Epigenetic response of endothelial cells to different wall shear stress magnitudes: A report of new mechano-miRNAs

Endothelial cells (ECs) respond to flow stress via a variety of mechanisms, leading to various intracellular responses that can modulate the vessel wall and lead to diseases if the flow is disturbed. Mechano-microRNAs (miRNAs) are a subset of miRNAs in the ECs that are flow responsive. Mechano-miRNAs were shown to be related to atherosclerosis pathophysiology, and a number of them were identified as pathologic. Here, we exposed human carotid ECs to different wall shear stresses (WSS), high and low, and evaluated the response of miRNAs by microarray and quantitative polymerase chain reaction analysis. We discovered five new mechano-miRNAs that were not reported in that context previously to the best of our knowledge. Moreover, functional pathway analysis revealed that under low WSS conditions, several pathways regulating apoptosis are affected. In addition, KLF2 and KLF4, known atheroprotective genes, were downregulated under low WSS and upregulated under high WSS. KLF2 and VCAM1, both angiogenic, were upregulated under high WSS. NOS3, which is vascular protective, was also upregulated with higher WSS. On the contrary, ICAM-1 and E-selectin, both atherogenic and proinflammatory, were upregulated with high WSS. Collectively, the epigenetic landscape with the gene expression analysis reveals that low WSS is associated with a proapoptotic state, while high WSS is associated with a proliferative and proinflammatory state.     

Preface

"Although few people die of pain,many people die in pain and even more live in pain"——IASP On October11,2004,International Association for the Study of Pain(IASP)     

The influence of RAMP1 overexpression on CGRP‐induced osteogenic differentiation in MG‐63 cells in vitro: An experimental study

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor‐like receptor (CRLR) in MG‐63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG‐63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG‐63 cells. Real‐time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post‐induction of differentiation. The CGRP‐induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP‐mediated differentiation of MG‐63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP‐induced MG‐63 differentiation. J. Cell. Biochem. 114: 314–322, 2013. © 2012 Wiley Periodicals, Inc.     

Demonstration of rod and cone photoreceptors in the lamprey retina by freeze-replication and immunofluorescence

Summary In common with other cyclostomata, the Japanese river lamprey (Lampetra japonica) has a retina consisting of distinct types of photoreceptor cells called long and short photoreceptor cells. After freeze-fracture, disc membranes of these photoreceptor cells were characterized in common by a homogeneous distribution of intramembrane particles on the protoplasmic fracture faces, in contrast to those of the myeloid bodies bearing scattering particles.Immunofluorescent examination was applied to the retina with monoclonal antibodies raised against bovine and chicken rhodopsins. Positive immunoreactivity was found to be limited to outer segments of the short cell, leaving the entire body of the long cell and all other components of the retina negative. The results suggest that the short cell is more closely related to a rod-type photoreceptor cell characterized by rhodopsin as its visual pigment.     

Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria

The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F₀F₁-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.     

Expression of γ-H2AX and patient prognosis in breast cancer cohort

H2AX phosphorylation is a novel marker of DNA double-stranded breaks. In the present study, we assessed the γ-H2AX expression, its association with other clinicopathologic characteristics, and the prognosis in a cohort of 97 patients with breast cancer. Ninety-seven specimens of tumor tissue and 77 adjacent normal tissues from patients with breast cancer were examined. All patients underwent modified radical mastectomy or local tumor resection without lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Patients were followed after surgery for a mean duration of 70.1 ± 18.7 months (range, 6-93 months). The γ-H2AX staining was positive in 27 (27.8%) patients. The positive rates of H2AX were 26.0% and 2.6% in tumor tissue and adjacent normal tissues, respectively. γ-H2AX positive status was negatively associated with TNM staging, with 24 positive cases (32.4%) in TNM staging I-II, while no positive cases in TNM staging III-IV (P = 0.026). Sixteen patients (16.5%) died during the follow-up. No significant association between γ-H2AX expression and patient survival was detected. The unadjusted HR (hazard ratio) for γ-H2AX positive was 0.84 (95% CI: 0.27, 2.60). In TNM staging subgroup analysis, death only occurred in γ-H2AX negative patients. Our study is the first study to demonstrate that expression of γ-H2AX is associated with TNM staging. Due to the small sample and limited follow-up time, we did not observe a significant association between γ-H2AX and patient survival. γ-H2AX expression could be a potential biomarker for cancer diagnosis and prediction, and further studies are in need.     

Serum profiling by MALDI-TOF mass spectrometry as a diagnostic tool for domoic acid toxicosis in California sea lions

Background

There are currently no reliable markers of acute domoic acid toxicosis (DAT) for California sea lions. We investigated whether patterns of serum peptides could diagnose acute DAT. Serum peptides were analyzed by MALDI-TOF mass spectrometry from 107 sea lions (acute DAT n = 34; non-DAT n = 73). Artificial neural networks (ANN) were trained using MALDI-TOF data. Individual peaks and neural networks were qualified using an independent test set (n = 20).

Results

No single peak was a good classifier of acute DAT, and ANN models were the best predictors of acute DAT. Performance measures for a single median ANN were: sensitivity, 100%; specificity, 60%; positive predictive value, 71%; negative predictive value, 100%. When 101 ANNs were combined and allowed to vote for the outcome, the performance measures were: sensitivity, 30%; specificity, 100%; positive predictive value, 100%; negative predictive value, 59%.

Conclusions

These results suggest that MALDI-TOF peptide profiling and neural networks can perform either as a highly sensitive (100% negative predictive value) or a highly specific (100% positive predictive value) diagnostic tool for acute DAT. This also suggests that machine learning directed by populations of predictive models offer the ability to modulate the predictive effort into a specific type of error.     

A Clinical Trial to Evaluate the Efficacy of α-Viniferin in Staphylococcus aureus – Specific Decolonization without Depleting the Normal Microbiota of Nares

Staphylococcus aureus is currently a significant multidrug-resistant bacterium, causing severe healthcare-associated and community-acquired infections worldwide. The current antibiotic regimen against this pathogen is becoming ineffective due to resistance, in addition, they disrupt the normal microbiota. It highlights the urgent need for a pathogen-specific drug with high antibacterial efficacy against S. aureus. α-Viniferin, a bioactive phytochemical compound, has been reported to have excellent anti-Staphylococcus efficacy as a topical agent. However, so far, there were no clinical trials that have been conducted to elucidate its efficacy. The present study aimed to investigate the antibacterial efficacy of α-viniferin against S. aureus in a ten-day clinical trial. Based on the results, α-viniferin showed 50% minimum inhibitory concentrations (MIC₅₀ values) of 7.8 μg/ml in culture broth medium. α-Viniferin was administered in the nares three times a day for ten days using a sterile cotton swab stick. Nasal swab specimens were collected before (0 days) and after finishing the trial (10^th day), and then analyzed. In the culture and RT-PCR-based analysis, S. ureus was reduced significantly: 0.01. In addition, 16S ribosomal RNA-based amplicon sequencing analysis showed that S. aureus reduced from 51.03% to 23.99% at the genus level. RNA-seq analysis was also done to gain insights into molecular mechanisms of α-viniferin against S. aureus, which revealed that some gene groups were reduced in 5-fold FC cutoff at two times MIC conditions. The study results demonstrate α-viniferin as a potential S. aureus-specific drug candidate.     

Oxidative stress, metabolism of ethanol and alcohol-related diseases

Alcohol-induced oxidative stress is linked to the metabolism of ethanol. Three metabolic pathways of ethanol have been described in the human body so far. They involve the following enzymes: alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS) and catalase. Each of these pathways could produce free radicals which affect the antioxidant system. Ethanol per se, hyperlactacidemia and elevated NADH increase xanthine oxidase activity, which results in the production of superoxide. Lipid peroxidation and superoxide production correlate with the amount of cytochrome P450 2E1. MEOS aggravates the oxidative stress directly as well as indirectly by impairing the defense systems. Hydroxyethyl radicals are probably involved in the alkylation of hepatic proteins. Nitric oxide (NO) is one of the key factors contributing to the vessel wall homeostasis, an important mediator of the vascular tone and neuronal transduction, and has cytotoxic effects. Stable metabolites--nitrites and nitrates--were increased in alcoholics (34.3 +/- 2.6 vs. 22.7 +/- 1.2 micromol/l, p < 0.001). High NO concentration could be discussed for its excitotoxicity and may be linked to cytotoxicity in neurons, glia and myelin. Formation of NO has been linked to an increased preference for and tolerance to alcohol in recent studies. Increased NO biosynthesis also via inducible NO synthase (NOS, chronic stimulation) may contribute to platelet and endothelial dysfunctions. Comparison of chronically ethanol-fed rats and controls demonstrates that exposure to ethanol causes a decrease in NADPH diaphorase activity (neuronal NOS) in neurons and fibers of the cerebellar cortex and superior colliculus (stratum griseum superficiale and intermedium) in rats. These changes in the highly organized structure contribute to the motor disturbances, which are associated with alcohol abuse. Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression, and they correlate significantly with the disease severity. The low-density lipoprotein (LDL) oxidation is supposed to be one of the most important pathogenic mechanisms of atherogenesis, and antibodies against oxidized LDL (oxLDL) are some kind of epiphenomenon of this process. We studied IgG oxLDL and four APA (anticardiolipin, antiphosphatidylserine, antiphosphatidylethanolamine and antiphosphatidylcholine antibodies). The IgG oxLDL (406.4 +/- 52.5 vs. 499.9 +/- 52.5 mU/ml) was not affected in alcoholic patients, but oxLDL was higher (71.6 +/- 4.1 vs. 44.2 +/- 2.7 micromol/l, p < 0.001). The prevalence of studied APA in alcoholics with mildly affected liver function was higher than in controls, but not significantly. On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide range of IgG oxLDL titers in a healthy population. These parameters do not appear to be very promising for the evaluation of the risk of atherosclerosis. Free radicals increase the oxidative modification of LDL. This is one of the most important mechanisms, which increases cardiovascular risk in chronic alcoholic patients. Important enzymatic antioxidant systems - superoxide dismutase and glutathione peroxidase - are decreased in alcoholics. We did not find any changes of serum retinol and tocopherol concentrations in alcoholics, and blood and plasma selenium and copper levels were unchanged as well. Only the zinc concentration was decreased in plasma. It could be related to the impairment of the immune system in alcoholics. Measurement of these parameters in blood compartments does not seem to indicate a possible organ, e.g. liver deficiency.