Isolation and characterization of microsatellite loci from two inbreeding bark beetle species (Coccotrypes)

We developed 14 microsatellite markers in Coccotrypes carpophagus and 14 in C. dactyliperda. These loci will be used for studying genetic structure and the level of inbreeding in populations in the Canary Islands and Madeira. As a result of long-term inbreeding, genetic variability is relatively low in these bark beetle species. We found one to five alleles per locus in 29 C. carpophagus and 41 C. dactyliperda from various localities. Eleven of the markers developed for C. carpophagus amplified in C. dactyliperda and seven of the markers developed for C. dactyliperda amplified in C. carpophagus

Isolation of polymorphic microsatellites in the stemless thistle (Cirsium acaule) and their utility in other Cirsium species

The genus Cirsium includes species with both widespread and restricted geographical distributions, several of which are serious weeds. Nine polymorphic microsatellite loci were isolated from the stemless thistle Cirsium acaule. Eight were polymorphic in C. acaule, six in C. arvense and seven in C. heterophyllum. One locus monomorphic in C. acaule showed polymorphism in C. heterophyllum. The mean number of alleles per locus was 4.1 in C. acaule, 6.2 in C. arvense and 2.9 in C. heterophyllum. These nine loci were also amplified in C. eriophorum and C. vulgare, suggesting that these markers may be of use throughout the genus

Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP-seq with in vitro biochemistry to characterize a putative NsrR homologue in the model organism Streptomyces venezuelae. ChIP seq analysis revealed that rather than regulating the nitrosative stress response like NsrR, Sven6563 binds to a different, much larger regulon of genes with a diverse range of functions, including a range of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the target gene regulon are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator

Detection of Ligation Products of DNA Linkers with 5′-OH Ends by Denaturing PAGE Silver Stain

To explore if DNA linkers with 5′-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5–1% of linkers A–B and E–F, and 0.13–0.5% of linkers C–D could be joined by T4 DNA ligases. About 0.25–0.77% of linkers A–B and E–F, and 0.06–0.39% of linkers C–D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025–0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5′-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025–0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3′-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5′-ends and thereby generated some 5′-phosphate ends, and then these 5′-phosphate ends could be joined to the 3′-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5′-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

The -786T>C promoter polymorphism of the NOS3 gene is associated with prostate cancer progression

<p>Abstract</p> <p>Background</p> <p>There is no biological or epidemiological data on the association between <it>NOS3 </it>promoter polymorphisms and prostate cancer. The polymorphisms in the promoter region of <it>NOS3 </it>gene may be responsible for variations in the plasma NO, which may promote cancer progression by providing a selective growth advantage to tumor cells by angiogenic stimulus and by direct DNA damage.</p> <p>Methods</p> <p>This study aimed evaluating the <it>NOS3 </it>promoter polymorphisms by PCR-SSCP and sequencing, associating genotypes and haplotypes with <it>NOS3 </it>expression levels through semi-quantitative RT-PCR, and with <it>PCA</it>3 mRNA detection, a specific tumor biomarker, in the peripheral blood of pre-surgical samples from 177 patients; 83 PCa and 94 BPH.</p> <p>Results</p> <p>Three novel SNPs were identified -764A>G, -714G>T and -649G>A in the <it>NOS3 </it>gene promoter region, which together with the -786T>C generated four haplotypes (N, T, C, A). <it>NOS3 </it>gene expression levels were affected by the -786T>C polymorphism, and there was a 2-fold increase in <it>NOS3 </it>levels favored by the incorporation of each C allele. <it>NOS3 </it>levels higher than 80% of the constitutive gene expression level (<it>B2M</it>) presented a 4-fold increase in PCa occurrence.</p> <p>Conclusion</p> <p>The -786T>C polymorphism was the most important promoter alteration of the <it>NOS3 </it>gene that may affect the PCa progression, but not its occurrence, and the incorporation of the C allele is associated with increased levels of <it>NOS3 </it>transcripts. The <it>NOS3 </it>transcript levels presented a bimodal behavior in tumor development and may be used as a biomarker together with the <it>PCA3 </it>marker for molecular staging of the prostate cancer.</p

Effect of polymorphisms in the Slc11a1 coding region on resistance to brucellosis by macrophages in vitro and after challenge in two Bos breeds (Blanco Orejinegro and Zebu)

The resistance/susceptibility of selected cattle breeds to brucellosis was evaluated in an F1 population generated by crossing animals classified as resistant (R) and susceptible (S) (R x R, R x S, S x R, S x S) based on challenges in vitro and in vivo. The association between single nucleotide polymorphisms identified in the coding region of the Slc11a1 gene and resistance/susceptibility was estimated. The trait resistance or susceptibility to brucellosis, evaluated by a challenge in vitro, showed a high heritable component in terms of additive genetic variance (h2 = 0.54 ± 0.11). In addition, there was a significant association (p < 0.05) between the control of bacterial survival and two polymorphisms (a 3'UTR and SNP4 located in exon 10). The antibody response of animals classified as resistant to infection by Brucella abortus differed significantly (p < 0.05) from that of susceptible animals. However, there was no significant association between single nucleotide polymorphisms located in the Slc11a1 gene and the antibody response stimulated by a challenge in vivo

Alterations of tumor suppressor gene p16(INK4a )in pancreatic ductal carcinoma

BACKGROUND: Cell cycle inhibitor and tumor suppressor gene p16 / MTS-1 has been reported to be altered in a variety of human tumors. The purpose of the study was to evaluate primary pancreatic ductal adenocarcinomas for potentially inactivating p16 alterations. METHODS: We investigated the status of p16 gene by polymerase chain reaction (PCR), nonradioisotopic single strand conformation polymorphism (SSCP), DNA sequencing and hypermethylation analysis in 25 primary resected ductal adenocarcinomas. In addition, we investigated p16 protein expression in these cases by immunohistochemistry (IHC) using a monoclonal antibody clone (MS-887-PO). RESULTS: Out of the 25 samples analyzed and compared to normal pancreatic control tissues, the overall frequency of p16 alterations was 80% (20/25). Aberrant promoter methylation was the most common mechanism of gene inactivation present in 52% (13/25) cases, followed by coding sequence mutations in 16% (4/25) cases and presumably homozygous deletion in 12% (3/25) cases. These genetic alterations correlated well with p16 protein expression as complete loss of p16 protein was found in 18 of 25 tumors (72%). CONCLUSION: These findings confirm that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behaviour of this tumor type