The causal relationship between metabolic factors, drinking, smoking and intrahepatic cholangiocarcinoma: a Mendelian randomization study.

Background: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. While multiple risk factors for iCCA have been established, metabolic diseases (obesity, diabetes, NAFLD, dyslipidemia, and hypertension) and other risk factors, including smoking and drinking, are still controversial due to their potential confounders. Here, Mendelian randomization (MR) analysis was performed to identify the causal relationship between them. Method: In this study, we obtained GWAS data related to exposures from corresponding large genome-wide association studies. Summary-level statistical data for iCCA were obtained from the UK Biobank (UKB). We performed a univariable MR analysis to identify whether genetic evidence of exposure was significantly associated with iCCA risk. A multivariable MR analysis was conducted to estimate the independent effects of exposures on iCCA. Results: Univariable and multivariable MR analysis based on the large GWAS data indicated that there is little evidence to support the genetic role of metabolic factors, smoking, drinking, and NAFLD in iCCA development (P >0.05). In contrast to most current studies, their impact on iCCA development, if any, might be smaller than we thought. The previous positive results might be due to the comorbidities between diseases and potentially unavoidable confounding factors. Conclusion: In this MR study, we found no strong evidence to support causal associations between metabolic factors, NAFLD, smoking, drinking, and iCCA risk.

Systematic analysis using a bioinformatics strategy identifies SFTA1P and LINC00519 as potential prognostic biomarkers for lung squamous cell carcinoma.

Lung cancer has high incidence and mortality rates, in which lung squamous cell carcinoma (LUSC) is a primary type of non-small cell lung carcinoma (NSCLC). The aim of our study was to discover long non-coding RNAs (lncRNAs) associated with diagnose and prognosis for LUSC. RNA sequencing data obtained from LUSC samples were extracted from The Cancer Genome Atlas database (TCGA). Two prognosis-associated lncRNAs (including SFTA1P and LINC00519) were selected from LUSC samples, and the expression levels were also verified to be associated abnormal in LUSC clinical samples. Our findings demonstrate that lncRNAs SFTA1P and LINC00519 exert important functions in human LUSC and may serve as new targets for LUSC diagnosis and therapy.

The role of iron in Parkinson's disease monkeys assessed by susceptibility weighted imaging and inductively coupled plasma mass spectrometry.

Mounting evidences indicated that elevated iron levels in the substantia nigra (SN) have been concerned as the underlying mechanisms of neurodegenerative diseases, including Parkinson's disease (PD). The present study used the 1-Methyl-4-phenyl-1, 2, 3, 6 -tetrahydropyridine (MPTP)-treated cynomolgus monkeys for PD to evaluate the usability of SWI for assessing iron deposition in the cerebral nuclei of PD. The results showed that susceptibility-weighted imaging (SWI) phase values of the ipsilateral (MPTP-lesion side) SN of MPTP-treated monkeys were lower than those in the contralateral SN of MPTP-treated monkeys and the same side of Control monkeys, suggesting that iron deposition were elevated in the affected side SN of MPTP-treated monkeys. Whereas MPTP has not effects on the SWI phase values in other detected brain regions of monkeys, including red nucleus (RN), putamen (PUT) and caudate nucleus (CA). Furthermore, ICP-MS results showed that MPTP increased the iron levels in MPTP injection side, but no in the ipsilateral striatum. Additionally, MPTP treatment did not affect the calcium and manganese levels in the detected brain regions of monkeys. However, Pearson correlation analysis results indicated that there were not relationship between SWI phase values in MPTP-lesion side of SN with the behavioral score, tyrosine hydroxylase (TH)-positive cells number and iron levels in the MPTP-lesion side of midbrain. Taken together, the results confirm the involvement of SN iron accumulations in the MPTP-treated monkey models for PD, and indirectly verify the usability of SWI for the measurement of iron deposition in the cerebral nuclei of PD.

MicroRNA expression profile and functional analysis reveal their roles in contact inhibition and its disruption switch of rat vascular smooth muscle cells.

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.

Stable isotope labeling - dispersive solid phase extraction - liquid chromatography - tandem mass spectrometry for quantitative analysis of transsulfuration pathway thiols in human serum.

Low-molecular-weight thiols play important roles in a variety of pathological processes and are closely associated with a wide range of diseases. In this study, a selective and sensitive method was developed for the simultaneous determination of all the 7 thiols occurring in the transsulfuration pathway (Cysteine (Cys), homocysteine (Hcys), glutathione (GSH), N-acetylcysteine (Nac), cysteinylglycine (CysGly), glutamylcysteine (GluCys) and cysteamine (CA)) in human serum by in-vitro stable isotope labeling - dispersive solid phase extraction - liquid chromatography - tandem mass spectrometry analysis (IL-DSPE-LC-MS/MS). In the proposed method, a pair of stable isotope-labeling reagents, BQB (ω-bromoacetonylquinolinium bromide) and BQB-D7, were utilized to label thiols in human serum samples and thiol standards, respectively. The BQB labeled thiols which carry a positive charge were extracted and purified with C8-SO3H-based DSPE followed by LC-MS/MS analysis. Good linearities for 7 thiols occurring in the transsulfuration pathway were obtained with the coefficient of determination (R2) >0.9901. The limits of detection (LODs) were in the range of 0.7-6.0 nmol/L. The method was further applied to investigate the contents change of 7 thiols in human serum samples of type 2 diabetes mellitus (T2DM) patients and breast cancer (BC) patients. The results showed that the contents of these thiols occurring in the transsulfuration pathway significantly changed and were highly diseases-related. In addition, partial least squares discriminant analysis (PLS-DA) suggested excellent classification performance between patients and healthy controls. The findings indicated that these significantly changed thiols occurring in the transsulfuration pathway in T2DM patients and BC patients might serve as the indicator for the diagnosis of T2DM and BC. Taken together, the developed IL-DSPE-LC-MS/MS method provides a promising tool for the sensitive analysis of thiols from complex biological samples, which may promote the in-depth investigation of the functions of thiols.

High expression of miR-493-5p positively correlates with clinical prognosis of non small cell lung cancer by targeting oncogene ITGB1.

Increasing evidence supports that microRNA (miRNA)-mediated gene regulation plays a significant functional role in cancer progression. To investigate the expression and clinical significance of ITGB1 in non small cell lung cancer (NSCLC), the expression levels of ITGB1 in NSCLC tissues and human normal lung tissues were analyzed in silico using genes microarray, KEGG pathway and hierarchical clustering analysis followed by validation with quantitative RT-PCR. Our results showed that ITGB1 was upregulated in NSCLC tissues when compared with normal lung tissues. Survival analysis based on the qRT-PCR data established that ITGB1 expression was attentively related to the prognosis of NSCLC, and patients with higher ITGB1 expression had shorter overall survival (OS). Moreover, ITGB1 was confirmed to be a direct target of miR-493-5p. Furthermore, concomitant high expression of ITGB1 and low expression of miR-493-5p correlated with a shorter median OS and PFS in NSCLC patients. In conclusion, our results provide the first evidence that ITGB1 is a direct target of miR-493-5p suggesting that ITGB1 and miR-493-5p may have potential prognostic value and may be useful as tumor biomarkers for the diagnosis of NSCLC patients.

Association of microRNA-33a Molecular Signature with Non-Small Cell Lung Cancer Diagnosis and Prognosis after Chemotherapy.

OBJECTIVE: This study aims to explore the expression pattern and prognostic significance of miR-33a in non-small cell lung cancer (NSCLC) treated with adjuvant chemotherapy. METHODS: MiR-33aexpression in NSCLC was analyzed in silico using the GEO database and was subsequently confirmed by quantitative RT-PCR in 147 NSCLC biopsies. Among these, 32 of these biopsies were paired with adjacent non-neoplastic tissues. The survival analysis of NSCLC by Kaplan-Meier estimates was stratified based on miR-33a expression. In addition, multivariate survival analysis in corresponding groups of NSCLC patients was conducted by Cox proportional hazards regression model. RESULTS: The in silico analysis of miR-33a expression in NSCLC resulted to its down-regulation in different tumor types. The expression level of miR-33a was lower in each grade of NSCLC tumor biopsies than in normal lung tissues. Univariate and multivariate survival analysis further established that low miR-33a expression was an important risk factor for overall survival and disease free survival in NSCLC patients. CONCLUSION: Our study implied that miR-33a expression levels may have an essential role in NSCLC progression, and could act as a specific and sensitive biomarker for NSCLC patients who have undergone adjuvant chemotherapy.

Increased N6-methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia.

Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N(6)-methyladenosine (m(6)A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m(6)A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m(6)A-selective-binding protein (YTHDF2). We found that m(6)A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m(6)A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 - 8.853, p = 0.023). Moreover, m(6)A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = -0.288, p = 0.038; non-progressive motility: r = -0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m(6)A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m(6)A contents in sperm RNA.

Interaction of human synovial phospholipase A2 with mixed lipid bilayers: a coarse-grain and all-atom molecular dynamics simulation study.

Human secreted phospholipase A2s have been shown to promote inflammation in mammals by catalyzing the first step of the arachidonic acid pathway by breaking down phospholipids, producing fatty acids, including arachidonic acid. They bind to the membrane water interface to access their phospholipid substrates from the membrane. Their binding modes on membrane surfaces are regulated by diverse factors, including membrane charge, fluidity, and heterogeneity. The influence of these factors on the binding modes of the enzymes is not well understood. Here we have studied several human synovial phospholipase A2 (hs-PLA2)/mixed bilayer systems through a combined coarse-grain and all-atom molecular dynamics simulation. It was found that hydrophobic residues Leu2, Val3, Ala18, Leu19, Phe23, Gly30, and Phe63 that form the edge of the entrance of the hydrophobic binding pocket in hs-PLA2 tend to penetrate into the hydrophobic area of lipid bilayers, and more than half of the total amino acid residues make contact with the lipid headgroups. Each enzyme molecule forms 19-38 hydrogen bonds with the bilayer to which it binds, most of which are with the phosphate groups. Analysis of the root-mean-square deviation (rmsd) shows that residues Val30-Thr40, Tyr66-Gln80, and Lys107-Arg118 have relatively large rmsds during all-atom molecular dynamics simulations, in accordance with the observation of an enlarged entrance region of the hydrophobic binding pocket. The amino acid sequences forming the entrance of the binding pocket prefer to interact with lipid molecules that are more fluid or negatively charged, and the opening of the binding pocket would be larger when the lipid components are more fluid.

A randomized controlled study of single-agent cisplatin and radiotherapy versus docetaxel/cisplatin and radiotherapy in high-risk early-stage cervical cancer after radical surgery.

BACKGROUND: This study explored whether docetaxel/cisplatin and radiotherapy (TP-R) increases overall survival (OS) and recurrence-free survival (RFS) compared to single-agent cisplatin and radiotherapy (C-R) in patients with high-risk early-stage cervical cancer post surgery. METHODS: Patients with clinical stage IB and IIA carcinoma of the cervix, initially treated with radical hysterectomy and pelvic lymphadenectomy, and who had positive pelvic lymph nodes and/or positive margins and/or the diameter of the primary tumor ≥4 cm and/or depth of interstitial infiltration ≥1/2 and/or lymphovascular space invasion were eligible for this study. Patients were randomized to receive C-R or TP-R. Radiotherapy in both groups was external radiation (46-54 Gy) followed by high-dose rate brachytherapy (12-24 Gy). Patients were given cisplatin (40 mg/m(2)) every week for five cycles (C-R group) or docetaxel (30 mg/m(2)) and cisplatin (30 mg/m(2)) every week for five cycles (TP-R group). RESULTS: Between 2003 and 2008, 320 patients were entered onto the study. Final analyses included 285 patients. One hundred and forty patients comprised the C-R group and 145 were in the TP-R group. The 5-year OS were 74.3 % in the C-R group and 82.8 % in the TP-R group. The hazard ratio (HR) for death was 0.65 in the TP-R group (95 % CI: 0.39-1.09, P = 0.098). The RFS were 69.3 % in the C-R group and 79.3 % in the TP-R group, and the HR for recurrence was 0.64 in the TP-R group (95 % CI: 0.40-1.03, P = 0.061). Recurrence rates were similar in both groups (27 in the C-R group and 18 in the TP-R group, P = 0.112). The seriousness of late side effects was similar in the two groups, with a higher rate of reversible hematological effects in the TP-R group. CONCLUSIONS: Compared with single-agent cisplatin and radiotherapy, docetaxel/cisplatin in combination with radiotherapy does not increase OS but has the trend of increasing RFS in patients with high-risk early-stage cervical cancer. However, docetaxel/cisplatin in combination with radiotherapy is associated with a higher incidence of side effects, this effect was reversible, and the incidence of late side effects was similar in the two treatment groups.

In situ unfolded lysozyme induces the lipid lateral redistribution of a mixed lipid model membrane.

Redistribution of charged lipids induced by polyelectrolytes is an intriguing phenomenon. Proteins, due to their nature as polyelectrolytes, should also have this capacity. But their highly ordered structures may bring about more complex mechanisms. We studied the interaction between positively charged lysozyme and liposomes consisting of neutral dipalmitoylphosphatidylcholine (DPPC) and negatively charged dioleoylphosphatidylglycerol (DOPG). Interestingly, the enrichment of DOPG cannot be induced by the native and ex situ unfolded (unfolded in the absence of liposomes) lysozyme, but requires that lysozyme undergo an in situ unfolding process (unfolding in the presence of liposomes). These observations suggest that, for proteins, the capacity to induce lipid redistribution relies on not only the net charges but also the spatial distribution of the charges. During the in situ unfolding process, lysozyme reorganizes its structure into a specially unfolded structure that is rich in flat ß-sheets and more "rigid" in the presence of lipid membrane. This special spatial structure changes the charge distribution and is advantageous to the protein-membrane electrostatic interaction and thus can induce lipid redistribution.

Structural and kinetic properties of alpha-tocopherol in phospholipid bilayers, a molecular dynamics simulation study.

Structural and kinetic properties of vitamin E in biomembranes provide the key to understanding the biological functions of this lipophilic vitamin. We report a series of molecular dynamics simulations of two alpha-tocopherol/phosphatidylcholine systems and two alpha-tocopherol/phosphatidylethanolamine systems in water at 280, 310, and 350 K. The preferential position, hydrogen bonding, orientation, and dynamic properties of the alpha-tocopherol molecule in the bilayers have been examined. In all the four systems simulated, the vitamin remains in one leaflet of lipid bilayer at 280 and 310 K but flips over from one side to the other at 350 K within 200 ns of the simulation. The hydroxyl oxygen in the headgroup of alpha-tocopherol preferred a location between the third and the fifth carbon atom in the sn-2 acyl chains of the lipids. Hydrogen bonding analysis shows that the hydrogen bonds are mainly with the oxygens of the fatty acid esters rather than with the phosphate oxygens of the lipid molecule, and those with the amino groups are trivial in the case of phosphatidylethanolamines, at all three temperatures. The hydrogen bonds with phosphatidylethanolamines are more stable than those with phosphatidylcholines at low temperatures. The orientation of alpha-tocopherol in the bilayers is relatively flexible: the chromanol ring takes various tilt angles with respect to the bilayer normal, and the isoprenyl chain is mobile and able to adopt many different conformers. Calculation of lateral diffusion coefficients of alpha-tocopherol and phospholipid molecules shows that alpha-tocopherol has a comparable diffusion rate with phospholipid molecules at the gel phase but diffuses more rapidly than lipid molecules at the liquid-crystal phase.

Structural characterization on the gel to liquid-crystal phase transition of fully hydrated DSPC and DSPE bilayers.

The structural properties of fully hydrated distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine (DSPE) bilayers near the main phase transition were investigated using molecular dynamics simulations on the basis of a united-atom model. Although largely similar in their molecular structures, the two lipids were found with different molecular packing modes at temperatures below the phase transition. For DSPC, three packing modes, namely, cross-tilt, partially interdigitated, and "mixed" gel phases, were observed, while, for DSPE, the lipid tails were almost perpendicular to the lipid surface. Above the main transition temperature, both lipid bilayers transformed into a disordered liquid-crystal phase with marked greater area per lipid and gauche % of the acyl chains and smaller bilayer thickness and order parameter, in comparison with the gel phase. The transformation process of liquid-crystal to gel phase was proved to experience the nucleation and growth stages in a hexagonal manner. The electron density profiles of some major components of both lipid bilayers at various temperatures have been calculated, and the results reveal that both lipid bilayers have less interdigitation around the main transition temperature.