MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Workshop #3 summary: adenoviruses/sv40/polyoma ii

Berries are a rich dietary source of bioactive polyphenols, including flavonoids, such as anthocyanins. Dietary flavonoids are known to impair Fe absorption and reduce non-haem-Fe transport across Caco-2 cell monolayers. Previously, we demonstrated that a flavonoidrich berry-extract decreases the expression of the divalent metal ion transporter (DMT1) and the haemochromatosis (HFE) genes, involved in the Fe-uptake pathway. The present study investigated the influence of this berry-extract on the gene expression and corresponding protein abundance of the apical ferrireductase, duodenal cytochrome B (DCYTB), the basolateral Fe efflux protein, ferroportin (FPN), the basolateral ferroxidase, hephaestin (HEPH) and DMT1 and HFE, which co-ordinate intestinal Fe transport. Human intestinal Caco-2 cells, cultured for 21 d, were then treated for 16 h with an anthocyanin-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.5% (w/v). RNA and protein were isolated for quantitative RT–PCR and western blotting, respectively. All gene expression data were normalised to 18S and GAPDH as housekeeping genes and presented as mean normalised expression ratio and SEM. All protein quantity data were normalised per mg of protein and presented as mean integral/{g protein and SEM. Statistical significance was determined by Student’s unpaired t-test with significance indicated at P £ 0.05. Following treatment with the berry-extract, there were significant decreases in DMT1 (mean 0.63 (SEM 0.07), p £ 0.039, n 6) and HFE (mean 0.52 (SEM 0.05), P £ 0.0001, n 6) mRNA expression. The abundance of the proteins HEPH and FPN, involved in the basolateral release of Fe, was significantly increased (Fig. 1). These results indicate that berry-flavonoids influence the expression of components of the Fe-uptake pathway. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the bioavailability of dietary Fe.

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Flavonoid-rich berry-extract influences expression of genes in the iron-uptake pathway in human intestinal Caco-2 cells

Non-haem iron bioavailability is regulated by a number of dietary components including, polyphenols which are thought to act through chelation of iron. The low solubility of iron/polyphenol complexes is regarded as the primary reason for reduced bioavailabilty. However, recent studies by our group and others suggest that the effects of polyphenols of iron absorption are complex and may involve not only control of iron solubility but also include nutrigenomic effects on iron transporter expression . In this present study we investigated the influence of quercetin, the most abundant flavonol in the diet on iron bioavailability by measuring transepithelial iron transport using radioactive Fe. The chronic effects of quercetin were assessed in fully differentiated Caco2 (intestinal) cells exposed for 24 h to quercetin (0, 10mM, and 100mM). In addition, the acute effects of polyphenols on iron bioavailability were also investigated. In these studies quercetin was added along with Fe at the start of the experimental period. Iron uptake was measured as the cellular accumulation of Fe over a 20 min time course. Efflux of iron from the cells into the basolateral medium was measured after 120 min. Data are mean S.E.M. of 5 observations in each group. Statistical analysis was carried out using One-way ANOVA. In the acute setting, the initial rate of iron uptake across the apical membrane of Caco-2 cells was significantly increased by the addition of quercetin (control, 5.7 0.9; 10mM quercetin, 11.0 1.6; 100mM quercetin 16.7 3.8 nmol/mg protein/20 min; P<0.03). In contrast, iron transport across the basolateral membrane was significantly decreased (control, 5.2 1.6; 10mM quercetin, 2.8 0.6; 100mM quercetin 0.12 0.03 nmol/mg protein/120 min; P<0.03). These data are in agreement with the reported effects of grape-seed polyphenols on iron transport and indicate that polyphenols may potentiate iron uptake across the apical membrane but act intracellularly to inhibit iron efflux via ferroportin. The mechanisms involved remain unresolved. Chronic exposure to quercetin for 24 h had no significant effect on iron uptake (P = 0.1), but iron efflux into the basolateral medium was significantly decreased (control, 1.0 0.3; 10mM quercetin, 0.7 0.1; 100mM quercetin 0.08 0.03 nmol/mg protein/120 min; P<0.002). These findings are consistent with our previous observation that quercetin significantly decreases the expression of the basolateral iron transporter ferroportin and its partner ferroxidase hephaestin .

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Effect of quercetin on non-haem iron transport in human intestinal Caco-2 cells

Science in general, and specifically the science of life, offers an intriguing path to walk along. As you deepen your understanding of a particular topic, you become aware of things you did not see at all when you started. Sometimes, this new seeing even drives you to reconsider and redefine very basic concepts that you learned along the way. This is exactly what happened after we discovered a process called RNA splicing. In RNA splicing, pieces of genetic instructions are cut and pasted together to form the final instructions for producing proteins. The discovery of RNA splicing has driven us to rethink what we previously believed about genes, which are the most fundamental units of information in biology. In this article, I will tell you what we discovered about RNA splicing, how it influenced our ideas about genes, and how we now use this knowledge to significantly improve people’s lives.

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RNA Splicing—Cutting and Pasting Genes

MicroRNAs (miRNAs) are short non-coding RNAs which regulate gene expression post-transcriptionally by triggering mRNA degradation or translation repression. Little is known about the role of miRNAs in the gastrointestinal tract. They may be instrumental in regulating gene expression in intestinal epithelial cells, particularly in response to nutrients. The present study investigated miRNA expression in response to a flavonoid-rich berry extract in human intestinal Caco-2 cell monolayers. Human intestinal Caco-2 cells, cultured for 19 d, were treated for 16 h with a flavonoid-rich berry extract (Optiberry; Interhealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.125% (w/v). Subsequently miRNA was extracted, pooled and used for the miRNA microarray analysis (Affymetrix1; UK) and validated by quantitative RT-PCR. MiRNA microarray data was analysed using the bioinformatic TargetScanHuman 5.1 database, which predicts miRNA targets. The results showed that 162 miRNA species were detected (P<0.01). Using quantile normalisation, 2 miRNAs were up-regulated and 27 were down-regulated in response to the berry extract. Previously we have shown that incubation with berry extract decreases expression of the intestinal sugar transporter GLUT2 in Caco-2 cells. Bioinformatic analysis identified nine of these differentially expressed miRNAs as potential regulators of the intestinal sugar transporter, GLUT2. Validation by qRT-PCR showed that two of these miRNAs, let-7a (P = 0.001) and miR-106b (P<0.001) were significantly up-regulated in Caco-2 cells in response to berry flavonoids. Studies are in progress, using RNA interference techniques, to investigate whether let-7a and miR-106b directly regulate GLUT2 expression.

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Phillip A. Sharp

Papers

Workshop #3 summary: adenoviruses/sv40/polyoma ii

Flavonoid-rich berry-extract influences expression of genes in the iron-uptake pathway in human intestinal Caco-2 cells

Effect of quercetin on non-haem iron transport in human intestinal Caco-2 cells

RNA Splicing—Cutting and Pasting Genes

MicroRNA expression in human intestinal Caco-2 cells in response to a flavonoid-rich berry extract