Integrated multiple microarray studies by robust rank aggregation to identify immune-associated biomarkers in Crohn's disease based on three machine learning methods.

Crohn's disease (CD) is a complex autoimmune disorder presumed to be driven by complex interactions of genetic, immune, microbial and even environmental factors. Intrinsic molecular mechanisms in CD, however, remain poorly understood. The identification of novel biomarkers in CD cases based on larger samples through machine learning approaches may inform the diagnosis and treatment of diseases. A comprehensive analysis was conducted on all CD datasets of Gene Expression Omnibus (GEO); our team then used the robust rank aggregation (RRA) method to identify differentially expressed genes (DEGs) between controls and CD patients. PPI (protein‒protein interaction) network and functional enrichment analyses were performed to investigate the potential functions of the DEGs, with molecular complex detection (MCODE) identifying some important functional modules from the PPI network. Three machine learning algorithms, support vector machine-recursive feature elimination (SVM-RFE), random forest (RF), and least absolute shrinkage and selection operator (LASSO), were applied to determine characteristic genes, which were verified by ROC curve analysis and immunohistochemistry (IHC) using clinical samples. Univariable and multivariable logistic regression were used to establish a machine learning score for diagnosis. Single-sample GSEA (ssGSEA) was performed to examine the correlation between immune infiltration and biomarkers. In total, 5 datasets met the inclusion criteria: GSE75214, GSE95095, GSE126124, GSE179285, and GSE186582. Based on RRA integrated analysis, 203 significant DEGs were identified (120 upregulated genes and 83 downregulated genes), and MCODE revealed some important functional modules in the PPI network. Machine learning identified LCN2, REG1A, AQP9, CCL2, GIP, PROK2, DEFA5, CXCL9, and NAMPT; AQP9, PROK2, LCN2, and NAMPT were further verified by ROC curves and IHC in the external cohort. The final machine learning score was defined as [Expression level of AQP9 × (2.644)] + [Expression level of LCN2 × (0.958)] + [Expression level of NAMPT × (1.115)]. ssGSEA showed markedly elevated levels of dendritic cells and innate immune cells, such as macrophages and NK cells, in CD, consistent with the gene enrichment results that the DEGs are mainly involved in the IL-17 signaling pathway and humoral immune response. The selected biomarkers analyzed by the RRA method and machine learning are highly reliable. These findings improve our understanding of the molecular mechanisms of CD pathogenesis.

[The mechanisms of inhibitory effect of adenovirus-mediated wild-type PTEN gene on the proliferation in activated hepatic stellate cells in vitro].

OBJECTIVE: Using an adenoviral vector, the wild-type PTEN gene was transduced into activated hepatic stellate cell (HSC) cultured in vitro and cell cycle markers and were detect. Thereby, the potential mechanisms of inhibitory effect of the wild-type PTEN overexpression on the proliferation in activated HSC was investigated. METHODS: The wild type PTEN gene was transduced into activated HSC (HSC-T6 ) cultured in vitro mediated by adenoviral vector. PTEN expression in HSC was measured by Western blot and Real-time fluorescent quantitation PCR. Flow cytometry (FCM) was then used to detect cell cycle phase of activated HSC. And the expressions of cyclinD1 and cyclin dependent kinase 4 (CDK4) in HSC were determined by Western blot. RESULTS: The data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC cultured in vitro. The over-expression of wild type PTEN resulted in the increased number of HSC at G0/G1 phase ( P less than 0.01), and the number of HSC at S phase and G2/M phase were decreased significantly, P less than 0.01. Furthermore, there were decreased cyclinD1 and CDK4 expression in HSC infected with Ad-PTEN, P less than 0.01. CONCLUSION: The over-expression of wild type PTEN inhibit transition of activated HSC in vitro from G1 to S phase and arrest cell cycle of them at G0/G1 phase via the down-regulated expressions of cyclinD1 and CDK4, and then inhibit HSC proliferation.

Serum Spondin-2 expression, tumor invasion, and antitumor immune response in patients with cervical cancer.

BACKGROUND: Cervical cancer is a gynecological malignancy common in middle-aged and older patients, with a high mortality rate. Spondin-2 is an extracellular matrix protein that involved in innate and acquired immune responses. Herein, we investigated the relationship between serum Spondin-2 expression, tumor invasion and infiltration, and immune response in patients with cervical cancer and provided a theoretical basis for clinical practice. AIM: To investigate the relationship between serum Spondin-2 expression and cervical cancer-related indicators. METHODS: Overall, 147 patients with cervical cancer who were admitted to our institution between January 2019 and August 2019 were assigned to the cervical cancer group, and 92 patients with benign uterine lesions and 86 healthy individuals were assigned to the benign and control groups, respectively. In each group, serum Spondin-2 expression was measured, and the receiver operating characteristic (ROC) curve was determined. Patients with cervical cancer were classified into high or low Spondin-2 groups depending on the Spondin-2 threshold value used for diagnosing cervical cancer. Patient's clinical data were collected to compare the clinicopathologic characteristics, immune cytokine levels, and prognosis of patients with varying Spondin-2 expression levels. RESULTS: The expression level of serum Spondin-2 was significantly higher in the cervical cancer group than in the benign and control groups (P < 0.05). According to the ROC curve, the cutoff value of Spondin-2 used in the diagnosis of cervical carcinoma was 25.68 ± 7.11 µg/L. The proportion of patients with Federation of Gynecology and Obstetrics stage III, nerve invasion, vascular invasion, and lymph node metastasis was higher in the high Spondin-2 group than in the low Spondin-2 group (P < 0.05). Interleukin-5 (IL-5) and IL-4 Levels were higher in the high Spondin-2 group than in the low Spondin-2 group. In contrast, IL-2 and tumor necrosis factor-α levels were lower in the high Spondin-2 group than in the low Spondin-2 group (P < 0.05). After 3 years of follow-up, progression-free survival and overall survival were significantly shorter in the high Spondin-2 group than in the low Spondin-2 group (P < 0.05). CONCLUSION: The expression of serum Spondin-2 is upregulated in patients with cervical carcinoma and is related to tumor invasion and infiltration, antitumor immune response, and prognosis.

Identification of a Ferroptosis-Related Signature Model Including mRNAs and lncRNAs for Predicting Prognosis and Immune Activity in Hepatocellular Carcinoma.

BACKGROUND: Ferroptosis is a novel form of regulated cell death involved in tumor progression. The role of ferroptosis-related lncRNAs in hepatocellular carcinoma (HCC) remains unclear. METHODS: RNA-seq and clinical data for HCC patients were downloaded from The Cancer Genome Atlas (TCGA) Genomic Data Commons (GDC) portal. Bioinformatics methods, including weighted gene coexpression network analysis (WGCNA), Cox regression, and least absolute shrinkage and selection operator (LASSO) analysis, were used to identify signature markers for diagnosis/prognosis. The tumor microenvironment, immune infiltration and functional enrichment were compared between the low-risk and high-risk groups. Subsequently, small molecule drugs targeting ferroptosis-related signature components were predicted via the L1000FWD and PubChem databases. RESULTS: The prognostic model consisted of 2 ferroptosis-related mRNAs (SLC1A5 and SLC7A11) and 8 ferroptosis-related lncRNAs (AC245297.3, MYLK-AS1, NRAV, SREBF2-AS1, AL031985.3, ZFPM2-AS1, AC015908.3, MSC-AS1). The areas under the curves (AUCs) were 0.830 and 0.806 in the training and test groups, respectively. Decision curve analysis (DCA) revealed that the ferroptosis-related signature performed better than all pathological characteristics. Multivariate Cox regression analysis showed that the risk score was an independent prognostic factor. The survival probability of low- and high-risk patients could be clearly distinguished by the principal component analysis (PCA) plot. The risk score divided HCC patients into two distinct groups in terms of immune status, especially checkpoint gene expression, which was further supported by the Gene Ontology (GO) biological process, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Finally, several small molecule drugs (SIB-1893, geldanamycin and PD-184352, etc) targeting ferroptosis-related signature components were identified for future reference. CONCLUSION: We constructed a new ferroptosis-related mRNA/lncRNA signature for HCC patients. The model can be used for prognostic prediction and immune evaluation, providing a reference for immunotherapies and targeted therapies.

[Dynamic changes of alpha-AR, beta1-AR and beta2-AR expression during hepatic fibrogenesis].

OBJECTIVE: To investigate the dynamic changes of a-AR, b1-AR and b2-AR expression in hepatic fibrosis. METHODS: Rat hepatic fibrosis model was established by bile duct ligation (BDL). HE and Masson staining were used to determine hepatic fibrosis levels. Immunohistochemistry was applied to detect alpha -smooth muscle actin (alpha -SMA), a marker of hepatic stellate cell (HSC) activation; Western blot and real-time RT-PCR were used to measure the dynamic changes of alpha -AR, beta(1)-AR, beta(2)-AR expression on protein and mRNA levels, respectively, during the development of hepatic fibrosis. RESULTS: (1) HE and Masson trichrome staining showed that the liver fibrosis models were established successfully. (2) At 1, 2, 3, 4 wk after BDL, alpha -SMA positive area density of the model group (10.58% +/- 1.75%, 24.14% +/- 2.02%, 29.74% +/- 2.59%, 34.28% +/- 2.01%) was significantly higher than that of the sham operation group (4.12% +/- 1.51%), P less than 0.01. (3) The expression of alpha -AR, beta(1)-AR, beta(2)-AR protein and mRNA was increased with the development of the hepatic fibrosis (P less than 0.05). (4) alpha -SMA expression was positively associated with alpha -AR, beta(1)-AR, beta(2)-AR, r values were 0.564, 0.753 and 0.606, respectively. CONCLUSION: The expression of alpha -SMA is increased dramatically during the fibrosis, and is positively associated with the expression of alpha -AR, beta(1)-AR and beta(2)-AR.

[The influence of down-regulation of focal adhesion kinase by RNA interference on the adhesion and migration of rat hepatic stellate cells in vitro].

OBJECTIVE: To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC). METHODS: Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN. RESULTS: The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively. CONCLUSIONS: FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.

[The effects of FRNK on expressions of MMP-2 mRNA and TIMP-2 mRNA in hepatic stellate cells].

OBJECTIVES: To investigate the effects of FAK-related non-kinase (FRNK) on expressions of type I collagen and matrix metalloproteinase-2 (MMP-2) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in rat hepatic stellate cells (HSC). METHODS: Using in vitro cell culture technique, FRNK plasmids were transfected into HSC mediated by cationic liposome. Type I collagen synthesis capability in HSC was examined by 3H-Pro incorporation assay. The levels of FRNK in HSC were assayed by Western blot, and the expressions of MMP-2 and TIMP-2 were assayed by RT-PCR on mRNA levels. RESULTS: The exposure of HSC to FRNK caused the expression of FRNK protein to be up-regulated, and the FRNK protein contents reached the highest point at 48 h after the transfection, P less than 0.05. The expressions of MMP-2 mRNA were up-regulated by FRNK; the expressions of TIMP-2 mRNA were down-regulated by FRNK; the ratios of MMP-2 mRNA/TIMP-2 mRNA were enhanced by FRNK. CONCLUSION: After FRNK was transfected, the capability of type I collagen synthesis in HSC was inhibited, which may be related to the up-regulation of MMP-2 mRNA/TIMP-2 mRNA.

Yeast Surface Display of Antheraea pernyi Lysozyme Revealed α-Helical Antibacterial Peptides in Its N-Terminal Domain.

The present study investigated a novel lysozyme ApLyz from the Chinese oak silkmoth, Antheraea pernyi, for its active expression with N- or C-terminus fused to the yeast cell surface, and the antimicrobial activities of the corresponding expressed lysozymes were evaluated. The bactericidal activity of C-terminal fusion of ApLyz surpassed that of the N-terminal fusion, which revealed the implication of an N-terminal stretch of ApLyz in the bactericidal function based on the structural mobility of this region. Two N-terminal peptides of ApLyz (residues 1-15 and 1-32), which primarily consist of amphiphilic α-helices, exerted similar bactericidal efficacy and had a strong preference for the Gram-negative strains. Further investigation revealed that the N-terminal peptides are membrane-targeting peptides causing cell permeabilization and also possess nonmembrane disturbing bactericidal mechanism. Overall, in addition to the key findings of novel bactericidal peptides from silkmoth lysozyme, this work laid the foundation for future improvement of ApLyz by protein engineering.

Current research of hepatic cirrhosis in China.

Hepatic cirrhosis is a common disease that poses a serious threat to public health, and is characterized by chronic, progressive and diffuse hepatic lesions preceded by hepatic fibrosis regardless of the exact etiologies. In recent years, considerable achievements have been made in China in research of the etiopathogenesis, diagnosis and especially the treatment of hepatic fibrosis, resulting in much improved prognosis of hepatic fibrosis and cirrhosis. In this paper, the authors review the current status of research in hepatic fibrosis, cirrhosis and their major complications.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats.

AIM: To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCl4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang. At wk 6, 10, 14 and 20 (baseline for CCl4 or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for alpha-SMA, type I collagen and in situ hybridization of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCl4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in microm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, alpha-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCl4- and pig serum-induced rat models.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells.

AIM: To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P<0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P<0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P<0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P<0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P<0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

[Distribution of respiratory nosocomial infection pathogen on geriatric (correction of geratic) people with chronic diseases and its antibiotic resistance].

OBJECTIVE: To detect respiratory nosocomial infection pathogen on geriatric (corrected) people with chronic disease and its antibiotic resistance. METHODS: Standard culture and antibiotic resistance method was used. RESULTS: Two hundred and fifty strains of species germ which caused nosocomial infection were cultured in 202 geratic people with chronic diseases, of which 144 strains were gram-negative bacill (55%); 44 strains were gram-positive coccus (17%); and 62 strains were Yeastlike Fungi (28%). The ESBL positive rate for Escherichiacol and Klebsiella was 37.8% and 39.6%. MRS positive rate for staphylococcus was 67%. Vancomycin resistance in Enterococcus was 6%. CONCLUSION: Geratic people with chronic disease are more easily to infect opportunistic pathogen and cause nosocomical infection. The more care we give for geratic people, the lower the nosocomial infection rate.

[The role of extracellular signal-regulated kinase in induction of apoptosis with salvia miltiorrhiza monomer IH764-3 in hepatic stellate cells].

OBJECTIVE: To explore the effect of Salvia miltiorrhiza monomer IH764-3 on apoptosis in hydrogen peroxide (H2O2)-stimulated hepatic stellate cells (HSCs). METHODS: HSCs were cultured in medium with different IH764-3 doses (10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L) and without IH764-3. Direct cell count, 3H-thymidine incorporation, Annexin-V/Propidium Iodide double-labeled flow cytometry, TUNEL and transmission electron microscopy were employed to estimate the influence of IH764-3 on proliferation and apoptosis of HSCs. The expression of extracellular signal-regulated kinase 1 (ERK1) mRNA and protein in HSCs were detected using RT-PCR and Western blot respectively. RESULTS: It was showed that H2O2 could promote HSC proliferation. In contrast, IH764-3 at concentrations of 10 mg/L, 20 mg/L, 30 mg/L and 40 mg/L inhibited its proliferation. The inhibition rates were 7.13%, 28.36%, 53.80% and 73.10% (P < 0.01). And the inhibition rates of IH764-3 at concentrations of 30 mg/L at 12 h, 24 h and 48 h were 22.24%, 40.51% and 61.65%. Furthermore, IH764-3 could also induce the HSC apoptosis in dose-dependent an dtime-dependent manners (P < 0.01). In addition, after exposed of HSCs to IH764-3 for 24 h, ERK production decreased and ERK1 mRNA was down-regulated earlier about 2 h after exposure to IH764-3. CONCLUSION: IH764-3 may inhibit the proliferation and induce apoptosis of HSCs in both dose-dependent and time-dependent manners, which may be related to down-regulation of ERK expression.