Gene expression profiling of endometrium versus bone marrow-derived mesenchymal stem cells: upregulation of cytokine genes

Postulated Stem/progenitor cells involved in endometrium regeneration are epithelial, mesenchymal, and endothelial. Bone marrow (BM) has been implicated in endometrial stem cells. We aimed at studying gene expression profiling of endometrial mesenchymal stem cells compared to BM MSCS to better understand their nature and functional phenotype. Endometrial tissues were obtained from premenopausal hysterectomies (n = 3), minced and enzymatically digested as well as Normal BM aspirates (n=3). Immunophenotyping, differentiation to mesoderm, and proliferation were studied. The expression profile of 84 genes relevant to mesenchymal stem cells was performed. Fold change calculations were determined with SA Biosciences data analysis software. VEGF, G-CSF, and GM-CSF in cultures supernatants of MSCs were assayed by Luminex immunoassay. Endo MSCs possess properties similar to BM MSCs. Cumulative population doubling was significantly higher in Endo MSCs compared to BM MSCs (p < 0.001). 52 core genes were shared between both generated MSCs including stemness, self-renewal, members of the Notch, TGFB, FGF, and WNT.16 downregulated genes (VCAM, IGF1)and 16 upregulated in Endo MSCs compared to BM (p < 0.05 → fourfolds). They included mostly cytokine and growth factor genes G-CSF, GM-CSF, VWF, IL1b, GDF15, and KDR. VEGF and G-CSF levels were higher in Endo MSCs supernatants (p < 0.0001). Cells sharing MSC and endothelial cell characteristics could be isolated from the human endometrium. Endo MSCs share a core genetic profile with BM MSCs including stemness. They show upregulation of genes involved in vasculogenesis, angiogenesis, cell adhesion, growth proliferation, migration, and differentiation of endothelial cells, all contributing to endometrial function.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

Size class homogeneity of repeat lengths and evolutionary divergence of ribosomal RNA genes in fishes as studied by restriction fragment length analysis

Fish ribosomal RNA genes (rDNA) have been compared by restriction endonuclease digestion followed by Southern hybridization using rRNA or cloned rRNA genes as labelled probes. In several species belonging to the orders Cypriniformes and Perciformes, the simple restriction patterns revealed a high degree of size class homogeneity among the rDNA repeats and similar restriction map within a species. Different species have different restriction patterns and fragment lengths arising mostly out of different length of the nontranscribed spacer. Polymorphic restriction sites are present in some species. The species-specific differences in fragment lengths produced in rDNA by some restriction enzymes can thus be used to study interspecific fish hybrids.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells

Cystic fibrosis transmembrane conductance regulator (CFTR) acts as a cAMP-dependent chloride channel, has been studied in various types of cells. CFTR is abundantly expressed in vascular smooth muscle cells and closely linked to vascular tone regulation. However, the functional significance of CFTR in basilar vascular smooth muscle cells (BASMCs) remains elusive. Accumulating evidence has shown the direct role of CFTR in cell apoptosis that contributes to several main pathological events in CF, such as inflammation, lung injury and pancreatic insufficiency. We therefore investigated the role of CFTR in BASMC apoptotic process induced by hydrogen peroxide (H₂O₂). We found that H₂O₂-induced cell apoptosis was parallel to a significant decrease in endogenous CFTR protein expression. Silencing CFTR with adenovirus-mediated CFTR specific siRNA further enhanced H₂O₂-induced BASMC injury, mitochondrial cytochrome c release into cytoplasm, cleaved caspase-3 and -9 protein expression and oxidized glutathione levels; while decreased cell viability, the Bcl-2/Bax ratio, mitochondrial membrane potential, total glutathione levels, activities of superoxide dismutase and catalase. The pharmacological activation of CFTR with forskolin produced the opposite effects. These results strongly suggest that CFTR may modulate oxidative stress-related BASMC apoptosis through the cAMP- and mitochondria-dependent pathway and regulating endogenous antioxidant defense system.     

Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.     

The effects of water flow and sedimentation on interactions between massive Porites and algal turf

Interactions between scleractinian corals and benthic algae can be an important process structuring reef communities, yet interaction dynamics are not fixed and may be influenced by abiotic factors such as sedimentation, a process often underlying reef degradation. However, rates of sedimentation and the effects of trapped sediments may be influenced by water flow. The first goal of this study was to quantify gradients in sedimentation and flow along fringing and back reefs of the north shore of Moorea, French Polynesia, and determine whether such gradients correlate with changes in the frequency and outcomes of massive Porites–algal turf interactions. On the back reef, the frequency of Porites–algal turf interactions and the competitive success of algal turfs increased significantly with decreasing flow. Sedimentation, however, was not a significant driver of the observed patterns. Along fringing reefs, in the absence of a flow gradient, it was the gradient in sedimentation that best explained spatial variation in Porites–algal turf interaction frequencies and the competitive success of algal turfs. The second goal was to quantify the separate and combined effects of flow and sedimentation on Porites–algal turf interactions in a laboratory setting. The combined effects of low flow and sedimentation significantly increased the area of Porites tissue damaged when in contact with algal turf, while high flow attenuated the negative effects of sedimentation. Together, these results implicate flow and sedimentation as important drivers of biological interactions between massive Porites and algal turf.     

Myeloid-derived suppressor cells and their role in CTLA-4 blockade therapy

Immune checkpoints are a series of inhibitory pathways that are crucial for modulating the intensity and duration of immune response. Among these checkpoints, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) has been shown to be a key regulator of the early activation of naïve and memory T cells. Immune checkpoint blockade is emerging as one of the most promising therapeutic approaches directed toward the activation of the immune response against tumors. The first of these therapies that has been FDA approved is ipilimumab, a fully human monoclonal antibody that blocks CTLA-4. The in cis effects that CTLA-4 blockade has on T cells have been properly described, but there are still questions to be answered regarding the indirect or in trans effects. One of the alternative cellular populations that may play a role in the outcome of CTLA-4 blockade therapy is myeloid-derived suppressor cells (MDSCs), which have recently been associated with clinical outcome in advanced melanoma. In addition to this, MDSCs have been shown to be decreased in number and functional potential after treatment with ipilimumab. A better clarification of what effects CTLA-4 blockade may have on these cellular populations is likely to provide insights on possible predictive biomarkers for CTLA-4 blockade therapy.     

PGC-1α is associated with C2C12 Myoblast differentiation

PGC-1α has been implicated as an important mediator of functional capacity of skeletal muscle. However, the role of PGC-1α in myoblast differentiation remains unexplored. In the present study, we observed a significant up-regulation of PGC-1α expression during the differentiation of murine C2C12 myoblast. To understand the biological significance of PGC-1α up-regulation in myoblast differentiation, C2C12 cells were transfected with murine PGC-1α cDNA and siRNA targeting PGC-1α, respectively. PGC-1α over-expressing clones fused to form typical myotubes with higher mRNA level of myosin heavy chain isoform I (MyHCI) and lower MyHCIIX. No obvious differentiation was observed in PGC-1α-targeted siRNA-transfected cells with marked decrement of mRNA levels of MyHCI and MyHCIIX. Furthermore, PGC-1α increased the expression of MyoD and MyoG in C2C12 cells, which controlled the commitment of precursor cells to myotubes. These results indicate that PGC-1α is associated with myoblast differentiation and elevates MyoD and MyoG expression levels in C2C12 cells.     

SNPs in MicroRNA-Binding Sites in the ITGB1 and ITGB3 3′-UTR Increase Colorectal Cancer Risk

The purpose of the study was to investigate the potential associations between single-nucleotide polymorphisms (SNPs) in microRNA (miRNA)-binding sites in the integrin beta-1 (ITGB1) gene and integrin beta-3 (ITGB3) gene 3′-untranslated regions, and colorectal cancer (CRC) susceptibility in a Chinese population. A hospital-based case–control study was performed in 200 patients with CRC and 200 matched healthy donors. Two SNPs in miRNA binding of ITGB1 and ITGB3 genes (rs17468 and rs2317676) were genotyped by polymerase chain reaction-restrict fragment length polymorphism assay. The association between genotypes and CRC risk was evaluated by computing the odds ratio (OR) and 95 % confidence interval (CI) from multivariate unconditional logistic regression analyses. The frequency of the T genotype in ITGB1 rs17468 and G genotype in ITGB3 rs2317676 occurred more frequently in CRC patients than in controls (P < 0.05). We found that CT and TT genotypes of rs17468 were associated with a significantly increased risk of CRC (OR = 1.67, 95 % CI = 1.090–2.559 for CT + TT vs. CC), also the AG and GG genotype in ITGB3 rs2317676 (OR = 1.65, 95 % CI = 1.114–2.458 for AG + GG vs. AA). In conclusion, our results showed that both the ITGB1 rs17468 SNP and ITGB3 rs2317676 SNP were associated with an increased risk of CRC, which suggests that these 2 SNPs might contribute to CRC risk in a Chinese population.     

Development of an immunoenzyme method for detection and quantitative determination of microcystins

An indirect competitive immunoenzyme method for the quantitative estimation of microcystins (MCs) in water (MC-ELISA) using prepared MC-specific polyclonal antibodies was developed. The threshold concentration of the most widespread and highly toxic MC-LR, which was reliably detected using MC-ELISA, was 0.05 ± 0.01 ng/mL; the 50% inhibition concentration was 0.41 ± 0.05 ng/mL; and the concentration range for the quantitative estimation of MC-LR was 0.1–5.0 ng/mL. The MC-ELISA made it possible to detect MC-LR in water at concentrations 10–20 times lower than the World Health Organization guideline level for drinking water and 100–200 times lower than the allowable MC concentrations in water bodies. A group of cross-reacting MCs and nodularin was detected using MC-ELISA. This method can be applied for monitoring MC concentrations in water bodies and drinking water.     

Expression profile of MAGI2 gene as a novel biomarker in combination with major deregulated genes in prostate cancer

Complex molecular changes that occur during prostate cancer (PCa) progression have been described recently. Whole genome sequencing of primary PCa samples has identified recurrent gene deletions and rearrangements in PCa. Specifically, these molecular events disrupt the gene loci of phosphatase and tensin homolog (PTEN) and membrane-associated guanylate kinase inverted-2 (MAGI2). In the present study, we analyzed the expression profile of MAGI2 gene in a cohort of clinical PCa (n = 45) and benign prostatic hyperplasia (BPH) samples (n = 36) as well as three PCa cell lines. We also studied the expression of PCa-related genes, including PTEN, NKX3.1, SPINK1, DD3, AMACR, ERG, and TMPRSS2-ERG fusion in the same samples. The expression of MAGI2 mRNA was significantly down-regulated in PC3, LNCaP and DU-145 PCa cell lines (p = 0.000), and also in clinical tumor samples (Relative expression = 0.307, p = 0.002, [95 % CI 0.002–12.08]). The expression of PTEN, NKX3.1, SPINK1, DD3, and AMACR genes was significantly deregulated in prostate tumor samples (p range 0.000–0.044). A significant correlation was observed between MAGI2 and NKX3.1 expression in tumor samples (p = 0.006). Furthermore, the inclusion of MAGI2 in the gene panel improved the accuracy for discrimination between PCa and BPH samples with the sensitivity and specificity of 0.88 [CI 0.76–0.95] and 0.83 [CI 0.68–0.92], respectively. The data presented here suggest that MAGI2 gene can be considered as a novel component of gene signatures for the detection of PCa.     

Hyperactivity and sensation seeking as autoregulatory attempts to stabilize brain arousal in ADHD and mania?

Hypoarousal as indicated by skin conductance and electroencephalography (EEG) has been discussed as a pathogenetic factor in attention-deficit/hyperactivity disorder (ADHD). The aim of this paper was to review these arousal-related pathogenetic concepts and to present the more recently proposed vigilance regulation model of affective disorders and ADHD. The latter builds on methodological advances in classifying short EEG segments into vigilance stages (Vigilance Algorithm Leipzig, VIGALL), indicating different states of global brain function (“brain arousal”). VIGALL allows the objective assessment of vigilance regulation under defined conditions, e.g. how fast vigilance declines to lower vigilance stages associated with drowsiness during 15–20-min EEG recordings under resting conditions with eyes closed. According to the vigilance regulation model, the hyperactivity and sensation seeking observed in overtired children, ADHD and mania may be interpreted as an autoregulatory attempt to create a stimulating environment in order to stabilize vigilance. The unstable regulation of vigilance observed in both mania and ADHD may thus explain the attention deficits, which become especially prominent in monotonous sustained attention tasks. Among the arguments supporting the vigilance regulation model are the facts that destabilizing vigilance (e.g. via sleep deprivation) can trigger or exacerbate symptoms of ADHD or mania, whereas stabilizing vigilance (e.g. via psychostimulants, reducing sleep deficits) alleviates these symptoms. The potential antimanic effects of methylphenidate are presently being studied in an international randomized controlled trial. We propose vigilance regulation as a converging biomarker, which could be useful for identifying treatment responders to psychostimulants and forming pathophysiologically more homogeneous ADHD subgroups for research purposes.     

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.