RATEmiRs: the rat atlas of tissue-specific and enriched miRNAs for discerning baseline expression exclusivity of candidate biomarkers.

MicroRNAs (miRNAs) are small RNAs that regulate mRNA expression and have been targeted as biomarkers of organ damage and disease. To explore the utility of miRNAs to assess injury to specific tissues, a tissue atlas of miRNA abundance was constructed. The Rat Atlas of Tissue-specific and Enriched miRNAs (RATEmiRs) catalogues miRNA sequencing data from 21 and 23 tissues in male and female Sprague-Dawley rats, respectively. RATEmiRs identifies tissue-enriched (TE), tissue-specific (TS), or organ-specific (OS) miRNAs via comparisons of one or more tissue or organ vs others. We provide a brief overview of RATEmiRs and present how to use it to detect miRNA expression abundance of candidate biomarkers as well as to compare the expression of miRNAs between rat and human. The database is available at https://www.niehs.nih.gov/ratemirs/.

RATEmiRs: the rat atlas of tissue-specific and enriched miRNAs database.

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression and have been targeted as indicators of environmental/toxicologic stressors. Using the data from our deep sequencing of miRNAs in an extensive sampling of rat tissues, we developed a database called RATEmiRs for the Rat Atlas of Tissue-specific and Enriched miRNAs to allow users to dynamically determine mature-, iso- and pre-miR expression abundance, enrichment and specificity in rat tissues and organs. RESULTS: Illumina sequencing count data from mapped reads and meta data from the miRNA body atlas consisting of 21 and 23 tissues (14 organs) of toxicologic interest from 12 to 13 week old male and female Sprague Dawley rats respectively, were managed in a relational database with a user-friendly query interface. Data-driven pipelines are available to tailor the identification of tissue-enriched (TE) and tissue-specific (TS) miRNAs. Data-driven organ-specific (OS) pipelines reveal miRNAs that are expressed predominately in a given organ. A user-driven approach is also available to assess the tissue expression of user-specified miRNAs. Using one tissue vs other tissues and tissue(s) of an organ vs other organs, we illustrate the utility of RATEmiRs to facilitate the identification of candidate miRNAs. As a use case example, RATEmiRs revealed two TS miRNAs in the liver: rno-miR-122-3p and rno-miR-122-5p. When liver is compared to just the brain tissues for example, rno-miR-192-5p, rno-miR-193-3p, rno-miR-203b-3p, rno-miR-3559-5p, rno-miR-802-3p and rno-miR-802-5p are also detected as abundantly expressed in liver. As another example, 55 miRNAs from the RATEmiRs query of ileum vs brain tissues overlapped with miRNAs identified from the same comparison of tissues in an independent, publicly available dataset of 10 week old male rat microarray data suggesting that these miRNAs are likely not age-specific, platform-specific nor pipeline-dependent. Lastly, we identified 10 miRNAs that have conserved tissue/organ-specific expression between the rat and human species. CONCLUSIONS: RATEmiRs provides a new platform for identification of TE, TS and OS miRNAs in a broad array of rat tissues. RATEmiRs is available at: https://www.niehs.nih.gov/ratemirs.

The Rat microRNA body atlas; Evaluation of the microRNA content of rat organs through deep sequencing and characterization of pancreas enriched miRNAs as biomarkers of pancreatic toxicity in the rat and dog.

BACKGROUND: MicroRNAs (miRNA) are ~19-25 nucleotide long RNA molecules that fine tune gene expression through the inhibition of translation or degradation of the mRNA through incorporation into the RNA induced silencing complex (RISC). MicroRNAs are stable in the serum and plasma, are detectable in a wide variety of body fluids, are conserved across veterinary species and humans and are expressed in a tissue specific manner. They can be detected at low concentrations in circulation in animals and humans, generating interest in the utilization of miRNAs as serum and/or plasma based biomarkers of tissue injury. MicroRNA tissue profiling in rodents has been published, but sample an insufficient number of organs of toxicologic interest using microarray or qPCR technologies for miRNA detection. Here we impart an improved rat microRNA body atlas consisting of 21 and 23 tissues of toxicologic interest from male and female Sprague Dawley rats respectively, using Illumina miRNA sequencing. Several of the authors created a dog miRNA body atlas and we collaborated to test miRNAs conserved in rat and dog pancreas in caerulein toxicity studies utilizing both species. RESULTS: A rich data set is presented that more robustly defines the tissue specificity and enrichment profiles of previously published and undiscovered rat miRNAs. We generated 1,927 sequences that mapped to mature miRNAs in rat, mouse and human from miRBase and discovered an additional 1,162 rat miRNAs as compared to the current number of rat miRNAs in miRBase version 21. Tissue specific and enriched miRNAs were identified and a subset of these miRNAs were validated by qPCR for tissue specificity or enrichment. As an example of the power of this approach, we have conducted rat and dog pancreas toxicity studies and examined the levels of some tissue specific and enriched miRNAs conserved between rat and dog in the serum of each species. The studies demonstrate that conserved tissue specific/enriched miRs-216a-5p, 375-3p, 148a-3p, 216b-5p and 141-3p are candidate biomarkers of pancreatic injury in the rat and dog. CONCLUSIONS: A microRNA body atlas for rat and dog was useful in identifying new candidate miRNA biomarkers of organ toxicity in 2 toxicologically relevant species.

Genomic structure and expression of the human serotonin 2A receptor gene (HTR2A) locus: identification of novel HTR2A and antisense (HTR2A-AS1) exons.

BACKGROUND: The serotonin 2A receptor is widely implicated in genetic association studies and remains an important drug target for psychiatric, neurological, and cardiovascular conditions. RNA sequencing redefined the architecture of the serotonin 2A receptor gene (HTR2A), revealing novel mRNA transcript isoforms utilizing unannotated untranslated regions of the gene. Expression of these untranslated regions is modulated by common single nucleotide polymorphisms (SNPs), namely rs6311. Previous studies did not fully capture the complexity of the sense- and antisense-encoded transcripts with respect to novel exons in the HTR2A gene locus. Here, we comprehensively catalogued exons and RNA isoforms for both HTR2A and HTR2A-AS1 using RNA-Seq from human prefrontal cortex and multiple mouse tissues. We subsequently tested associations between expression of newfound gene features and common SNPs in humans. RESULTS: We find that the human HTR2A gene spans ~66 kilobases and consists of 7, rather than 4 exons. Furthermore, the revised human HTR2A-AS1 gene spans ~474 kilobases and consists of 18, rather than 3 exons. Three HTR2A exons directly overlap with HTR2A-AS1 exons, suggesting potential for complementary nucleotide interactions. The repertoire of possible mouse Htr2a splice isoforms is remarkably similar to humans and we also find evidence for overlapping sense-antisense transcripts in the same relative positions as the human transcripts. rs6311 and SNPs in high linkage disequilibrium are associated with HTR2A-AS1 expression, in addition to previously described associations with expression of the extended 5' untranslated region of HTR2A. CONCLUSIONS: Our proposed HTR2A and HTR2A-AS1 gene structures dramatically differ from current annotations, now including overlapping exons on the sense and anti-sense strands. We also find orthologous transcript isoforms expressed in mice, providing opportunities to elucidate the biological roles of the human isoforms using a model system. Associations between rs6311 and expression of HTR2A and HTR2A-AS1 suggest this polymorphism is capable of modulating the expression of the sense or antisense transcripts. Still unclear is whether these SNPs act directly on the expression of the sense or antisense transcripts and whether overlapping exons are capable of interacting through complimentary base-pairing. Additional studies are necessary to determine the extent and nature of interactions between the SNPs and the transcripts prior to interpreting these findings in the context of phenotypes associated with HTR2A.

An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease.

Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3ß, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3ß using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

Genome-wide screen for modulation of hepatic apolipoprotein A-I (ApoA-I) secretion.

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Paenibacillus lentus sp. nov., a ß-mannanolytic bacterium isolated from mixed soil samples in a selective enrichment using guar gum as the sole carbon source.

A novel bacterial strain, CMG1240(T), was isolated in 1988 from mixed soil samples collected from the United States and South America in a selective enrichment medium with guar gum as the sole carbon source. This microbial isolate showed ß-mannanolytic activity to hydrolyse the galactomannans present in guar gum. Strain CMG1240(T) was aerobic, Gram-stain-variable, non-motile, rod-shaped and endospore-forming. It was further examined based on a combination of phenotypic, physiological and genetic characterization. On the basis of 16S rRNA gene sequence similarity, cellular lipid profile and fatty acid composition, strain CMG1240(T) was shown to belong unequivocally to the genus Paenibacillus. Quinone analysis showed that MK-7 was the only menaquinone detected. The main cell-wall sugar was xylose with trace amounts of mannose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unknown glycolipids, phospholipids, phosphoglycolipids and other lipids. The peptidoglycan structure was A1γ (meso-diaminopimelic acid-direct). The major fatty acids were anteiso-C15 : 0 and C16 : 0. The DNA G+C content was 46 mol% as determined experimentally and by analysis of the genomic sequence. The 16S rRNA gene sequence of strain CMG1240(T) shared highest similarity with that of Paenibacillus fonticola ZL(T) (97.6 %) while all other tested Paenibacillus strains showed lower sequence similarities (≤95.3 %). The results of DNA-DNA hybridization and chemotaxonomic tests enabled the genotypic and phenotypic differentiation of strain CMG1240(T) from P. fonticola. Based on these results, strain CMG1240(T) ( = ATCC BAA-2594(T) = DSM 25539(T)) should be designated the type strain of a novel species within the genus Paenibacillus, for which the name Paenibacillus lentus sp. nov. is proposed.

Large eQTL meta-analysis reveals differing patterns between cerebral cortical and cerebellar brain regions.

The availability of high-quality RNA-sequencing and genotyping data of post-mortem brain collections from consortia such as CommonMind Consortium (CMC) and the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) Consortium enable the generation of a large-scale brain cis-eQTL meta-analysis. Here we generate cerebral cortical eQTL from 1433 samples available from four cohorts (identifying >4.1 million significant eQTL for >18,000 genes), as well as cerebellar eQTL from 261 samples (identifying 874,836 significant eQTL for >10,000 genes). We find substantially improved power in the meta-analysis over individual cohort analyses, particularly in comparison to the Genotype-Tissue Expression (GTEx) Project eQTL. Additionally, we observed differences in eQTL patterns between cerebral and cerebellar brain regions. We provide these brain eQTL as a resource for use by the research community. As a proof of principle for their utility, we apply a colocalization analysis to identify genes underlying the GWAS association peaks for schizophrenia and identify a potentially novel gene colocalization with lncRNA RP11-677M14.2 (posterior probability of colocalization 0.975).

Molecular dissection of CRC primary tumors and their matched liver metastases reveals critical role of immune microenvironment, EMT and angiogenesis in cancer metastasis.

Metastasis is the primary cause of cancer mortality. The primary tumors of colorectal cancer (CRC) often metastasize to the liver. In this study, we have collected 122 samples from 45 CRC patients. Among them, 32 patients have primary tumors, adjacent normal tissues, and matched liver metastases. Thirteen patients have primary tumors without distant metastasis and matched normal tissues. Characterization of these samples was conducted by whole-exome and RNA sequencing and SNP6.0 analysis. Our results revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and metastatic tumors. We then assembled gene co-expression networks and identified metastasis-correlated gene networks of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets has verified that these specific gene networks are up regulated throughout the tumor progression. The gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely correlated with the poor patient outcome and intrinsic anti-PD-1 resistance. These results offer insights of combinational strategy for the treatment of metastatic CRC.

Genomic characterization of intrinsic and acquired resistance to cetuximab in colorectal cancer patients.

Anti-EGFR antibodies are effective in therapies for late-stage colorectal cancer (CRC); however, many tumours are unresponsive or develop resistance. We performed genomic analysis of intrinsic and acquired resistance to anti-EGFR therapy in prospectively collected tumour samples from 25 CRC patients receiving cetuximab (an EGFR inhibitor). Of 25 CRC patients, 13 displayed intrinsic resistance to cetuximab; 12 were intrinsically sensitive. We obtained six re-biopsy samples at acquired resistance from the intrinsically sensitive patients. NCOA4-RET and LMNA-NTRK1 fusions and NRG1 and GNAS amplifications were found in intrinsic-resistant patients. In cetuximab-sensitive patients, we found KRAS K117N and A146T mutations in addition to BRAF V600E, AKT1 E17K, PIK3CA E542K, and FGFR1 or ERBB2 amplifications. The comparison between baseline and acquired-resistant tumours revealed an extreme shift in variant allele frequency of somatic variants, suggesting that cetuximab exposure dramatically selected for rare resistant subclones that were initially undetectable. There was also an increase in epithelial-to-mesenchymal transition at acquired resistance, with a reduction in the immune infiltrate. Furthermore, characterization of an acquired-resistant, patient-derived cell line showed that PI3K/mTOR inhibition could rescue cetuximab resistance. Thus, we uncovered novel genomic alterations that elucidate the mechanisms of sensitivity and resistance to anti-EGFR therapy in metastatic CRC patients.

Microarray probe mapping and annotation in cross-species comparative toxicogenomics.

Genomics-based tools, such as microarrays, do appear to offer promise in evaluating the relevance of one species to another in terms of molecular and cellular response to a given treatment. However, to fulfill this promise the individual end points (i.e., the genes, proteins, or mRNAs) measured in one species must be mapped to corresponding end points in another species. Several approaches, along with their strengths and weaknesses, are described in this chapter. A sequential approach is described that first makes use of a "Genome To Genome Through Orthology" method, where probe sequences for a given species are mapped into full-length sequences for that species, associated with the locus for those sequences and then into a second species by consulting orthology resources. The second step supplements these results by mapping the probe sequences for the given species into the best matching transcript from any organism, which then are mapped into the appropriate native locus and finally into the second species via an orthology resource. The results of this method are given for an experiment comparing the transcriptional response of canine liver to phenobarbital with that of rat liver.

Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses.

Oxidation of monoclonal antibodies (mAb) is a common chemical modification with potential impact on a therapeutic protein's activity and immunogenicity. In a previous study, it was found that tryptophan oxidation (Trp-ox) levels of two mAb produced in Chinese hamster ovary (CHO) cells were significantly lowered by modifying cell culture medium/feed. In this study, transcriptome analysis by RNA-Seq is applied to further elucidate the underlying mechanism of those changes in lowering the Trp-ox levels. Cell samples from the 5L fed-batch conditions are harvested and subjected to RNA-Seq analysis. The results showed that the cell culture changes had little impact on neither the expression of the mAb transgenes nor genes related to glycosylation. However, those changes did significantly alter the expression of multiple genes (p-value ≤0.05 and absolute fold change ≥1.5 or adjusted p-value ≤0.1) involved in transport of copper, regulation of glutathione, iron storage, heme reduction, oxidative phosphorylation, and Nrf2-mediated antioxidative response. These findings suggest a key underlying mechanism in lowering Trp-ox levels by CDM was likely to be collectively controlling ROS levels through regulation of those genes' expression. This is the first example, to our knowledge, applying transcriptomic analysis to mechanistically understand the impact of cell culture on mAb oxidation.

Underlying mechanisms of pharmacology and toxicity of a novel PPAR agonist revealed using rodent and canine hepatocytes.

Marked species-specific responses to agonists of the peroxisome proliferator-activated alpha receptor (PPAR alpha) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR alpha and -gamma coagonist, LY465608, relative to fenofibrate, a prototypical PPAR alpha agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial beta-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity but did demonstrate increased MBox activity. Analysis of peroxisomal beta-oxidation gene expression markers by quantitative real-time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl-coenzyme A (CoA) oxidase (Acox), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat hepatocytes, but not dog hepatocytes. Expression of mRNA for the mitochondrial beta-oxidation gene hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial beta-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single-cell necrosis following 30 days of dosing exclusively in dogs, but not in rats. Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) and flow cytometry. At doses of 0.1 microM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 microM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development and propose an underlying mechanism for these species-specific effects.

A flexible integration and visualisation system for biomarker discovery.

Biological data have accumulated at an unprecedented pace as a result of improvements in molecular technologies. However, the translation of data into information, and subsequently into knowledge, requires the intricate interplay of data access, visualisation and interpretation. Biological data are complex and are organised either hierarchically or non-hierarchically. For non-hierarchically organised data, it is difficult to view relationships among biological facts. In addition, it is difficult to make changes in underlying data storage without affecting the visualisation interface. Here, we demonstrate a platform where non-hierarchically organised data can be visualised through the application of a customised hierarchy incorporating medical subject headings (MeSH) classifications. This platform gives users flexibility in updating and manipulation. It can also facilitate fresh scientific insight by highlighting biological impacts across different hierarchical branches. An example of the integration of biomarker information from the curated Proteome database using MeSH and the StarTree visualisation tool is presented.

The acyl coenzymeA:monoacylglycerol acyltransferase 3 (MGAT3) gene is a pseudogene in mice but encodes a functional enzyme in rats.

Triglyceride (TAG) absorption involves its initial hydrolysis to fatty acids and monoacylglycerol (MAG), which are resynthesized back to diacylglycerol (DAG) and TAG within enterocytes. The resynthesis of DAG is facilitated by fatty acyl-CoA dependent monoacylglycerol acyltransferases (MGATs). Three MGAT enzymes have been isolated in humans and the expression of MGAT2 and MGAT3 in the intestines suggests their functional role in the TAG absorption. In this paper, we report that the Mogat3 gene appears to be a pseudogene in mice while it is a functional gene in rats. Examination of the mouse genomic Mogat3 sequence revealed multiple changes that would result in a translational stop codon or frameshifts. The rat Mogat3 gene, however, is predicted to encode a functional enzyme of 362 amino acids. Expression of rat MGAT3 in human embryonic kidney 293 (HEK293) cells led to the formation of a 36-kDa protein that displayed significant MGAT but not DGAT activity. Tissue expression analysis of rat MGAT3 by real-time PCR analysis indicated that rat MGAT3 has a high level of expression in intestines and pancreas. Our results thus provide the molecular basis to understand the relative functional role of MGAT2 and MGAT3 and also for future exploration of MGAT3 function in animal models.

Assessment of diet-induced obese rats as an obesity model by comparative functional genomics.

OBJECTIVE: We applied a comparative functional genomics approach to evaluate whether diet-induced obese (DIO) rats serve as an effective obesity model. METHODS AND PROCEDURES: Gene-expression profiles of epididymal fat from DIO and lean rats were generated using microarrays and compared with the published array data of obese and non-obese human subcutaneous adipocytes. RESULTS: Caloric intake and fuel efficiency were significantly higher in DIO rats, which resulted in increased body weight and adiposity. Circulating glucose, cholesterol, triglyceride, insulin, and leptin levels in DIO rats were significantly higher than those in the lean controls. DIO rats also exhibited impaired insulin sensitivity. A direct comparison of gene-expression profiles from DIO and lean rats and those from obese and non-obese humans revealed that global gene-expression patterns in DIO rat fat resemble those of obese human adipocytes. Differentially expressed genes between obese and non-obese subjects in both human and rat studies were identified and associated with biological pathways by mapping genes to Gene Ontology (GO) categories. Immune response-related genes and angiogenesis-related genes exhibited significant upregulation in both obese humans and DIO rats when compared with non-obese controls. However, genes in fatty acid metabolism and oxidation exhibited a broad downregulation only in obese human adipocytes but not in DIO rat epididymal fat. DISCUSSION: Our study based on gene-expression profiling suggested that DIO rats in general represent an appropriate obesity model. However, the discrepancies in gene-expression alterations between DIO rats and obese humans, particularly in the metabolic pathways, may explain the limitations of using DIO rodent models in obesity research and drug discovery.

Expression of Xenopus XlSALL4 during limb development and regeneration.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization.

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.