High Resolution Melt analysis for mutation screening in PKD1 and PKD2

<p>Abstract</p> <p>Background</p> <p>Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. <it>PKD1 </it>and <it>PKD2 </it>have been implicated in ADPKD pathogenesis but genetic features and the size of <it>PKD1 </it>make genetic diagnosis tedious.</p> <p>Methods</p> <p>We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in <it>PKD1 </it>and <it>PKD2 </it>with HRM in 37 unrelated patients with ADPKD.</p> <p>Results</p> <p>We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in <it>PKD1 </it>and 3 in <it>PKD2 </it>) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in <it>PKD1 </it>and two in <it>PKD2</it>.</p> <p>Conclusion</p> <p>HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.</p

The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus

<p>Abstract</p> <p>Background</p> <p>Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.</p> <p>Results</p> <p>We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug <it>Oncopeltus fasciatus</it>, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing <it>O. fasciatus </it>accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in <it>de novo </it>transcriptome analyses.</p> <p>Conclusions</p> <p>Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.</p> <p>[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (<url>http://www.ncbi.nlm.nih.gov/sra?term=SRP002610</url>). Custom scripts generated are available at <url>http://www.extavourlab.com/protocols/index.html</url>. Seven Additional files are available.]</p

Rapid Dissemination of SIV Follows Multisite Entry after Rectal Inoculation

Receptive ano-rectal intercourse is a major cause of HIV infection in men having sex with men and in heterosexuals. Current knowledge of the mechanisms of entry and dissemination during HIV rectal transmission is scarce and does not allow the development of preventive strategies. We investigated the early steps of rectal infection in rhesus macaques inoculated with the pathogenic isolate SIVmac251 and necropsied four hours to nine days later. All macaques were positive for SIV. Control macaques inoculated with heat-inactivated virus were consistently negative for SIV. SIV DNA was detected in the rectum as early as four hours post infection by nested PCR for gag in many laser-microdissected samples of lymphoid aggregates and lamina propria but never in follicle-associated epithelium. Scarce SIV antigen positive cells were observed by immunohistofluorescence in the rectum, among intraepithelial and lamina propria cells as well as in clusters in lymphoid aggregates, four hours post infection and onwards. These cells were T cells and non-T cells that were not epithelial cells, CD68+ macrophages, DC-SIGN+ cells or fascin+ dendritic cells. DC-SIGN+ cells carried infectious virus. Detection of Env singly spliced mRNA in the mucosa by nested RT-PCR indicated ongoing viral replication. Strikingly, four hours post infection colic lymph nodes were also infected in all macaques as either SIV DNA or infectious virus was recovered. Rapid SIV entry and dissemination is consistent with trans-epithelial transport. Virions appear to cross the follicle-associated epithelium, and also the digestive epithelium. Viral replication could however be more efficient in lymphoid aggregates. The initial sequence of events differs from both vaginal and oral infections, which implies that prevention strategies for rectal transmission will have to be specific. Microbicides will need to protect both digestive and follicle-associated epithelia. Vaccines will need to induce immunity in lymph nodes as well as in the rectum

Measurement of inclusive very forward jet cross sections in proton-lead collisions at \sqrt{sNN} = 5:02 TeV

Measurements of differential cross sections for inclusive very forward jet production in proton-lead collisions as a function of jet energy are presented. The data were collected with the CMS experiment at the LHC in the laboratory pseudorapidity range −6.6 < η < −5.2. Asymmetric beam energies of 4 TeV for protons and 1.58 TeV per nucleon for Pb nuclei were used, corresponding to a center-of-mass energy per nucleon pair of \sqrt{sNN} = 5:02 TeV. Collisions with either the proton (p+Pb) or the ion (Pb+p) traveling towards the negative η hemisphere are studied. The jet cross sections are unfolded to stable-particle level cross sections with p_{T} ≳ 3 GeV, and compared to predictions from various Monte Carlo event generators. In addition, the cross section ratio of p+Pb and Pb+p data is presented. The results are discussed in terms of the saturation of gluon densities at low fractional parton momenta. None of the models under consideration describes all the data over the full jet-energy range and for all beam configurations. Discrepancies between the differential cross sections in data and model predictions of more than two orders of magnitude are observed

Inter-ventricular heterogeneity in rat heart responses to hypoxia: the tuning of glucose metabolism, ion gradients and function

The matching of energy supply and demand under hypoxic conditions is critical for sustaining myocardial function. Numerous reports indicate that basal energy requirements and ion handling may differ between the ventricles. We hypothesized that ventricular response to hypoxia shows inter-ventricular differences caused by the heterogeneity in glucose metabolism and expression and activity of ion transporters. Thus we assessed glucose utilization rate, ATP, sodium and potassium concentrations, Na,K-ATPase activity and tissue reduced:oxidised glutathione (GSH/GSSG) content in the right and left ventricles before and after the exposure of either the whole animals or isolated blood-perfused hearts to hypoxia. The hypoxia-induced boost in glucose utilization was more pronounced in the left ventricle compared to the right one. ATP levels in the right ventricle of hypoxic heart were lower than those in the left ventricle. Left ventricular sodium content was higher and hydrolytic Na,K-ATPase activity was reduced compared with the right ventricle. Administration of the Na, K-ATPase blocker ouabain caused rapid increase in the right ventricular Na+ and elimination of the inter-ventricular Na(+) gradients. Exposure of the hearts to hypoxia made the inter-ventricular heterogeneity in the Na(+) distribution even more pronounced. Furthermore, systemic hypoxia caused oxidative stress that was more pronounced in the right ventricle as revealed by GSH/GSSG ratios. Based on these findings, we suggest that the right ventricle is more prone to hypoxic damage as it is less efficient in recruiting glucose as an alternative fuel and is particularly dependent on the efficient Na, K-ATPase function