Primary muscle cells cultivated in medium conditioned by spinal cord cells show changes in messenger RNA as detected by translation in ovo accompanied by synthesis of extracellular matrix components

Primary muscle cell cultures were prepared from rat embryos and maintained in normal (unconditioned) and spinal cord (conditioned) media. In order to relate translational regulation to the morphological changes associated with each culture condition, mRNA was isolated from both culture variants and subjected to in ovo translation. Xenopus oocytes were injected with mRNA and their incubation media checked for the presence of newly formed proteins coded by the mRNAs and secreted into the medium. Electrophoretograms indicated mRNA-dependent synthesis of several proteins in the molecular mass range of 30-260 kD. To further characterize these proteins, antisera directed against several extracellular matrix proteins were used for immunoprecipitation: antigens recognized by anti-heparan-sulfate-proteoglycan and anti-fibronectin were enhanced in conditioned cells, whereas laminin was found to be reduced.     

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.     

The Systemic Immune Network in Recent Onset Type 1 Diabetes: Central Role of Interleukin-1 Receptor Antagonist (DIATOR Trial)

Background

The hypothesis was tested that the systemic immune milieu in recent-onset type 1 diabetes is associated with residual beta cell function and other metabolic patient characteristics.

Methods and Findings

All patients (n = 89, 40% female) of the Diabetes and Atorvastatin (DIATOR) Trial were analyzed at recruitment, i.e. prior to receiving the study medication. Inclusion criteria were insulin dependent diabetes for 2 weeks to 3 months, age range 18–39 years, and islet cell autoantibodies. Blood samples were analyzed for 14 immune mediators by standard methods. Concentrations of all mediators correlated with at least one other mediator (p<0.05, Spearman correlation) giving rise to a network. Interleukin 1 receptor antagonist (IL1-RA) held a central position and was associated with both pro- and anti-inflammatory mediators. Further central elements were the pro-inflammatory mediators CRP and IL-6, the soluble adhesion molecules sICAM-1 and E-selectin, and MCP-4 which held a central position in the chemokine network. The two Th1-associated mediators IFNγ and IP-10 remained outside the network but correlated with each other. All correlations were positive (r = 0.25–0.72), i.e., high levels of pro-inflammatory mediators were accompanied by increased levels of anti-inflammatory mediators. IL-1RA was the only mediator associated with fasting and liquid mixed meal stimulated C-peptide concentrations (r = 0.31 and 0.24, p = 0.003 and 0.025, after adjustment for age, sex, BMI). There were associations between the immune mediator network and BMI (IL-1RA, CRP, IL-6, MCP-4, MIP-1ß) but few or no associations with HbA1c, insulin dose, lipid parameters, age or sex.

Conclusions

In patients with recent onset type 1 diabetes, systemic acute phase proteins, cytokines, chemokines and soluble adhesion molecules form a network. Among the few central elements IL-1RA has a dominant role. IL-1RA is associated with all other groups of mediators and is the only mediator which correlates (positively) with residual beta cell function.

Trial registration

ClinicalTrials.gov registration number: {"type":"clinical-trial","attrs":{"text":"NCT00974740","term_id":"NCT00974740"}}NCT00974740     

Interrelationships among life-history traits in three California oaks

Life-history traits interact in important ways. Relatively few studies, however, have explored the relationships between life-history traits in long-lived taxa such as trees. We examined patterns of energy allocation to components of reproduction and growth in three species of California oaks (Quercus spp.) using a combination of annual acorn censuses, dendrometer bands to measure radial increment, and litterfall traps. Our results are generally consistent with the hypothesis that energy invested in reproduction detracts from the amount of energy available for growth in these long-lived taxa; i.e., there are trade-offs between these traits. The relationships between reproduction and growth varied substantially among specific trait combinations and tree species, however, and in some cases were in the direction opposite that expected based on the assumption of trade-offs between them. This latter finding appears to be a consequence of the pattern of resource use across years in these long-lived trees contrasting with the expected partitioning of resource use within years in short-lived taxa. Thus, the existence and magnitude of putative trade-offs varied depending on whether the time scale considered was within or across years. Collectively, our results indicate that negative relationships between fundamental life-history traits can be important at multiple levels of modular organization and that energy invested in reproduction can have measurable consequences in terms of the amount of energy available for future reproduction and both current and future growth.     

Fine genetic mapping of RXopJ4, a bacterial spot disease resistance locus from Solanum pennellii LA716

The RXopJ4 resistance locus from the wild accession Solanum pennellii (Sp) LA716 confers resistance to bacterial spot disease of tomato (S. lycopersicum, Sl) caused by Xanthomonas perforans (Xp). RXopJ4 resistance depends on recognition of the pathogen type III effector protein XopJ4. We used a collection of Sp introgression lines (ILs) to narrow the RXopJ4 locus to a 4.2-Mb segment on the long arm of chromosome 6, encompassed by the ILs 6-2 and 6-2-2. We then adapted or developed a collection of 14 molecular markers to map on a segregating F₂ population from a cross between the susceptible parent Sl FL8000 and the resistant parent RXopJ4 8000 OC₇. In the F₂ population, a 190-kb segment between the markers J350 and J352 cosegregated with resistance. This fine mapping will enable both the identification of candidate genes and the detection of resistant plants using cosegregating markers. The RXopJ4 resistance gene(s), in combination with other recently characterized genes and a quantitative trait locus (QTL) for bacterial spot disease resistance, will likely be an effective tool for the development of durable resistance in cultivated tomato.     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.     

Objectively Measured Walking Duration and Sedentary Behaviour and Four-Year Mortality in Older People

BackgroundPhysical activity is an important component of health. Recommendations based on sensor measurements are sparse in older people. The aim of this study was to analyse the effect of objectively measured walking and sedentary duration on four-year mortality in community-dwelling older people.MethodsBetween March 2009 and April 2010, physical activity of 1271 participants (≥65 years, 56.4% men) from Southern Germany was measured over one week using a thigh-worn uni-axial accelerometer (activPAL; PAL Technologies, Glasgow, Scotland). Mortality was assessed during a four-year follow-up. Cox-proportional-hazards models were used to estimate the associations between walking (including low to high intensity) and sedentary duration with mortality. Models were adjusted for age and sex, additional epidemiological variables, and selected biomarkers.ResultsAn inverse relationship between walking duration and mortality with a minimum risk for the 3rd quartile (102.2 to128.4 minutes walking daily) was found even after multivariate adjustment with HRs for quartiles 2 to 4 compared to quartile 1 of 0.45 (95%-CI: 0.26; 0.76), 0.18 (95%-CI: 0.08; 0.41), 0.39 (95%-CI: 0.19; 0.78), respectively. For sedentary duration an age- and sex-adjusted increased mortality risk was observed for the 4th quartile (daily sedentary duration ≥1137.2 min.) (HR 2.05, 95%-CI: 1.13; 3.73), which diminished, however, after full adjustment (HR 1.63, 95%-CI: 0.88; 3.02). Furthermore, our results suggest effect modification between walking and sedentary duration, such that in people with low walking duration a high sedentary duration was noted as an independent factor for increased mortality.ConclusionsIn summary, walking duration was clearly associated with four-year overall mortality in community-dwelling older people.