Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).España, Junta de Andalucía grant P07-CVI-02792 and group BIO-284España, MINECO y Fondo Social Europeo grant BFU2013-41712-

Genetic testing for hepatocellular carcinoma: An ambitious goal still to achieve

Background & Aims: A single nucleotide polymorphism 61⁄G (rs4444903) in the epidermal growth factor (EGF) gene has been associated, in two case–control studies, with hepatocellular carcinoma (HCC). We tested associations between demographic, clinical, and genetic data and development of HCC, and developed a simple predictive model in a cohort of patients with chronic hepatitis C and advanced fibrosis. Methods: Black and white subjects from the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) trial (n = 816) were followed up prospectively for development of a definite or presumed case of HCC for a median time period of 6.1 years. We used the Cox proportional hazards regression model to determine the hazard ratio for risk of HCC and to develop prediction models. Results: Subjects with EGF genotype G/G had a higher adjusted risk for HCC than those with genotype A/A (hazard ratio, 2.10; 95% confidence interval, 1.05–4.23; P = .03). After adjusting for EGF genotype, blacks had no increased risk of HCC risk compared with whites. Higher serum levels of EGF were observed among subjects with at least one G allele (P = .08); the subset of subjects with EGF G/G genotype and above-median serum levels of EGF had the highest risk of HCC. We developed a simple prediction model that included the EGF genotype to identify patients at low, intermediate, and high risk for HCC; 6-year cumulative HCC incidences were 2.3%, 10.4%, and 26%, respectively. Conclusions: We associated the EGF genotype G/G with increased risk for HCC; differences in its frequency among black and white subjects might account for differences in HCC incidence between these Journal of Hepatology 2

EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON ENERGY METABOLISM

Type 2 diabetes mellitus (T2DM) is the most common metabolic disease in the world. Maintenance of glucose homeostasis depends on a complex interplay between the insulin responsiveness of skeletal muscle, liver, adipose tissue and glucose-stimulated insulin secretion by pancreatic beta cells. Defects in these organs are responsible for insulin resistance and progression to hyperglycemia. Understanding the integrated pathophysiology initiating the development of insulin resistance should extend our capacity to identify novel therapeutic targets for the prevention and/or treatment of T2DM. This biology remains incompletely characterized, in part, due to the interaction of multiple organ systems. The complexity of this biology is further underscored by the progressive changes in the systemic milieu including the onset of hyperinsulinemia, elevated circulating free fatty acids and triglycerides, hyperglycemia, and the activation of systemic immune system during the development of T2DM. In skeletal muscle, loss of mitochondrial function is evident in some insulin-resistant subjects years before they develop diabetes. Mitochondria are particularly important for skeletal muscle function, given the high oxidative demands imposed on this tissue by intermittent contraction. Moreover, muscle cells must maintain metabolic flexibility, defined as the ability to rapidly modulate substrate oxidation as a function of hormonal and energetic conditions. The molecular mechanisms that control mitochondrial number and function remain poorly understood, and only a few transcription factors or coactivators (e.g., PGC-1\u3b1, NRF1, Tfam) have been associated with this process. Notably, skeletal muscle differentiation and remodelling are also controlled at the epigenetic level, via transcriptional modulation of key genes in mitochondrial biogenesis and oxidative metabolism, involving enzymes, such as members of the histone deacetylase (HDAC) family, in particular those belonging to class I and class II, which modulate post-translational modifications on target proteins. The current knowledge on HDACs is that they function in general as transcriptional repressors, however their role in vivo is likely more complex. Less is known on the effects of HDACs modulators on energy metabolism, however a recent study reported that supplementation with sodium butyrate, a dietary component active as HDAC inhibitor, promotes energy expenditure and mitochondrial function in mice fed with a high fat diet212. Given the importance of skeletal muscle metabolism in insulin resistance/diabetes, and given the role of HDACs in skeletal muscle biology, it is reasonable to speculate that modulation of these enzymes would play a role in this pathology that deserves to be investigated more deeply. Based on the evidences that mitochondrial dysfunction is often associated to whole body metabolic dysregulation, aim of this study is to better understand the role of histone deacetylases in the regulation of mitochondrial biogenesis and in the modulation of all these mechanisms underlying the pathophisiology of insulin resistance. In C2C12 myotubes treated with pan and class selective HDAC inhibitors (HDACi), such as SAHA(pan-inhibitor), MS275 (Class I HDAC inhibitor) and MC1568 (Class II HDAC inhibitor), transcriptome analysis revealed an increase of OXPHOS genes and of genes encoding fatty acid catabolic enzymes, following treatment with pan or class I HDACi. Moreover, staining of myotubes treated with SAHA and MS275 showed an increase of mitochondrial density and activity, coupled with an increase in mitochondrial DNA content. In Db/Db obese and diabetic mice we observed that treatments with SAHA and MS275 reduce glycemia, triglycerides, plasma insulin land transaminases levels and improves glucose clearance and insulin sensitivity. In vivo metabolic study revealed an increase of oxygen consumption, a net decrease of the respiratory exchange ratio and an increased heat production, implying a more oxidative metabolism, in the MS275 treated group. In order to characterize the effects of HDACi in different tissues, we performed microarray analysis in skeletal muscles, liver, brown and white adipose tissues and histological analysis. In the skeletal muscle of mice treated with MS275, and with SAHA to a lesser degree, we observed a general increase in OXPHOS genes and in genes involved in lipid and glucose metabolism. These observations were also supported by the increased oxidative capacity highlighted by SDH staining in gastrocnemius sections. In brown fat we found an increased expression of brown adipocytes markers , such as PRDM16, CIDEA, UCP1, ELOVL3 and DIO2, and an induction of mitochondrial biogenesis, after MS275 administration. In parallel, adypocyte size appeared smaller respect to the adipocytes of the control group. In white adipose tissue, we observed an increase of adipocytes markers and, even in this case, an induction of mitochondrial biogenesis; adipocytes, beyond being smaller, also showed a reduced macrophages infiltration. Gene expression also revealed an increased expression of brown adipocytes markers, such as UCP1, also confirmed by immunohistochemistry assay, ADRB3, ELOVL3 and DIO2. Nevertheless, white adipocytes remained PRDM16 negative. Collectively, our results suggest that HDACs, and in particular Class I HDACs, play an unexpected role in energy metabolism, induce mitochondrial biogenesis in different tissues and may represent key regulators in diseases based on metabolic alterations

Post-liver transplantation graft biopsies should not be used to assess the IL28B donor genotype in HCV recipients.

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Problematic internet use and study motivation in higher education

The current study explored the relationship between problematic internet use (PIU) and motivation to learn, and examined psychological and social factors mediating this relationship. Two hundred and eighty-five students in an Italian University were recruited for the current study. There was a negative relationship between PIU and motivation to study: a negative impact on learning strategies, meaning that the students found it harder to organize their learning productively; and PIU also positively associated with test anxiety. The current results also demonstrated that there was partial mediation of this effect of PIU on learning strategies in terms of loneliness. This suggest at those with high levels of PIU may be particularly at risk from lower motivations to study, and, hence, lower actual generalized academic performance due to a number of consequences of PIU

NADPH-thioredoxin reductase c mediates the response to oxidative stress and thermotolerance in the cyanobacterium anabaena sp. pcc7120

NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2- Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (LintrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatment

Analyzing the Complex Regulatory Landscape of Hfq – an Integrative, Multi-Omics Approach

The ability of bacteria to respond to environmental change is based on the ability to coordinate, redirect and fine-tune their genetic repertoire as and when required. While we can learn a great deal from reductive analysis of individual pathways and global approaches to gene regulation, a deeper understanding of these complex signaling networks requires the simultaneous consideration of several regulatory layers at the genome scale. To highlight the power of this approach we analyzed the Hfq transcriptional/translational regulatory network in the model bacterium Pseudomonas fluorescens. We first used extensive ‘omics’ analyses to assess how hfq deletion affects mRNA abundance, mRNA translation and protein abundance. The subsequent, multi-level integration of these datasets allows us to highlight the discrete contributions by Hfq to gene regulation at different levels. The integrative approach to regulatory analysis we describe here has significant potential, for both dissecting individual signaling pathways and understanding the strategies bacteria use to cope with external challenges

In vitro characterization of mitochondrial function and structure in rat and human cells with a deficiency of the NADH:ubiquinone oxidoreductase Ndufc2 subunit

Ndufc2, a subunit of the NADH:ubiquinone oxidoreductase, plays a key role in the assembly and activity of complex I within the mitochondrial OXPHOS chain. Its deficiency has been shown to be involved in diabetes, cancer and stroke. To improve our knowledge on the mechanisms underlying the increased disease risk due to Ndufc2 reduction, we performed the present in vitro study aimed at the fine characterization of the derangements in mitochondrial structure and function consequent to Ndufc2 deficiency. We found that both fibroblasts obtained from skin of heterozygous Ndufc2 knock-out rat model showed marked mitochondrial dysfunction and PBMC obtained from subjects homozygous for the TT genotype of the rs11237379/NDUFC2 variant, previously shown to associate with reduced gene expression, demonstrated increased generation of reactive oxygen species and mitochondrial damage. The latter was associated with increased oxidative stress and significant ultrastructural impairment of mitochondrial morphology with a loss of internal cristae. In both models the exposure to stress stimuli, such as high-NaCl concentration or LPS, exacerbated the mitochondrial damage and dysfunction. Resveratrol significantly counteracted the ROS generation. These findings provide additional insights on the role of an altered pattern of mitochondrial structure-function as a cause of human diseases. In particular, they contribute to underscore a potential genetic risk factor for cardiovascular diseases, including stroke

Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells

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