COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (<it>EGFR</it>) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect <it>KRAS </it>mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for <it>KRAS </it>mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.</p> <p>Methods</p> <p>We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in <it>KRAS </it>codons 12 and 13 using the Roche LightCycler^®instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (<it>T_c</it>) of 81°C increased the sensitivity of the assay >10-fold for the majority of <it>KRAS </it>mutations.</p> <p>Results</p> <p>We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant <it>KRAS </it>mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.</p> <p>Conclusions</p> <p>We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for <it>KRAS </it>mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.</p

Genotyping faecal samples of Bengal tiger Panthera tigris tigris for population estimation: A pilot study

BACKGROUND: Bengal tiger Panthera tigris tigris the National Animal of India, is an endangered species. Estimating populations for such species is the main objective for designing conservation measures and for evaluating those that are already in place. Due to the tiger's cryptic and secretive behaviour, it is not possible to enumerate and monitor its populations through direct observations; instead indirect methods have always been used for studying tigers in the wild. DNA methods based on non-invasive sampling have not been attempted so far for tiger population studies in India. We describe here a pilot study using DNA extracted from faecal samples of tigers for the purpose of population estimation. RESULTS: In this study, PCR primers were developed based on tiger-specific variations in the mitochondrial cytochrome b for reliably identifying tiger faecal samples from those of sympatric carnivores. Microsatellite markers were developed for the identification of individual tigers with a sibling Probability of Identity of 0.005 that can distinguish even closely related individuals with 99.9% certainty. The effectiveness of using field-collected tiger faecal samples for DNA analysis was evaluated by sampling, identification and subsequently genotyping samples from two protected areas in southern India. CONCLUSION: Our results demonstrate the feasibility of using tiger faecal matter as a potential source of DNA for population estimation of tigers in protected areas in India in addition to the methods currently in use

Prevention of Apoptosis by Mitochondrial Phosphatase PGAM5 in the Mushroom Body Is Crucial for Heat Shock Resistance in Drosophila melanogaster

The heat shock (HS) response is essential for survival of all organisms. Although the machinery of the HS response has been extensively investigated at the cellular level, it is poorly understood at the level of the organism. Here, we show the crucial role of the mushroom body (MB) in the HS response in Drosophila. Null mutants of the mitochondrial phosphatase Drosophila PGAM5 (dPGAM5) exhibited increased vulnerability to HS, which was reversed by MB-specific expression of the caspase inhibitor p35, and similar vulnerability was induced in wild-type flies by knockdown of MB dPGAM5. Elimination of the MB did not affect the HS response of wild-type flies, but did increase the resistance of dPGAM5-deficient flies to HS. Thus, the MB may possess an apoptosis-dependent toxic function, the suppression of which by dPGAM5 appears to be crucial for HS resistance

Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model

<p>Abstract</p> <p>Background</p> <p>Hematopoietic stem cell transplantation is increasingly performed for hematologic diseases. As a major side effect, acute graft versus host disease (GvHD) with serious gastrointestinal symptoms including diarrhea, gastrointestinal bleeding and high mortality can be observed. Because surveillance and biopsies of human gastrointestinal GvHD are difficult to perform, rare information of the alterations of the gastrointestinal barrier exists resulting in a need for systematic animal models.</p> <p>Methods</p> <p>To investigate the effects of GvHD on the intestinal barrier of the small intestine we utilized an established acute semi allogenic GvHD in C57BL/6 and B6D2F1 mice.</p> <p>Results</p> <p>By assessing the differential uptake of lactulose and mannitol in the jejunum, we observed an increased paracellular permeability as a likely mechanism for disturbed intestinal barrier function. Electron microscopy, immunohistochemistry and PCR analysis indicated profound changes of the tight-junction complex, characterized by downregulation of the tight junction protein occludin without any changes in ZO-1. Furthermore TNF-α expression was significantly upregulated.</p> <p>Conclusions</p> <p>This analysis in a murine model of GvHD of the small intestine demonstrates serious impairment of intestinal barrier function in the jejunum, with an increased permeability and morphological changes through downregulation and localization shift of the tight junction protein occludin.</p

Utilization of Renewable Biomass and Waste Materials for Production of Environmentally-Friendly, Bio-based Composites

The introduction of renewable biomass into a polymer matrix is an option competing with other possibilities, such as energy recovery and/or re-use in the carbonized state, or production of chemicals, such as, in the case of ligno-cellulosic waste, concentrates on the production of simple sugars, then possibly leading to the development of biopolymers. These competitive applications have also some interest and market, however with a considerable energy, water and materials consumption, due also to the not always high yielding. Other possibilities for renewable biomass are therefore being used as fillers to increase mechanical performance of polymers or to allow e.g., the absorption of toxic chemicals. This review concentrates on the use of biomass as close as possible to the “as received” state, therefore avoiding whenever suitable any thermal treatment. More specifically, it focuses on its introduction into the three categories of oil-based (or bio-based replacement) of engineered polymers, into industrial biopolymers, such as poly(lactic acid) (PLA) and self-developed biopolymers, such as thermoplastic starch (TPS)

In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats

<p>Abstract</p> <p>Background</p> <p>Environmental estrogens are exogenous estrogen-mimicking compounds that can interfere with endogenous endocrine systems. Several of these endocrine disruptors have been shown to alter normal development and influence tumorigenesis in experimental models. N-butyl benzyl phthalate (BBP), a widely used plasticizer, is a well-known endocrine disruptor. The aim of this study was to elucidate the effect of prenatal exposure to BBP on the morphology, proliferative index, and genomic signature of the rat mammary gland at different ages.</p> <p>Methods</p> <p><it>In utero </it>exposure was performed by gavage of pregnant Sprague Dawley CD rats with 120mg or 500mg BBP/kg/day from day 10 post-conception to delivery. Female litters were euthanized at 21, 35, 50 and 100 days. The morphology and proliferative index of the mammary gland were studied from whole mount preparations and BrdU incorporation, respectively. Gene expression profile was assessed by microarrays. Several genes found differentially expressed and related to different functional categories were further validated by real time RT-PCR.</p> <p>Results</p> <p>Prenatal exposure of BBP induced delayed vaginal opening and changes in the post-natal mammary gland long after the end of the treatment, mainly by 35 days of age. Exposure to the high dose resulted in modifications in architecture and proliferative index of the mammary gland, mostly affecting the undifferentiated terminal end buds. Moreover, the expression profiles of this gland in the exposed rats were modified in a dose-dependent fashion. Analysis of functional categories showed that modified genes were related to immune function, cell signaling, proliferation and differentiation, or metabolism.</p> <p>Conclusions</p> <p>Our data suggest that <it>in utero </it>exposure to BBP induced a delayed pubertal onset and modified morphology of the mammary gland. These alterations were accompanied by modifications in gene expression previously associated with an increased susceptibility to carcinogenesis.</p