Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes

Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers. We profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes. The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation. We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions

Thermoelectric properties of lead chalcogenide core-shell nanostructures

We present the full thermoelectric characterization of nanostructured bulk PbTe and PbTe-PbSe samples fabricated from colloidal core-shell nanoparticles followed by spark plasma sintering. An unusually large thermopower is found in both materials, and the possibility of energy filtering as opposed to grain boundary scattering as an explanation is discussed. A decreased Debye temperature and an increased molar specific heat are in accordance with recent predictions for nanostructured materials. On the basis of these results we propose suitable core-shell material combinations for future thermoelectric materials of large electric conductivities in combination with an increased thermopower by energy filtering.Comment: 12 pages, 8 figure

Submillimeter Emission from Water in the W3 Region

We have mapped the submillimeter emission from the 1(10)-1(01) transition of ortho-water in the W3 star-forming region. A 5'x5' map of the W3 IRS4 and W3 IRS5 region reveals strong water lines at half the positions in the map. The relative strength of the Odin lines compared to previous observations by SWAS suggests that we are seeing water emission from an extended region. Across much of the map the lines are double-peaked, with an absorption feature at -39 km/s; however, some positions in the map show a single strong line at -43 km/s. We interpret the double-peaked lines as arising from optically thick, self-absorbed water emission near the W3 IRS5, while the narrower blue-shifted lines originate in emission near W3 IRS4. In this model, the unusual appearance of the spectral lines across the map results from a coincidental agreement in velocity between the emission near W3 IRS4 and the blue peak of the more complex lines near W3 IRS5. The strength of the water lines near W3 IRS4 suggests we may be seeing water emission enhanced in a photon-dominated region.Comment: Accepted to A&A Letters as part of the special Odin issue; 4 page

Biosynthesis of Mitochondrial Porin and Insertion into the Outer Mitochondrial Membrane of Neuruspora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4°C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane

Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence

First detection of NH3 (1,0 - 0,0) from a low mass cloud core: On the low ammonia abundance of the rho Oph A core

Odin has successfully observed the molecular core rho Oph A in the 572.5 GHz rotational ground state line of ammonia, NH3 (J,K = 1,0 - 0,0). The interpretation of this result makes use of complementary molecular line data obtained from the ground (C17O and CH3OH) as part of the Odin preparatory work. Comparison of these observations with theoretical model calculations of line excitation and transfer yields a quite ordinary abundance of methanol, X(CH3OH) = 3e-9. Unless NH3 is not entirely segregated from C17O and CH3OH, ammonia is found to be significantly underabundant with respect to typical dense core values, viz. X(NH3) = 8e-10.Comment: 4 pages, 2 figures, 2 tables, to appear in Astron. Astrophys. Letter

Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes.

AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins. METHODS: We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats. RESULTS: We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro. CONCLUSIONS: Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.JB-J was supported by a grant from the Danish PhD School for Molecular Metabolism. The study was supported by grants from the Danish Medical Research Council, the Danish Strategic Research Council. The Novo Nordisk Foundation, the Danish Ministry of Science, Technology and Innovation. DSF-T was supported by the Biotechnology and Biological Sciences Research Council project grant BB/F-15364/1. SEO is a British Heart Foundation Senior Fellow (FS/09/029/27902).This is the final version of the article. It was first published by Springer at http://link.springer.com/article/10.1007%2Fs00125-014-3434-

Psychometric properties of an instrument to measure the clinical learning environment

Objectives: The clinical learning environment is an influential factor in work-based learning. Evaluation of this environment gives insight into the educational functioning of clinical departments. The Postgraduate Hospital Educational Environment Measure (PHEEM) is an evaluation tool consisting of a validated questionnaire with 3 subscales. In this paper we further investigate the psychometric properties of the PHEEM. We set out to validate the 3 subscales and test the reliability of the PHEEM for both clerks (clinical medical students) and registrars (specialists in training). Methods: Clerks and registrars from different hospitals and specialties filled out the PHEEM. To investigate the construct validity of the 3 subscales, we used an exploratory factor analysis followed by varimax rotation, and a cluster analysis known as Mokken scale analysis. We estimated the reliability of the questionnaire by means of variance components according to generalisability theory. Results: A total of 256 clerks and 339 registrars filled out the questionnaire. The exploratory factor analysis plus varimax rotation suggested a 1-dimensional scale. The Mokken scale analysis confirmed this result. The reliability analysis showed a reliable outcome for 1 department with 14 clerks or 11 registrars. For multiple departments 3 respondents combined with 10 departments provide a reliable outcome for both groups. Discussion: The PHEEM is a questionnaire measuring 1 dimension instead of the hypothesised 3 dimensions. The sample size required to achieve a reliable outcome is feasible. The instrument can be used to evaluate both single and multiple departments for both clerks and registrars. © 2007 Blackwell Publishing Ltd

Bradyrhizobium japonicum and soybean symbiotic response to glyphosate in glyphosate-tolerant soybeans

Soybean (Glycine max) grain contains approximately 40% protein and 6.5% nitrogen (N) on an elemental basis. Therefore, the plant requires an abundant N supply throughout its life cycle, and symbiotic N fixation of soybean with Bradyrhizobium japonicum provides 40 to 85% of the soybean N. Although soybean cultivars have been genetically engineered to withstand the herbicide glyphosate, B. japonicum grown in culture is sensitive to glyphosate. We hypothesized that glyphosate applied to glyphosate-tolerant soybean would inhibit nodulation by B. japonicum unless B. japonicum could also be selected for glyphosate tolerance. Cultures of B. japonicum were challenged with sublethal doses of glyphosate, and individual colonies were selected for growth in the presence of glyphosate. Of the 40 isolates that were originally selected for glyphosate tolerance, all isolates in subsequent experiments had similar sensitivity to glyphosate as wild-type B. japonicum. To determine if glyphosate affected B. japonicum in plants, soybean seeds were imbibed with differing levels of glyphosate and water and then planted and inoculated with B. japonicum. After several weeks of growth the plants were harvested and nodules were scanned and analyzed by digital imagery. Glyphosate application to glyphosate-tolerant soybean did not affect the ability of B. japonicum to form nodules and fix nitrogen. These data do not agree with previous responses of small soybean plants sprayed with glyphosate, which showed delayed nodulation and decreased nodule size. It may be that the dosage applied to plants and the timing of the application affect the response of glyphosate on symbiotic effectiveness

Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (,23%) than the other two, COPI (,9%) and COPII (,8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest major roles for this structural property in shaping the differences of evolutionary adaptability in the three routes