Defective Mitochondrial Function In Vivo in Skeletal Muscle in Adults with Down’s Syndrome: A 31P-MRS Study

Down’s syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy (³¹P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7±0.1 min⁻¹ vs 2.1±0.1 min⁻¹ respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using ³¹P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.     

Expression and Antimicrobial Function of Beta-Defensin 1 in the Lower Urinary Tract

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1 ^-/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1 ^-/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

HER2 Phosphorylates and Destabilizes Pro-Apoptotic PUMA,Leading to Antagonized Apoptosis in Cancer Cells

HER2 is overexpressed in 15–20% of breast cancers. HER2 overexpression is known to reduce apoptosis but the underlying mechanisms for this association remain unclear. To elucidate the mechanisms for HER2-mediated survival, we investigated the relationship between HER2 and p53 upregulated modulator of apoptosis (PUMA), a potent apoptosis inducer. Our results showed that HER2 interacts with PUMA, which was independent of HER2 activation. In addition, we observed that HER2 interacted with PUMA in both mitochondrial and non-mitochondrial compartments. We next examined whether HER2 phosphorylates PUMA. Notably, PUMA tyrosine phosphorylation has never been reported. Using an intracellular assay, we found PUMA to be phosphorylated in breast cancer cells with activated HER2. Via cell-free HER2 kinase assay, we observed that PUMA was directly phosphorylated by HER2. Activation of HER2 decreased PUMA protein half-life. To identify which of the three tyrosines within PUMA are targeted by HER2, we generated three PUMA non-phosphorylation mutants each with a single Tyr→Phe substitution. Results indicated that each PUMA single mutant had lost some, but not all phosphorylation by HER2 indicating that HER2 targets all three tyrosines. Consequently, we created an additional PUMA mutant with all three tyrosines mutated (TM-PUMA) that could not be phosphorylated by HER2. Importantly, TM-PUMA was found to have a longer half-life than PUMA. An inverse association was observed between HER2 and PUMA in 93 invasive breast carcinoma samples. We further found that TM-PUMA suppressed growth of breast cancer cells to a greater degree than PUMA. Also, TM-PUMA had a stronger propensity to induce apoptosis than PUMA. Together, our results demonstrate, for the first time, that PUMA can be tyrosine phosphorylated and that HER2-mediated phosphorylation destabilizes PUMA protein. The HER2-PUMA interplay represents a novel mechanism by which PUMA is regulated and a new molecular basis for HER2-mediated growth and survival of cancer cells.     

Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD⁺- or NADP⁺-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies.     

Terrestrial support of pelagic consumers: patterns and variability revealed by a multilake study

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National‐scale analyses of habitat associations of Marsh Tits Poecile palustris and Blue Tits Cyanistes caeruleus: two species with opposing population trends in Britain

Capsule Marsh Tits were strongly associated with both the amount and species diversity of woodland understorey; Blue Tits were associated with large trees and deadwood.

Aims To gather quantitative information on the habitat requirements of Marsh Tits, in comparison with those of Blue Tits, across a large number of sites in England and Wales, and secondly to evaluate the range of habitat conditions likely to encourage the presence, and increase the abundance of, each species.

Methods Counts of birds were made at each of 181 woods across England and Wales, and habitat data were collected from the same locations in each woodland. Marsh Tit and Blue Tit presence and abundance were related to habitat characteristics, interspecific competition and deer impact.

Results Shrub cover and species diversity were important for the presence and abundance of Marsh Tits, across their geographical range in Britain. Blue Tits were associated with large trees and deadwood.

Conclusion Our results support the hypothesis that changes in woodland management, leading to canopy closure and a decline in the understorey available, could have had an impact on Marsh Tits, and may have led to the observed population decline. These same changes were also consistent with population increase in Blue Tits.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.