PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-6 (6)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Whole transcriptome RNA-Seq allelic expression in human brain 
BMC Genomics  2013;14:571.
Background
Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.
Results
We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.
Conclusions
These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.
doi:10.1186/1471-2164-14-571
PMCID: PMC3765493  PMID: 23968248
RNA-Seq; Whole transcriptome; Allele expression; mRNA expression; Functional genetics; Regulatory polymorphism; eQTL; Read alignment; Next generation sequencing; Bioinformatics
2.  Distinct cellular pools of perilipin 5 point to roles in lipid trafficking 
Biochimica et biophysica acta  2011;1821(2):268-278.
The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.
doi:10.1016/j.bbalip.2011.10.017
PMCID: PMC3740563  PMID: 22063271
Perilipin; Lipolysis; Lipid trafficking; Lipid droplets; Muscle; Adipose
3.  Cholesteryl Ester Transfer Protein (CETP) Polymorphisms Affect mRNA Splicing, HDL Levels, and Sex-Dependent Cardiovascular Risk 
PLoS ONE  2012;7(3):e31930.
Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5–7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4×10−5, allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8×10−10) and intron 8 polymorphism rs9930761-T>C (5.6×10−8) (in high linkage disequilibrium with allele frequencies 6–7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.
The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6×10−28 and rs5883 p = 8.6×10−10, adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29–4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.
doi:10.1371/journal.pone.0031930
PMCID: PMC3293889  PMID: 22403620
4.  Testing and Comparison of Non-Opioid Analgesics in Amphibians 
Because of the lack of information about effective analgesics in non-mammalian vertebrates, the potency of various non-opioid agents were tested in a model of analgesia by using Northern grass frogs (Rana pipiens). This alternative model has been used widely for investigating opioid analgesic action. Potential non-opioid analgesics tested included antipsychotic, benzodiazepine, barbiturate, antihistamine, non-steroidal anti-inflammatory (NSAID), and partial opioid agents. Northern grass frogs were acclimated to lab conditions in individual cages. Drugs were administered systemically through the dorsal lymph sac, and analgesic effects were estimated by using the acetic acid test (AAT). The AAT is done by placing logarithmic dilutions of acid dropwise on the dorsum of the animal’s thigh until a wiping response is obtained. At various doses, chlorpromazine and haloperidol (antipsychotics), chlordiazepoxide (a benzodiazepine), buprenorphine (a partial opioid agonist), and diphenhydramine (a histamine antagonist) produced moderate to strong analgesic effects. Indomethacin and ketorolac (NSAIDs), butorphanol (a partial opioid agonist), and pentobarbital (a barbiturate) produced weaker but noticeable analgesic effects. Our results are the first to document the effectiveness of a wide array of pharmacologically active agents in a novel amphibian model for analgesia. These findings provide needed data regarding the use of alternative, non-opioid agents for the treatment of pain in amphibians and other poikilothermic species.
PMCID: PMC3075466  PMID: 11451391
5.  Characterization of µ, κ, and δ Opioid Binding in Amphibian Whole Brain Tissue Homogenates 
Opioid agonists produce analgesia in mammals through the activation of µ, κ, or δ opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that, µ, κ, or δ opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) (µ-selective), [3H]U65953 [(5α,7α,8β)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] (κ-selective), and [3H]DPDPE ([d-Pen2,d-Pen5]-enkephalin) (δ-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (KD) in the low nanomolar range. The density of opioid binding sites (Bmax) was 224.4, 118.6, and 268.9 fmol/mg for µ, κ, and δ opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the κ radioligand bound to significantly fewer sites than did the µ or δ radioligands. Ki values for unlabeled µ, κ, and δ competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile. The present data suggest that µ, κ, and δ opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.
PMCID: PMC3066634  PMID: 11907194
6.  Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord 
Brain research  2000;884(1--2):184-191.
Opioids elicit antinociception in mammals through three distinct types of receptors designated as μ, κ and δ. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A Bmax value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (Ki) of unlabeled μ, κ and δ ranged from 2.58 nM to 84 μM. The highly-selective opioid antagonists yielded similar Ki values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective μ, κ and δ opioid agonists.
PMCID: PMC3062938  PMID: 11082500
Amphibian; [3H]-Naloxone; Antinociception; Opioid; β-FNA; nor-BNI; NTI

Results 1-6 (6)