Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.     

A mitochondrial stratigraphy for island southeast Asia

Island Southeast Asia (ISEA) was first colonized by modern humans at least 45,000 years ago, but the extent to which the modern inhabitants trace their ancestry to the first settlers is a matter of debate. It is widely held, in both archaeology and linguistics, that they are largely descended from a second wave of dispersal, proto-Austronesian-speaking agriculturalists who originated in China and spread to Taiwan approximately 5,500 years ago. From there, they are thought to have dispersed into ISEA approximately 4,000 years ago, assimilating the indigenous populations. Here, we demonstrate that mitochondrial DNA diversity in the region is extremely high and includes a large number of indigenous clades. Only a fraction of these date back to the time of first settlement, and the majority appear to mark dispersals in the late-Pleistocene or early-Holocene epoch most likely triggered by postglacial flooding. There are much closer genetic links to Taiwan than to the mainland, but most of these probably predated the mid-Holocene "Out of Taiwan" event as traditionally envisioned. Only approximately 20% at most of modern mitochondrial DNAs in ISEA could be linked to such an event, suggesting that, if an agriculturalist migration did take place, it was demographically minor, at least with regard to the involvement of women.     

Association between endometritis diagnosis using a novel intravaginal device and reproductive performance in dairy cattle

Endometritis reduces reproductive performance in dairy cattle. Diagnosis of endometritis is undertaken using a variety of techniques including vaginoscopy, manual examination, cytology and ultrasonography. The current studies compared a novel test device ("metricheck") that is inserted into the vagina with vaginoscopy and then examined the relationship between the metricheck test score at 35 days before the start of the seasonal breeding programme and subsequent reproductive performance. Cows (n = 191; Study 1) with a history of a peripartum disease were examined by both vaginoscopy and the metricheck device and any material viewed within the vagina (using vaginoscopy) or retrieved (by the metricheck device) was scored on a 0 (no material) to 5 (grossly purulent and with an odour) scale. Within each herd the order of examination was randomized with sequentially presented pairs of cows. All cows (n = 2793; Study 2) from nine herds were examined and scored using the metricheck device 35 days before the start of the seasonal breeding programme. All cows were pregnancy tested to determine date of conception. In Study 1, more cows were defined as infected (i.e. score > 1) following metricheck than vaginoscopic examination (60% versus 43%, respectively; P < 0.05) and the level of agreement between the two tests was moderate (kappa = 0.45). The metricheck device had a higher sensitivity, but lower specificity, than vaginoscopy. Endometritis (i.e. score > 1) was detected in 21.2% of cows examined in Study 2. The prevalence of endometritis varied among herds, declined with time postpartum (P < 0.05) and was higher in cows recorded as having a peripartum disease (P < 0.01). Cows diagnosed with endometritis were at higher risk of not being detected in oestrus before the start of breeding (P < 0.01), took longer to be inseminated after the start of the seasonal breeding programme (P < 0.01), had a lower first service conception rate (P < 0.01), had lower 56-day and final pregnancy rates (P < 0.05) and took longer to conceive than cows without endometritis (P < 0.05). It is concluded that examination with the metricheck device is more sensitive in detecting endometritis than vaginoscopy. Diagnosis of endometritis with the metricheck device was associated with poorer subsequent reproductive performance.     

No evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males

The first complete mitochondrial DNA (mtDNA) sequences (approximately 16,569 bp) in 20 patients with asthenozoospermia and a comparison with 23 new complete mtDNA sequences in teratoasthenozoospermic individuals, confirmed no sharing of specific polymorphisms or specific mitochondrial lineages between these individuals. This is strong evidence against the accepted claim of a major role played by mtDNA in male fertility, once supported by haplogroup association studies based on the screening of hypervariable region I. The hypothesis of maternally driven selection acting in male reproductive success must thus be treated with caution.     

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.     

Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α) in glucose metabolism: effect of strain variation

Background

Over-activity and elevated expression of glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3β) is not known. Recently, we demonstrated that an out-bred strain of mice (ICR) lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver) inactivation of GSK-3β conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6).

Methodology/Principal Findings

Here, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform.

Conclusions/Significance

From these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin-sensitization observed in the out-bred strain of mice lacking GSK-3α is mediated by indirect means that do not require intrinsic function of GSK-3α in skeletal muscle and liver tissues.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Differential regulation of adiponectin receptor gene expression by adiponectin and leptin in myotubes derived from obese and diabetic individuals

Objective: This study aimed to investigate the regulation of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) gene expression in primary skeletal muscle myotubes, derived from human donors, after exposure to globular adiponectin (gAd) and leptin. Research Methods and Procedures: Four distinct primary cell culture groups were established [Lean, Obese, Diabetic, Weight Loss (Wt Loss); n = 7 in each] from rectus abdominus muscle biopsies obtained from surgical patients. Differentiated myotube cultures were exposed to gAd (0.1 μg/mL) or leptin (2.5 μg/mL) for 6 hours. AdipoR1 and AdipoR2 gene expression was measured by real‐time polymerase chain reaction analysis. Results: AdipoR1 mRNA expression in skeletal muscle myotubes derived from Lean subjects (p < 0.05) was stimulated 1.8‐fold and 2.5‐fold with gAd and leptin, respectively. No increase in AdipoR1 gene expression was measured in myotubes derived from Obese, Diabetic, or Wt Loss subjects. AdipoR2 mRNA expression was unaltered after gAd and leptin exposure in all myotube groups. Discussion: Adiponectin and leptin are rapid and potent stimulators of AdipoR1 in myotubes derived from lean healthy individuals. This effect was abolished in myotubes derived from obese, obese diabetic subjects, and obese‐prone individuals who had lost significant weight after bariatric surgery. The incapacity of skeletal muscle of obese and diabetic individuals to respond to exogenous adiponectin and leptin may be further suppressed as a result of impaired regulation of the AdipoR1 gene.