Replacing the wild type loxP site in BACs from the public domain with lox66 using a lox66 transposon

<p>Abstract</p> <p>Background</p> <p>Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.</p> <p>Findings</p> <p>A general procedure to replace the <it>loxP </it>site located at one end of genomic DNA inserts in BACs with <it>lox66 </it>is described. Truncating insert DNA from the <it>loxP </it>end with a Tn10 transposon carrying a <it>lox66 </it>site simultaneously substitutes the <it>loxP </it>with a <it>lox66 </it>sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of <it>loxP </it>with <it>lox66 </it>or <it>lox71 </it>was found to be as efficient as another <it>loxP </it>site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a <it>lox66 </it>transposon occurred at no more than 20% the efficiency observed with a <it>loxP </it>transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical <it>lox</it>-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.</p> <p>Conclusions</p> <p>The <it>loxP/lox66 </it>replacement procedure should allow targeting BACs to a pre-positioned <it>lox71 </it>site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.</p

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5–16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg−/−) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT

Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumours

Chromosomal rearrangements occur constitutionally in the general population and somatically in the majority of cancers. Detection of balanced rearrangements, such as reciprocal translocations and inversions, is troublesome, which is particularly detrimental in oncology where rearrangements play diagnostic and prognostic roles. Here we describe the use of Hi-C as a tool for detection of both balanced and unbalanced chromosomal rearrangements in primary human tumour samples, with the potential to define chromosome breakpoints to bp resolution. In addition, we show copy number profiles can also be obtained from the same data, all at a significantly lower cost than standard sequencing approaches

Probing forces of menisci: what levels are safe for arthroscopic surgery

Purpose To facilitate effective learning, feedback on performance during arthroscopic training is essential. Less attention has been paid to feedback on monitoring safe handling of delicate tissues such as meniscus. The goal is to measure in vitro probing forces of menisci and compare them with a theoretical maximum probing force (TMPF). Method Menisci samples of ten cadavers were mounted on force platforms to measure probing forces up to 20 N in three directions. Nineteen subjects participated: six novices (experience 60 arthroscopies), and three faculty (>250 a year). All had to perform three tasks on each meniscus sample with an arthroscopic probe: push three times on the superior meniscal surface, perform one continuous run on the superior meniscal surface, and push three times on the inferior meniscal surface. The absolute maximum probing force (AMPF) was determined for each condition. A multivariable linear regression analysis was performed to assess the influence of experience on the force magnitude (P < 0.05). AMPFs were compared to the TMPF (estimated to be 8.5 N). Results The AMPF of the push task was on average 2.8 N (standard deviation (SD) of 0.8 N), of the continuous run task 2.5 N (SD 0.9 N), and of the pull task 3.9 N (SD 2.0 N). Significant difference was present between experts and novices (P < 0.05). The AMPFs are in the same order of magnitude as the TMPF. Conclusion The results indicate the necessity of using a safety level for tissue manipulation when training arthroscopy and a value for is magnitude.Biomechanical EngineeringMechanical, Maritime and Materials Engineerin

Adherence to colorectal cancer screening guidelines in Canada

<p>Abstract</p> <p>Background</p> <p>To identify correlates of adherence to colorectal cancer (CRC) screening guidelines in average-risk Canadians.</p> <p>Methods</p> <p>2003 Canadian Community Health Survey Cycle 2.1 respondents who were at least 50 years old, without past or present CRC and living in Ontario, Newfoundland, Saskatchewan, and British Columbia were included. Outcomes, defined according to current CRC screening guidelines, included adherence to: i) fecal occult blood test (FOBT) (in prior 2 years), ii) endoscopy (colonoscopy/sigmoidoscopy) (prior 10 years), and iii) adherence to CRC screening guidelines, defined as either (i) or (ii). Generalized estimating equations regression was employed to identify correlates of the study outcomes.</p> <p>Results</p> <p>Of the 17,498 respondents, 70% were non-adherent CRC screening to guidelines. Specifically, 85% and 79% were non-adherent to FOBT and endoscopy, respectively. Correlates for all outcomes were: having a regular physician (OR = (i) 2.68; (ii) 1.91; (iii) 2.39), getting a flu shot (OR = (i) 1.59; (ii) 1.51; (iii) 1.55), and having a chronic condition (OR = (i) 1.32; (ii) 1.48; (iii) 1.43). Greater physical activity, higher consumption of fruits and vegetables and smoking cessation were each associated with at least 1 outcome. Self-perceived stress was modestly associated with increased odds of adherence to endoscopy and to CRC screening guidelines (OR = (ii) 1.07; (iii) 1.06, respectively).</p> <p>Conclusion</p> <p>Healthy lifestyle behaviors and factors that motivate people to seek health care were associated with adherence, implying that invitations for CRC screening should come from sources that are independent of physicians, such as the government, in order to reduce disparities in CRC screening.</p

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia.

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects

Differences in bleeding behavior after endoscopic band ligation: a retrospective analysis

<p>Abstract</p> <p>Background</p> <p>Endoscopic band ligation (EBL) is generally accepted as the treatment of choice for bleeding from esophageal varices. It is also used for secondary prophylaxis of esophageal variceal hemorrhage. However, there is no data or guidelines concerning endoscopic control of ligation ulcers. We conducted a retrospective study of EBL procedures analyzing bleeding complications after EBL.</p> <p>Methods</p> <p>We retrospectively analyzed data from patients who underwent EBL. We analyzed several data points, including indication for the procedure, bleeding events and the time interval between EBL and bleeding.</p> <p>Results</p> <p>255 patients and 387 ligation sessions were included in the analysis. We observed an overall bleeding rate after EBL of 7.8%. Bleeding events after elective treatment (3.9%) were significantly lower than those after treatment for acute variceal hemorrhage (12.1%). The number of bleeding events from ligation ulcers and variceal rebleeding was 14 and 15, respectively. The bleeding rate from the ligation site in the group who underwent emergency ligation was 7.1% and 0.5% in the group who underwent elective ligation. Incidence of variceal rebleeding did not vary significantly. Seventy-five percent of all bleeding episodes after elective treatment occurred within four days after EBL. 20/22 of bleeding events after emergency ligation occured within 11 days after treatment. Elective EBL has a lower risk of bleeding from treatment-induced ulceration than emergency ligation.</p> <p>Conclusions</p> <p>Patients who underwent EBL for treatment of acute variceal bleeding should be kept under medical surveillance for 11 days. After elective EBL, it may be reasonable to restrict the period of surveillance to four days or even perform the procedure in an out-patient setting.</p

Serum CD26 is related to histopathological polyp traits and behaves as a marker for colorectal cancer and advanced adenomas

<p>Abstract</p> <p>Background</p> <p>Serum CD26 (sCD26) levels were previously found diminished in colorectal cancer (CRC) patients compared to healthy donors, suggesting its potential utility for early diagnosis. Therefore we aimed to estimate the utility of the sCD26 as a biomarker for CRC and advanced adenomas in a high-risk group of patients. The relationship of this molecule with polyp characteristics was also addressed.</p> <p>Methods</p> <p>sCD26 levels were measured by ELISA in 299 symptomatic and asymptomatic patients who had undergone a colonoscopy. Patients were diagnosed as having no colorectal pathology, non-inflammatory or inflammatory bowel disease, polyps (hyperplastic, non-advanced and advanced adenomas) or CRC.</p> <p>Results</p> <p>At a 460 ng/mL cut-off, the sCD26 has a sensitivity and specificity of 81.8% (95% CI, 64.5-93.0%) and 72.3% (95% CI, 65.0-77.2%) for CRC regarding no or benign colorectal pathology. Clinicopathological analysis of polyps showed a relationship between the sCD26 and the grade of dysplasia and the presence of advanced adenomas. Hence, a 58.0% (95% CI, 46.5-68.9%) sensitivity detecting CRC and advanced adenomas was obtained, with a specificity of 75.5% (95% CI, 68.5-81.0%).</p> <p>Conclusions</p> <p>Our preliminary results show that measurement of the sCD26 is a non-invasive and reasonably sensitive assay, which could be combined with others such as the faecal occult blood test for the early diagnosis and screening of CRC and advanced adenomas. Additional comparative studies in average-risk populations are necessary.</p

Context dependent function of APPb enhancer identified using enhancer trap-containing BACs as transgenes in zebrafish

An enhancer within intron 1 of the amyloid precursor protein gene (APPb) of zebrafish is identified functionally using a novel approach. Bacterial artificial chromosomes (BACs) were retrofitted with enhancer traps, and expressed as transgenes in zebrafish. Expression from both transient assays and stable lines were used for analysis. Although the enhancer was active in specific nonneural cells of the notochord when placed with APPb gene promoter proximal elements its function was restricted to, and absolutely required for, specific expression in neurons when juxtaposed with additional far-upstream promoter elements of the gene. We demonstrate that expression of green fluorescent protein fluorescence resembling the tissue distribution of APPb mRNA requires both the intron 1 enhancer and ∼28 kb of DNA upstream of the gene. The results indicate that tissue-specificity of an isolated enhancer may be quite different from that in the context of its own gene. Using this enhancer and upstream sequence, polymorphic variants of APPb can now more closely recapitulate the endogenous pattern and regulation of APPb expression in animal models for Alzheimer's disease. The methodology should help functionally map multiple noncontiguous regulatory elements in BACs with or without gene-coding sequences