Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error

Hermatypic Coral Growth Banding as Environmental Recorder

Study of incremental banding in coral skeletons was initiated by Ma and later extended by Wells and others. More recently, discrete annual banding in the skeletons of certain hermatypic corals has been described. Here we present an analysis of annual band width measurements from Bermuda corals which relates, through regression techniques, coral band time series to air temperature and air pressure variations. Our results indicate that coral bands record important aspects of their environment and therefore become useful where reconstruction of palaeoclimatic variables is of interest. Specifically the coral time series may be used as a palaeotemperature indicator or, when coupled with relatively well established palaeotemperatures, for palaeobarometric pressure determinations. Derived series of otherwise unobtainable palaeovariables are important not only for work on coral physiology, but also for construction and testing of climatic models; in this later instance information on palaeopressure is particularly desirable

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV

Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

Transposable elements (TEs) have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C(chromosomeconformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r=0.9, P<2.2×1016; IMR90 fibroblasts: r = 0.94, P < 2.2 × 1016) and also have a significant positive correlation withsomeremote functional DNA elements like enhancers and promoters (Enhancer: hESC: r=0.997, P=2.3×10−4; IMR90: r=0.934, P=2×10−2; Promoter: hESC: r = 0.995, P = 3.8 × 10−4; IMR90: r = 0.996, P = 3.2 × 10−4). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes

The relation between Lyman-alpha absorbers and gas--rich galaxies in the local universe

We use high-resolution hydrodynamical simulations to investigate the spatial correlation between weak (N(HI) <10^{15} cm^{-2}) Ly-alpha absorbers and gas-rich galaxies in the local universe. We confirm that Ly-alpha absorbers are preferentially expected near gas-rich galaxies and that the degree of correlation increases with the column density of the absorber. The real-space galaxy auto-correlation is stronger than the cross-correlation (correlation lengths r_{0,gg}=3.1 \pm 0.1 Mpc/h and r_{0,ag}=1.4 \pm 0.1 Mpc/h, respectively), in contrast with the recent results of Ryan-Weber (2006, RW06), and the auto-correlation of absorbers is very weak. These results are robust to the presence of strong galactic winds in the hydrodynamical simulations. In redshift-space a further mismatch arises since at small separations the distortion pattern of the simulated galaxy-absorber cross-correlation function is different from the one measured by RW06. However, when sampling the intergalactic medium along a limited number of lines-of-sight, as in the real data, uncertainties in the cross correlation estimates are large enough to account for these discrepancies. Our analysis suggests that the statistical significance of difference between the cross-correlation and auto-correlation signal in current datasets is ~ 1-sigma only.Comment: 11+ pages, 9 figures. MNRAS in pres

Spatio-temporal Models of Lymphangiogenesis in Wound Healing

Several studies suggest that one possible cause of impaired wound healing is failed or insufficient lymphangiogenesis, that is the formation of new lymphatic capillaries. Although many mathematical models have been developed to describe the formation of blood capillaries (angiogenesis), very few have been proposed for the regeneration of the lymphatic network. Lymphangiogenesis is a markedly different process from angiogenesis, occurring at different times and in response to different chemical stimuli. Two main hypotheses have been proposed: 1) lymphatic capillaries sprout from existing interrupted ones at the edge of the wound in analogy to the blood angiogenesis case; 2) lymphatic endothelial cells first pool in the wound region following the lymph flow and then, once sufficiently populated, start to form a network. Here we present two PDE models describing lymphangiogenesis according to these two different hypotheses. Further, we include the effect of advection due to interstitial flow and lymph flow coming from open capillaries. The variables represent different cell densities and growth factor concentrations, and where possible the parameters are estimated from biological data. The models are then solved numerically and the results are compared with the available biological literature.Comment: 29 pages, 9 Figures, 6 Tables (39 figure files in total

Altered Arterial Stiffness and Subendocardial Viability Ratio in Young Healthy Light Smokers after Acute Exercise

Studies showed that long-standing smokers have stiffer arteries at rest. However, the effect of smoking on the ability of the vascular system to respond to increased demands (physical stress) has not been studied. The purpose of this study was to estimate the effect of smoking on arterial stiffness and subendocardial viability ratio, at rest and after acute exercise in young healthy individuals.Healthy light smokers (n = 24, pack-years = 2.9) and non-smokers (n = 53) underwent pulse wave analysis and carotid-femoral pulse wave velocity measurements at rest, and 2, 5, 10, and 15 minutes following an exercise test to exhaustion. Smokers were tested, 1) after 12h abstinence from smoking (chronic condition) and 2) immediately after smoking one cigarette (acute condition). At rest, chronic smokers had higher augmentation index and lower aortic pulse pressure than non-smokers, while subendocardial viability ratio was not significantly different. Acute smoking increased resting augmentation index and decreased subendocardial viability ratio compared with non-smokers, and decreased subendocardial viability ratio compared with the chronic condition. After exercise, subendocardial viability ratio was lower, and augmentation index and aortic pulse pressure were higher in non-smokers than smokers in the chronic and acute conditions. cfPWV rate of recovery of was greater in non-smokers than chronic smokers after exercise. Non-smokers were also able to achieve higher workloads than smokers in both conditions.Chronic and acute smoking appears to diminish the vascular response to physical stress. This can be seen as an impaired 'vascular reserve' or a blunted ability of the blood vessels to accommodate the changes required to achieve higher workloads. These changes were noted before changes in arterial stiffness or subendocardial viability ratio occurred at rest. Even light smoking in young healthy individuals appears to have harmful effects on vascular function, affecting the ability of the vascular bed to respond to increased demands

Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells.

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein-protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future

Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library

<p>Abstract</p> <p>Background</p> <p>To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population.</p> <p>Results</p> <p>The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme <it>Hae</it>III; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts.</p> <p>Conclusion</p> <p>The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.</p

Search for anomalous t t-bar production in the highly-boosted all-hadronic final state

A search is presented for a massive particle, generically referred to as a Z', decaying into a t t-bar pair. The search focuses on Z' resonances that are sufficiently massive to produce highly Lorentz-boosted top quarks, which yield collimated decay products that are partially or fully merged into single jets. The analysis uses new methods to analyze jet substructure, providing suppression of the non-top multijet backgrounds. The analysis is based on a data sample of proton-proton collisions at a center-of-mass energy of 7 TeV, corresponding to an integrated luminosity of 5 inverse femtobarns. Upper limits in the range of 1 pb are set on the product of the production cross section and branching fraction for a topcolor Z' modeled for several widths, as well as for a Randall--Sundrum Kaluza--Klein gluon. In addition, the results constrain any enhancement in t t-bar production beyond expectations of the standard model for t t-bar invariant masses larger than 1 TeV.Comment: Submitted to the Journal of High Energy Physics; this version includes a minor typo correction that will be submitted as an erratu