In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes

Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model, few causal gene variants have been identified. Here we show that RNA interference (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein. Using lentiviral transgenesis, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.     

Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells

Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ～100?picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10?megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.     

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation.

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T' parallel order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T' parallel for a 3-h period at 37 degrees C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.     

Different life histories of brook trout populations invading mid-elevation and high-elevation cutthroat trout streams in Colorado

Brook Trout ( Salvelinus fontinalis ), native to eastern North America, have invaded many montane cold-water systems of western North America, and these invasions are implicated in the decline of native cutthroat trout ( Oncorhynchus clarki ). If fisheries biologists are to be effective in managing brook trout invasions, demographic models that predict invasion success will need to incorporate life history variation in different environments. We tested whether brook trout populations invading streams at 2 different elevations varied in life history characteristics that influence population dynamics and potential invasion success. In the high-elevation stream (3195 m), water temperatures were colder and brook trout apparently grew more slowly (i.e., had shorter lengths-at-age), became sexually mature 2 years later, and had life spans 2 to 3 times longer than those in the mid-elevation stream (2683 m). This flexibility in life history may allow brook trout to maximize their chance of establishment and invasion success among elevations. We propose that in mid-elevation streams fast growth and early maturity maximize fitness and can lead to rapid establishment and high population growth rates. In high-elevation streams, slow growth, later maturity, and a long reproductive life span may allow brook trout to successfully establish populations in these marginal habitats where recruitment is often poor.     

Distributional records for the Colorado flora II

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Single-channel currents in isolated patches of plasma membrane from basal surface of pancreatic acini

Precise localization and characterization of conductance pathways in glandular epithelia have so far proved difficult. The patch-clamp technique for high resolution current recording, which has already been applied successfully to a number of electrically excitable cells, can in principle overcome these difficulties. We now report measurements of single-channel currents from isolated patches of plasma membrane (inside-out) from the baso-lateral surface of collagenase-isolated rat and mouse pancreatic acini. We have identified a cation channel having a conductance of approximately 30 pS and a mean open time in the range 0.3-1 s which is dependent on internal calcium. The single-channel current-voltage relationship is linear and the mean open time independent of the membrane potential. These channels may, at least in part, account for the Ca2+-mediated neural and hormonal control of pancreatic acinar membrane conductance, which is probably responsible for the Ca2+-dependent acinar fluid secretion.