MethylAction: detecting differentially methylated regions that distinguish biological subtypes

DNA methylation differences capture substantial information about the molecular and gene-regulatory states among biological subtypes. Enrichment-based next generation sequencing methods such as MBD-isolated genome sequencing (MiGS) and MeDIP-seq are appealing for studying DNA methylation genome-wide in order to distinguish between biological subtypes. However, current analytic tools do not provide optimal features for analyzing three-group or larger study designs. MethylAction addresses this need by detecting all possible patterns of statistically significant hyper- and hypo- methylation in comparisons involving any number of groups. Crucially, significance is established at the level of differentially methylated regions (DMRs), and bootstrapping determines false discovery rates (FDRs) associated with each pattern. We demonstrate this functionality in a four-group comparison among benign prostate and three clinical subtypes of prostate cancer and show that the bootstrap FDRs are highly useful in selecting the most robust patterns of DMRs. Compared to existing tools that are limited to two-group comparisons, MethylAction detects more DMRs with strong differential methylation measurements confirmed by whole genome bisulfite sequencing and offers a better balance between precision and recall in cross-cohort comparisons. MethylAction is available as an R package at http://jeffbhasin.github.io/methylaction.     

FTO and MC4R gene variants are associated with obesity in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women. It is also associated with metabolic disturbances that place women at increased risk for obesity and type 2 diabetes. There is strong evidence for familial clustering of PCOS and a genetic predisposition. However, the gene(s) responsible for the PCOS phenotypes have not been elucidated. This two-phase family-based and case-control genetic study was designed to address the question of whether SNPs identified as susceptibility loci for obesity in genome-wide association studies (GWAS) are also associated with PCOS and elevated BMI. Members of 439 families having at least one offspring with PCOS were genotyped for 15 SNPs previously shown to be associated with obesity. Linkage and association with PCOS was assessed using the transmission/disequilibrium test (TDT). These SNPs were also analyzed in an independent case-control study involving 395 women with PCOS and 176 healthy women with regular menstrual cycles. Only one of these 15 SNPs (rs2815752 in NEGR1) was found to have a nominally significant association with PCOS (χ² = 6.11, P = 0.013), but this association failed to replicate in the case-control study. While not associated with PCOS itself, five SNPs in FTO and two in MC4R were associated with BMI as assessed with a quantitative-TDT analysis, several of which replicated association with BMI in the case-control cohort. These findings demonstrate that certain SNPs associated with obesity contribute to elevated BMI in PCOS, but do not appear to play a major role in PCOS per se. These findings support the notion that PCOS phenotypes are a consequence of an oligogenic/polygenic mechanism.     

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Saponins in aerial parts of Helleborus viridis L.

In the search of natural compounds inhibiting methane production in ruminants three novel steroidal saponins have been isolated from the aerial parts of Helleborus viridis L. Their structures have been established based on spectral analyses as: (25R)-26-O-β-d-glucopyranosyl-5β-furostan-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1 → 6)-O-β-d-glucopyranoside, (25R)-26-O-β-d-glucopyranosyl-5α-furostan-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1 → 6)-O-β-d-glucopyranoside and (25R)-26-O-β-d-glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetraol 1-O-{α-l-rhamnopyranosyl-(1 → 2)-O-[β-d-glucopyranosyl-(1 → 3)]-6-O-acetoxy-β-d-glucopyranoside}.     

High cell-free virus load and robust autologous humoral immune responses in breast milk of simian immunodeficiency virus-infected african green monkeys

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4(+) T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.     

Inhibition of enveloped virus infection of cultured cells by valproic acid

Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.