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Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα) + breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress.

RNA-binding proteins (RBPs) play a critical role in the regulation of alternative splicing (AS), a prevalent mechanism for generating transcriptomic and proteomic diversity in eukaryotic cells. Studies have shown that AS can be regulated by RBPs in a binding-site-position dependent manner. Depending on where RBPs bind, splicing of an alternative exon can be enhanced or suppressed. Therefore, spatial analyses of RBP motifs and binding sites around alternative exons will help elucidate splicing regulation by RBPs. The development of high-throughput sequencing technologies has allowed transcriptome-wide analyses of AS and RBP-RNA interactions. Given a set of differentially regulated alternative exons obtained from RNA sequencing (RNA-seq) experiments, the rMAPS web server (http://rmaps.cecsresearch.org) performs motif analyses of RBPs in the vicinity of alternatively spliced exons and creates RNA maps that depict the spatial patterns of RBP motifs. Similarly, rMAPS can also perform spatial analyses of RBP-RNA binding sites identified by cross-linking immunoprecipitation sequencing (CLIP-seq) experiments. We anticipate rMAPS will be a useful tool for elucidating RBP regulation of alternative exon splicing using high-throughput sequencing data.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral genome is an RNA virus consisting of approximately 30,000 bases. As part of testing efforts, whole genome sequencing of human isolates has resulted in over 1,600 complete genomes publicly available from GenBank. We have performed a comparative analysis of the sequences, in order to detect common mutations within the population. Analysis of variants occurring within the assembled genomes yields 417 variants occurring in at least 1% of the completed genomes, including 229 within the 5’ untranslated region (UTR), 152 within the 3’UTR, 2 within intergenic regions and 34 within coding sequences.

The length of untranslated regions at the 3' end of transcripts (3'UTRs) is regulated by alternate polyadenylation (APA). 3'UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3'UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3'UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals-CSIs). This approach facilitated (1) profiling of 3'UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3'UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3'UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation. Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR.

A mutation in STAT3 has been linked to the incidence of autosomal dominant hyper IgE syndrome, a disease characterized by elevated serum IgE Ab. However, how this genetic mutation leads to the phenotype has not been fully understood. We investigated the specific role of STAT3 in the germinal center (GC) B cells and plasma cells for IgE class switching. Through the use of STAT3 conditional knockout (cKO) mice in a Th2-type immunization model, we demonstrated that CD2-Cre–driven STAT3 cKO mice showed elevated IgE and decreased IgG1 in the serum and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased, whereas IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of Ab-producing plasma cells. Subsequent experiments using a CD19-Cre cKO mouse established this effect to be B cell–intrinsic. Transcription factors critical for GC and plasma cell differentiation, including Bcl-6 and Aicda, were shown to function as downstream signals of STAT3 regulation. Chromatin immunoprecipitation sequencing analysis revealed that many genes, including Bcl3 and Crtc2, were among the direct STAT3 regulated targets. Mice with STAT3 deficiency in B cells also demonstrated an increase in lung inflammation when used in an asthma-like disease model. This model suggests a negative role for STAT3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a therapeutic target for treatment of autosomal dominant hyper IgE syndrome and other immune disorders.

Anacardic acid (AnAc), a potential dietary agent for preventing and treating breast cancer, inhibited the proliferation of estrogen receptor α (ERα) positive MCF-7 and MDA-MB-231 triple negative breast cancer cells. To characterize potential regulators of AnAc action, MCF-7 and MDA-MB-231 cells were treated for 6 h with purified AnAc 24:1n5 congener followed by next generation transcriptomic sequencing (RNA-seq) and network analysis. We reported that AnAc-differentially regulated miRNA transcriptomes in each cell line and now identify AnAc-regulated changes in mRNA and lncRNA transcript expression. In MCF-7 cells, 80 AnAc-responsive genes were identified, including lncRNA MIR22HG. More AnAc-responsive genes (886) were identified in MDA-MB-231 cells. Only six genes were commonly altered by AnAc in both cell lines: SCD, INSIG1, and TGM2 were decreased and PDK4, GPR176, and ZBT20 were increased. Modeling of AnAc-induced gene changes suggests that AnAc inhibits monounsaturated fatty acid biosynthesis in both cell lines and increases endoplasmic reticulum stress in MDA-MB-231 cells. Since modeling of downregulated genes implicated NFκB in MCF-7, we confirmed that AnAc inhibited TNFα-induced NFκB reporter activity in MCF-7 cells. These data identify new targets and pathways that may account for AnAc’s anti-proliferative and pro-apoptotic activity.

Breast cancer (BC) is increasing in incidence and resistance to treatment worldwide. The challenges in limited therapeutic options and poor survival outcomes in BC subtypes persist because of its molecular heterogeneity and resistance to standard endocrine therapy. Recently, high throughput RNA sequencing (RNA-seq) has been used to identify biomarkers of disease progression and signaling pathways that could be amenable to specific therapies according to the BC subtype. However, there is no single generally accepted pipeline for the analysis of RNA-seq data in biomarker discovery due, in part, to the needs of simultaneously satisfying constraints of sensitivity and specificity. We proposed a combined approach using gene-wise normalization, UQ-pgQ2, followed by a Wald test from DESeq2. Our approach improved the analysis based on within-group comparisons in terms of the specificity when applied to publicly available RNA-seq BC datasets. In terms of identifying differentially expressed genes (DEGs), we combined an optimized log2 fold change cutoff with a nominal false discovery rate of 0.05 to further minimize false positives. Using this method in the analysis of two GEO BC datasets, we identified 797 DEGs uniquely expressed in triple negative BC (TNBC) and significantly associated with T cell and immune-related signaling, contributing to the immunotherapeutic efficacy in TNBC patients. In contrast, we identified 1403 DEGs uniquely expressed in estrogen positive and HER2 negative BC (ER+HER2-BC) and significantly associated with eicosanoid, notching and FAK signaling while a common set of genes was associated with cellular growth and proliferation. Thus, our approach to control for false positives identified two distinct gene expression profiles associated with these two subtypes of BC which are distinguishable by their molecular and functional attributes.

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose–course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE64375. Keywords: Ionizing radiation, Radiation exposure, Astronaut, Long duration space travel

Recent studies implicate Poly (ADP-ribose) polymerase 1 (PARP1) in alternative splicing regulation, and PARP1 may be an RNA-binding protein. However, detailed knowledge of RNA targets and the RNA-binding region for PARP1 are unknown. Here we report the first global study of PARP1–RNA interactions using PAR–CLIP in HeLa cells. We identified a largely overlapping set of 22 142 PARP1–RNA-binding peaks mapping to mRNAs, with 20 484 sites located in intronic regions. PARP1 preferentially bound RNA containing GC-rich sequences. Using a Bayesian model, we determined positional effects of PARP1 on regulated exon-skipping events: PARP1 binding upstream and downstream of the skipped exons generally promotes exon inclusion, whereas binding within the exon of interest and intronic regions closer to the skipped exon promotes exon skipping. Using truncation mutants, we show that removal of the Zn1Zn2 domain switches PARP1 from a DNA binder to an RNA binder. This study represents a first step into understanding the role of PARP1–RNA interaction. Continued identification and characterization of the functional interplay between PARPs and RNA may provide important insights into the role of PARPs in RNA regulation.

Correction Following the publication of the original supplement article [1], it was brought to our attention that the supplement title unfortunately contains an error: “Proceedings of the 16th Annual UT-KBRIN Bioinformatics Summit 2016: proceedings” should instead read “Proceedings of the 16th Annual UT-KBRIN Bioinformatics Summit 2017: proceedings”. The correct title has been included in this correction.

Long duration space missions expose astronauts to ionizing radiation events associated with highly energetic and charged heavy particles. Such exposure can result in chromosomal aberrations increasing the likelihood of the development of cancer. Early detection and mitigation of these events is critical in providing positive outcomes. In order to aid in the development of portable devices used to measure radiation exposure, we constructed a genome-wide screen to detect transcriptional changes in peripheral blood lymphocytes shortly after (approximately 1 hour) radiation exposure at low (0.3 Gy), medium (1.5 Gy) and high (3.0 Gy) doses compared to control (0.0 Gy) using Affymetrix®Human Gene 1.0 ST v1 microarrays. Our results indicate a number of sensitive and specific transcriptional profiles induced by radiation exposure that can potentially be implemented as biomarkers for radiation exposure as well as dose effect. For overall immediate radiation exposure,KDELC1,MRPS30,RARS, andHEXIM1were determined to be effective biomarkers whilePRDM9,CHST4, andSLC26A10were determined to be biomarkers specific to 0.3 Gy exposure;RPH,CCDC96,WDYHV1, andIFNA16were identified for 1.5 Gy exposure; andCWC15,CHCHD7, andDNAAF2were determined to be sensitive and specific to 3.0 Gy exposure. The resulting raw and analyzed data are publicly available through NCBI's Gene Expression Ominibus via accession GSE64375.

Dietary copper-fructose interactions contribute to the development of nonalcoholic fatty liver disease (NAFLD). Gut microbiota play critical roles in the pathogenesis of NAFLD. The aim of this study was to determine the effect of different dietary doses of copper and their interactions with high fructose on gut microbiome. Male weanling Sprague-Dawley rats were fed diets with adequate copper (6 ppm CuA), marginal copper (1.5 ppm CuM) (low copper), or supplemented copper (20 ppm CuS) (high copper) for 4 wk. Deionized water or deionized water containing 30% fructose (wt/vol) was given ad libitum. Copper status, liver enzymes, gut barrier function, and gut microbiome were evaluated. Both low- and high-copper diets led to liver injury in high-fructose-fed rats, and this was associated with gut barrier dysfunction, as shown by the markedly decreased tight junction proteins and increased gut permeability. 16S rDNA sequencing analysis revealed distinct alterations of the gut microbiome associated with dietary low- and high-copper/high-fructose feeding. The common features of the alterations of the gut microbiome were the increased abundance of Firmicutes and the depletion of Akkermansia. However, they differed mainly within the phylum Firmicutes. Our data demonstrated that a complex interplay among host, microbes, and dietary copper-fructose interaction regulates gut microbial metabolic activity, which may contribute to the development of liver injury and hepatic steatosis. The distinct alterations of gut microbial activity, which were associated with the different dietary doses of copper and fructose, imply that separate mechanism(s) may be involved.NEW & NOTEWORTHY First, dietary low- and high-copper/high-fructose-induced liver injury are associated with distinct alterations of gut microbiome. Second, dietary copper level plays a critical role in maintaining the gut barrier integrity, likely by acting on the intestinal tight junction proteins and the protective commensal bacteria Akkermansia. Third, the alterations of gut microbiome induced by dietary low and high copper with or without fructose differ mainly within the phylum Firmicutes.

Alternative splicing refers to the production of multiple mRNA isoforms from a single gene due to alternative selection of exons or splice sites during pre-mRNA splicing. While canonical alternative splicing produces a linear form of RNA by joining an upstream donor site (5' splice site) with a downstream acceptor site (3' splice site), a special form of alternative splicing produces a non-coding circular form of RNA (circular RNA) by ligating a downstream donor site (5' splice site) with an upstream acceptor site (3' splice site); i.e., back-splicing. Over the past two decades, many studies have discovered this special form of alternative splicing that produces a circular form of RNA. Although these circular RNAs have garnered considerable attention in the scientific community for their biogenesis and functions, the focus of these studies has been on exonic circular RNAs (circRNAs: donor site and acceptor site are from exon boundaries) and circular intronic RNAs (ciRNAs: donor and acceptor are from a single intron). This type of approach was conducted in the relative absence of methods for searching another group of circular RNAs, or circular complex RNAs (ccRNAs: either the donor site or acceptor site is not from exon boundaries), that contains at least one exon and one or more flanking introns. Studies of ccRNAs would serve as a significant first step in filling this void. In this paper, we developed a new computational algorithm that can detect all three types of circular RNAs. We applied our algorithm on a set of RNA-seq data to examine the composition of circular RNAs in the given dataset. Surprisingly, our results showed that the new type of circular RNA (ccRNA) was the second most common type of circular RNA while circRNA was the most common type as expected.

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.

The Florida manatee (Trichechus manatus latirotris) is considered a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (PVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). In this study, we identified DNA sequences with the potential to form G-quadruplex structures in all three PVs. G-quadruplex structures (G4) are guanine-rich nucleic acid sequences capable of forming secondary structures in DNA and RNA. In humans, G4 are known to regulate molecular processes such as transcription and translation. Although G4 have been identified in several viral genomes, including human PVs, no attempt has been made to identify G4 in animal PVs. We found that sequences capable of forming G4 were present on both DNA strands and across coding and non-coding regions on all PVs. The vast majority of the identified sequences would allow the formation of non-canonical structures with only two G-tetrads. The formation of one such structure was supported through biophysical analysis. Computational analysis demonstrated enrichment of G4 sequences on the reverse strand in the E2/E4 region on all manatee PVs and on the forward strand in the E2/E4 region on one genital PV. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all PVs, G4 sequences were located near putative E2 binding sites in the non-coding region. Together, these findings suggest that G4 are likely regulatory elements in manatee PVs.Author summaryG-quadruplex structures (G4) are found in the DNA and RNA of many species and are known to regulate the expression of genes and the synthesis of proteins, among other important molecular processes. Recently, these structures have been identified in several viruses, including the human papillomavirus (PV). As regulatory structures, G4 are of great interest to researchers as drug targets for viral control. In this paper, we identify the first G4 sequences in three PVs infecting a non-human animal, the Florida manatee. Through computational and biophysical analysis, we find that a greater variety of sequence patterns may underlie the formation of these structures than previously identified. The sequences are found in all protein coding regions of the virus and near sites for viral replication in non-coding regions. Furthermore, the distribution of these sequences across the PV genomes supports the notion that sequences are conserved across PV types, suggesting they are under selective pressure. This paper extends previous research on G4 in human PVs with additional evidence for their role as regulators. The G4 sequences we identified also provide potential regulatory targets for researchers interested in controlling this virus in the Florida manatee, a threatened aquatic mammal.

Background: Current non-invasive diagnostics for acute myocardial infarction (MI) identify myocardial necrosis rather than the primary cause and therapeutic target—plaque disruption and resultant thrombosis. Aim: The aim of this study is to identify change specific to plaque disruption and pathological thrombosis. Methods: We quantified 1,032 plasma metabolites by mass spectrometry in 11 thrombotic MI, 12 non-thrombotic MI and 15 stable CAD subjects at two acute phase [time of catheterization (T0), six hours (T6)] and one quiescent (>3 months follow-up) time points. A statistical classifier was constructed utilizing (T0) abundances of a parsimonious set of metabolites that demonstrated a significant change between quiescent phase and the acute phase that was distinct from any change seen in non-thrombotic MI or stable CAD subjects. Classifier performance as estimated by 10-fold cross-validation was suggestive of high sensitivity and specificity for differentiating thrombotic from non-thrombotic MI and stable CAD subjects. Results: Nineteen metabolites (Table 1) with an intra-subject fold change from time of acute thrombotic MI presentation to the quiescent state were distinct from any change measured in both the non-thrombotic MI and stable CAD subjects undergoing cardiac catheterization over the same time course (false discovery rate Conclusions: We have identified a candidate metabolic signature that differentiates acute thrombotic MI from quiescent state and from acute non-thrombotic MI and stable CAD. Further validation of these metabolites is warranted given their potential as diagnostic biomarkers and novel therapeutic targets for the prevention or treatment of acute MI.

BackgroundSince the introduction of microarrays in 1995, researchers world-wide have used both commercial and custom-designed microarrays for understanding differential expression of transcribed genes. Public databases such as ArrayExpress and the Gene Expression Omnibus (GEO) have made millions of samples readily available. One main drawback to microarray data analysis involves the selection of probes to represent a specific transcript of interest, particularly in light of the fact that transcript-specific knowledge (notably alternative splicing) is dynamic in nature.ResultsWe therefore developed a framework for reannotating and reassigning probe groups for Affymetrix® GeneChip® technology based on functional regions of interest. This framework addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome knowledge and grouping probes into gene, transcript and region-based (UTR, individual exon, CDS) probe sets. Updated gene and transcript probe sets provide more specific analysis results based on current genomic and transcriptomic knowledge. The framework selects unique probes, aligns them to gene annotations and generates a custom Chip Description File (CDF). The analysis reveals only 87% of the Affymetrix® GeneChip® HG-U133 Plus 2 probes uniquely align to the current hg38 human assembly without mismatches. We also tested new mappings on the publicly available data series using rat and human data from GSE48611 and GSE72551 obtained from GEO, and illustrate that functional grouping allows for the subtle detection of regions of interest likely to have phenotypical consequences.ConclusionThrough reanalysis of the publicly available data series GSE48611 and GSE72551, we profiled the contribution of UTR and CDS regions to the gene expression levels globally. The comparison between region and gene based results indicated that the detected expressed genes by gene-based and region-based CDFs show high consistency and regions based results allows us to detection of changes in transcript formation.

Peak lists derived from nuclear magnetic resonance (NMR) spectra are commonly used as input data for a variety of computer assisted and automated analyses. These include automated protein resonance assignment and protein structure calculation software tools. Prior to these analyses, peak lists must be aligned to each other and sets of related peaks must be grouped based on common chemical shift dimensions. Even when programs can perform peak grouping, they require the user to provide uniform match tolerances or use default values. However, peak grouping is further complicated by multiple sources of variance in peak position limiting the effectiveness of grouping methods that utilize uniform match tolerances. In addition, no method currently exists for deriving peak positional variances from single peak lists for grouping peaks into spin systems, i.e. spin system grouping within a single peak list. Therefore, we developed a complementary pair of peak list registration analysis and spin system grouping algorithms designed to overcome these limitations. We have implemented these algorithms into an approach that can identify multiple dimension-specific positional variances that exist in a single peak list and group peaks from a single peak list into spin systems. The resulting software tools generate a variety of useful statistics on both a single peak list and pairwise peak list alignment, especially for quality assessment of peak list datasets. We used a range of low and high quality experimental solution NMR and solid-state NMR peak lists to assess performance of our registration analysis and grouping algorithms. Analyses show that an algorithm using a single iteration and uniform match tolerances approach is only able to recover from 50 to 80% of the spin systems due to the presence of multiple sources of variance. Our algorithm recovers additional spin systems by reevaluating match tolerances in multiple iterations. To facilitate evaluation of the algorithms, we developed a peak list simulator within our nmrstarlib package that generates user-defined assigned peak lists from a given BMRB entry or database of entries. In addition, over 100,000 simulated peak lists with one or two sources of variance were generated to evaluate the performance and robustness of these new registration analysis and peak grouping algorithms. Electronic supplementary material The online version of this article (doi:10.1007/s10858-017-0126-5) contains supplementary material, which is available to authorized users.

The high throughput RNA sequencing (RNA-seq) technology has become the popular method of choice for transcriptomics and the detection of differentially expressed genes. Sample size calculations for RNA-seq experimental design are an important consideration in biological research and clinical trials. Currently, the sample size formulas derived from the Wald and the likelihood ratio statistical tests with a Poisson distribution to model RNA-seq data have been developed. However, since the mean read counts in the real RNA-seq data are not equal to the variance, an extended method to calculate sample sizes based on a negative binomial distribution using an exact test statistic was proposed by Li et al. in 2013. In this study, we alternatively derive five sample size calculation methods based on the negative binomial distribution using the Wald test, the log-transformed Wald test and the log-likelihood ratio test statistics. A comparison of our five methods and an existing method was performed by calculating the sample sizes and the simulated power in different scenarios. We first calculated the sample sizes for testing a single gene using the six methods given a nominal significance level α at 0.05 and 80% power. Then, we calculated the sample sizes for testing multiple genes given a false discovery rate (FDR) at 0.05 and 0.10. The empirical power and true prognostic genes for differential gene expression analysis corresponding to the estimated sample sizes from the six methods are also estimated via the simulation studies. Using the sample size formulas derived from log-transformed and Wald-based tests, we observed smaller sample properties while maintaining the nominal power close to or higher than 80% in all the settings compared to other methods. Moreover, the Wald test based sample size calculation method is easier to compute and faster in an RNA-seq experimental design.

Aims Current non-invasive diagnostics for acute myocardial infarction (MI) identify myocardial necrosis rather than the primary cause and therapeutic target—plaque disruption and resultant thrombosis. The aim of this study was to identify changes specific to plaque disruption and pathological thrombosis that are distinct from acute myocardial necrosis. Methods and results We quantified 1,032 plasma metabolites by mass spectrometry in 11 thrombotic MI, 12 non-thrombotic MI, and 15 stable coronary artery disease (CAD) subjects at two acute phase (time of catheterization [T0], six hours [T6]) and one quiescent (>3 months follow-up) time points. A statistical classifier was constructed utilizing baseline (T0) abundances of a parsimonious set of 17 qualifying metabolites. Qualifying metabolites were those that demonstrated a significant change between the quiescent phase and the acute phase and that were distinct from any change seen in non-thrombotic MI or stable CAD subjects. Classifier performance as estimated by 10-fold cross-validation was suggestive of high sensitivity and specificity for differentiating thrombotic from non-thrombotic MI and stable CAD subjects at presentation. Nineteen metabolites demonstrated an intra-subject change from time of acute thrombotic MI presentation to the quiescent state that was distinct from any change measured in both the non-thrombotic MI and stable CAD subjects undergoing cardiac catheterization over the same time course (false discovery rate

Exposure to ionizing radiation associated with highly energetic and charged heavy particles is an inherent risk astronauts face in long duration space missions. We have previously considered the transcriptional effects that three levels of radiation (0.3 Gy, 1.5 Gy, and 3.0 Gy) have at an immediate time point (1 hr) post-exposure [1]. Our analysis of these results suggest effects on transcript levels that could be modulated at lower radiation doses [2]. In addition, a time dependent effect is likely to be present. Therefore, in order to develop a lab-on-a-chip approach for detection of radiation exposure in terms of both radiation level and time since exposure, we developed a time- and dose-course study to determine appropriate sensitive and specific transcript biomarkers that are detectable in blood samples. The data described herein was developed from a study measuring exposure to 0.15 Gy, 0.30 Gy, and 1.5 Gy of radiation at 1 hr, 2 hr, and 6 hr post-exposure using Affymetrix® GeneChip® PrimeView™ microarrays. This report includes raw gene expression data files from the resulting microarray experiments representing typical radiation exposure levels an astronaut may experience as part of a long duration space mission. The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE63952. Keywords: Radiation exposure, Microarrays, Space flight, Gene expression, Lab-on-a-chip

Calcium/calmodulin-dependent protein kinase 4 (gene and transcript: CaMK4; protein: CaMKIV) is the nuclear effector of the Ca(2+) /calmodulin kinase (CaMK) pathway where it coordinates transcriptional responses. However, CaMKIV is present in the cytoplasm and axons of subpopulations of neurons, including some sensory neurons of the dorsal root ganglia (DRG), suggesting an extranuclear role for this protein. We observed that CaMKIV was expressed strongly in the cytoplasm and axons of a subpopulation of small-diameter DRG neurons, most likely cutaneous nociceptors by virtue of their binding the isolectin IB4. In IB4+ spinal nerve axons, 20% of CaMKIV was colocalized with the endocytic marker Rab7 in axons that highly expressed CAM-kinase-kinase (CAMKK), an upstream activator of CaMKIV, suggesting a role for CaMKIV in signaling though signaling endosomes. Using fluorescent in situ hybridization (FISH) with riboprobes, we also observed that small-diameter neurons expressed high levels of a novel 3' untranslated region (UTR) variant of CaMK4 mRNA. Using rapid amplification of cDNA ends (RACE), reverse-transcription polymerase chain reaction (RT-PCR) with gene-specific primers, and cDNA sequencing analyses we determined that the novel transcript contains an additional 10 kb beyond the annotated gene terminus to a highly conserved alternate polyadenylation site. Quantitative PCR (qPCR) analyses of fluorescent-activated cell sorted (FACS) DRG neurons confirmed that this 3'-UTR-extended variant was preferentially expressed in IB4-binding neurons. Computational analyses of the 3'-UTR sequence predict that UTR-extension introduces consensus sites for RNA-binding proteins (RBPs) including the embryonic lethal abnormal vision (ELAV)/Hu family proteins. We consider the possible implications of axonal CaMKIV in the context of the unique properties of IB4-binding DRG neurons.

Commercially developed microarrays, such as those from Agilent® and Affymetrix®, allow for the analysis of differential gene expression changes on a genome-wide scale. Publicly repositories of microarray data, most notably ArrayExpress and the Gene Expression Omnibus (GEO) have made available millions of microarray samples to researchers worldwide. One of the drawbacks of microarray technology is the static construction of probes based on current genomic knowledge and gene annotation information available at the design phase. As the knowledge base about genes expands, including alternative isoform formation and alternative polyadenylation signaling, the need for a dynamically changing approach to microarray expression analysis has become apparent. We have therefore designed a framework for the reanalysis of publicly available microarray datasets by updating probe set construction based on gene, transcript, and region-based (UTR, exon, CDS) annotations. Our analysis of two publicly available GEO series, GSE48611 and GSE72551, illustrate that the analysis of expression changes using different annotation groupings yields additional insight into changes in transcript expression, in particular, 3' UTR dynamics, which are likely to present phenotypical differences.

Metalloproteins bind and utilize metal ions for a variety of biological purposes. Due to the ubiquity of metalloprotein involvement throughout these processes across all domains of life, how proteins coordinate metal ions for different biochemical functions is of great relevance to understanding the implementation of these biological processes. Toward these ends, we have improved our methodology for structurally and functionally characterizing metal binding sites in metalloproteins. Our new ligand detection method is statistically much more robust, producing estimated false positive and false negative rates of ∼0.11% and ∼1.2%, respectively. Additional improvements expand both the range of metal ions and their coordination number that can be effectively analyzed. Also, the inclusion of additional quality control filters has significantly improved structure‐function Spearman correlations as demonstrated by rho values greater than 0.90 for several metal coordination analyses and even one rho value above 0.95. Also, improvements in bond‐length distributions have revealed bond‐length modes specific to chemical functional groups involved in multidentation. Using these improved methods, we analyzed all single metal ion binding sites with Zn, Mg, Ca, Fe, and Na ions in the wwPDB, producing statistically rigorous results supporting the existence of both a significant number of unexpected compressed angles and subsequent aberrant metal ion coordination geometries (CGs) within structurally known metalloproteins. By recognizing these aberrant CGs in our clustering analyses, high correlations are achieved between structural and functional descriptions of metal ion coordination. Moreover, distinct biochemical functions are associated with aberrant CGs versus nonaberrant CGs. Proteins 2017; 85:885–907. © 2016 Wiley Periodicals, Inc.

Recent papers highlight the presence of large numbers of compressed angles in metal ion coordination geometries for metalloprotein entries in the worldwide Protein Data Bank, due mainly to multidentate coordination. The prevalence of these compressed angles has raised the controversial idea that significantly populated aberrant or even novel coordination geometries may exist. Some of these papers have undergone severe criticism, apparently due to views held that only canonical coordination geometries exist in significant numbers. While criticism of controversial ideas is warranted and to be expected, we believe that a line was crossed where unfair criticism was put forth to discredit an inconvenient result that compressed angles exist in large numbers, which does not support the dogmatic canonical coordination geometry view. We present a review of the major controversial results and their criticisms, pointing out both good suggestions that have been incorporated in new analyses, but also unfair criticism that was put forth to support a particular view. We also suggest that better science is enabled through: (i) a more collegial and collaborative approach in future critical reviews and (ii) the requirement for a description of methods and data including source code and visualizations that enables full reproducibility of results. Proteins 2017; 85:938-944. © 2016 Wiley Periodicals, Inc.

Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the ratin vivospared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structurein vitrorevealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA+/RAB5+signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons.SIGNIFICANCE STATEMENTGrowth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes.

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway plays a central role in defining various cellular fates. Scaffold proteins modulating ERK1/2 activity control growth factor signals transduced by the pathway. Here, we analyzed signals transduced by Shoc2, a critical positive modulator of ERK1/2 activity. We found that loss of Shoc2 results in impaired cell motility and delays cell attachment. As ERKs control cellular fates by stimulating transcriptional response, we hypothesized that the mechanisms underlying changes in cell adhesion could be revealed by assessing the changes in transcription of Shoc2-depleted cells. Using quantitative RNA-seq analysis, we identified 853 differentially expressed transcripts. Characterization of the differentially expressed genes showed that Shoc2 regulates the pathway at several levels, including expression of genes controlling cell motility, adhesion, crosstalk with the transforming growth factor beta (TGFβ) pathway, and expression of transcription factors. To understand the mechanisms underlying delayed attachment of cells depleted of Shoc2, changes in expression of the protein of extracellular matrix (lectin galactoside-binding soluble 3-binding protein; LGALS3BP) were functionally analyzed. We demonstrated that delayed adhesion of the Shoc2-depleted cells is a result of attenuated expression and secretion of LGALS3BP. Together our results suggest that Shoc2 regulates cell motility by modulating ERK1/2 signals to cell adhesion.

G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.

Recent tools to detect RNA editing have expanded our understanding of epitranscriptomics, linking changes in RNA editing to both disease and normal cellular processes. However, the research community currently lacks tools for determining if change in RNA editing or "differential editing" has occurred. To meet this need, we present DRETools, a command-line tool-set for finding differential editing among samples, editing islands, and editing sites.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Microarrays provide a means of studying expression level of tens of thousands of genes by providing one or more oligonucleotide probe(s) for each transcript studied. Affymetrix(R) GeneChiptrade mark platforms historically pair each 25-base perfect match (PM) probe with a mismatch probe (MM) differing by a complementary base located in the 13(th) position to quantify and deflate effects of cross-hybridization. Analytical routines for analyzing these arrays take into account difference in expression levels of MM and PM probes to determine which ones are useful for further study. If a single nucleotide polymorphism (SNP) occurs at the 13(th) base, a probe with a higher MM expression level may be incorrectly omitted. In order to examine SNP affects on PM and MM expression levels, known human SNPs from dbSNP were mapped to probe sets within the Affymetrix(R) HG-U133A platform. Probe sets containing one or more probe pairs with a single SNP at the 13(th) position were extracted. A set of twelve microarray experiments were analyzed for the PM and MM expression levels for these probe sets. Over 6,000,000 human SNPs and their flanking regions were extracted from dbSNP. These sequences were aligned against each of the 247,965 probe pair sequences from the Affymetrix(R) HG-U133A platform. A total of 915 probe sets containing a single probe sequence with a SNP mapped to the 13(th) base were extracted. A subset containing 166 probe sets result in complementary base SNPs. Comparison of gene expression levels for the SNP to non-SNP PM and MM probes does not yield a significant difference using chi2 analysis. Thus, omission of probes with MM expression levels higher than PM expression levels does not appear to result in a loss of information concerning SNPs for these regions.

Phosphorylase kinase (PhK), the key enzyme that regulates glycogenolysis, has traditionally been thought to be expressed predominantly in muscle and liver. In this study, we show by two different database searches (Expressed Sequence Tag and UniGene) that PhK gene expression occurs in at least 28-36 different tissues, and that the genes encoding the alpha, beta, and gamma subunits of PhK undergo extensive transcriptional processing. In particular, we have identified exon 6 of PHKG1 as a 3' composite terminal exon due to the presence of a weak polyadenylation and cleavage site in intron 6. We have verified biochemically that transcriptional processing of PHKG1 does occur in vivo; mRNA corresponding to the alternate variant is expressed in skeletal muscle, brain, heart, and tongue. In silico translation of this mRNA yields a PhK gamma subunit that contains the first 181 residues of the protein, followed by an additional 21 amino acids. The implication of this alternate processing is discussed within the context of gamma catalysis and regulation.

Cells in the developing embryo must integrate complex signals from the genome and environment to make decisions about their behavior or fate. The ability to understand the fundamental biology of the decision-making process, and how these decisions may go awry during abnormal development, requires a systems biology paradigm. Presently, the ability to build models with predictive capability in birth defects research is constrained by an incomplete understanding of the fundamental parameters underlying embryonic susceptibility, sensitivity, and vulnerability. Key developmental milestones must be parameterized in terms of system structure and dynamics, the relevant control methods, and the overall design logic of metabolic and regulatory networks. High-content data from genome-based studies provide some comprehensive coverage of these operational processes but a key research challenge is data integration. Analysis can be facilitated by data management resources and software to reveal the structure and function of bionetwork motifs potentially associated with an altered developmental phenotype. Borrowing from applied mathematics and artificial intelligence, we conceptualize a system that can help address the new challenges posed by the transformation of birth defects research into a data-driven science. Birth Defects Research (Part C) 81:1–19, 2007. © 2007 Wiley-Liss, Inc.

Primary afferent collateral sprouting is a process whereby non-injured primary afferent neurons respond to some stimulus and extend new branches from existing axons. Neurons of both the central and peripheral nervous systems undergo this process, which contributes to both adaptive and maladaptive plasticity (e.g., [1], [2], [3], [4], [5], [6], [7], [8], [9]). In the model used here (the "spared dermatome" model), the intact sensory neurons respond to the denervation of adjacent areas of skin by sprouting new axon branches into that adjacent denervated territory. Investigations of gene expression changes associated with collateral sprouting can provide a better understanding of the molecular mechanisms controlling this process. Consequently, it can be used to develop treatments to promote functional recovery for spinal cord injury and other similar conditions. This report includes raw gene expression data files from microarray experiments in order to study the gene regulation in spared sensory ganglia in the initiation (7 days) and maintenance (14 days) phases of the spared dermatome model relative to intact ("naïve") sensory ganglia. Data has been deposited into GEO (GSE72551).

Metalloproteins are essential to all domains of life. The structure of the metalloproteins, especially the metal ion's coordination geometry (CG), needs to be carefully studied, because it provides a strong bridge between metal-binding protein sequences and their functions. Our previous study has shown that new CGs can be classified in zinc metalloproteins via a less biased analysis, and these new CGs also demonstrated distinct functional propensity. Applying the methodology to all metalloproteins will greatly help us in better understanding metalloproteins' structure and their contribution to the protein functions.

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone marks, CTCF and DNase hypersensitive sites. Additionally, the binding of PARP1 to chromatin genome-wide is mutually exclusive with DNA methylation pattern suggesting a functional interplay between PARP1 and DNA methylation. Indeed, inhibition of PARylation results in genome-wide changes in DNA methylation patterns. Our results suggest that PARP1 controls the fidelity of gene transcription and marks actively transcribed gene regions by selectively binding to transcriptionally active chromatin. These studies provide a platform for developing our understanding of PARP1's role in gene regulation.

Zinc metalloproteins are involved in many biological processes and play crucial biochemical roles across all domains of life. Local structure around the zinc ion, especially the coordination geometry (CG), is dictated by the protein sequence and is often directly related to the function of the protein. Current methodologies in characterizing zinc metalloproteins' CG consider only previously reported CG models based mainly on nonbiological chemical context. Exceptions to these canonical CG models are either misclassified or discarded as “outliers.” Thus, we developed a less‐biased method that directly handles potential exceptions without pre‐assuming any CG model. Our study shows that numerous exceptions could actually be further classified and that new CG models are needed to characterize them. Also, these new CG models are cross‐validated by strong correlation between independent structural and functional annotation distance metrics, which is partially lost if these new CGs models are ignored. Furthermore, these new CG models exhibit functional propensities distinct from the canonical CG models. Proteins 2015; 83:1470–1487. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

After publication of this supplement [1], it was brought to our attention that the wrong year was used in the title of the supplement. The 16th Annual UT-KBRIN Bioinformatics Summit was held in 2017.