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MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.

BACKGROUND: Recent withdrawals of major drugs have highlighted the critical importance of drug safety surveillance in the postmarketing phase. Limitations of spontaneous report data have led drug safety professionals to pursue alternative postmarketing surveillance approaches based on healthcare administrative claims data. These data are typically analysed by comparing the adverse event rates associated with a drug of interest to those of a single comparable reference drug. OBJECTIVE: The aim of this study was to determine whether adverse event detection can be improved by incorporating information from multiple reference drugs. We developed a pharmacological network model that implemented this approach and evaluated its performance. METHODS: We studied whether adverse event detection can be improved by incorporating information from multiple reference drugs, and describe two approaches for doing so. The first, reported previously, combines a set of related drugs into a single reference cohort. The second is a novel pharmacoepidemiological network model, which integrates multiple pair-wise comparisons across an entire set of related drugs into a unified consensus safety score for each drug. We also implemented a single reference drug approach for comparison with both multi-drug approaches. All approaches were applied within a sequential analysis framework, incorporating new information as it became available and addressing the issue of multiple testing over time. We evaluated all these approaches using statin (HMG-CoA reductase inhibitors) safety data from a large healthcare insurer in the US covering April 2000 through March 2005. RESULTS: We found that both multiple reference drug approaches offer earlier detection (6-13 months) than the single reference drug approach, without triggering additional false positives. CONCLUSIONS: Such combined approaches have the potential to be used with existing healthcare databases to improve the surveillance of therapeutics in the postmarketing phase over single-comparator methods. The proposed network approach also provides an integrated visualization framework enabling decision makers to understand the key high-level safety relationships amongst a group of related drugs.

BACKGROUND: The molecular mechanisms that mediate the ischemia-reperfusion (I/R) injury in kidney are not completely understood. It is also largely unknown whether such mechanisms overlap with those governing the early development of kidney. METHODOLOGY/PRINCIPAL FINDINGS: We performed gene expression analysis to investigate the transcriptome changes during regeneration after I/R injury in the rat (0 hr, 6 hr, 24 hr, and 120 hr after reperfusion) and early development of mouse kidney (embryonic day 16 p.c. and postnatal 1 and 7 day). Pathway analysis revealed a wide spectrum of molecular functions that may participate in the regeneration and developmental processes of kidney as well as the functional association between them. While the genes associated with cell cycle, immunity, inflammation, and apoptosis were globally activated during the regeneration after I/R injury, the genes encoding various transporters and metabolic enzymes were down-regulated. We also observed that these injury-associated molecular functions largely overlap with those of early kidney development. In particular, the up-regulation of kinases and kinesins with roles in cell division was common during regeneration and early developmental kidney as validated by real-time PCR and immunohistochemistry. CONCLUSIONS: In addition to the candidate genes whose up-regulation constitutes an overlapping expression signature between kidney regeneration and development, this study lays a foundation for studying the functional relationship between two biological processes.

A transcriptional regulatory network represents a molecular framework in which developmental or environmental cues are transformed into differential expression of genes. Transcriptional regulation is mediated by the combinatorial interplay between cis-regulatory DNA elements and trans-acting transcription factors, and is perhaps the most important mechanism for controlling gene expression. Recent innovations, most notably the method for detecting protein-DNA interactions genome-wide, can help provide a comprehensive catalog of cis-regulatory elements and their interaction with given trans-acting factors in a given condition. A transcriptional regulatory network that integrates such information can lead to a systems-level understanding of regulatory mechanisms. In this review, we will highlight the key aspects of current knowledge on eukaryotic transcriptional regulation, especially on known transcription factors and their interacting regulatory elements. Then we will review some recent technical advances for genome-wide mapping of DNA-protein interactions based on high-throughput sequencing. Finally, we will discuss the types of biological insights that can be obtained from a network-level understanding of transcription regulation as well as future challenges in the field.

PURPOSE: Numerous preclinical studies have demonstrated that angiogenesis inhibitors can increase the efficacy of radiotherapy (RT). We sought to examine the safety and efficacy of bevacizumab (BV) and RT in soft tissue sarcomas and explore biomarkers to help determine the treatment response. METHODS AND MATERIALS: Patients with ≥5 cm, intermediate- or high-grade soft tissue sarcomas at significant risk of local recurrence received neoadjuvant BV alone followed by BV plus RT before surgical resection. Correlative science studies included analysis of the serial blood and tumor samples and serial perfusion computed tomography scans. RESULTS: The 20 patients had a median tumor size of 8.25 cm, with 13 extremity, 1 trunk, and 6 retroperitoneal/pelvis tumors. The neoadjuvant treatment was well tolerated, with only 4 patients having Grade 3 toxicities (hypertension, liver function test elevation). BV plus RT resulted in ≥80% pathologic necrosis in 9 (45%) of 20 tumors, more than double the historical rate seen with RT alone. Three patients had a complete pathologic response. The median microvessel density decreased 53% after BV alone (p <.05). After combination therapy, the median tumor cell proliferation decreased by 73%, apoptosis increased 10.4-fold, and the blood flow, blood volume, and permeability surface area decreased by 62-72% (p <.05). Analysis of gene expression microarrays of untreated tumors identified a 24-gene signature for treatment response. The microvessel density and circulating progenitor cells at baseline and the reduction in microvessel density and plasma soluble c-KIT with BV therapy also correlated with a good pathologic response (p <.05). After a median follow-up of 20 months, only 1 patient had developed local recurrence. CONCLUSIONS: The results from the present exploratory study indicated that BV increases the efficacy of RT against soft tissue sarcomas and might reduce the incidence of local recurrence. Thus, this regimen warrants additional investigation. Gene expression profiles and other tissue and circulating biomarkers showed promising correlations with treatment response.

BACKGROUND AND AIMS: The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation. METHODS: Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting. RESULTS: mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16. CONCLUSIONS: This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.

The recent development of next-generation sequencing technology has enabled significant progress in chromatin structure analysis. Here, we review the experimental and bioinformatic approaches to studying nucleosome positioning and histone modification profiles on a genome scale using this technology. These studies advanced our knowledge of the nucleosome positioning patterns of both epigenetically modified and bulk nucleosomes and elucidated the role of such patterns in regulation of gene expression. The identification and analysis of large sets of nucleosome-bound DNA sequences allowed better understanding of the rules that govern nucleosome positioning in organisms of various complexity. We also discuss the existing challenges and prospects of using next-generation sequencing for nucleosome positioning analysis and outline the importance of such studies for the entire chromatin structure field.

Structural variations are widespread in the human genome and can serve as genetic markers in clinical and evolutionary studies. With the advances in the next-generation sequencing technology, recent methods allow for identification of structural variations with unprecedented resolution and accuracy. They also provide opportunities to discover variants that could not be detected on conventional microarray-based platforms, such as dosage-invariant chromosomal translocations and inversions. In this review, we will describe some of the sequencing-based algorithms for detection of structural variations and discuss the key issues in future development.

CONTEXT: Fatty liver is an important complication of obesity; however, regulatory mechanisms mediating altered gene expression patterns have not been identified. OBJECTIVE: The aim of the study was to identify novel transcriptional changes in human liver that could contribute to hepatic lipid accumulation and associated insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis. DESIGN: We evaluated gene expression in surgical liver biopsies from 13 obese (nine with type 2 diabetes) and five control subjects using Affymetrix U133A microarrays. PCR validation was performed in liver biopsies using an additional 16 subjects. We also tested thyroid hormone responses in mice fed chow or high-fat diet. SETTING: Recruitment was performed in an academic medical center. PARTICIPANTS: Individuals undergoing elective surgery for obesity or gallstones participated in the study. RESULTS: The top-ranking gene set, down-regulated in obese subjects, was comprised of genes previously demonstrated to be positively regulated by T(3) in human skeletal muscle (n = 399; P < 0.001; false discovery rate = 0.07). This gene set included genes related to RNA metabolism (SNRPE, HNRPH3, TIA1, and SFRS2), protein catabolism (PSMA1, PSMD12, USP9X, IBE2B, USP16, and PCMT1), and energy metabolism (ATP5C1, COX7C, UQCRB). We verified thyroid hormone regulation of these genes in the liver after injection of C57BL/6J mice with T(3) (100 microg/100 g body weight); furthermore, T(3)-induced increases in expression of these genes were abolished by high-fat diet. In agreement, expression of these genes inversely correlated with liver fat content in humans. CONCLUSIONS: These data suggest that impaired thyroid hormone action may contribute to altered patterns of gene expression in fatty liver.

Uterine leiomyomata (UL), the most common neoplasm in reproductive-age women, have recurrent cytogenetic abnormalities including interstitial deletion of 7q. To develop a molecular signature, matched del(7q) and non-del(7q) tumors identified by FISH or karyotyping from 11 women were profiled with expression arrays. Our analysis using paired t tests demonstrates this matched design is critical to eliminate the confounding effects of genotype and environment that underlie patient variation. A gene list ordered by genome-wide significance showed enrichment for the 7q22 target region. Modification of the gene list by weighting each sample for percent of del(7q) cells to account for the mosaic nature of these tumors further enhanced the frequency of 7q22 genes. Pathway analysis revealed two of the 19 significant functional networks were associated with development and the most represented pathway was protein ubiquitination, which can influence tumor development by stabilizing oncoproteins and destabilizing tumor suppressor proteins. Array CGH (aCGH) studies determined the only consistent genomic imbalance was deletion of 9.5 megabases from 7q22-7q31.1. Combining the aCGH data with the del(7q) UL mosaicism-weighted expression analysis resulted in a list of genes that are commonly deleted and whose copy number is correlated with significantly decreased expression. These genes include the proliferation inhibitor HPB1, the loss of expression of which has been associated with invasive breast cancer, as well as the mitosis integrity-maintenance tumor suppressor RINT1. This study provides a molecular signature of the del(7q) UL subgroup and will serve as a platform for future studies of tumor pathogenesis.

Hierarchical clustering is a common procedure for identifying structure in a data set, and this is frequently used for organizing genomic data. Although more advanced clustering algorithms are available, the simplicity and visual appeal of hierarchical clustering has made it ubiquitous in gene expression data analysis. Hence, even minor improvements in this framework would have significant impact. There is currently no simple and systematic way of assessing and displaying the significance of various clusters in a resulting dendrogram without making certain distributional assumptions or ignoring gene-specific variances. In this work, we introduce a permutation test based on comparing the within-cluster structure of the observed data with those of sample datasets obtained by permuting the cluster membership. We carry out this test at each node of the dendrogram using a statistic derived from the singular value decomposition of variance matrices. The p-values thus obtained provide insight into the significance of each cluster division. Given these values, one can also modify the dendrogram by combining non-significant branches. By adjusting the cut-off level of significance for branches, one can produce dendrograms with a desired level of detail for ease of interpretation. We demonstrate the usefulness of this approach by applying it to illustrative data sets.

PURPOSE: Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). METHODS AND MATERIALS: Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy x 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. RESULTS: The mean fluorescence in the tumors was not decreased by RT but decreased 38-44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm(3) after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. CONCLUSION: SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS.

Array comparative genomic hybridization (aCGH) is a technique for measuring chromosomal aberrations in genomic DNA. With the availability of high-resolution microarrays, detailed characterization of the cancer genome has become possible. In this review, we discuss several issues in the generation and interpretation of aCGH data, including array platforms, experimental design, and data analysis. Due to the complexity of the data, application of appropriate statistical methods is crucial for avoiding false positive findings. We also describe integration of copy number data with other types of data to identify functional significance of observed aberrations.

Next-generation sequencing is poised to unleash dramatic changes in every area of molecular biology. In the past few years, chromatin immunoprecipitation (ChIP) on tiled microarrays (ChIP-chip) has been an important tool for genome-wide mapping of DNA-binding proteins or histone modifications. Now, ChIP followed by direct sequencing of DNA fragments (ChIP-seq) offers superior data with less noise and higher resolution and is likely to replace ChIP-chip in the near future. We will describe advantages of this new technology and outline some of the issues in dealing with the data. ChIP-seq generates considerably larger quantities of data and the most challenging aspect for investigators will be computational and statistical analysis necessary to uncover biological insights hidden in the data.

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.