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BACKGROUND Despite overt insulin resistance, adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids. AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide, since it is produced by monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in adipocytes. METHODS 3H-2-deoxyglucose uptake (2-DG) was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine. 14C-tyramine oxidation and binding of imidazolinic radioligands [3H-Idazoxan, 3H-(2-benzofuranyl)-2-imidazoline] were studied in adipocytes, the liver, and muscle. The influence of in vivo administration of tyramine + vanadium on glucose handling was assessed in lean and obese rats. RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats, when compared to their lean littermates. Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited, while MAO was increased and SSAO decreased. These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat, when compared to the lean. In vitro, tyramine precluded the binding to I2 sites, while in vivo, its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese rats. CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number. However, probably as a consequence of SSAO down-regulation, the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes. The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

BACKGROUND Liver cancer is one of the most highly malignant cancers, characterized by easy metastasis and chemoradiotherapy resistance. Emerging evidence indicates that long noncoding RNAs (LncRNAs), including Lnc524369, are highly involved in the initiation, progression, radioresistance, and chemoresistance of hepatocellular carcinoma (HCC). However, the function of Lnc524369 remains unclear. AIM To explore the function of Lnc524369 in HCC. METHODS To investigate the effect of Lnc524369, tissue from 41 HCC patients were analyzed using CCK8, migration, and invasion assays. Lnc524369 and YWHAZ (also named 14-3-3ζ) mRNA were detected by qPCR, and YWHAZ and RAF1 proteins were detected by western blot in liver cancer cell lines and human HCC tissues. The Cancer Cell Line Encyclopedia (CCLE) databases, STRING database, Human Protein Atlas database, and the TCGA database were used for bioinformatic analysis. RESULTS Lnc524369 was significantly upregulated in the nucleus of liver cancer cells and human HCC tissues. Overexpression of Lnc524369 was associated with the proliferation, migration, and invasion of liver cancer cells. YWHAZ and RAF1 proteins and YWHAZ mRNA were overexpressed in liver cancer, which could be attenuated by overexpression of Lnc524369. Lnc524369 and its downstream target YWHAZ and RAF1 proteins were negatively associated with overall survival time. CONCLUSION Lnc524369 might be a promising target of HCC as it can enhance liver cancer progression and decrease the overall survival time of HCC by activating the YWHAZ/RAF1 pathway.

International audience; BACKGROUNDWhen combined with vanadium salts, catecholamines strongly activate glucose uptake in rat and mouse adipocytes.AIMTo test whether catecholamines activate glucose transport in human adipocytes.METHODSThe uptake of 2-deoxyglucose (2-DG) was measured in adipocytes isolated from pieces of abdominal subcutaneous tissue removed from women undergoing reconstructive surgery. Pharmacological approaches with amine oxidase inhibitors, adrenoreceptor agonists and antioxidants were performed to unravel the mechanisms of action of noradrenaline or adrenaline (also named epinephrine).RESULTSIn human adipocytes, 45-min incubation with 100 µmol/L adrenaline or noradrenaline activated 2-DG uptake up to more than one-third of the maximal response to insulin. This stimulation was not reproduced with millimolar doses of dopamine or serotonin and was not enhanced by addition of vanadate to the incubation medium. Among various natural amines and adrenergic agonists tested, no other molecule was more efficient than adrenaline and noradrenaline in stimulating 2-DG uptake. The effect of the catecholamines was not impaired by pargyline and semicarbazide, contrarily to that of benzylamine or methylamine, which are recognized substrates of semicarbazide-sensitive amine oxidase. Hydrogen peroxide at 1 mmol/L activated hexose uptake but not pyrocatechol or benzoquinone, and only the former was potentiated by vanadate. Catalase and the phosphoinositide 3-kinase inhibitor wortmannin inhibited adrenaline-induced activation of 2-DG uptake.CONCLUSIONHigh doses of catecholamines exert insulin-like actions on glucose transport in human adipocytes. At submillimolar doses, vanadium did not enhance this catecholamine activation of glucose transport. Consequently, this dismantles our previous suggestion to combine the metal ion with catecholamines to improve the benefit/risk ratio of vanadium-based antidiabetic approaches.

BACKGROUND Children with attention-deficit/hyperactivity disorder (ADHD) often exhibit behaviour challenges and deficits in executive functions (EF). Psychostimulant medications [e.g., methylphenidate (MPH)] are commonly prescribed for children with ADHD and are considered effective in 70% of the cases. Furthermore, only a handful of studies have investigated the long-term impact of MPH medication on EF and behaviour. AIM To evaluate behaviour and EF challenges in children with ADHD who were involved in an MPH treatment trial across three-time points. METHODS Thirty-seven children with ADHD completed a stimulant medication trial to study the short- and long-term impact of medication. Children with ADHD completed three neuropsychological assessments [Continuous Performance Test (CPT)-II, Digit Span Backwards and Spatial Span Backwards]. Parents of children with ADHD completed behaviour rating scales [Behaviour Rating Inventory of Executive Functioning (BRIEF) and Behaviour Assessment System for Children-Second Edition (BASC-2)]. Participants were evaluated at: (1) Baseline (no medication); and (2) Best-dose (BD; following four-week MPH treatment). Additionally, 18 participants returned for a long-term naturalistic follow up (FU; up to two years following BD). RESULTS Repeated measure analyses of variance found significant effects of time on two subscales of BRIEF and four subscales of BASC-2. Neuropsychological assessments showed some improvement, but not on all tasks following the medication trial. These improvements did not sustain at FU, with increases in EF and behaviour challenges, and a decline in performance on the CPT-II task being observed. CONCLUSION Parents of children with ADHD reported improvements in EF and behaviours during the MPH trial but were not sustained at FU. Combining screening tools and neuropsychological assessments may be useful for monitoring medication responses.

BACKGROUND Burnout and compassion fatigue are affecting the quality of professional life. AIM To investigate the levels of vocational satisfaction, burnout, and compassion fatigue and factors that may be related to health professionals working in children’s clinics. METHODS The study sample was in the west of Turkey. Data were collected using the questionnaire form and the quality of life scale for employees. RESULTS The findings obtained in this study showed that the level of vocational satisfaction of female health professionals and the burnout level of male health professionals were higher. The professional satisfaction of the doctors was lower than that of the nurses and midwives, and the mean score of burnout and fatigue was high. CONCLUSION Further studies are needed on this topic to help improve the factors that may affect the professional quality of life of health professionals.

BACKGROUND Hepatic overload of gut-derived lipopolysaccharide dictates the progression of alcoholic liver disease (ALD) by inducing oxidative stress and activating Kupffer cells and hepatic stellate cells through toll-like receptor 4 signaling. Therefore, targeting the maintenance of intestinal barrier integrity has attracted attention for the treatment of ALD. Zinc acetate and rifaximin, which is a nonabsorbable antibiotic, had been clinically used for patients with cirrhosis, particularly those with hepatic encephalopathy, and had been known to improve intestinal barrier dysfunction. However, only few studies focused on their efficacies in preventing the ALD-related fibrosis development. AIM To investigate the effects of a combined zinc acetate with rifaximin on liver fibrosis in a mouse ALD model. METHODS To induce ALD-related liver fibrosis, female C57BL/6J mice were fed a 2.5% (v/v) ethanol-containing Lieber-DeCarli liquid diet and received intraperitoneal carbon tetrachloride (CCl4) injection twice weekly (1 mL/kg) for 8 wk. Zinc acetate (100 mg/L) and/or rifaximin (100 mg/L) were orally administered during experimental period. Hepatic steatosis, inflammation and fibrosis as well as intestinal barrier function were evaluated by histological and molecular analyses. Moreover, the direct effects of both agents on Caco-2 barrier function were assessed by in vitro assays. RESULTS In the ethanol plus CCl4-treated mice, combination of zinc acetate and rifaximin attenuated oxidative lipid peroxidation with downregulation of Nox2 and Nox4. This combination significantly inhibited the Kupffer cells expansion and the proinflammatory response with blunted hepatic exposure of lipopolysaccharide and the toll-like receptor 4/nuclear factor kB pathway. Consequently, liver fibrosis and hepatic stellate cells activation were efficiently suppressed with downregulation of Mmp-2, -9, -13, and Timp1. Both agents improved the atrophic changes and permeability in the ileum, with restoration of tight junction proteins (TJPs) by decreasing the expressions of tumor necrosis factor α and myosin light chain kinase. In the in vitro assay, both agents directly reinforced ethanol or lipopolysaccharide-stimulated paracellular permeability and upregulated TJPs in Caco-2 cells. CONCLUSION Dual therapy with zinc acetate and rifaximin may serve as a strategy to prevent ALD-related fibrosis by maintaining intestinal barrier integrity.

BACKGROUND Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. The association of hepatitis B virus (HBV) infection with HCC is hitherto documented. Exosomal miRNAs contribute to cancer progression and chemoresistance. HBV X protein has been known to modulate miRNAs that facilitate cell proliferation and the process of hepatocarcinogenesis. However, there has been no report on hepatitis B core antigen (HBc) regulating exosomal miRNAs to induce drug resistance of HCC cells. AIM To elucidate the mechanism by which HBc promotes Doxorubicin hydrochloride (Dox) resistance in HCC. METHODS Exosomes were isolated by ultracentrifugation. The morphology and size of exosomes were evaluated by Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM). The miRNAs differentially expressed in HCC were identified using The Cancer Genome Atlas (TCGA) database. The level of miR-135a-5p in patient tissue samples was detected by quantitative polymerase chain reaction. TargetScan and luciferase assay were used to predict and prove the target gene of miR-135a-5p. Finally, we identified the effects of miR-135a-5p on anti-apoptosis and the proliferation of HCC in the presence or absence of Dox using flow cytometry, Cell counting kit 8 (CCK-8) assay and western blot. RESULTS We found that HBc increased the expression of exosomal miR-135a-5p. Integrated analysis of bioinformatics and patient samples found that miR-135a-5p was increased in HCC tissues in comparison with paracancerous tissues. Bioinformatic analysis and in vitro validation identified vesicle-associated membrane protein 2 (VAMP2) as a novel target gene of miR-135a-5p. Functional assays showed that exosomal miR-135a-5p induced apoptosis protection, cell proliferation, and chemotherapy resistance in HCC. In addition, the rescue experiment demonstrated that VAMP2 reversed apoptosis protection, cell growth, and drug resistance by miR-135a-5p. Finally, HBc promoted HCC anti-apoptosis, proliferation, and drug resistance and prevented Dox-induced apoptosis via the miR-135a-5p/VAMP2 axis. CONCLUSION These data suggested that HBc upregulated the expression of exosomal miR-135a-5p and promoted anti-apoptosis, cell proliferation, and chemical resistance through miR-135a-5p/VAMP2. Thus, our work indicated an essential role of the miR-135a-5p/VAMP2 regulatory axis in chemotherapy resistance of HCC and a potential molecular therapeutic target for HCC.

BACKGROUND As a cellular mode of therapy, bone marrow mesenchymal stem cells (BMSCs) are used to treat stroke. However, their mechanisms in stroke treatment have not been established. Recent evidence suggests that regulation of dysregulated gut flora after stroke affects stroke outcomes. AIM To investigate the effects of BMSCs on gut microbiota after ischemic stroke. METHODS A total of 30 Sprague-Dawley rats were randomly divided into three groups, including sham operation control group, transient middle cerebral artery occlusion (MCAO) group, and MCAO with BMSC treatment group. The modified Neurological Severity Score (mNSS), beam walking test, and Morris water maze test were used to evaluate neurological function recovery after BMSC transplantation. Nissl staining was performed to elucidate on the pathology of nerve cells in the hippocampus. Feces from each group of rats were collected and analyzed by 16s rDNA sequencing. RESULTS BMSC transplantation significantly reduced mNSS (P < 0.01). Rats performed better in the beam walking test in the BMSC group than in the MCAO group (P < 0.01). The Morris water maze test revealed that the BMSC treatment group exhibited a significant improvement in learning and memory. Nissl staining for neuronal damage assessment after stroke showed that in the BMSC group, cells were orderly arranged with significantly reduced necrosis. Moreover, BMSCs regulated microbial structure composition. In rats treated with BMSCs, the abundance of potential short-chain fatty acid producing bacteria and Lactobacillus was increased. CONCLUSION BMSC transplantation is a potential therapeutic option for ischemic stroke, and it promotes neurological functions by regulating gut microbiota dysbiosis.

BACKGROUND Chromofungin (CHR: chromogranin-A 47-66) is a chromogranin-A derived peptide with anti-inflammatory and anti-microbial properties. Ulcerative colitis (UC) is characterized by a colonic decrease of CHR and a dysregulation of dendritic CD11c+ cells. AIM To investigate the association between CHR treatment and dendritic cells (DCs)-related markers in different immune compartments in colitis. METHODS A model of acute UC-like colitis using dextran sulphate sodium (DSS) was used in addition to biopsies collected from UC patients. RESULTS Intrarectal CHR treatment reduced the severity of DSS-induced colitis and was associated with a significant decrease in the expression of CD11c, CD40, CD80, CD86 and interleukin (IL)-12p40 in the inflamed colonic mucosa and CD11c, CD80, CD86 IL-6 and IL-12p40 within the mesenteric lymph nodes and the spleen. Furthermore, CHR treatment decreased CD80 and CD86 expression markers of splenic CD11c+ cells and decreased NF-κB expression in the colon and of splenic CD11c+ cells. In vitro, CHR decreased CD40, CD80, CD86 IL-6 and IL-12p40 expression in naïve bone marrow-derived CD11c+ DCs stimulated with lipopolysaccharide. Pharmacological studies demonstrated an impact of CHR on the NF-κB pathway. In patients with active UC, CHR level was reduced and showed a negative linear relationship with CD11c and CD86. CONCLUSION CHR has protective properties against intestinal inflammation via the regulation of DC-related markers and CD11c+ cells. CHR could be a potential therapy of UC.

BACKGROUND Bile duct ligation (BDL) in animals is a classical method for mimicking cholestatic fibrosis. Although different surgical techniques have been described in rats and rabbits, mouse models can be more cost-effective and reproducible for investigating cholestatic fibrosis. Magnetic resonance imaging (MRI) has made great advances for noninvasive assessment of liver fibrosis. More comprehensive liver fibrotic features of BDL on MRI are important. However, the utility of multiparameter MRI to detect liver fibrosis in a BDL mouse model has not been assessed. AIM To evaluate the correlation between the pathological changes and multiparameter MRI characteristics of liver fibrosis in a BDL mouse model. METHODS Twenty-eight healthy adult male balb/c mice were randomly divided into four groups: sham, week 2 BDL, week 4 BDL, and week 6 BDL. Multiparameter MRI sequences, included magnetic resonance cholangiopancreatography, T1-weighted, T2-weighted, T2 mapping, and pre- and post-enhanced T1 mapping, were performed after sham and BDL surgery. Peripheral blood and liver tissue were collected after MRI. For statistical analysis, Student’s t-test and Pearson’s correlation coefficient were used. RESULTS Four mice died after BDL surgery; seven, six, five and six mice were included separately from the four groups. Signal intensities of liver parenchyma showed no difference on TI- and T2-weighted images. Bile duct volume, ΔT1 value, T2 value, and the rate of liver fibrosis increased steadily in week 2 BDL, week 4 BDL and week 6 BDL groups compared with those in the sham group (P < 0.01). Alanine aminotransferase and aspartate transaminase levels initially surged after surgery, followed by a gradual decline over time. Strong correlations were found between bile duct volume (r = 0.84), T2 value (r = 0.78), ΔT1 value (r = 0.62), and hepatic fibrosis rate (all P < 0.01) in the BDL groups. CONCLUSION The BDL mouse model induces changes that can be observed on MRI. The MRI parameters correlate with the hepatic fibrosis rate and allow for detection of cholestatic fibrosis.

BACKGROUND Over the past decade, resting-state functional magnetic resonance imaging (rs-fMRI) has concentrated on brain networks such as the default mode network (DMN), the salience network (SN), and the central executive network (CEN), allowing for a better understanding of cognitive deficits observed in mental disorders, as well as other characteristic psychopathological phenomena such as thought and behavior disorganization. AIM To investigate differential patterns of effective connectivity across distributed brain networks involved in schizophrenia (SCH) and mood disorders. METHODS The sample comprised 58 patients with either paranoid syndrome in the context of SCH (n = 26) or depressive syndrome (Ds) (n = 32), in the context of major depressive disorder or bipolar disorder. The methods used include rs-fMRI and subsequent dynamic causal modeling to determine the direction and strength of connections to and from various nodes in the DMN, SN and CEN. RESULTS A significant excitatory connection from the dorsal anterior cingulate cortex to the anterior insula (aI) was observed in the SCH patient group, whereas inhibitory connections from the precuneus to the ventrolateral prefrontal cortex and from the aI to the precuneus were observed in the Ds group. CONCLUSION The results delineate specific patterns associated with SCH and Ds and offer a better explanation of the underlying mechanisms of these disorders, and inform differential diagnosis and precise treatment targeting.

BACKGROUND Kallmann syndrome (KS) is a hypogonadotropic hypogonadism accompanied by anosmia or hyposmia. It is associated with the low secretion of gonadotropins which can lead to other abnormal endocrine metabolism disorders such as diabetes. Through genetic and molecular biological methods, more than 10 KS pathogenic genes have been found. AIM To identify the existing mutation sites of KS with diabetes and reveal the relationship between genotype and phenotype. METHODS We studied KS pathogenesis through high-throughput exome sequencing on four diabetes’ patients with KS for screening the potential pathogenic sites and exploring the genotype-phenotype correlation. Clinical data and peripheral blood samples were collected from the patients. White blood cells were separated and genomic DNA was extracted. High-throughput sequencing of all exons in the candidate pathogenic genes of probands was performed, and the results obtained were analyzed. RESULTS Sequencing revealed mutations in the KLB p.T313M, ANOS1 p.C172F, and IGSF10 gene (p.Lys1819Arg and p.Arg1035Thr) at different sites, which may have been associated with disease onset. CONCLUSION The diagnosis of KS is challenging, especially in early puberty, and the clinical manifestations reflect physical delays in development and puberty. Timely diagnosis and treatment can induce puberty, thereby improving sexual, bone, metabolic and mental health.

BACKGROUND BRAFV600E mutated colorectal cancer (CRC) is prone to peritoneal and distant lymph node metastasis and this correlates with a poor prognosis. The BRAFV600E mutation is closely related to the formation of an immunosuppressive microenvironment. However, the correlation between BRAFV600E mutation and changes in local immune microenvironment of CRC is not clear. AIM To explore the effect and mechanism of BRAFV600E mutant on the immune microenvironment of CRC. METHODS Thirty patients with CRC were included in this study: 20 in a control group and 10 in a treatment group. The density of microvessels and microlymphatic vessels, and M2 subtype macrophages in tumor tissues were detected by immunohistochemistry. Screening and functional analysis of exosomal long noncoding RNAs (lncRNAs) were performed by transcriptomics. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were detected by CCK-8 assay and scratch test, respectively. The tube-forming ability of endothelial cells was detected by tube formation assay. The macrophage subtypes were obtained by flow cytometry. The expression of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, VEGF-C, claudin-5, occludin, zonula occludens (ZO)-1, fibroblast activation protein, and α-smooth muscle actin was assessed by western blot analysis. The levels of cytokines interleukin (IL)-6, TGF-β1, and VEGF were assessed by enzyme-linked immunosorbent assay. RESULTS BRAFV600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen 19-9 (P < 0.05), and with poor tumor tissue differentiation in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAFV600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163+ M2 macrophages in BRAFV600E mutant CRC tumor tissue was markedly increased (P < 0.05). Compared with exosomes from CRC cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes from BRAFV600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs were closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment. The proliferation, migration, and tube formation ability of HUVECs and HLECs induced by exosomes in the 1627 cell group (HT29 cells with BRAF gene silencing) was greatly reduced compared with the HT29 cell group (P < 0.05). Compared with the HT29 cell group, the expression levels of VEGF-A, bFGF, TGF-β1, and VEGF-C in the exosomes derived from 1627 cells were reduced. The expression of ZO-1 in HUVECs, and claudin-5, occludin, and ZO-1 in HLECs of the 1627 cell group was higher. Compared with the 1627 cell group, the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages (P < 0.05). IL-6 secretion by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-β1 was decreased (P < 0.05). The levels of IL-6, TGF-β1, and VEGF secreted by fibroblasts in the 1627 cell group decreased, compared with the HT29 cell group (P < 0.05). CONCLUSION BRAFV600E mutant CRC cells can reach the tumor microenvironment by releasing exosomal lncRNAs, and induce the formation of an immunosuppressive microenvironment.

BACKGROUND Sepsis is a major medical challenge. Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects, but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear. AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms. METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and regulated on activation, normal T-cell expressed and secreted (RANTES) levels in serum and ileal tissue in animal studies. The histopathological changes of the ileal mucosa in different groups were observed under a microscope. Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide (LPS)-induced decrease in permeability. Immunofluorescence and Western blot assays were used to detect the levels of RANTES, inhibitor of nuclear factor kappa-B kinase β (IKKβ), phosphorylated IKKβ (p-IKKβ), inhibitor of nuclear factor kappa-B kinase α (IκBα), p65, and p-p65 proteins in different groups in vitro. RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol, magnolol inhibited the expression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α in a dose-dependent manner. In addition, magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats. Moreover, in vitro studies suggested that magnolol inhibited the increase of p65 nucleation, thereby markedly downregulating the production of the phosphorylated form of IKKβ in LPS-treated Caco2 cells. Specifically, magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B (NF-κB) from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells. CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways, thereby suppressing IL-1β, IL-6, and TNF-α expression to alleviate the mucosal barrier dysfunction in sepsis.

BACKGROUND Acute lung injury (ALI) is a common and life-threatening complication of severe acute pancreatitis (SAP). There are currently limited effective treatment options for SAP and associated ALI. Calycosin (Cal), a bioactive constituent extracted from the medicinal herb Radix Astragali exhibits potent anti-inflammatory properties, but its effect on SAP and associated ALI has yet to be determined. AIM To identify the roles of Cal in SAP-ALI and the underlying mechanism. METHODS SAP was induced via two intraperitoneal injections of L-arg (4 g/kg) and Cal (25 or 50 mg/kg) were injected 1 h prior to the first L-arg challenge. Mice were sacrificed 72 h after the induction of SAP and associated ALI was examined histologically and biochemically. An in vitro model of lipopolysaccharide (LPS)-induced ALI was established using A549 cells. Immunofluorescence analysis and western blot were evaluated in cells. Molecular docking analyses were conducted to examine the interaction of Cal with HMGB1. RESULTS Cal treatment substantially reduced the serum amylase levels and alleviated histopathological injury associated with SAP and ALI. Neutrophil infiltration and lung tissue levels of neutrophil mediator myeloperoxidase were reduced in line with protective effects of Cal against ALI in SAP. Cal treatment also attenuated the serum levels and mRNA expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, IL-1β, HMGB1 and chemokine (CXC motif) ligand 1 in lung tissue. Immunofluorescence and western blot analyses showed that Cal treatment markedly suppressed the expression of HMGB1 and phosphorylated nuclear factor-kappa B (NF-κB) p65 in lung tissues and an in vitro model of LPS-induced ALI in A549 cells suggesting a role for HGMB1 in the pathogenesis of ALI. Furthermore, molecular docking analysis provided evidence for the direct interaction of Cal with HGMB1. CONCLUSION Cal protects mice against L-arg-induced SAP and associated ALI by attenuating local and systemic neutrophil infiltration and inflammatory response via inhibition of HGMB1 and the NF-κB signaling pathway.

BACKGROUND Studies correlating specific genetic mutations and treatment response are ongoing to establish an effective treatment strategy for gastric cancer (GC). To facilitate this research, a cost- and time-effective method to analyze the mutational status is necessary. Deep learning (DL) has been successfully applied to analyze hematoxylin and eosin (H and E)-stained tissue slide images. AIM To test the feasibility of DL-based classifiers for the frequently occurring mutations from the H and E-stained GC tissue whole slide images (WSIs). METHODS From the GC dataset of The Cancer Genome Atlas (TCGA-STAD), wild-type/mutation classifiers for CDH1, ERBB2, KRAS, PIK3CA, and TP53 genes were trained on 360 × 360-pixel patches of tissue images. RESULTS The area under the curve (AUC) for the receiver operating characteristic (ROC) curves ranged from 0.727 to 0.862 for the TCGA frozen WSIs and 0.661 to 0.858 for the TCGA formalin-fixed paraffin-embedded (FFPE) WSIs. The performance of the classifier can be improved by adding new FFPE WSI training dataset from our institute. The classifiers trained for mutation prediction in colorectal cancer completely failed to predict the mutational status in GC, indicating that DL-based mutation classifiers are incompatible between different cancers. CONCLUSION This study concluded that DL could predict genetic mutations in H and E-stained tissue slides when they are trained with appropriate tissue data.

BACKGROUND Severe acute pancreatitis (SAP) is a deadly inflammatory disease with complex pathogenesis and lack of effective therapeutic options. N6-methyladenosine (m6A) modification of circRNAs plays important roles in physiological and pathological processes. However, the roles of m6A circRNA in the pathological process of SAP remains unknown. AIM To identify transcriptome-wide map of m6A circRNAs and to determine their biological significance and potential mechanisms in SAP. METHODS The SAP in C57BL/6 mice was induced using 4% sodium taurocholate salt. The transcriptome-wide map of m6A circRNAs was identified by m6A-modified RNA immunoprecipitation sequencing. The biological significance of circRNAs with differentially expressed m6A peaks was evaluated through gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The underlying mechanism of m6A circRNAs in SAP was analyzed by constructing of m6A circRNA-microRNA networks. The expression of demethylases was determined by quantitative polymerase chain reaction and western blot to deduce the possible mechanism of reversible m6A process in SAP. RESULTS Fifty-seven circRNAs with differentially expressed m6A peaks were identified by m6A-modified RNA immunoprecipitation sequencing, of which 32 were upregulated and 25 downregulated. Functional analysis of these m6A circRNAs in SAP found some important pathways involved in the pathogenesis of SAP, such as regulation of autophagy and protein digestion. In m6A circRNA–miRNA networks, several important miRNAs participated in the occurrence and progression of SAP were found to bind to these m6A circRNAs, such as miR-24-3p, miR-26a, miR-92b, miR-216b, miR-324-5p and miR-762. Notably, the total m6A level of circRNAs was reduced, while the demethylase alkylation repair homolog 5 was upregulated in SAP. CONCLUSION m6A modification of circRNAs may be involved in the pathogenesis of SAP. Our findings may provide novel insights to explore the possible pathogenetic mechanism of SAP and seek new potential therapeutic targets for SAP.

BACKGROUND Circulating tumor cells (CTCs) and survivin are indicators for tumor stage and metastasis, as well as epitheliomesenchymal transition, in various cancers, including hepatocellular cancer (HCC). AIM To explore the potential of survivin-positive CTCs, specifically, as a marker for tumor progression in HCC patients. METHODS We examined the survivin expression pattern in CTCs obtained from 179 HCC patients, and investigated the in vitro effects of survivin silencing and overexpression on the proliferation and invasion of HCC cells. CTC count and survivin expression in patient samples were examined using RNA in situ hybridization. RESULTS All 179 patients were positive for CTC markers, and 94.41% of the CTCs were positive for survivin. The CTC and survivin-positive CTC counts were significantly higher in the HCC patients than in the normal controls, and were significantly associated with tumor stage and degree of differentiation. Further, survivin overexpression was found to induce HepG2 cell proliferation, reduce apoptosis, and improve invasive ability. CONCLUSION Survivin shows upregulated expression (indicative of anti-apoptotic effects) in HCC. Thus, survivin-positive CTCs are promising as a predictor of HCC prognosis and metastasis, and their accurate measurement may be useful for the management of this cancer.

BACKGROUND Serum small extracellular vesicles (sEVs) and their small RNA (sRNA) cargoes could be promising biomarkers for the diagnosis of liver injury. However, the dynamic changes in serum sEVs and their sRNA components during liver injury have not been well characterized. Given that hepatic macrophages can quickly clear intravenously injected sEVs, the effect of liver injury-related serum sEVs on hepatic macrophages deserves to be explored. AIM To identify the characteristics of serum sEVs and the sRNAs during liver injury and explore their effects on hepatic macrophages. METHODS To identify serum sEV biomarkers for liver injury, we established a CCL4-induced mouse liver injury model in C57BL/6 mice to simulate acute liver injury (ALI), chronic liver injury (CLI) and recovery. Serum sEVs were obtained and characterized by transmission electron microscopy and nanoparticle tracking analysis. Serum sEV sRNAs were profiled by sRNA sequencing. Differentially expressed microRNAs (miRNAs) were compared to mouse liver-enriched miRNAs and previously reported circulating miRNAs related to human liver diseases. The biological significance was evaluated by Ingenuity Pathway Analysis of altered sEV miRNAs and conditioned cultures of ALI serum sEVs with primary hepatic macrophages. RESULTS We found that both ALI and CLI changed the concentration and morphology of serum sEVs. The proportion of serum sEV miRNAs increased upon liver injury, with the liver as the primary contributor. The altered serum sEV miRNAs based on mouse studies were consistent with human liver disease-related circulating miRNAs. We established serum sEV miRNA signatures for ALI and CLI and a panel of miRNAs (miR-122-5p, miR-192-5p, and miR-22-3p) as a common marker for liver injury. The differential serum sEV miRNAs in ALI contributed mainly to liver steatosis and inflammation, while those in CLI contributed primarily to hepatocellular carcinoma and hyperplasia. ALI serum sEVs decreased both CD86 and CD206 expression in monocyte-derived macrophages but increased CD206 expression in resident macrophages in vitro. CONCLUSION Serum sEVs acquired different concentrations, sizes, morphologies and sRNA contents upon liver injury and could change the phenotype of liver macrophages. Serum sEVs therefore have good diagnostic and therapeutic potential for liver injury.

BACKGROUND Schizophrenia afflicts 1% of the world population. Clinical studies suggest that schizophrenia patients may have an imbalance of mitochondrial energy metabolism via inhibition of mitochondrial complex I activity. Moreover, recent studies have shown that ERVWE1 is also a risk factor for schizophrenia. Nevertheless, there is no available literature concerning the relationship between complex I deficits and ERVWE1 in schizophrenia. Identifying risk factors and blood-based biomarkers for schizophrenia may provide new guidelines for early interventions and prevention programs. AIM To address novel potential risk factors and the underlying mechanisms of mitochondrial complex I deficiency caused by ERVWE1 in schizophrenia. METHODS Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay were used to detect differentially expressed risk factors in blood samples. Clinical statistical analyses were performed by median analyses and Mann-Whitney U analyses. Spearman’s rank correlation was applied to examine the correlation between different risk factors in blood samples. qPCR, western blot analysis, and luciferase assay were performed to confirm the relationship among ERVWE1, cytoplasmic polyadenylation element-binding protein 1 (CPEB1), NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), and NDUFV2 pseudogene (NDUFV2P1). The complex I enzyme activity microplate assay was carried out to evaluate the complex I activity induced by ERVWE1. RESULTS Herein, we reported decreasing levels of CPEB1 and NDUFV2 in schizophrenia patients. Further studies showed that ERVWE1 was negatively correlated with CPEB1 and NDUFV2 in schizophrenia. Moreover, NDUFV2P1 was increased and demonstrated a significant positive correlation with ERVWE1 and a negative correlation with NDUFV2 in schizophrenia. In vitro experiments disclosed that ERVWE1 suppressed NDUFV2 expression and promoter activity by increasing NDUFV2P1 level. The luciferase assay revealed that ERVWE1 could enhance the promoter activity of NDUFV2P1. Additionally, ERVWE1 downregulated the expression of CPEB1 by suppressing the promoter activity, and the 400 base pair sequence at the 3′ terminus of the promoter was the minimum sequence required. Advanced studies showed that CPEB1 participated in regulating the NDUFV2P1/NDUFV2 axis mediated by ERVWE1. Finally, we found that ERVWE1 inhibited complex I activity in SH-SY5Y cells via the CPEB1/NDUFV2P1/NDUFV2 signaling pathway. CONCLUSION In conclusion, CPEB1 and NDUFV2 might be novel potential blood-based biomarkers and pathogenic factors in schizophrenia. Our findings also reveal a novel mechanism of ERVWE1 in the etiology of schizophrenia.

BACKGROUND Pancreatic cancer (PC) is one of the most lethal malignancies worldwide. It is known that the proliferation of PC cells is a critical process in the disease. Previous studies have failed to identify the key genes associated with PC cell proliferation, using bioinformatic analysis, genome-wide association studies, and candidate gene testing. AIM To investigate the function of the chromobox 8 (CBX8)/receptor substrate 1 (IRS1)/AKT axis in PC. METHODS A genome-wide CRISPR-Cas9 screening was performed to select genes that could facilitate PC cell proliferation. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression of CBX8 in PC tissues and cells. The regulatory roles of CBX8 in cell proliferation, migration, and invasion were verified by in vivo and in vitro functional assays. RESULTS CBX8 was upregulated in PC tissues and shown to drive PC cell proliferation. Higher expression of CBX8 was correlated with worse outcomes of PC patients from two independent cohorts comprising a total of 116 cases. CBX8 was also proved to serve as a promising therapeutic target for a PC xenograft model. We demonstrated that hypoxia-inducible factor (HIF)-1a induced CBX8 transcription by binding to the promoter of CBX8. CBX8 efficiently activated the PI3K/AKT signaling by upregulating insulin IRS1. CONCLUSION CBX8 is a key gene regulated by HIF-1α, and activates the IRS1/AKT pathway, which suggests that targeting CBX8 may be a promising therapeutic strategy for PC.

BACKGROUND Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients’ sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.

BACKGROUND Hepatic stellate cells (HSCs) are the key effector cells mediating the occurrence and development of liver fibrosis, while aerobic glycolysis is an important metabolic characteristic of HSC activation. Transforming growth factor-β1 (TGF-β1) induces aerobic glycolysis and is a driving factor for metabolic reprogramming. The occurrence of glycolysis depends on a high glucose uptake level. Glucose transporter 1 (GLUT1) is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism, thus affecting cell proliferation and growth. However, little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs. AIM To investigate the mechanisms of action of GLUT1, TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis. METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue. A Seahorse extracellular flux (XF) analyzer was used to examine changes in aerobic glycolytic flux, lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation. The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothers-against-decapentaplegic-homolog 2/3 (Smad2/3) signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. In addition, GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs. Finally, a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis. RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues. In addition, immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins, indicating that GLUT1 expression was related to the development of liver fibrosis. TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad, p38 MAPK and P13K/AKT signaling pathways. The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression. GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration. A GLUT1 inhibitor was administered in a mouse model of liver fibrosis, and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis. CONCLUSION TGF-β1 induces GLUT1 expression in HSCs, a process related to liver fibrosis progression. In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs. In addition, in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

BACKGROUND Senescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known. AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells. METHODS We compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured. RESULTS SA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells. CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.

Background Diabetes mellitus (DM) is a progressively increasing metabolic disorder and a significant public health burden that demands immediate global attention. However, there is a paucity of data about adherence to antidiabetic drugs among patients with type-2 (T2)DM in Uttarakhand, India. Outpatient research reported that more than 50% of patients do not adhere to the correct administration and appropriate medicine dosage. It has been reported that patients with chronic diseases who adhere to treatment may experience improvement in quality of life (QoL) and vice versa. Aim To assess the adherence to antidiabetic medication and QoL among patients with T2DM. Methods This cross-sectional descriptive study was conducted at a tertiary care hospital in Uttarakhand, India. The Medication Adherence Rating Scale and World Health Organization QoL-BREF scale were used to assess medication adherence and QoL. Results Two hundred seventy-seven patients suffering from T2DM participated in the study. Their mean age was 50.80 (± 10.6) years, 155 (56%) had a poor adherence level and 122 (44%) had a good adherence level to antidiabetic medications. After adjusting for sociodemographic factors, multiple linear regression analysis found patients who were adherent to antidiabetic medications had significantly higher mean overall perception of QoL and overall perception of health, with beta scores of 0.36 and 0.34, respectively (both P = 0.000) points compared with nonadherent patients. Conclusion There was an association between medication adherence and QoL in patients with T2DM. Hence, there is a need to plan awareness and counseling programs followed by regular follow-up to motivate patient adherence to recommended treatment and lifestyle regimens.

Background Pancreatic cancer is a highly malignant tumor of the gastrointestinal system whose emerging resistance to chemotherapy has necessitated the development of novel antitumor treatments. Scoparone, a traditional Chinese medicine monomer with a wide range of pharmacological properties, has attracted considerable attention for its antitumor activity. Aim To explore the potential antitumor effect of scoparone on pancreatic cancer and the possible molecular mechanism of action. Methods The target genes of scoparone were determined using both the bioinformatics and multiplatform analyses. The effect of scoparone on pancreatic cancer cell proliferation, migration, invasion, cell cycle, and apoptosis was detected in vitro. The expression of hub genes was tested using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the molecular mechanism was analyzed using Western blot. The in vivo effect of scoparone on pancreatic cancer cell proliferation was detected using a xenograft tumor model in nude mice as well as immunohistochemistry. Results The hub genes involved in the suppression of pancreatic cancer by scoparone were obtained by network bioinformatics analyses using publicly available databases and platforms, including SwissTargetPrediction, STITCH, GeneCards, CTD, STRING, WebGestalt, Cytoscape, and Gepia; AKT1 was confirmed using qRT-PCR to be the hub gene. Cell Counting Kit-8 assay revealed that the viability of Capan-2 and SW1990 cells was significantly reduced by scoparone treatment exhibiting IC50 values of 225.2 μmol/L and 209.1 μmol/L, respectively. Wound healing and transwell assays showed that scoparone inhibited the migration and invasion of pancreatic cancer cells. Additionally, flow cytometry confirmed that scoparone caused cell cycle arrest and induced apoptosis. Scoparone also increased the expression levels of Bax and cleaved caspase-3, decreased the levels of MMP9 and Bcl-2, and suppressed the phosphorylation of Akt without affecting total PI3K and Akt. Moreover, compared with the control group, xenograft tumors, in the 200 μmol/L scoparone treatment group, were smaller in volume and lighter in weight, and the percentages of Ki65- and PCNA-positive cells were decreased. Conclusion Our findings indicate that scoparone inhibits pancreatic cancer cell proliferation in vitro and in vivo, inhibits migration and invasion, and induces cycle arrest and apoptosis in vitro through the PI3K/Akt signaling pathway.

Study Design: Animal case control study.Purpose: To create a simple, reproducible disc degeneration model for mouse coccygeal vertebrae. Overview of Literature: Back pain due to disc degeneration is probably the most common problem encountered in neurosurgical practice. An easily reproducible animal model for disc degeneration will help in understanding its pathophysiology, and serve as a platform for examining various therapeutic options.Methods: A total of 18 mice were divided into injured (n=12) and non-injured (n=6) groups. The disc height index (DHI%) at coccygeal 4–5 level was measured by computed tomography (CT) scan for all mice. Coccygeal 4–5 discs of the injury group were injured using a 32G needle fixed to a novel tool and confirmed by CT. The non-injury group underwent no procedure. DHI% was measured by CT at 2-, 4-, and 6-week post-injury, and all mice tails were sectioned for histopathology grading of disc degeneration at the respective time intervals.Results: The injured group showed significant variation in DHI% at 2, 4, and 6 weeks, whereas there was no change in the noninjured group. Histopathologic evaluation with Safranin O stain showed a worsening of the disc degeneration score at 2, 4, and 6 weeks in the injured group, but in the non-injured group there was no change. Percutaneous needle injury technique with our novel tool provided 100% accuracy and uniform degeneration.Conclusions: A simple, easily reproducible mouse model for disc degeneration was created using a simple, cost-effective, novel tool and technique, its advantage being high precision and user friendly.

BACKGROUND Intestinal barrier breakdown, a frequent complication of intestinal ischemia-reperfusion (I/R) including dysfunction and the structure changes of the intestine, is characterized by a loss of tight junction and enhanced permeability of the intestinal barrier and increased mortality. To develop effective and novel therapeutics is important for the improvement of outcome of patients with intestinal barrier deterioration. Recombinant human angiopoietin-like protein 4 (rhANGPTL4) is reported to protect the blood-brain barrier when administered exogenously, and endogenous ANGPTL4 deficiency deteriorates radiation-induced intestinal injury. AIM To identify whether rhANGPTL4 may protect intestinal barrier breakdown induced by I/R. METHODS Intestinal I/R injury was elicited through clamping the superior mesenteric artery for 60 min followed by 240 min reperfusion. Intestinal epithelial (Caco-2) cells and human umbilical vein endothelial cells were challenged by hypoxia/ reoxygenation to mimic I/R in vitro. RESULTS Indicators including fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) clearance, ratio of phosphorylated myosin light chain/total myosin light chain, myosin light chain kinase and loss of zonula occludens-1, claudin-2 and VE-cadherin were significantly increased after intestinal I/R or cell hypoxia/reoxygenation. rhANGPTL4 treatment significantly reversed these indicators, which were associated with inhibiting the inflammatory and oxidative cascade, excessive activation of cellular autophagy and apoptosis and improvement of survival rate. Similar results were observed in vitro when cells were challenged by hypoxia/reoxygenation, whereas rhANGPTL4 reversed the indicators close to normal level in Caco-2 cells and human umbilical vein endothelial cells significantly. CONCLUSION rhANGPTL4 can function as a protective agent against intestinal injury induced by intestinal I/R and improve survival via maintenance of intestinal barrier structure and functions.

BACKGROUND MMP-2 also known as gelatinase A and MMP-7 (matrilysin) are members of the zinc-dependent family of MMPs (Matrix metalloproteinase). MMP-2 and MMP-7 are remodeling enzymes that digest extracellular matrix; MMP-2 is extensively expressed during development and is upregulated at sites of tissue damage, inflammation, and in stromal cells of metastatic tumors. MMP-7 is expressed in the epithelial cells and in a variety of cancers including colon tumors. Plasma MMP-2 and MMP-7 levels were assessed before and after minimally invasive colorectal resection for cancer pathology. AIM To determine plasma MMP-2 and MMP-7 levels before and after minimally invasive colorectal resection for cancer pathology. METHODS Patients enrolled in a plasma bank for whom plasma was available were eligible. Plasma obtained from preoperative (Preop) and postoperative blood samples was used. Only colorectal cancer (CRC) patients who underwent elective minimally invasive cancer resection with preop, post-operative day (POD) 1, 3 and at least 1 late postop sample (POD 7-34) were included. Late samples were bundled into 7 d blocks (POD 7-13, 14-20, etc.) and treated as single time points. Plasma MMP-2 and MMP-7 levels were determined via enzyme-linked immunosorbent assay in duplicate. RESULTS Total 88 minimally invasive CRC resection CRC patients were studied (right colectomy, 37%; sigmoid, 24%; and LAR/AR 18%). Cancer stages were: 1, 31%; 2, 30%; 3, 34%; and 4, 5%. Mean Preop MMP-2 plasma level (ng/mL) was 179.3 ± 40.9 (n = 88). Elevated mean levels were noted on POD1 (214.3 ± 51.2, n = 87, P < 0.001), POD3 (258.0 ± 63.9, n = 80, P < 0.001), POD7-13 (229.9 ± 62.3, n = 65, P < 0.001), POD 14-20 (234.9 ± 47.5, n = 25, P < 0.001), POD 21-27 (237.0 ± 63.5, n = 17, P < 0.001,) and POD 28-34 (255.4 ± 59.7, n = 15, P < 0.001). Mean Preop MMP-7 level was 3.9 ± 1.9 (n = 88). No significant differences were noted on POD 1 or 3, however, significantly elevated levels were noted on POD 7-13 (5.7 ± 2.5, n = 65, P < 0.001), POD 14-20 (5.9 ± 2.5, n = 25, P < 0.001), POD 21-27 (6.1 ± 3.6, n = 17, P = 0.002) and on POD 28-34 (6.8 ± 3.3, n = 15 P < 0.001,) vs preop levels. CONCLUSION MMP-2 levels are elevated for 5 wk and MMP-7 levels elevated for weeks 2-6. The etiology of these changes in unclear, trauma and wound healing likely play a role. These changes may promote residual tumor growth and metastasis.

BACKGROUND Chronic stress during pregnancy may increase visceral hyperalgesia of offspring in a sex-dependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen in exacerbating visceral hypersensitivity in female rodents in preclinical models, we predicted that chronic prenatal stress (CPS) + chronic adult stress (CAS) will maximize visceral hyperalgesia; and that estrogen plays an important role in colonic hyperalgesia. AIM The aim was to illuminate the role of estrogen in colonic hyperalgesia and its underlying mechanisms. METHODS We established a CPS plus CAS rodent model in which the balloon was used to distend the colorectum. The single-fiber recording in vivo and patch clamp experiments in vitro were used to monitor the colonic neuron’s activity. The reverse transcription-polymerase chain reaction, western blot, and immunofluorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity. We used ovariectomy and letrozole to reduce estrogen levels of female rats respectively in order to assess the role of estrogen in female-specific enhanced primary afferent sensitization. RESULTS Spontaneous activity and single fiber activity were significantly greater in females than in males. The enhanced sensitization in female rats mainly came from low-threshold neurons. CPS significantly increased single-unit afferent fiber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. In addition, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increased in CPS + CAS rats and was associated with a decrease in transient A-type K+ currents. Compared with ovariectomy, treatment with the aromatase inhibitor letrozole significantly reduced estrogen levels in female rats, confirming the gender difference. Moreover, mice treated with letrozole had decreased colonic DRG neuron excitability. The intrathecal infusion of estrogen increased brain-derived neurotrophic factor (BDNF) protein levels and contributed to the response to visceral pain. Western blotting showed that nerve growth factor protein was upregulated in CPS + CAS mice. CONCLUSION This study adds to the evidence that estrogen-dependent sensitization of primary afferent colon neurons is involved in the development of chronic stress-induced visceral hypersensitivity in female rats.

BACKGROUND Approximately 90% of new cases of noncardiac gastric cancer (GC) are related to Helicobacter pylori (H. pylori), and cytotoxin-associated gene A (CagA) is one of the main pathogenic factors. Recent studies have shown that the pharmacological effects of cryptotanshinone (CTS) can be used to treat a variety of tumors. However, the effects of CTS on H. pylori, especially CagA+ strain-induced gastric mucosal lesions, on the development of GC is unknown. AIM To assess the role of CTS in CagA-induced proliferation and metastasis of GC cells, and determine if CagA+ H. pylori strains causes pathological changes in the gastric mucosa of mice. METHODS The effects of CTS on the proliferation of GC cells were assessed using the Cell Counting Kit-8 (CCK-8) assay, and the abnormal growth, migration and invasion caused by CagA were detected by CCK-8 and transwell assays. After transfection with pSR-HA-CagA and treatment with CTS, proliferation and metastasis were evaluated by CCK-8 and transwell assays, respectively, and the expression of Src homology 2 (SH2) domain–containing phosphatase 2 (SHP2) and phosphorylated SHP2 (p-SHP2) was detected using western blotting in AGS cells. The enzyme-linked immunosorbent assay was used to determine the immunoglobulin G (IgG) level against CagA in patient serum. Mice were divided into four groups and administered H. pylori strains (CagA+ or CagA-) and CTS (or PBS) intragastrically, and establishment of the chronic infection model was verified using polymerase chain reaction and sequencing of isolated strains. Hematoxylin and eosin staining was used to assess mucosal erosion in the stomach and toxicity to the liver and kidney. RESULTS CTS inhibited the growth of GC cells in dose- and time-dependent manners. Overexpression of CagA promoted the growth, migration, and invasion of GC cells. Importantly, we demonstrated that CTS significantly inhibited the CagA-induced abnormal proliferation, migration, and invasion of GC cells. Moreover, the expression of p-SHP2 protein in tumor tissue was related to the expression of IgG against CagA in the serum of GC patients. Additionally, CTS suppressed the protein expression levels of both SHP2 and p-SHP2 in GC cells. CTS suppressed CagA+ H. pylori strain-induced mucosal erosion in the stomach of mice but had no obvious effects on the CagA- H. pylori strain group. CONCLUSION CTS inhibited CagA-induced proliferation and the epithelial-mesenchymal transition of GC cells in vitro, and CagA+ H. pylori strains caused mucosal erosions of the stomach in vivo by decreasing the protein expression of SHP2.

Background As one of the major microvascular complications of diabetes, diabetic retinopathy (DR) is the leading cause of blindness in the working age population. Because the extremely complex pathogenesis of DR has not been fully clarified, the occurrence and development of DR is closely related to tissue ischemia and hypoxia and neovascularization The formation of retinal neovascularization (RNV) has great harm to the visual acuity of patients. Aim To investigate the expression of P-element-induced wimpy testis-interacting RNA (piRNA) in proliferative DR mice and select piRNA related to RNV. Methods One hundred healthy C57BL/6J mice were randomly divided into a normal group as control group (CG) and proliferative DR (PDR) group as experimental group (EG), with 50 mice in each group. Samples were collected from both groups at the same time, and the lesions of mice were evaluated by hematoxylin and eosin staining and retinal blood vessel staining. The retinal tissues were collected for second-generation high-throughput sequencing, and the differentially expressed piRNA between the CG and EG was detected, and polymerase chain reaction (PCR) was conducted for verification. The differentially obtained piRNA target genes and expression profiles were enrichment analysis based on gene annotation (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes. Results In the CG there was no perfusion area, neovascularization and endothelial nucleus broke through the inner boundary membrane of retinap. In the EG, there were a lot of nonperfused areas, new blood vessels and endothelial nuclei breaking through the inner boundary membrane of the retina. There was a statistically significant difference in the number of vascular endothelial nuclei breaking through the inner retinal membrane between the two groups. High-throughput sequencing analysis showed that compared with the CG, a total of 79 piRNAs were differentially expressed in EG, among which 43 piRNAs were up-regulated and 36 piRNAs were down-regulated. Bioinformatics analysis showed that the differentially expressed piRNAs were mainly concentrated in the signaling pathways of angiogenesis and cell proliferation. Ten piRNAs were selected for PCR, and the results showed that the expression of piR-MMU-40373735, piR-MMU-61121420, piR-MMU-55687822, piR-MMU-1373887 were high, and the expression of piR-MMU-7401535, piR-MMU-4773779, piR-MMU-1304999, and piR-MMU-5160126 were low, which were consistent with the sequencing results. Conclusion In the EG, the abnormal expression of piRNA is involved in the pathway of angiogenesis and cell proliferation, suggesting that piRNAs have some regulatory function in proliferative diabetic-retinopathy.

BACKGROUND Norcantharidin (NCTD) is suitable for the treatment of primary liver cancer, especially early and middle primary liver cancer. This compound can reduce tumors and improve immune function. However, the side effects of NCTD have limited its application. There is a marked need to reduce the side effects and increase the efficacy of NCTD. AIM To develop a nanomaterial carrier, NCTD-loaded metal-organic framework IRMOF-3 coated with a temperature-sensitive gel (NCTD-IRMOF-3-Gel), aiming to improve the anticancer activity of NCTD and reduce the drug dose. METHODS NCTD-IRMOF-3-Gel was obtained by a coordination reaction. The apparent characteristics and in vitro release of NCTD-IRMOF-3-Gel were investigated. Cell cytotoxicity assays, flow cytometry, and apoptosis experiments in mouse hepatoma (Hepa1-6) cells were used to determine the anti-liver cancer activity of NCTD-IRMOF-3-Gel in in vitro models. RESULTS The particle size of NCTD-IRMOF-3-Gel was 50-100 nm, and the particle size distribution was uniform. The release curve showed that NCTD-IRMOF-3-Gel had an obvious sustained-release effect. The cytotoxicity assays showed that the free drug NCTD and NCTD-IRMOF-3-Gel treatments markedly inhibited Hepa1-6 cell proliferation, and the inhibition rate increased with increasing drug concentration. By flow cytometry, NCTD-IRMOF-3-Gel was observed to block the Hepa1-6 cell cycle in the S and G2/M phases, and the thermosensitive gel nanoparticles may inhibit cell proliferation by inducing cell cycle arrest. Apoptosis experiments showed that NCTD-IRMOF-3-Gel induced the apoptosis of Hepa1-6 cells. CONCLUSION Our results indicated that the NCTD-IRMOF-3-Gel may be beneficial for liver cancer disease treatment.

Background Ubiquitin-specific protease 15 (USP15) is an important member of the ubiquitin-specific protease family, the largest deubiquitinase subfamily, whose expression is dysregulated in many types of cancer. However, the biological function and the underlying mechanisms of USP15 in gastric cancer (GC) progression have not been elucidated. Aim To explore the biological role and underlying mechanisms of USP15 in GC progression. Methods Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC. Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC. A loss- and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis. RNA sequencing, immunofluorescence, and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions. Results USP15 was up-regulated in GC tissue and cell lines. The expression level of USP15 was positively correlated with clinical characteristics (tumor size, depth of invasion, lymph node involvement, tumor-node-metastasis stage, perineural invasion, and vascular invasion), and was related to poor prognosis. USP15 knockdown significantly inhibited cell proliferation, invasion and epithelial-mesenchymal transition (EMT) of GC in vitro, while overexpression of USP15 promoted these processes. Knockdown of USP15 inhibited tumor growth in vivo. Mechanistically, RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC. Western blotting confirmed that USP15 silencing led to significant down-regulation of β-catenin and Wnt/β-catenin downstream genes (c-myc and cyclin D1), while overexpression of USP15 yielded an opposite result and USP15 mutation had no change. Immunofluorescence indicated that USP15 promoted nuclear translocation of β-catenin, suggesting activation of the Wnt/β-catenin signaling pathway, which may be the critical mechanism promoting GC progression. Finally, rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway. Conclusion USP15 promotes cell proliferation, invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway, which suggests that USP15 is a novel potential therapeutic target for GC.

BACKGROUND The poor prognosis and rising incidence of esophageal cancer highlight the need for improved therapeutics that are essential prior to treatment. LCL161 is an SMAC (second mitochondrial activator of caspases) mimic and inhibitor of apoptosis protein (IAP) antagonist which exhibits anti-tumor effects and improves the chemical sensitivity of many cancers. AIM To ascertain the effects and mechanisms of the SMAC analog LCL161 on esophageal cancer cells. METHODS MTT assay and TUNEL assay were used to detect cell proliferation and apoptosis, respectively. Western blot analysis was used to study the molecular mechanisms of LCL161-induced death of ECA109 cells. RESULTS LCL161 decreased ECA109 cell proliferation in dose- and time-dependent manner and induced apoptosis of ECA109 cells in a dose-dependent manner. Also, LCL161 induced a significant decrease in the expression of the XIAP and significant increase in the expression of Caspase-3. In addition, Bax increased significantly with increasing concentrations of LCL161, and the relative expression of Bax was significantly different between groups. CONCLUSION These findings support the hypothesis that LCL161 can inhibit proliferation and induce apoptosis in esophageal cancer cells by regulating the expression of IAP family members, suggesting that it has potential to be an effective treatment for esophageal squamous cell carcinoma.

Study Design: The study examined and compared four artificial cervical disks using validated finite element models.Purpose: To compare and contrast the biomechanical behavior of four artificial cervical disks by determining the external (range of motion) and internal (facet force and intradiscal pressure) responses following cervical disc arthroplasty (CDA) and to elucidate any device design effects on cervical biomechanics.Overview of Literature: Despite CDA’s increasing popularity most studies compare the CDA procedure with anterior cervical discectomy and fusion. There is little comparative evaluation of different artificial disks and, therefore, little understanding of how varying disk designs may influence spinal biomechanics.Methods: A validated C2–T1 finite element model was subjected to flexion-extension. CDAs were simulated at the C5–C6 level with the Secure-C, Mobi-C, Prestige LP, and Prodisc C prosthetic disks. We used a hybrid loading protocol to apply sagittal moments. Normalized motions at the index and adjacent levels, and intradiscal pressures and facet column loads were also obtained.Results: The ranges of motion at the index level increased after CDA. The Mobi-C prosthesis demonstrated the highest amount of flexion, followed by the Secure-C, Prestige LP, and Prodisc C. The Secure-C demonstrated the highest amount of extension, followed by the Mobi-C, Prodisc C, and Prestige LP. The motion decreased at the rostral and caudal adjacent levels. Facet forces increased at the index level and decreased at the rostral and caudal adjacent levels following CDA. Intradiscal pressures decreased at the adjacent levels for the Mobi-C, Secure-C, and Prodisc C. Conversely, the use of the Prestige LP increased intradiscal pressure at both adjacent levels.Conclusions: While all artificial disks were useful in restoring the index level motion, the Secure-C and Mobi-C translating abilities allowed for lower intradiscal pressures at the adjacent segments and may be the driving mechanism for minimizing adjacent segment degenerative arthritic changes. The facet joint integrity should also be considered in the clinical decision-making process for CDA selection.

Background The drug resistance rate of clinical Helicobacter pylori (H. pylori) isolates has increased. However, the mechanism of drug resistance remains unclear. In this study, drug-resistant H. pylori strains were isolated from different areas and different populations of Chinese for genomic analysis. Aim To investigate drug-resistant genes in H. pylori and find the genes for the early diagnosis of clarithromycin resistance. Methods Three drug-resistant H. pylori strains were isolated from patients with gastritis in Bama County, China. Minimal inhibitory concentrations of clarithromycin, metronidazole, and levofloxacin were determined and complete genome sequencing was performed with annotation. Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction. The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains. The homology of the strains with hp26695 was assessed through complete genome detection and identification. Differences in genome sequences, gene quantity, and gene characteristics were detected amongst the three strains. Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains, which was the same in the artificially induced clarithromycin-resistant bacteria. After gene knockout, the drug sensitivity of the strains was assessed. Results The strains showing a high degree of homology with hp26695, hp1181, and hp1184 genes were found in these strains; the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance. Conclusion Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance, and they may be the potential target genes for the diagnosis, prevention, and treatment of clarithromycin resistance.

Background Fasudil, as a Ras homology family member A (RhoA) kinase inhibitor, is used to improve brain microcirculation and promote nerve regeneration clinically. Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis. Aim To evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide (TAA). Methods C57BL/6 mice were administered TAA once every 3 d for 12 times. At 1 wk after induction with TAA, Fasudil was intraperitoneally injected once a day for 3 wk, followed by hematoxylin and eosin staining, sirius red staining, western blotting, and quantitative polymerase chain reaction (qPCR), and immune cell activation was assayed by fluorescence-activated cell sorting. Furthermore, the effects of Fasudil on hepatic stellate cells and natural killer (NK) cells were assayed in vitro. Results First, we found that TAA-induced liver injury was protected, and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment. Furthermore, western blot and qPCR assays showed that the levels of alpha smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor beta 1 (TGF-β1) were inhibited by Fasudil. Moreover, flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro. Furthermore, Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasing α-SMA and TGF-β1. Conclusion Fasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation, thereby providing a feasible solution for the clinical treatment of liver fibrosis.

BACKGROUND Heat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed. AIM To evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration. METHODS We used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism. RESULTS This study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSION We found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.

AIM To detect the mechanisms of Helicobacter pylori (H. pylori) infection in the invasion and metastasis of gastric cancer (GC). METHODS Specimens from 99 patients with GC were collected. The correlation among H. pylori infection, heparanase (HPA) and mitogen-activated protein kinase (MAPK) expression, which was determined by immunohistochemistry, and the clinical features of GC was analysed using SPSS 22.0. Overall survival (OS) and relapse-free survival (RFS) of GC patients were estimated by the Kaplan-Meier method. Independent and multiple factors of HPA and MAPK with prognosis were determined with COX proportional hazards models. HPA and MAPK expression in MKN-45 cells infected with H. pylori was analysed using Western blot. RESULTS H. pylori infection was observed in 70 of 99 patients with GC (70.7%), which was significantly higher than that in healthy controls. H. pylori infection was related to lymph metastasis and expression of HPA and MAPK (P < 0.05); HPA expression was relevant to MAPK expression (P = 0.024). HPA and MAPK expression in MKN-45 cells was significantly upregulated following H. pylori infection and peaked at 24 h and 60 min, before decreasing (P < 0.05). SB203580, an inhibitor of MAPK, significantly decreased HPA expression. HPA was related to lymph metastasis and invasive depth. HPA positive GC cases and H. pylori positive GC cases showed poorer prognosis than HPA negative cases (P < 0.05). COX models showed that the prognosis of GC was connected with HPA expression, lymph metastasis, tissue differentiation, and invasive depth. CONCLUSION H. pylori may promote the invasion and metastasis of GC by increasing HPA expression that may associate with MAPK activation, thus causing a poorer prognosis of GC.

Background The coronavirus disease 2019 (COVID-19), a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide, was described to be frequently accompanied by extrapulmonary manifestations, including liver dysfunction. Liver dysfunction and elevated liver enzymes were observed in about 53% of COVID-19 patients. Aim To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction. Methods The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed. Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways. The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis, fibrosis, non-alcoholic fatty liver disease (NAFLD), and hepatitis A/B/C. Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR. Results Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes, DNAJB1, IGF2, EGFR, and HDGF. Concordantly, the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling (liver cirrhosis, Fibrosis, NAFLD, and hepatitis A/B/C). Moreover, we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function, including cytochrome P450 family members, ACAD11, CIDEB, GNMT, and GPAM. Consequently, drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity. In correspondence with the RNA-seq data analysis, we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction. Conclusion Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling, mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.

Background Hepatocellular carcinoma (HCC) is a malignancy found globally. Accumulating studies have shown that long noncoding RNAs (lncRNAs) play critical roles in HCC. However, the function of lncRNA in HCC remains poorly understood. Aim To understand the effect of lncRNA W42 on HCC and dissect the underlying molecular mechanisms. Methods We measured the expression of lncRNA W42 in HCC tissues and cells (Huh7 and SMMC-7721) by quantitative reverse transcriptase polymerase chain reaction. Receiver operating characteristic curves were used to assess the sensitivity and specificity of lncRNA W42 expression. HCC cells were transfected with pcDNA3.1-lncRNA W42 or shRNA-lncRNA W42. Cell functions were detected by cell counting Kit-8 (CCK-8), colony formation, flow cytometry and Transwell assays. The interaction of lncRNA W42 and DBN1 was confirmed by RNA immunoprecipitation and RNA pull down assays. An HCC xenograft model was used to assess the role of lncRNA W42 on tumor growth in vivo. The Kaplan-Meier curve was used to evaluate the overall survival and recurrence-free survival after surgery in patients with HCC. Results In this study, we identified a novel lncRNA (lncRNA W42), and investigated its biological functions and clinical significance in HCC. LncRNA W42 expression was upregulated in HCC tissues and cells. Overexpression of lncRNA W42 notably promoted the proliferative and invasion of HCC, and inhibited cell apoptosis. LncRNA W42 directly bound to DBN1 and activated the downstream pathway. LncRNA W42 knockdown suppressed HCC xenograft tumor growth in vivo. The clinical investigation revealed that HCC patients with high lncRNA W42 expression exhibited shorter survival times. Conclusion In vitro and in vivo results suggested that the novel lncRNA W42, which is upregulated in HCC, may serve as a potential candidate prognostic biomarker and therapeutic target in HCC patients.

BACKGROUND In recent years, we created and employed a new anastomosis method, “bridging” pancreaticogastrostomy, to treat patients with extremely severe pancreatic injury. This surgery has advantages such as short length of surgery, low secondary trauma, rapid construction of shunts for pancreatic fluid, preventing second surgeries, and achieving good treatment outcomes in clinical practice. However, due to the limited number of clinical cases, there is a lack of strong evidence to support the feasibility and safety of this surgical procedure. Therefore, we carried out animal experiments to examine this procedure, which is reported here. AIM To examine the feasibility and safety of a new rapid method of pancreaticogastrostomy, “bridging” pancreaticogastrostomy. METHODS Ten Landrace pigs were randomized into the experimental and control groups, with five pigs in each group. “Bridging” pancreaticogastrostomy was performed in the experimental group, while routine mucosa-to-mucosa pancreaticogastrostomy was performed in the control group. After surgery, the general condition, amylase levels in drainage fluid on Days 1, 3, 5, and 7, fasting and 2-h postprandial blood glucose 6 mo after surgery, fasting, 2-h postprandial peripheral blood insulin, and portal vein blood insulin 6 mo after surgery were assessed. Resurgery was carried out at 1 and 6 mo after the former one to examine the condition of the abdominal cavity and firmness and tightness of the pancreaticogastric anastomosis and pancreas. RESULTS After surgery, the general condition of the animals was good. One in the control group did not gain weight 6 mo after surgery, whereas significant weight gain was present in the others. There were significant differences on Days 1 and 3 after surgery between the two groups but no differences on Days 5 and 7. There were no differences in fasting and 2-h postprandial blood glucose and fasting and 2-h insulin values of postprandial peripheral blood and portal vein blood 6 mo after surgery between the two groups. One month after surgery, the sinus tract orifice/anastomosis was patent in the two groups. Six months after surgery, the sinus tract orifice/anastomosis was sealed, and pancreases in both groups presented with chronic pancreatitis. CONCLUSION “Bridging” pancreaticogastrostomy is a feasible and safe a means of damage control surgery during the early stage of pancreatic injury.

BACKGROUND The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI. AIM To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs). METHODS Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle. RESULTS Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones. CONCLUSION Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.

Background Breast cancer is the most common cause of the majority of cancer-related deaths in women, among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has been extensively studied as a potent anticancer molecule against various types of cancers. Honeybee products such as the royal jelly (RJ), the nutritive secretion fed to honeybee queens, exhibit a variety of biological activities besides its anticancer effect. However, the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown. Aim To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ, RJ, and their combinations in the MDA-MB-231 cell line. Methods Cells were treated with TQ, RJ, and their combinations for 24 h. Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we determined the half-maximal inhibitory concentration of TQ. Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions. Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ, RJ, and their combination. Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation. Results TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration. RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5 µg/mL. High doses of RJ (200 µg/mL) had greater toxicity against MDA-MB-231 cells. Interestingly, the inhibition of cell viability was most pronounced in response to 15 µmol/L TQ and 5 µg/mL RJ. A dose of 15 µmol/L TQ caused a significant increase in the PreG1 population, while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations. TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ. In contrast, no significant regulation of Ki67 expression was observed, indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation. Conclusion This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells, which could confer an advantage for cancer therapy.

BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis (SAP)-related acute lung injury (ALI). Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI. However, the precise mechanism associated with p38 is unclear, particularly in pulmonary microvascular endothelial cell (PMVEC) injury. AIM To determine its role in the tumor necrosis factor-alpha (TNF-α)-induced inflammation and apoptosis of PMVECs in vitro. We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI. METHODS In vitro, PMVEC were transfected with mitogen-activated protein kinase kinase 6 (Glu), which constitutively activates p38, and then stimulated with TNF-α. Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels, respectively. In vivo, SAP-ALI was induced by 5% sodium taurocholate and three different doses of SB203580 (2.5, 5.0 or 10.0 mg/kg) were intraperitoneally injected prior to SAP induction. SAP-ALI was assessed by performing pulmonary histopathology assays, measuring myeloperoxidase activity, conducting arterial blood gas analyses and measuring TNF-α, interleukin (IL)-1β and IL-6 levels. Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration, Evans blue extravasation and ultrastructural changes in PMVECs. The apoptotic death of pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2, Bax, Bim and cle-caspase3 levels. The proteins levels of P-p38, NFκB, IκB, P-signal transducer and activator of transcription-3, nuclear factor erythroid 2-related factor 2, HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580. RESULTS In vitro, mitogen-activated protein kinase (Glu) transfection resulted in higher apoptotic rates and cytokine (IL-1β and IL-6) levels in TNF-α-treated PMVECs. In vivo, SB2035080 attenuated lung histopathological injury, decreased inflammatory activity (TNF-α, IL-1β, IL-6 and myeloperoxidase) and preserved pulmonary function. Furthermore, SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration, Evans blue accumulation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers, apoptosis-related proteins (cle-caspase3, Bim and Bax) and endothelial microstructure. Moreover, SB203580 significantly reduced the pulmonary P-p38, NFκB, P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels. CONCLUSION p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.

Background Esophageal cancer is a malignant tumor of the digestive tract that is difficult to diagnose early. CPI-455 has been reported to inhibit various cancers, but its role in esophageal squamous cell carcinoma (ESCC) is unknown. Aim To investigate the effects and mechanism of the lysine demethylase 5C inhibitor, CPI-455, on ESCC cells. Methods A methyl tetrazolium assay was used to detect the inhibitory effect of CPI-455 on the proliferation of Eca-109 cells. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were assessed by flow cytometry. Laser confocal scanning and transmission electron microscopy were used to observe changes in Eca-109 cell morphology. The protein expression of P53, Bax, lysine-specific demethylase 5C (KDM5C), cleaved Caspase-9, and cleaved Caspase-3 were assayed by western blotting. Results Compared with the control group, CPI-455 significantly inhibited Eca-109 cell proliferation. Gemcitabine inhibited Eca-109 cell proliferation in a concentration- and time-dependent manner. CPI-455 caused extensive alteration of the mitochondria, which appeared to have become atrophied. The cell membrane was weakly stained and the cytoplasmic structures were indistinct and disorganized, with serious cavitation when viewed by transmission electron microscopy. The flow cytometry and western blot results showed that, compared with the control group, the mitochondrial membrane potential was decreased and depolarized in Eca-109 cells treated with CPI-455. CPI-455 significantly upregulated the ROS content, P53, Bax, Caspase-9, and Caspase-3 protein expression in Eca-109 cells, whereas KDM5C expression was downregulated. Conclusion CPI-455 inhibited Eca-109 cell proliferation via mitochondrial apoptosis by regulating the expression of related genes.

BACKGROUND Gastric carcinoma (GC) is a digestive system disease with high morbidity and mortality. However, early clinical detection is difficult, and the therapeutic effect for advanced disease is not satisfactory. Thus, finding new tumor markers and therapeutic targets conducive to the treatment of GC is imperative. MRPL35 is a member of the large subunit family of mitochondrial ribosomal protein. MRPL35 shows the characteristic of oncogene in colorectal cancer and esophageal cancer, which promotes the exploration of the correlation between MRPL35 and GC. We proposed that the expression of MRPL35 might be critical in GC. AIM To study the effect of MRPL35 knockdown on GC cell proliferation. METHODS The expression of MRPL35 in GC was evaluated based on data from the public tumor database UALCAN (www.ualcan.path.uab.edu). The effect of the expression of MRPL35 on the prognosis was evaluated with KMplot (www.kmplot.com). The expression of MRPL35 was assessed on the tissue microarray by immunohistochemistry and the level of MRPL35 mRNA in 25 pairs of clinical GC tissues and matched adjacent tissues was detected by quantitative reverse transcription-polymerase chain reaction. Celigo cell count assay, colony formation assay, and flow cytometry were used to assess the role of MRPL35 in GC cell proliferation and apoptosis in vitro. Additionally, tumor formation experiment in BALB/c nude mice was utilized to determine the effect of MRPL35 on GC cell proliferation. After knockdown of MRPL35, related proteins were identified by isobaric tags for relative and absolute quantification analysis, and the expression of related proteins was detected by Western blot. RESULTS The expression of MRPL35 was up-regulated in GC (P = 1.77 × 10-4). The Kaplan-Meier plots of the overall survival indicated that high expression of MRPL35 was associated with a poor survival in GC. Compared with adjacent tissues, the expression of MRPL35 in GC tissues was increased, which was related to age (P = 0.03), lymph node metastasis (P = 0.007), and pathological tumor-node-metastasis stage (P = 0.024). Knockdown of MRPL35 inhibited GC cell proliferation and colony formation and induced apoptosis. Animal experiment results showed that knockdown of MRPL35 inhibited tumor formation in BALB/c nude mice. Western blotting analysis showed that after knockdown of MRPL35, the expression of PICK1 and BCL-XL proteins decreased, and that of AGR2 protein increased. CONCLUSION Collectively, our findings demonstrate that knockdown of MRPL35 inhibits GC cell proliferation through related proteins including PICK1, BCL-XL, and AGR2.

BACKGROUND As human placenta-derived mesenchymal stem cells (hP-MSCs) exist in a physiologically hypoxic microenvironment, various studies have focused on the influence of hypoxia. However, the underlying mechanisms remain to be further explored. AIM The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs. METHODS A hypoxic cell incubator (2.5% O2) was used to mimic a hypoxic microenvironment. Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs. The cell cycle was profiled by flow cytometry. Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing. CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction. Small interfering RNA-mediated hypoxia-inducible factor 1α (HIF-1α) or CD99 knockdown of hP-MSCs, luciferase reporter assays, and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis. Protein expression was assayed by western blotting; immunofluorescence assays were conducted to evaluate changes in expression levels. RESULTS Hypoxia enhanced hP-MSC proliferation, increased the expression of cyclin E1, cyclin-dependent kinase 2, and cyclin A2, and decreased the expression of p21. Under hypoxia, CD99 expression was increased by HIF-1α. CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation. CONCLUSION Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death among cancers, it is characterized by poor prognosis and strong chemoresistance. In the PDAC microenvironment, stromal cells release different extracellular components, including CXCL12. The CXCL12 is a chemokine promoting the communication between tumour and stromal cells. Six different splicing isoforms of CXCL12 are known (α, β, γ, δ, e, θ) but their role in PDAC has not yet been characterized. Aim To investigate the specific role of α, β, and γ CXCL12 isoforms in PDAC onset. Methods We used hTERT-HPNE E6/E7/KRasG12D (Human Pancreatic Nestin-Expressing) cell line as a pancreatic pre-tumour model and exposed it to the α, β, and γ CXCL12 isoforms. The altered expression profiles were assessed by microarray analyses and confirmed by Real-Time polymerase chain reaction. The functional enrichment analyses have been performed by Enrichr tool to highlight Gene Ontology enriched terms. In addition, wound healing assays have been carried out to assess the phenotypic changes, in terms of migration ability, induced by the α, β, and γ CXCL12 isoforms. Results Microarray analysis of hTERT-HPNE cells treated with the three different CXCL12 isoforms highlighted that the expression of only a few genes was altered. Moreover, the α and β isoforms showed an alteration in expression of different genes, whereas γ isoform affected the expression of genes also common with α and β isoforms. The β isoform altered the expression of genes mainly involved in cell cycle regulation. In addition, all isoforms affected the expression of genes associated to cell migration, adhesion and cytoskeleton. In vitro cell migration assay confirmed that CXCL12 enhanced the migration ability of hTERT-HPNE cells. Among the CXCL12 splicing isoforms, the γ isoform showed higher induction of migration than α and β isoforms. Conclusion Our data suggests an involvement and different roles of CXCL12 isoforms in PDAC onset. However, more investigations are needed to confirm these preliminary observations.