Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications

SUMMARY: A combination of bisulfite treatment of DNA and high-throughput sequencing (BS-Seq) can capture a snapshot of a cell's epigenomic state by revealing its genome-wide cytosine methylation at single base resolution. Bismark is a flexible tool for the time-efficient analysis of BS-Seq data which performs both read mapping and methylation calling in a single convenient step. Its output discriminates between cytosines in CpG, CHG and CHH context and enables bench scientists to visualize and interpret their methylation data soon after the sequencing run is completed. Availability and implementation: Bismark is released under the GNU GPLv3+ licence. The source code is freely available from www.bioinformatics.bbsrc.ac.uk/projects/bismark/.     

Common and specific responses to iron and phosphorus deficiencies in roots of apple tree (Malus × domestica)

Plant Molecular Biology - Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for...     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

Testing the role of P2X7 receptors in the development of type 1 diabetes in nonobese diabetic mice

Although P2rx7 has been proposed as a type 1 diabetes (T1D) susceptibility gene in NOD mice, its potential pathogenic role has not been directly determined. To test this possibility, we generated a new NOD stock deficient in P2X(7) receptors. T1D development was not altered by P2X(7) ablation. Previous studies found CD38 knockout (KO) NOD mice developed accelerated T1D partly because of a loss of CD4(+) invariant NKT (iNKT) cells and Foxp3(+) regulatory T cells (Tregs). These immunoregulatory T cell populations are highly sensitive to NAD-induced cell death activated by ADP ribosyltransferase-2 (ART2)-mediated ADP ribosylation of P2X(7) receptors. Therefore, we asked whether T1D acceleration was suppressed in a double-KO NOD stock lacking both P2X(7) and CD38 by rescuing CD4(+) iNKT cells and Tregs from NAD-induced cell death. We demonstrated that P2X(7) was required for T1D acceleration induced by CD38 deficiency. The CD38 KO-induced defects in homeostasis of CD4(+) iNKT cells and Tregs were corrected by coablation of P2X(7). T1D acceleration in CD38-deficient NOD mice also requires ART2 expression. If increased ADP ribosylation of P2X(7) in CD38-deficient NOD mice underlies disease acceleration, then a comparable T1D incidence should be induced by coablation of both CD38 and ART2, or CD38 and P2X(7). However, a previously established NOD stock deficient in both CD38 and ART2 expression is T1D resistant. This study demonstrated the presence of a T1D resistance gene closely linked to the ablated Cd38 allele in the previously reported NOD stock also lacking ART2, but not in the newly generated CD38/P2X(7) double-KO line.     

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Background  

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.     

Expression and high glucose-mediated regulation of K channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K⁺ channels K_V4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K_V4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K⁺ current and an alteration in the inactivation of the macroscopic outward K⁺ currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K_V4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.     

Examination of Apoptosis Signaling in Pancreatic Cancer by Computational Signal Transduction Analysis

Background

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of cancer death. Changes in apoptosis signaling in pancreatic cancer result in chemotherapy resistance and aggressive growth and metastasizing. The aim of this study was to characterize the apoptosis pathway in pancreatic cancer computationally by evaluation of experimental data from high-throughput technologies and public data bases. Therefore, gene expression analysis of microdissected pancreatic tumor tissue was implemented in a model of the apoptosis pathway obtained by computational protein interaction prediction.

Methodology/Principal Findings

Apoptosis pathway related genes were assembled from electronic databases. To assess expression of these genes we constructed a virtual subarray from a whole genome analysis from microdissected native tumor tissue. To obtain a model of the apoptosis pathway, interactions of members of the apoptosis pathway were analysed using public databases and computational prediction of protein interactions. Gene expression data were implemented in the apoptosis pathway model. 19 genes were found differentially expressed and 12 genes had an already known pathophysiological role in PDAC, such as Survivin/BIRC5, BNIP3 and TNF-R1. Furthermore we validated differential expression of IL1R2 and Livin/BIRC7 by RT-PCR and immunohistochemistry. Implementation of the gene expression data in the apoptosis pathway map suggested two higher level defects of the pathway at the level of cell death receptors and within the intrinsic signaling cascade consistent with references on apoptosis in PDAC. Protein interaction prediction further showed possible new interactions between the single pathway members, which demonstrate the complexity of the apoptosis pathway.

Conclusions/Significance

Our data shows that by computational evaluation of public accessible data an acceptable virtual image of the apoptosis pathway might be given. By this approach we could identify two higher level defects of the apoptosis pathway in PDAC. We could further for the first time identify IL1R2 as possible candidate gene in PDAC.     

Y Chromosomal Variation Tracks the Evolution of Mating Systems in Chimpanzee and Bonobo

The male-specific regions of the Y chromosome (MSY) of the human and the chimpanzee (Pan troglodytes) are fully sequenced. The most striking difference is the dramatic rearrangement of large parts of their respective MSYs. These non-recombining regions include ampliconic gene families that are known to be important for male reproduction,and are consequently under significant selective pressure. However, whether the published Y-chromosomal pattern of ampliconic fertility genes is invariable within P. troglodytes is an open but fundamental question pertinent to discussions of the evolutionary fate of the Y chromosome in different primate mating systems. To solve this question we applied fluorescence in situ hybridisation (FISH) of testis-specific expressed ampliconic fertility genes to metaphase Y chromosomes of 17 chimpanzees derived from 11 wild-born males and 16 bonobos representing seven wild-born males. We show that of eleven P. troglodytes Y-chromosomal lines, ten Y-chromosomal variants were detected based on the number and arrangement of the ampliconic fertility genes DAZ (deleted in azoospermia) and CDY (chromodomain protein Y)—a so-far never-described variation of a species'' Y chromosome. In marked contrast, no variation was evident among seven Y-chromosomal lines of the bonobo, P. paniscus, the chimpanzee''s closest living relative. Although, loss of variation of the Y chromosome in the bonobo by a founder effect or genetic drift cannot be excluded, these contrasting patterns might be explained in the context of the species'' markedly different social and mating behaviour. In chimpanzees, multiple males copulate with a receptive female during a short period of visible anogenital swelling, and this may place significant selection on fertility genes. In bonobos, however, female mate choice may make sperm competition redundant (leading to monomorphism of fertility genes), since ovulation in this species is concealed by the prolonged anogenital swelling, and because female bonobos can occupy high-ranking positions in the group and are thus able to determine mate choice more freely.     

Dealing with Uncertainty in Spatially Explicit Population Models

It has been argued that spatially explicit population models (SEPMs) cannot provide reliable guidance for conservation biology because of the difficulty of obtaining direct estimates for their demographic and dispersal parameters and because of error propagation. We argue that appropriate model calibration procedures can access additional sources of information, compensating the lack of direct parameter estimates. Our objective is to show how model calibration using population-level data can facilitate the construction of SEPMs that produce reliable predictions for conservation even when direct parameter estimates are inadequate. We constructed a spatially explicit and individual-based population model for the dynamics of brown bears (Ursus arctos) after a reintroduction program in Austria. To calibrate the model we developed a procedure that compared the simulated population dynamics with distinct features of the known population dynamics (=patterns). This procedure detected model parameterizations that did not reproduce the known dynamics. Global sensitivity analysis of the uncalibrated model revealed high uncertainty in most model predictions due to large parameter uncertainties (coefficients of variation CV 0.8). However, the calibrated model yielded predictions with considerably reduced uncertainty (CV 0.2). A pattern or a combination of various patterns that embed information on the entire model dynamics can reduce the uncertainty in model predictions, and the application of different patterns with high information content yields the same model predictions. In contrast, a pattern that does not embed information on the entire population dynamics (e.g., bear observations taken from sub-areas of the study area) does not reduce uncertainty in model predictions. Because population-level data for defining (multiple) patterns are often available, our approach could be applied widely.