Gut bacteria and necrotizing enterocolitis: cause or effect?

Development of necrotising enterocolitis (NEC) is considered to be dependent on the bacterial colonisation of the gut. With little concordance between published data and a recent study failing to detect a common strain in infants with NEC, more questions than answers are arising about our understanding of this complex disease

Molecular structure and genetic mapping of the mouse gastrin gene

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/117035/1/feb20014579396004309.pd

microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

BACKGROUND: Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles. RESULTS: The expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction. CONCLUSIONS: miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity

TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cellsPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43859/1/11248_2004_Article_172516.pd

Somatostatin analogue treatment primarily induce miRNA expression changes and up-regulates growth inhibitory miR-7 and miR-148a in neuroendocrine cells

Somatostatin (SST) analogues are used to control the proliferation and symptoms of neuroendocrine tumors (NETs). MicroRNAs (miRNA) are small non-coding RNAs that modulate posttranscriptional gene expression. We wanted to characterize the miRNAs operating under the control of SST to elucidate to what extent they mediate STT actions. NCI-H727 carcinoid cell line was treated with either a chimeric SST/dopamine analogue; a SST or dopamine analogue for proliferation assays and for identifying differentially expressed miRNAs using miRNA microarray. The miRNAs induced by SST analogue treatment are investigated in carcinoid cell lines NCI-H727 and CNDT2 using in situ hybridization, qPCR and proliferation assays. SST analogues inhibited the growth of carcinoid cells more potently compared to the dopamine analogue. Principal Component Analysis (PCA) of the samples based on miRNA expression clearly separated the samples based on treatment. Two miRNAs which were highly induced by SST analogues, miR-7 and miR-148a, were shown to inhibit the proliferation of NCI-H727 and CNDT2 cells. SST analogues also produced a general up-regulation of the let-7 family members. SST analogues control and induce distinct miRNA expression patterns among which miR-7 and miR-148a both have growth inhibitory properties

The optimal cut-off value in fit-based colorectal cancer screening:An observational study

Abstract Background Colorectal cancer (CRC) screening programs using fecal immunochemical test (FIT) have to choose a cut‐off value to decide which citizens to recall for colonoscopy. The evidence on the optimal cut‐off value is sparse and based on studies with a low number of cancer cases. Methods This observational study used data from the Danish Colorectal Cancer Screening Database. Sensitivity and specificity were estimated for various cut‐off values based on a large number of cancers. Traditionally optimal cut‐off values are found by weighting sensitivity and specificity equally. As this might result in too many unnecessary colonoscopies we also provide optimal cut‐off values for different weighting of sensitivity and specificity/number of needed colonoscopies to detect one cancer. Results Weighting sensitivity and specificity equally gives an optimal cut‐off value of 45 ng Hb/ml. This, however, means making 24 colonoscopies to detect one cancer. Weighting sensitivity lower and for example, aiming at making about 16 colonoscopies to detect one cancer, gives an optimal cut‐off value of 125 ng Hb/ml. Conclusions The optimal cut‐off value in an FIT population‐based screening program is 45 ng Hb/ml, when as traditionally sensitivity and specificity are weighted equally. If, however, 24 colonoscopies needed to detect one cancer is too huge a burden on the health care system and the participants, 80, 125, 175, and 350 ng Hb/ml are optimal cut‐off values when only 19/16/14/10 colonoscopies are accepted to find one cancer