[Effects of titanium dioxide nanoparticles on circRNA expression profiles in human hepatocellular carcinoma cells HepG2].

OBJECTIVE: To investigate the effect of titanium dioxide nanoparticles (TiO2 NPs) on the expression profile of circular ribonucleic acid (circRNA) in human hepatocytes through in vitro cell experiments, and to attempt to understand the potential mechanism of hepatotoxicity through bioinformatics analysis. METHODS: TiO2 NPs were characterized from the aspects of particle size, shape and agglomeration state. The cell counting kit-8 (CCK8) was used to detect the cytotoxicity of TiO2 NPs against human hepatocellular carcinoma cells (HepG2) after exposure to 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 mg/L TiO2 NPs for 24 h or 48 h. The cells were treated at doses of 0 mg/L TiO2 NPs (control group) and 100 mg/L TiO2 NPs (treatment group), and collected after exposure for 48 h, and then RNA from the extracted cell samples was collected and sequenced. The differential circRNAs between the control and the TiO2 NPs treatment groups were screened, and then the enrichment pathway of the differential circRNA target gene was analyzed by multivariate statistics. According to the sequencing results, significantly altered genes and important genes in the significant enrichment pathways were screened, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was performed to verify the results. RESULTS: TiO2 NPs were spherical anatase with a hydrated particle size of (323.50±85.44) nm and a Zeta potential of (-21.00±0.72) mV in a serum-free medium. The results of the CCK8 cytotoxicity assay showed that with the increase of TiO2 NPs concentration, cell viability gradually decreased. A total of 11 478 circRNAs were found by RNA sequencing. Compared with the control groups, TiO2 NPs treatment groups (100 mg/L) had a total of 89 differential circRNAs, of which 59 were up-regulated and 30 were down-regulated. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that the targeted genes of differential circRNAs were mainly enriched in fatty acid degradation, Fanconi anemia pathway, and fatty acid metabolism. The expression levels of circRNA.6730, circRNA.3650 and circRNA.4321 were significantly different between the TiO2 NPs treatment group and the control group, which were consistent with the sequencing results. CONCLUSION: TiO2 NPs can induce changes in circRNA expression profile, and epigenetics may play an important role in the mechanism of hepatotoxicity.

[Effects of nano titanium dioxide on gut microbiota based on human digestive tract microecology simulation system in vitro].

OBJECTIVE: To explore the effects of oral exposure to titanium dioxide nanoparticles (TiO2 NPs) on the composition and structure of human gut microbiota. METHODS: The particle size, shape, crystal shape and degree of agglomeration in ultrapure water of TiO2 NPs were characterized. The in vitro human digestive tract microecological simulation system was established by simulating the fluid environment and physical conditions of stomach, small intestine and colon, and the stability of the simulation system was evaluated. The bacterial communities were extracted from human feces and cultured stably in the simulated system. They were exposed to 0, 20, 100 and 500 mg/L TiO2 NPs, respectively, and the bacterial fluids were collected after 24 h of exposure. The effect of TiO2 NPs on the composition and structure of human gut microbiota was analyzed by 16S rRNA sequencing technology. Linear discriminant analysis effect size (LEfSe) was used to screen differential bacteria, and the Kyoto encyclopedia of genes and genomes (KEGG) database for functional prediction. RESULTS: The spherical and anatase TiO2 NPs were (25.12±5.64) nm in particle size, while in ultra-pure water hydrated particle size was (609.43±60.35) nm and Zeta potential was (-8.33±0.22) mV. The in vitro digestive tract microecology simulation system reached a relatively stable state after 24 hours, and the counts of Enterococci, Enterobacte-rium, and Lactobacillus reached (1.6±0.85)×107, (5.6±0.82)×107 and (2.7±1.32)×107, respectively. 16S rRNA sequencing results showed that compared with the control group, the number and evenness of gut microbiota were not significantly affected at phylum, class, order, family and genus levels in TiO2 NPs groups (20, 100 and 500 mg/L). The relative abundance of some species was significantly changed, and a total of 42 different bacteria were screened between the TiO2 NPs groups (20, 100 and 500 mg/L) and the control group [linear discriminant analysis(LDA) score>3], represented by Enterobacter, Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae and Clostridium. Further predictive analysis of gut microbiota function showed that TiO2 NPs might affect oxidative phosphorylation, energy meta-bolism, phosphonate and phosphonate metabolism, and methane metabolism (P < 0.05). CONCLUSION: In human digestive tract microecological simulation system, TiO2 NPs could significantly change the composition and structure of human gut microbiota, represented by Enterobacter and probiotics, and may further affect a variety of metabolism and function of the body.

Circular RNA_LARP4 inhibits the progression of non-small-cell lung cancer by regulating the expression of SMAD7.

OBJECTIVE: Researchers have uncovered the importance of circular RNAs (circ) in malignant tumors. Circ LARP4 has been found to serve as a tumor suppressor gene in gastric cancer. However, the exact function of circ LARP4 in non-small-cell lung cancer (NSCLC) has not been fully elucidated. The aim of this study was to uncover the role of circ LARP4 in the tumorigenesis of NSCLC. PATIENTS AND METHODS: Expression level of circ LARP4 in NSCLC tissues was detected through Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Subsequently, the association between expression and patients' prognosis was analyzed. Circ LARP4 lentivirus was constructed and transfected into NSCLC cells. The effect of circ LARP4 on NSCLC cell migration and invasion was detected by function assays. Furthermore, Western blot was performed to analyze the expression of predicted protein of circ LARP4. RESULTS: Compared with adjacent tissues, circ LARP4 was lowly expressed in NSCLC tissues. Meanwhile, expression of circ LARP4 was associated with the prognosis of NSCLC patients. Downregulated circ LARP4 was found in NSCLC cell lines as well. The migration and invasion abilities of NSCLC cells were significantly inhibited via overexpression of circ LARP4. SMAD7, the predicted protein of circ LARP4, increased remarkably via overexpression of circ LARP4. CONCLUSIONS: Circ LARP4 could suppress the metastasis of NSCLC by up-regulating SMAD7.

Hypoxia promotes migration and invasion of gastric cancer cells by activating HIF-1α and inhibiting NDRG2 associated signaling pathway.

OBJECTIVE: Gastric cancer has been become the fourth most prevalent cancer in whole world and the third most common cancer in Asian countries. This study aimed to discuss the invasive and migration mechanisms of gastric cells. MATERIALS AND METHODS: Human gastric cancer line, BGC-823 cell, was treated with hypoxia and divided into Hypoxia-12 h, Hypoxia-24 h, Hypoxia-36 h, Hypoxia-48 h and Hypoxia-72 group. Meanwhile, blank BGC-823 cells were assigned as Normal group. mRNA and protein expression of N-myc downstream-regulated gene 2 (NDRG2), Twist, E-cadherin and hypoxia-inducible factor 1α (HIF-1α) were evaluated by using quantitative Real-time PCR (qRT-PCR) and Western blot assay, respectively. Invasion and migration of BGC-823 cells were also examined in this study. RESULTS: Hypoxia treatment significantly enhanced invasion and migration ability of BGC-823 cells compared to that of Normal group (p<0.05). Hypoxia treatment significantly reduced E-cadherin and NDRG2 expression compared to that of Normal group (p<0.05). Hypoxia treatment significantly increased Twist and HIF-1α expression compared to that of Normal group (p<0.05). HIF-1α inhibitor, YC-1, significantly suppressed the effects of hypoxia treatment on E-cadherin and Twist expression (p<0.05). Meanwhile, YC-1 treatment also significantly suppressed the effects of hypoxia treatment on NDRG2 and HIF-1α expression. CONCLUSIONS: Hypoxia promoted the migration and invasion of gastric cancer cell BGC-823 by activating HIF-1α and inhibiting NDRG2 associated signaling pathway.

Mixed-effects Gaussian process functional regression models with application to dose-response curve prediction.

We propose a new semiparametric model for functional regression analysis, combining a parametric mixed-effects model with a nonparametric Gaussian process regression model, namely a mixed-effects Gaussian process functional regression model. The parametric component can provide explanatory information between the response and the covariates, whereas the nonparametric component can add nonlinearity. We can model the mean and covariance structures simultaneously, combining the information borrowed from other subjects with the information collected from each individual subject. We apply the model to dose-response curves that describe changes in the responses of subjects for differing levels of the dose of a drug or agent and have a wide application in many areas. We illustrate the method for the management of renal anaemia. An individual dose-response curve is improved when more information is included by this mechanism from the subject/patient over time, enabling a patient-specific treatment regime.

Acute toxicity and n-octanol/water partition coefficients of substituted thiophenols: determination and QSAR analysis.

The acute toxicity (-log EC(50)) to Photobacterium phosphoreum and the n-octanol/water partition coefficient (log K(ow)) of 31 kinds of substituted thiophenols were determined at 298.15K. The -log EC(50) values of studied chemicals are between 4.26 and 5.89. Their log K(ow) values are between 1.34 and 4.02. Comparative molecular field (CoMFA) and comparative molecular similarity index analysis (CoMSIA) models established were successful in predicting -log EC(50) and log K(ow) values of halogenated, methylic, amino and methoxy thiophenols. The size of molecule is the main factor influencing the properties. No correlation was found between the properties and their structural and thermodynamic descriptors from DFT calculation.

Penalized gaussian process regression and classification for high-dimensional nonlinear data.

The model based on Gaussian process (GP) prior and a kernel covariance function can be used to fit nonlinear data with multidimensional covariates. It has been used as a flexible nonparametric approach for curve fitting, classification, clustering, and other statistical problems, and has been widely applied to deal with complex nonlinear systems in many different areas particularly in machine learning. However, it is a challenging problem when the model is used for the large-scale data sets and high-dimensional data, for example, for the meat data discussed in this article that have 100 highly correlated covariates. For such data, it suffers from large variance of parameter estimation and high predictive errors, and numerically, it suffers from unstable computation. In this article, penalized likelihood framework will be applied to the model based on GPs. Different penalties will be investigated, and their ability in application given to suit the characteristics of GP models will be discussed. The asymptotic properties will also be discussed with the relevant proofs. Several applications to real biomechanical and bioinformatics data sets will be reported.

Development and characterization of microsatellite markers to study population genetic diversity of Przewalski's naked carp Gymnocypris przewalskii in China.

Two populations of Przewalski's naked carp Gymnocypris przewalskii, 30 individuals per population, were screened for 10 microsatellite loci. Moderate allele variation was found in these loci with two to eight alleles per locus. The expected and observed heterozygosity ranged from 0.019 to 0.805 and from 0.160 to 0.575, respectively.

Gaussian process functional regression modeling for batch data.

A Gaussian process functional regression model is proposed for the analysis of batch data. Covariance structure and mean structure are considered simultaneously, with the covariance structure modeled by a Gaussian process regression model and the mean structure modeled by a functional regression model. The model allows the inclusion of covariates in both the covariance structure and the mean structure. It models the nonlinear relationship between a functional output variable and a set of functional and nonfunctional covariates. Several applications and simulation studies are reported and show that the method provides very good results for curve fitting and prediction.

Maximum likelihood estimation of two-level latent variable models with mixed continuous and polytomous data.

Two-level data with hierarchical structure and mixed continuous and polytomous data are very common in biomedical research. In this article, we propose a maximum likelihood approach for analyzing a latent variable model with these data. The maximum likelihood estimates are obtained by a Monte Carlo EM algorithm that involves the Gibbs sampler for approximating the E-step and the M-step and the bridge sampling for monitoring the convergence. The approach is illustrated by a two-level data set concerning the development and preliminary findings from an AIDS preventative intervention for Filipina commercial sex workers where the relationship between some latent quantities is investigated.

A sensitivity analysis for publication bias in systematic reviews.

There is no simple method of correcting for publication bias in systematic reviews. We suggest a sensitivity analysis in which different patterns of selection bias can be tested against the fit to the funnel plot. Publication bias leads to lower values, and greater uncertainty, in treatment effect estimates. Two examples are discussed. An appendix lists the S-plus code needed for carrying out the analysis.

[Changes of capillarity and blood flow of skeletal muscle in rat during acclimatization to hypoxia].

AIM: To study the changes of capillary density and blood flow of skeletal muscle in rat during acclimatization to hypoxia. METHODS: Myosin-ATPase histochemistry was used to assay the size and capillarity of skeletal muscle. Biomicrospheres method was used to determine blood flow in the rats at 5-, 15-or 30-day hypobaric hypoxic exposure (simulated 5 000 m altitude). RESULTS: It was found that cross-sectional area of skeletal muscle fibers (including type I and type II) were decreased during acclimatization to hypoxia. Capillary density (CD) was increased in 15-day and 30-day groups but the capillary/fiber ratio (C/F) remained unchanged among all of the groups observed. There was no obvious difference in blood flow between hypoxic rats and control rats. CONCLUSION: Capillary neoformation does not occur in skeletal muscle of rat during acclimatization to hypobaric hypoxia. Increased capillary density due to atrophy of skeletal muscle fibre might benefit oxygen delivery at high altitude.

Correlation of bFGF, FGFR-1 and VEGF expression with vascularity and malignancy of human astrocytomas.

OBJECTIVE: To investigate the correlation of angiogenic factor expression levels with the degrees of malignancy and vascularity and their clinicopathologic significance in astrocytomas. STUDY DESIGN: Factor VIII-related antigen (FVIII-RAg) was used as the marker of endothelia and basic fibroblast growth factor (bFGF); FGF receptor (FGFR)-1 and vascular endothelial growth factor (VEGF) were qualitatively and quantitatively detected with immunohistochemistry and image analysis in 61 brain astrocytomas. The correlation with tumor grades, angiogenesis and prognosis was studied. RESULTS: Measurement of FVIIIRAg expression could describe endothelial proliferation and vascularity, which were related to grade of tumor and prognosis. bFGF and VEGF expression levels in neoplastic astrocytes and endothelia were significantly different in various grades of astrocytoma. These angiogenic factors affected the positive reaction areas and integral optical densities of FVIII-RAg as well as survival time. In contrast, the expression of FGFR-1 was related to neither bFGF nor FVIIIRAg and had no significant effect on tumor malignancy. CONCLUSION: Positive regulation by bFGF and autocrine/paracrine VEGF contributes to the growth and angiogenesis of astrocytomas. Measurement of endothelial cell proliferation with FVIIIRAg in tumor stroma and quantitative detection of angiogenic factor levels in neoplastic cells had prognostic value in brain astrocytomas. The results also indicate that inhibiting bFGF and VEGF expression and/or blocking their effects could be a very useful therapeutic strategy for malignant gliomas.

Reanalysis of epidemiological evidence on lung cancer and passive smoking.

OBJECTIVE: To assess the epidemiological evidence for an increase in the risk of lung cancer resulting from exposure to environmental tobacco smoke. DESIGN: Reanalysis of 37 published epidemiological studies previously included in a meta-analysis allowing for the possibility of publication bias. MAIN OUTCOME MEASURE: Relative risk of lung cancer among female lifelong non-smokers, according to whether her partner was a current smoker or a lifelong non-smoker. RESULTS: If it is assumed that all studies that have ever been carried out are included, or that those selected for review are truly representative of all such studies, then the estimated excess risk of lung cancer is 24%, as previously reported (95% confidence interval 13% to 36%, P<0.001). However, a significant correlation between study outcome and study size suggests the presence of publication bias. Adjustment for such bias implies that the risk has been overestimated. For example, if only 60% of studies have been included, the estimate of excess risk falls from 24% to 15%. CONCLUSION: A modest degree of publication bias leads to a substantial reduction in the relative risk and to a weaker level of significance, suggesting that the published estimate of the increased risk of lung cancer associated with environmental tobacco smoke needs to be interpreted with caution.

Pathologic significance of proliferative activity and oncoprotein expression in astrocytic tumors.

OBJECTIVE: To clarify the clinicopathologic significance of immunohistochemistry for proliferative activity and oncoprotein expression in astrocytic tumors. STUDY DESIGN: Ninety-seven cases of brain astrocytic tumors with histologic grading and follow-up data were investigated with immunohistochemistry and image analyzer to detect the expression of proliferating cell nuclear antigen (PCNA), silver-binding nucleolar organizer regions (AgNORs) and several oncoproteins. RESULTS: PCNA was significantly related to AgNORs, grading and prognosis of astrocytomas. The frequency of mutant p53 protein expression was higher in grade 2-4 astrocytomas than in grade 1. Epidermal growth factor (EGF) (37.1%), EGF receptor (83.5%) and p21ras (42.3%) expression levels were related to neither the grade nor prognosis of the tumors. The positive ratios of p53 antibodies were higher in grades 2-4 than in grade 1, and the intensities correlated with PCNA but not with prognosis. CONCLUSION: Aberrations of c-erbB-2, p21ras, EGF and EGF receptor might be early events in the initiation and progression of astrocytomas, whereas p53 overexpression is involved in all the stages. Immunohistochemical detection had no prognostic value. PCNA could be important to the evaluation of astrocytoma malignancy.

Meta-analysis, funnel plots and sensitivity analysis.

Publication bias is a major problem, perhaps the major problem, in meta-analysis (or systematic reviews). Small studies are more likely to be published if their results are 'significant' than if their results are negative or inconclusive, and so the studies available for review are biased in favour of those with positive outcomes. Correcting for this bias is not possible without making untestable assumptions. In this paper, a sensitivity analysis is suggested which is based on fitting a model to the funnel plot. Some examples are discussed.

Immunohistochemical detection of proliferating cell nuclear antigen in hepatocellular carcinoma.

AIM: To investigate the expression levels of proliferating cell nuclear antigen (PCNA) and its correlation with the mitotic count, histological grade, and metastasis of human hepatocellular carcinoma (HCC). METHODS: PCNA expression was detected immunohistochemically with PC10 monoclonal antibody for 80 cases of HCC. The PCNA labeling index (LI) was assessed by counting the positively-stained nuclei per 500 cells. RESULTS: PCNA LI ranged from 1.8% to 91.4% (mean = 33.9%) in cancerous tissues, and was significantly related to the tumor size, histological grade, mitotic count and the metastases found in HCC samples. CONCLUSION: HCC Proliferation activity, as defined by PCNA immunohistochemical analysis, is strongly correlated with tumor invasiveness. These findings hold potential prognostic value for HCC patients.

[A comparative study of the expression of ras oncogenic protein P21 and oncofetal protein AFP, CEA in human hepatocellular carcinoma (HCC)].

The expression of P21, AFP and CEA were detected in human hepatocellular carcinoma and its surrounding nontumor tissues by immunohistochemical staining on serial sections. The significance of AFP and P21 in HCC auxiliary diagnosis was discussed. The results are as follows: (1) AFP and P21 were more closely related with HCC than CEA. Both AFP and P21 were expressed simultaneously in more than half the cases. The distribution of these two antigens in HCC was similar. (2) The positive incidence of AFP in tumor tissues was higher than that in surrounding nontumor tissues, but vice versa for P21. Based on the results of this study, we regard that AFP is a relatively specific marker of HCC, better than P21 in auxiliary diagnosis of HCC. The hepatocytes which surround the tumor and can simultaneously express AFP and P21, are likely to be precancerous cells which have been initiated and possess the ability of neoplastic growth but lack phenotype transformation.

Detection of antigens associated with lung carcinoma in sera by monoclonal antibodies WLA-2C4 and CL-3.

The levels of tumor-associated antigens (TAAS) corresponded to monoclonal antibodies WLA-2C4 and CL-3 in sera of 57 lung cancer patients, 100 healthy adults and 50 non-tumor disease patients were assayed with SABC-ELISA of immunobinding inhibition test. The threshold values of WLA-2C4 and CL-3 (RBI) were 12% and 36%, respectively. The positive results of lung carcinomas with at least one of the two TAAS were as follows: squamous cell carcinoma 89%; adenocarcinoma 83%; small cell carcinoma 67% and their mean positive rate was 79%. Whereas the positive rate in healthy adults and non-tumor disease patients was only 6%. These results indicate that using monoclonal antibodies WLA-2C4 and CL-3 simultaneously may be helpful to the serological diagnosis of lung carcinoma.