activity were investigated. Methods and Results Echocardiography measurements showed that the cardioprotective effect of DTβ4 in myocardial infarction mice was significantly higher than that of wild-type Tβ4. Corresponding in vitro analyses demonstrated that the enhanced cardioprotection provided by DTβ4 was largely due to increased stimulation of angiogenesis. HPLC analysis, western blotting and qRT-PCR indicated that the enhanced pro-angiogenesis activity of DTβ4 was independent of the protein half-life and the known downstream pathways of wild-type Tβ4. Transcriptome deep sequencing (RNA-seq), BrdU incorporation assays, flow cytometry analysis and RNA interference demonstrated that the enhanced angiogenic activity of DTβ4 depended on MALAT1 (metastasis-associated lung adenocarcinoma transcript 1)-induced proliferation of vascular endothelial cells, which has not been reported for wild-type Tβ4. Moreover, transcription factor activation screening, luciferase promoter reporter assay and immunoprecipitation assay demonstrated that DTβ4 enhanced MALAT1 transcription by inhibiting the degradation of prospero-related homeobox 1 (PROX1). Conclusion This study demonstrates the potential applications and the novel bioactivity of the Tβ4 dimer. Moreover, to construct the dimer represents a new method for production of bioactive peptides that may have novel activities.

Immunotherapy is gathering momentum as a kind of important therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only in a small subset of patients. Combination therapies targeting multiple pathways often can augment an immune response to improve survival further. Here, the tumoricidal effects of the dual hPD-L1(human programmed cell death ligand 1) vaccination/HER2(human epidermal growth factor receptor 2) gene vaccination immunotherapy against the established HER2-expressed cancers were observed. Animals treated with combination therapy using hPD-L1 vaccine and HER2 gene vaccine had significantly improved survival in a mammary carcinoma model. We observed an increase in tumor growth inhibition following treatment. The percentage of the tumor-free mice (%) was much higher in the combined PD-L1/HER2 group. Furthermore, under the tumor-burden condition, hPD-L1 vaccine enhanced humoral immunity of HER2 gene vaccine. And the combination treatment increased the IFN-γ-producing effector T cells. Additionally, splenocytes from the combined PD-L1/HER2 group immunized mice possessed higher CTL activity. Notably, vaccination with combination therapy induced a significant decrease in the percentage of CD4+CD25+ Treg cells. Collectively, these data demonstrate that PD-L1/HER2 gene vaccine combination therapy synergistically generates marked tumoricidal effects against established HER2-expressing cancers.

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SIGNIFICANCE: gC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

Duck enteritis virus (DEV) can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of DEV vaccine strain pE1 (pDEV-EF1) has been constructed in our previous study. Based on pE1, a recombinant mutated clone pDL (pVP26CFP-gCRFP), which carries a red fluorescent protein (mRFP) gene fused to the viral envelope protein gC in combination with a cyan fluorescent protein (CFP) gene fused to the viral capsid VP26, was constructed by two-step Red/ET recombination and the recombinant virus rDL (rVP26CFP-gCRFP) was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate transfection. Western blot analysis revealed that VP26-CFP and gC-mRFP were both expressed in fusion forms in rDL-infected CEFs, and subcellular localization study showed that gC-mRFP was mainly localized in whole cell at 36, 48 h post infection (p.i.); and then mostly migrated to the cytoplasm after 60 h.p.i., ; whereas VP26-CFP was localized in the nucleus in all stages of virus infection. Additionally, viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells by transmission electron microscopy, indicating that exogenous gene insertion has no effect on virus assembly. This study has laid a foundation for visually studying localization, transportation of DEV capsid proteins and envelope glycoproteins as well as virus assembly, virion movement and virus-cell interaction.

Elevated adipogenesis of bone marrow stromal cells (BMSCs) is closely associated with non-traumatic osteonecrosis of femoral head (ONFH). Our previous studies have shown that Huogu (HG) formula was effective both in clinic experience and experimental ONFH. How HG impacts the differentiation of BMSCs and what is the underlying molecular mechanism remain largely unknown. Our results showed that ethyl acetate extract of HG (HGE) significantly decreased the adipocyte differentiation as determined by oil red staining, while slightly increased the ALP activity. Investigation of the molecular mechanism revealed that HGE could inhibit the mRNA and protein expression of peroxisome proliferators-activated receptor (PPAR)γ, lipoprotein lipase (LPL) and adipocyteprotein2 (AP2). Interestingly, the inhibition of adipogenic differentiation in BMSCs by HGE could be restored by DKK-1, an inhibitor of Wnts. However, Noggin (an inhibitor of BMPs) displayed an additive role with HGE in suppressing the expression of PPARγ, LPL, and AP2. Furthermore, the bone marrow fat formation, as well as the expression of Wnt3a and PPARγ, was effectively regulated by HGE in the steroid-induced ONFH rats. Our results demonstrated that HGE treatment significantly inhibited adipogenesis and slightly promoted osteogenesis of BMSCs through regulating the BMP and Wnt pathways. The findings shed lights on the molecular mechanism of HGE in the inhibition of adipogenesis and provide scientific rationale for its clinical application of HGE in the treatment of ONFH.

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.

Our recent studies have shown that Huogu (HG) formula was effective both in clinic experience and in experimental osteonecrosis of the femoral head (ONFH). Given that defective of bone marrow stromal cells (MSCs) contribute to the development of osteonecrosis and MSCs show enormous potential in the treatment of ONFH, especially to aging people. How HG impacts the differentiation of MSCs and what is the underlying cellular and molecular mechanism remains largely unknown. Here, we found that an aqueous fraction of HG (HGA) significantly increased the alkaline phosphatase (ALP) activity, mineralized nodules, and migration of MSCs in a dose-dependent manner. Meanwhile, HGA could enhance the mRNA and protein expression of Runt-related transcription factor 2 (Runx2), Alp, Bmp2, osteocalcin (Ocn), and Osterix (Osx). Further investigation of the molecular mechanisms revealed that HGA treatment obviously increased expression, secretion, and activation of bone morphogenetic protein (BMP) 2 and β-catenin, two key regulators of the BMP or Wnt signaling pathway. Furthermore, osteogenic differentiation of MSCs could be blocked by using pharmacological inhibitors for these signaling pathways such as Noggin and Dkk-1. Besides, HGA could inhibit adipogenic differentiation of MSCs. Our study reveals that HGA promotes the osteogenesis of MSCs via the BMP and Wnt signaling pathways. Our findings provide mechanistic insights into the role of HG in treating ONFH.

Traditional Chinese Medicines (TCMs) have been used for more than 3,000 years. However, most TCMs are used based on previous experience, and their exact mechanisms are still unclear. Proteomics, as a major component of systems biology, is an appropriate tool that could bring breakthroughs in the study of pharmacological mechanisms of TCMs. Researchers made substantial progress by using the proteomics approach to investigate the mechanism of TCMs. In this review, we summarize the current applications of proteomics in the mechanistic study of TCMs. Proteomics technologies and strategies that might be used in the future to enhance the study of TCMs are also discussed.

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)–stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence. Bacterial DNA from Listeria monocytogenes infection is packaged within extracellular vesicles and induces paracrine cGAS–STING signalling in bystander cells to subvert host responses.