Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

A Mouthful of Diversity: Knowledge of Cider Apple Cultivars in the United Kingdom and Northwest United States

A Mouthful of Diversity: Knowledge of Cider Apple Cultivars in the United Kingdom and Northwest United States. There is a general assumption in the study of folk taxonomy that those people who have been interacting with a given crop the longest have the most knowledge about the crop’s names. We treated this as an hypothesis which can be tested with knowledge of cider apples. This use of apples extends back many generations in some places, while in other regions people are just learning to make cider. The experimental design is to assess quantitatively the cider apple diversity being used compared to the knowledge of this diversity by cider makers. The test involves two populations of cider makers: those who come from a long-standing tradition of cider making and those who recently learned to make cider. Research was conducted in parts of England, Wales, Northern Ireland, and Washington State, U.S.A. Semistructured interviews and questionnaires were used to elicit cider apple variety names. Traditional knowledge associated with cider production was also collected. Eighty-two cider apple variety names were obtained. In addition, it is estimated that between 111 and 328 varieties were recognized but could not be named. There was a significant difference between the cider apple cultivars that cider makers could name and those that they could discern. On average, cider makers could name eight varieties, but discern 16 varieties of cider apples largely on the basis of appearance, taste, and smell. There was no significant difference in the knowledge of cider apple variety names between long-standing cider makers and those that recently learned to make cider. As with cider apples, we would expect that farmers of other culturally-significant crops would not always know named diversity if there are other cues to let them track varietal difference, such as appearance, taste, or smell.     

Caenorhabditis elegans unc-82 Encodes a Serine/Threonine Kinase Important for Myosin Filament Organization in Muscle During Growth

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.THE contractile apparatus of striated muscle is a highly ordered cytoskeletal structure (Figure 1) composed of actin and myosin filaments, the filament anchoring structures, and a host of regulatory proteins. During Caenorhabditis elegans embryogenesis, the body-wall muscle cells polarize and assemble their cytoskeletons in response to contact with the epidermal cells, to which they attach through focal-adhesion-like structures. The epidermal cells respond in a similar fashion and assemble attachment structures and fibrous organelles at the sites of muscle-cell contact (reviewed in Moerman and Williams 2006). The coordination of the cytoskeletons of the two tissue types provides the physical attachment that transmits the force of muscle-cell contraction to the epidermis and its secreted cuticle and allows the worm to locomote through its environment. The patterning of the contractile apparatus occurs through integrin-mediated signaling at the plasma membrane where muscle cells contact the epidermis. The assembly of more interior (membrane-distal) components of the contractile apparatus follows and requires the membrane-proximal events (Hresko et al. 1994). Failure to assemble functional epidermal–muscle-cell contacts or failure to make contractile muscle cells prevents elongation of the embryo from an egg shape into a long tube. Many genes required for these early patterning events, as well as those essential for muscle contraction, have been identified by screening for embryonic lethal mutations that produce the Pat phenotype (paralyzed, arrested elongation at two-fold) (Williams and Waterston 1994).Open in a separate windowFigure 1.—unc-82 mutants show dramatic defects in localization of thick-filament and M-line components, but normal patterning of membrane and dense-body proteins. A diagram of the sarcomere (top) is highlighted to indicate those components affected in unc-82 mutants. Structures represented include the actin filaments anchored to the dense body (the Z-line analog) and myosin-containing thick filaments associated with the M-line. The components represented in white exhibit abnormal staining patterns in unc-82 mutants; those represented in gray are relatively unaffected. (A–N) Adult fragments from wild-type (left column) and unc-82 mutant worms (right column) were stained with antibodies specific for components of the contractile apparatus. Thick-filament proteins myosin A (A and B) and paramyosin (C and D) are grossly mislocalized in unc-82 mutants, as is the M-line component UNC-89/obscurin (E and F). White arrows (B, D, F, and H) indicate abnormal accumulations of thick-filament and M-line proteins, and asterisks (A and K) mark a cell border. Actin staining (G and H) is mildly disrupted, but does not appear in large clumps. The distribution of α-actinin, vinculin, and integrin (I–N) (organized lines of puncta, solid arrows) is similar in mutant and wild type. Antibodies: myosin A, 5.6; paramyosin, 5.23; UNC-89, EU30; actin, C4; α-actinin, MH35; vinculin, MH24; integrin MH25. Bar, 10 μm.However, proteins that act subsequent to the early patterning events or are not essential for contraction would not have been identified in the Pat screens. Mutations in the unc-82 gene were isolated by screening apparently normal animals for muscle-cell disorganization using polarized light microscopy (Waterston et al. 1980). Animals homozygous for unc-82 mutations exhibit patchy, bright birefringence rather than the uniform bright bands of signal that mark the areas of organized myosin-containing thick filaments in wild-type worms. To define the mechanisms underlying filament organization within the contractile apparatus, we undertook molecular and phenotypic analyses of unc-82 mutants. Our data suggest that UNC-82 is a kinase, orthologous to human ARK5 and SNARK, that is required specifically for myosin filament reorganization during cellular elongation in normal development.     

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

Background  

Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum).     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

The expression patterns of c-kit and Sl in chicken embryos suggest unexpected roles for these genes in somite and limb development

We have used whole-mount in situ hybridization to investigate the patterns of c-kit and Sl expression in stage 11-22 chicken embryos. Our analysis shows that c-kit and Sl are expressed quite differently in chicken embryos compared to the reported expression patterns of these genes in embryos of other taxa. Most notably, chicken c-kit is expressed in primordial germ cells as well as in the developing somite, the apical ectodermal ridge, and in the early foregut endoderm. Sl is expressed in the lateral and intermediate mesoderm and in extraembryonic membranes. These data suggest that chicken c-kit and Sl may play novel and unexpected roles in somitogenesis, limb development, and foregut development in avian embryos.     

Trimetazidine improves left ventricular function in diabetic patients with coronary artery disease: a double-blind placebo-controlled study

Background

Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy.

Methods

32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months.

Results

Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo.

Conclusion

in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.     

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.