Symmetry of computerised tomography of the brain in traumatic brain injury: a quality improvement audit.

BACKGROUND: Non-contrast Computerised Tomography (NCCT) of brain is the gold standard investigation for diagnosis and management of Traumatic brain injury (TBI). Asymmetrical CT brain images as a result of improper head positioning in the CT gantry will compromise the diagnostic value. Therefore, this audit aimed to assess the degree of asymmetry in CT brain studies carried out in TBI patients. METHODS: This audit was carried out at a level one trauma centre and included CT scans of TBI patients with a Glasgow come scale (GCS) score ≤ 13, admitted to the Neurological intensive care unit (NICU). The first cycle involved a period of three months. The data collected included demographic data and variables such as GCS at the time of the scan and whether the patient was intubated or not. The visualisation of bilateral internal auditory meatuses was used as landmark to determine scan symmetry. If the internal auditory meatus on both sides were visible on the same slice of CT scan, it was considered symmetric. The degree of asymmetry was gauged based on the axial slice difference between bilateral meatuses. The data collected was tabulated and presented to Neurosurgery residents and a checklist was formulated which had to be followed while positioning the patient on CT table prior to imaging. RESULTS: The first cycle of the audit showed that 83.8% of scans were asymmetric and among them 44.1% revealed gross asymmetry affecting interpretation of the scan. Following, implementation of the checklist the percentage of gross asymmetry dropped to 21.86% in the second and to 22.22% in the third audit. CONCLUSION: The use of checklist prior to CT brain studies showed sustainable improvement in reducing gross asymmetry and in acquisition of symmetrical CT brain images.

NR2F1 controls tumour cell dormancy via SOX9- and RARß-driven quiescence programmes.

Metastases can originate from disseminated tumour cells (DTCs), which may be dormant for years before reactivation. Here we find that the orphan nuclear receptor NR2F1 is epigenetically upregulated in experimental head and neck squamous cell carcinoma (HNSCC) dormancy models and in DTCs from prostate cancer patients carrying dormant disease for 7-18 years. NR2F1-dependent dormancy is recapitulated by a co-treatment with the DNA-demethylating agent 5-Aza-C and retinoic acid across various cancer types. NR2F1-induced quiescence is dependent on SOX9, RARß and CDK inhibitors. Intriguingly, NR2F1 induces global chromatin repression and the pluripotency gene NANOG, which contributes to dormancy of DTCs in the bone marrow. When NR2F1 is blocked in vivo, growth arrest or survival of dormant DTCs is interrupted in different organs. We conclude that NR2F1 is a critical node in dormancy induction and maintenance by integrating epigenetic programmes of quiescence and survival in DTCs.

MiST: a new approach to variant detection in deep sequencing datasets.

MiST is a novel approach to variant calling from deep sequencing data, using the inverted mapping approach developed for Geoseq. Reads that can map to a targeted exonic region are identified using exact matches to tiles from the region. The reads are then aligned to the targets to discover variants. MiST carefully handles paralogous reads that map ambiguously to the genome and clonal reads arising from PCR bias, which are the two major sources of errors in variant calling. The reduced computational complexity of mapping selected reads to targeted regions of the genome improves speed, specificity and sensitivity of variant detection. Compared with variant calls from the GATK platform, MiST showed better concordance with SNPs from dbSNP and genotypes determined by an exonic-SNP array. Variant calls made only by MiST confirm at a high rate (>90%) by Sanger sequencing. Thus, MiST is a valuable alternative tool to analyse variants in deep sequencing data.

Antibodies reactive to non-HLA antigens in transplant glomerulopathy.

Although T and B cell alloimmunity contribute to transplant injury, autoimmunity directed at kidney-expressed, non-HLA antigens may also participate. Because the specificity, prevalence, and importance of antibodies to non-HLA antigens in late allograft injury are poorly characterized, we used a protein microarray to compare antibody repertoires in pre- and post-transplant sera from several cohorts of patients with and without transplant glomerulopathy. Transplantation routinely induced changes in antibody repertoires, but we did not identify any de novo non-HLA antibodies common to patients with transplant glomerulopathy. The screening studies identified three reactivities present before transplantation that persisted after transplant and strongly associated with transplant glomerulopathy. ELISA confirmed that reactivity against peroxisomal-trans-2-enoyl-coA-reductase strongly associated with the development of transplant glomerulopathy in independent validation sets. In addition to providing insight into effects of transplantation on non-HLA antibody repertoires, these results suggest that pretransplant serum antibodies to peroxisomal-trans-2-enoyl-coA-reductase may predict prognosis in kidney transplantation.

Web-based tools for studying RNA structure and function.

Like protein coding sequences, functional motifs in RNA elements are frequently conserved, but this conservation is most often at the structure level rather than sequence based. Proper characterization of these structural RNA motifs is both the key and the limiting step to understanding the nature of RNA-protein interactions. The discovery of elements targeted by RNA-binding proteins and how they function remains one of the most active, yet elusive areas of RNA biology. Only a limited number of these elements have been well characterized with many of the fundamental rules yet to be discovered. Here we present a comprehensive list of web based resources that can be used in the study and identification of RNA-based structural and regulatory motifs and provide a survey of the informatic resources that can have been developed to facilitate this research.

RIP-Chip analysis: RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling.

Post-transcriptional regulation of gene expression plays an important role in complex cellular processes. Just like transcription factors regulate gene expression through combinatorial binding to multiple, physically dispersed cis elements, mRNA binding proteins can regulate the translation of functionally related gene products by coordinately binding to subsets of mRNAs. The networks of mRNA binding proteins that facilitate this fine-tuning of gene expression are poorly understood. By combining genomic technologies with standard molecular biology tools, we have helped pioneer the development of high-throughput technologies for the global analysis of subsets of mRNAs bound to RNA-binding proteins. This technique is termed RIP-Chip and stands for RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling. This approach is also referred to as "ribonomic profiling" and has revealed valuable information about the workings of mRNP networks in the cell and the regulation of gene expression. In this chapter, we describe the latest advances that we have made in the RIP-CHIP technology.

Geoseq: a tool for dissecting deep-sequencing datasets.

BACKGROUND: Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. RESULTS: Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. CONCLUSIONS: Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

RIP-CHIP in drug development.

Microarrays are extensively used to evaluate the effects of compounds on gene expression in the cells. Most of the studies so far have analyzed the transcriptome of the cell. The basic assumption of this approach is that the changes in gene expression occur at the level of transcription of a gene. However, changes often occur at the posttranscriptional level and are not reflected in the analysis of whole transcriptome. We have pioneered the development of "ribonomic profiling" as a high-throughput method to study posttranscriptional regulation of gene expression in the cell. This method is also often referred to as RIP-CHIP. In this chapter, we describe how to use the RIP-CHIP technology to assess the posttranscriptional changes occurring in the cell in response to treatment with a drug.

Computational identification of a p38SAPK-regulated transcription factor network required for tumor cell quiescence.

The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.

Informatic resources for identifying and annotating structural RNA motifs.

Post-transcriptional regulation of genes and transcripts is a vital aspect of cellular processes, and unlike transcriptional regulation, remains a largely unexplored domain. One of the most obvious and most important questions to explore is the discovery of functional RNA elements. Many RNA elements have been characterized to date ranging from cis-regulatory motifs within mRNAs to large families of non-coding RNAs. Like protein coding genes, the functional motifs of these RNA elements are highly conserved, but unlike protein coding genes, it is most often the structure and not the sequence that is conserved. Proper characterization of these structural RNA motifs is both the key and the limiting step to understanding the post-transcriptional aspects of the genomic world. Here, we focus on the task of structural motif discovery and provide a survey of the informatics resources geared towards this task.

Systems based mapping demonstrates that recovery from alkylation damage requires DNA repair, RNA processing, and translation associated networks.

The identification of cellular responses to damage can promote mechanistic insight into stress signalling. We have screened a library of 3968 Escherichia coli gene-deletion mutants to identify 99 gene products that modulate the toxicity of the alkylating agent methyl methanesulfonate (MMS). We have developed an ontology mapping approach to identify functional categories over-represented with MMS-toxicity modulating proteins and demonstrate that, in addition to DNA re-synthesis (replication, recombination, and repair), proteins involved in mRNA processing and translation influence viability after MMS damage. We have also mapped our MMS-toxicity modulating proteins onto an E. coli protein interactome and identified a sub-network consisting of 32 proteins functioning in DNA repair, mRNA processing, and translation. Clustering coefficient analysis identified seven highly connected MMS-toxicity modulating proteins associated with translation and mRNA processing, with the high connectivity suggestive of a coordinated response. Corresponding results from reporter assays support the idea that the SOS response is influenced by activities associated with the mRNA-translation interface.

Bioinformatic tools for studying post-transcriptional gene regulation : The UAlbany TUTR collection and other informatic resources.

The untranslated regions (UTRs) of many mRNAs contain sequence and structural motifs that are used to regulate the stability, localization, and translatability of the mRNA. It should be possible to discover previously unidentified RNA regulatory motifs by examining many related nucleotide sequences, which are assumed to contain a common motif. This is a general practice for discovery of DNA-based sequence-based patterns, in which alignment tools are heavily exploited. However, because of the complexity of sequential and structural components of RNA-based motifs, simple-alignment tools are frequently inadequate. The consensus sequences they find frequently have the potential for significant variability at any given position and are only loosely characterized. The development of RNA-motif discovery tools that infer and integrate structural information into motif discovery is both necessary and expedient. Here, we provide a selected list of existing web-accessible algorithms for the discovery of RNA motifs, which, although not exhaustive, represents the current state of the art. To facilitate the development, evaluation, and training of new software programs that identify RNA motifs, we created the UAlbany training UTR (TUTR) database, which is a collection of validated sets of sequences containing experimentally defined regulatory motifs. Presently, eleven training sets have been generated with associated indexes and "answer sets" provided that identify where the previously characterized RNA motif [the iron responsive element (IRE), AU-rich class-2 element (ARE), selenocysteine insertion sequence (SECIS), etc.] resides in each sequence. The UAlbany TUTR collection is a shared resource that is available to researchers for software development and as a research aid.

Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling.

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.

Genome-wide analysis identifies interleukin-10 mRNA as target of tristetraprolin.

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP(-/-) mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.

Microarray analysis in B cells among siblings with/without MS - role for transcription factor TCF2.

BACKGROUND: We investigated if global gene expression and transcription networks in B-lymphocytes of siblings with multiple sclerosis (MS) were different from healthy siblings. RESULTS: Using virus-transformed immortalized B cells and human whole genome bioarrays with validation using RT-qPCR, we found that in siblings with MS, genes for CXCL10, serpin B1 and FUT4 were up regulated whereas CDC5L, TNFRSF19 and HLA-DR were down regulated, among others; transcription analysis showed two intersecting clusters of transcriptional factors - the larger, governed by the upregulated transcription factor 2 (TCF2) and the smaller network regulated by the downregulated CDC5L. CONCLUSION: No study has linked TCF2 to MS and to better understand the role of TCF2 in MS, studies in larger cohorts are required.

MicroRNA modulation of RNA-binding protein regulatory elements.

We propose that microRNAs could modulate RNA-binding protein binding sites in a dynamic manner. We suggest that the cis-regulatory code targeted by microRNAs is, at least in part, the same as that read by mRNA-binding proteins. Our hypothesis predicts that microRNAs indirectly or directly bind to RNA-binding protein binding sites. Alternatively, the microRNA-mRNA interactions can themselves be the target of an mRNA-binding protein. Lastly, we envision examples where multiple mRNA regulatory elements are simultaneously influenced by microRNA-mRNA interactions such that the binding of one or more microRNA results in conformational changes in the structure of the mRNA, thereby, either revealing or masking a second regulatory element.