Evolution of Testing Algorithms at a University Hospital for Detection of Clostridium difficile Infections

We present the evolution of testing algorithms at our institution in which the C. Diff Quik Chek Complete immunochromatographic cartridge assay determines the presence of both glutamate dehydrogenase and Clostridium difficile toxins A and B as a primary screen for C. difficile infection and indeterminate results (glutamate dehydrogenase positive, toxin A and B negative) are confirmed by the GeneXpert C. difficile PCR assay. This two-step algorithm is a cost-effective method for highly sensitive detection of toxigenic C. difficile

Molecular markers and genomic resources for disease resistance in peanut-A review

Recent polyploidation of peanut genome and geographical isolation has rendered peanut to be a highly monomorphic species. Due to its narrow genetic base, cultivated peanut has been susceptible to various diseases, causing economic loss to farmers. Availability of only a few disease resistance sources in cultivated peanut has resulted in limited success using the conventional breeding practices. Also, scarcity of markers has been the major limiting factor to precisely identify the disease resistance genomic regions. Recentidentification of largenumber ofmolecular markers using advancedgenomic resources and high throughput sequencing technologies has and will continue to assist in improvement of peanut diversity and breeding. This review gives an update on recent discovery of molecular markers associated with major diseases and the available genomic resources in peanut

Nested‐association mapping (NAM)‐based genetic dissection uncovers candidate genes for seed and pod weights in peanut ( Arachis hypogaea )

Multiparental genetic mapping populations such as nested-association mapping (NAM) havegreat potential for investigating quantitative traits and associated genomic regions leading torapid discovery of candidate genes and markers. To demonstrate the utility and power of thisapproach, two NAM populations, NAM_Tifrunner and NAM_Florida-07, were used for dissectinggenetic control of 100-pod weight (PW) and 100-seed weight (SW) in peanut. Two high-densitySNP-based genetic maps were constructed with 3341 loci and 2668 loci for NAM_Tifrunner andNAM_Florida-07, respectively. The quantitative trait locus (QTL) analysis identified 12 and 8major effect QTLs for PW and SW, respectively, in NAM_Tifrunner, and 13 and 11 major effectQTLs for PW and SW, respectively, in NAM_Florida-07. Most of the QTLs associated with PW andSW were mapped on the chromosomes A05, A06, B05 and B06. A genomewide associationstudy (GWAS) analysis identified 19 and 28 highly significant SNP–trait associations (STAs) inNAM_Tifrunner and 11 and 17 STAs in NAM_Florida-07 for PW and SW, respectively. Thesesignificant STAs were co-localized, suggesting that PW and SW are co-regulated by severalcandidate genes identified on chromosomes A05, A06, B05, and B06. This study demonstratesthe utility of NAM population for genetic dissection of complex traits and performing high-resolution trait mapping in peanut

Identification of expressed resistance gene analogs from peanut (Arachis hypogaea L.) expressed sequence tags

Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and 4 that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two PCR-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVE: The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested. RESEARCH DESIGN AND METHODS: The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions. RESULTS: HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates. CONCLUSIONS: ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction

Genetic Mapping of QTLs Controlling Fatty Acids Provided Insights into the Genetic Control of Fatty Acid Synthesis Pathway in Peanut (Arachis hypogaea L.)

Peanut, a high-oil crop with about 50% oil content, is either crushed for oil or used as edible products. Fatty acid composition determines the oil quality which has high relevance to consumer health, flavor, and shelf life of commercial products. In addition to the major fatty acids, oleic acid (C18:1) and linoleic acid (C18:2) accounting for about 80% of peanut oil, the six other fatty acids namely palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0) are accounted for the rest 20%. To determine the genetic basis and to improve further understanding on effect of FAD2 genes on these fatty acids, two recombinant inbred line (RIL) populations namely S-population (high oleic line ‘SunOleic 97R’ × low oleic line ‘NC94022’) and T-population (normal oleic line ‘Tifrunner’ × low oleic line ‘GT-C20’) were developed. Genetic maps with 206 and 378 marker loci for the S- and the T-population, respectively were used for quantitative trait locus (QTL) analysis. As a result, a total of 164 main-effect (M-QTLs) and 27 epistatic (E-QTLs) QTLs associated with the minor fatty acids were identified with 0.16% to 40.56% phenotypic variation explained (PVE). Thirty four major QTLs (>10% of PVE) mapped on five linkage groups and 28 clusters containing more than three QTLs were also identified. These results suggest that the major QTLs with large additive effects would play an important role in controlling composition of these minor fatty acids in addition to the oleic and linoleic acids in peanut oil. The interrelationship among these fatty acids should be considered while breeding for improved peanut genotypes with good oil quality and desired fatty acid composition

Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using simple sequence repeat markers

Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent with the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies

A recombination bin-map identified a major QTL for resistance to Tomato Spotted Wilt Virus in peanut (Arachis hypogaea)

Tomato spotted wilt virus (TSWV) is a devastating disease to peanut growers in the South-eastern region of the United States. Newly released peanut cultivars in recent years are crucial as they have some levels of resistance to TSWV. One mapping population of recombinant inbred line (RIL) used in this study was derived from peanut lines of SunOleic 97R and NC94022. A whole genome re-sequencing approach was used to sequence these two parents and 140 RILs. A recombination bin-based genetic map was constructed, with 5,816 bins and 20 linkage groups covering a total length of 2004 cM. Using this map, we identified three QTLs which were colocalized on chromosome A01. One QTL had the largest effect of 36.51% to the phenotypic variation and encompassed 89.5 Kb genomic region. This genome region had a cluster of genes, which code for chitinases, strictosidine synthase-like, and NBS-LRR proteins. SNPs linked to this QTL were used to develop Kompetitive allele specific PCR (KASP) markers, and the validated KASP markers showed expected segregation of alleles coming from resistant and susceptible parents within the population. Therefore, this bin-map and QTL associated with TSWV resistance made it possible for functional gene mapping, map-based cloning, and marker-assisted breeding. This study identified the highest number of SNP makers and demonstrated recombination bin-based map for QTL identification in peanut. The chitinase gene clusters and NBS-LRR disease resistance genes in this region suggest the possible involvement in peanut resistance to TSWV

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

<p>Abstract</p> <p>Background</p> <p>Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231).</p> <p>Methods</p> <p>Stable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion.</p> <p>Results</p> <p>Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis.</p> <p>Conclusions</p> <p>Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.</p