Liver microRNA hsa-miR-125a-5p in HBV Chronic Infection: Correlation with HBV Replication and Disease Progression

To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak''s scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p<0.0001). Higher liver hsa-miR-125a-5p concentrations were observed in patients with HBV-DNA plasma level >10³ IU/ml (p<0.02), in those with HAI >6 (p = 0.02) and those with fibrosis score >2 (p<0.02) than in patients with lower scores. Higher HBV-DNA liver concentrations were found in patients with abnormal AST (p = 0.005) and ALT serum levels (p = 0.05), in those with serum HBV DNA higher than 10E3 IU/mL (p = 0.001) and those with fibrosis score >2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95%  = 1.08–16.43, p<0.05, for HAI >6; O.R. = 3.12, C.I. 95%  = 1.17–8.27, p<0.05, for fibrosis score >2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression.     

Increased FGF3 and FGF4 gene dosage is a risk factor for craniosynostosis

Interstitial duplications involving chromosome 11q have rarely been reported in the literature and mainly represent large, cytogenetically detectable rearrangements associated with a wide and variable spectrum of neurodevelopmental disorders. We report on a patient affected by intellectual disability, craniosynostosis, and microcephaly. Array-CGH analysis identified a de novo 290 kb interstitial duplication of chromosome 11q13.3 including the FGF3 and FGF4 genes. Clinical comparison of our patient with those previously reported with overlapping 11q duplications allows us to define the minimal duplicated region associated with craniosynostosis and strongly supports the hypothesis that the constitutional increased dosage of the FGF3 and FGF4 genes is a risk factor for craniosynostosis in humans.     

Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

microRNA-377-3p downregulates the oncosuppressor T-cadherin in colorectal adenocarcinoma cells

Colorectal cancer (CRC) is the second leading cause of death of malignant tumors worldwide. Recent studies point to a role for the adiponectin-receptor axis in colorectal carcinogenesis, and in particular to the oncosuppressive properties of the T-cadherin receptor. In addition, the loss of T-cadherin expression in tumor tissues has been linked to cancer progression and attributed to aberrant methylation of its promoter. Recognizing the pivotal role of microRNAs in CRC, this study explores their possible contribution to the downregulation of T-cadherin. A systematic bioinformatics analysis, restricted by microRNA expression data in the colon or in cultured colorectal cell lines, predicted twelve top-ranking target miRNA sites within the 3ʹ UTR of T-cadherin. Experimental validation analyses based on luciferase reporter constructs and miRNA mimic or miRNA inhibitor transfections toward colorectal adenocarcinoma cell lines indicated that miR-377-3p was able to directly bind to the T-cadherin sequence, and thus downregulating its expression. Given the oncogenic activity of miR-377 and the oncosuppressive activity of T-cadherin in CRC, the regulatory circuit highlighted in this study may add new insights into molecular mechanisms driving colorectal carcinogenesis, and perspectively it could be exploited to identify novel biomarkers and therapeutic targets.     

Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.     

The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging

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Sickle cell disease in mice is associated with sensitization of sensory nerve fibers

The pain phenotype in sickle cell disease (SCD) patients is highly variable. A small percentage of SCD patients experience many vaso-occlusive crises/year, 5% of patients account for over 30% of pain episodes, while 39% report few episodes of severe pain. Clearly, a better understanding of the pathobiology of SCD is needed to improve its therapy. Humanized sickle cell mice recapitulate several phenotypes of SCD patients and provide a model for the study of SCD pain. Researchers have shown that one strain of humanized SCD mice, the BERK strain, has abnormal pain phenotype. However, the nociception phenotype of another humanized SCD mouse strain, the Townes strain, has not been described. In a large cross-sectional study of BERK and Townes SCD mice, we examined thermosensory response and sensory nerve fiber function using sine-wave electrical stimulation at 2000, 250, and 5 Hz to stimulate preferentially Aβ, Aδ, and C sensory nerve fibers, respectively. We found that BERK and Townes mice, compared to respective controls, had decreases in 2000, 250, and 5 Hz current vocalization thresholds in patterns that suggest sensitization of a broad spectrum of sensory nerve fibers. In addition, the pattern of sensitization of sensory fibers varied according to strain, sex, age, and mouse genotype. In a similarly variable pattern, Townes and BERKs also had significantly altered sensitivity to noxious thermal stimuli in agreement with what has been shown by others. In summary, the analysis of somatosensory function using sine-wave electrical stimulation in humanized sickle cell mice suggests that in SCD, both myelinated and unmyelinated, fibers are sensitized. The pattern of sensory fiber sensitization is distinct from that observed in pain models of neuropathic and inflammatory pain. These findings raise the possibility that sensitization of a broad spectrum of sensory fibers might contribute to the altered and variable nociception phenotype in SCD.     

Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

Background

The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs.

Results

A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology.

Conclusions

A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.     

Reward-related dorsal striatal activity differences between former and current cocaine dependent individuals during an interactive competitive game

Cocaine addiction is characterized by impulsivity, impaired social relationships, and abnormal mesocorticolimbic reward processing, but their interrelationships relative to stages of cocaine addiction are unclear. We assessed blood-oxygenation-level dependent (BOLD) signal in ventral and dorsal striatum during functional magnetic resonance imaging (fMRI) in current (CCD; n?=?30) and former (FCD; n?=?28) cocaine dependent subjects as well as healthy control (HC; n?=?31) subjects while playing an interactive competitive Domino game involving risk-taking and reward/punishment processing. Out-of-scanner impulsivity-related measures were also collected. Although both FCD and CCD subjects scored significantly higher on impulsivity-related measures than did HC subjects, only FCD subjects had differences in striatal activation, specifically showing hypoactivation during their response to gains versus losses in right dorsal caudate, a brain region linked to habituation, cocaine craving and addiction maintenance. Right caudate activity in FCD subjects also correlated negatively with impulsivity-related measures of self-reported compulsivity and sensitivity to reward. These findings suggest that remitted cocaine dependence is associated with striatal dysfunction during social reward processing in a manner linked to compulsivity and reward sensitivity measures. Future research should investigate the extent to which such differences might reflect underlying vulnerabilities linked to cocaine-using propensities (e.g., relapses).     

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head–head (8), tail–tail (9) or head–tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2–BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail–tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.