Analysis of transcription factor CREM dependent gene expression during mouse spermatogenesis

Computational methods are getting increasingly important for the analysis of large data sets in molecular biology. The data sets analyzed in this thesis are derived from experiments measuring the changes of expression levels in response to the transcription factor CREM (cAMP Responsive Element Modulator) during mouse spermatogenesis. In the course of this analysis new computational methods were developed and used that will also be of value in other projects in Bioinformatics. CREM belongs to a family of cAMP-responsive nuclear factors. The activator splice-isoform CREM is exclusively expressed at high levels in post-meiotic germ cells during mouse spermiogenesis. Mutant male mice lacking CREM expression are sterile due to lack of maturation of the germ cells. In order to find CREM target genes the mRNA expression levels in testes of CREM-deficient mice and wild-type mice were compared using the suppression subtractive hybridization (SSH) technique as well as oligonucleotide DNA microarrays. SSH was used to selectively amplify the differentially expressed genes. 12,000 clones, which contain sequence fragments of genes expressed stronger in wild-type as in the CREM (-/-) mutant, were analyzed by a combination of sequencing and hybridization. Sequence analysis methods were used to characterize 956 unique sequences. Homologies to 158 known mouse genes and 99 known genes from other organisms were detected. 296 sequences show homologies to sequences of expressed sequence tags (ESTs). 199 novel sequences have been found. The sequences not corresponding to full length genes of known function were characterized using publicly available EST data. To make EST databases useful for data analysis all of the publicly available ESTs have been grouped into clusters and methods to analyze and visualize EST data were developed. Nylon cDNA microarrays containing the unique sequences from the CREM SSH library were constructed to determine expression levels of those sequences. Most of the sequences from the CREM SSH library are shown to be expressed in wild-type but are down-regulated in CREM deficient mice. Statistical methods to standardize microarray expression data were developed and software was implemented to perform comparisons. Further CREM dependent genes were detected comparing the mRNA expression levels in testes of CREM deficient mice and wild-type mice using Affymetrix oligonucleotide microarrays containing 10,000 mouse sequences. Comparison of the different techniques (SSH, nylon cDNA arrays and Affymetrix oligonucleotide microarrays) shows that the results are complementing each other. The unique sequences from the CREM SSH library were further analyzed by determining the spermatogenic stage specific expression profiles. cDNA from prepubertal mice at certain stages of spermatogenesis were hybridized on nylon cDNA arrays. Several important functional groups of genes like transcription factors, signal transduction proteins and metabolic enzymes are shown to be coexpressed at the latest stages of spermatogenesis. Expression profiles were arranged to find similar profile shapes and co-regulation of functionally related genes. An algorithm to arrange the profiles in an optimal linear order was developed. The linear order is constructed in a way that similar expression profiles end up close together in the linear order, i.e. the sum over all distances of neighboring profiles is minimized. This corresponds to the solution of a traveling salesman problem (TSP), which is well known in computer science. A fast algorithm that computes a heuristic solution to a TSP was adapted to be used in expression profile analysis

Extending pathways based on gene lists using InterPro domain signatures

<p>Abstract</p> <p>Background</p> <p>High-throughput technologies like functional screens and gene expression analysis produce extended lists of candidate genes. Gene-Set Enrichment Analysis is a commonly used and well established technique to test for the statistically significant over-representation of particular pathways. A shortcoming of this method is however, that most genes that are investigated in the experiments have very sparse functional or pathway annotation and therefore cannot be the target of such an analysis. The approach presented here aims to assign lists of genes with limited annotation to previously described functional gene collections or pathways. This works by comparing InterPro domain signatures of the candidate gene lists with domain signatures of gene sets derived from known classifications, e.g. KEGG pathways.</p> <p>Results</p> <p>In order to validate our approach, we designed a simulation study. Based on all pathways available in the KEGG database, we create test gene lists by randomly selecting pathway genes, removing these genes from the known pathways and adding variable amounts of noise in the form of genes not annotated to the pathway. We show that we can recover pathway memberships based on the simulated gene lists with high accuracy. We further demonstrate the applicability of our approach on a biological example.</p> <p>Conclusion</p> <p>Results based on simulation and data analysis show that domain based pathway enrichment analysis is a very sensitive method to test for enrichment of pathways in sparsely annotated lists of genes. An R based software package <it>domainsignatures</it>, to routinely perform this analysis on the results of high-throughput screening, is available via Bioconductor.</p

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

<p>Abstract</p> <p>Background</p> <p>Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.</p> <p>Results</p> <p>A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins.</p> <p>Conclusions</p> <p>Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.</p

A global microRNA screen identifies regulators of the ErbB receptor signaling network

Background: The growth factor heregulin (HRG) potently stimulates epithelial cell survival and proliferation through the binding of its cognate receptor ErbB3 (also known as HER3). ErbB3-dependent signal transmission relies on the dimerization partner ErbB2, a receptor tyrosine kinase that is frequently overexpressed and/or amplified in breast cancer cells. Substantial evidence suggests that deregulated ErbB3 expression also contributes to the transformed phenotype of breast cancer cells. Results: By genome-wide screening, we identify 43 microRNAs (miRNAs) that specifically impact HRG-induced activation of the PI3K-Akt pathway. Bioinformatic analysis combined with experimental validation reveals a highly connected molecular miRNA-gene interaction network particularly for the negative screen hits. For selected miRNAs, namely miR-149, miR-148b, miR-326, and miR-520a-3p, we demonstrate the simultaneous downregulation of the ErbB3 receptor and multiple downstream signaling molecules, explaining their efficient dampening of HRG responses and ascribing to these miRNAs potential context-dependent tumor suppressive functions. Conclusions: Given the contribution of HRG signaling and the PI3K-Akt pathway in particular to tumorigenesis, this study not only provides mechanistic insight into the function of miRNAs but also has implications for future clinical applications

A new analysis approach of epidermal growth factor receptor pathway activation patterns provides insights into cetuximab resistance mechanisms in head and neck cancer

The pathways downstream of the epidermal growth factor receptor (EGFR) have often been implicated to play crucial roles in the development and progression of various cancer types. Different authors have proposed models in cell lines in which they study the modes of pathway activities after perturbation experiments. It is prudent to believe that a better understanding of these pathway activation patterns might lead to novel treatment concepts for cancer patients or at least allow a better stratification of patient collectives into different risk groups or into groups that might respond to different treatments. Traditionally, such analyses focused on the individual players of the pathways. More recently in the field of systems biology, a plethora of approaches that take a more holistic view on the signaling pathways and their downstream transcriptional targets has been developed. Fertig et al. have recently developed a new method to identify patterns and biological process activity from transcriptomics data, and they demonstrate the utility of this methodology to analyze gene expression activity downstream of the EGFR in head and neck squamous cell carcinoma to study cetuximab resistance. Please see related article: http://www.biomedcentral.com/1471-2164/13/16

Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

Differences in MYCN/c-MYC target gene expression are associated with distinct neuroblastoma subtypes and clinical outcome

Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.</p> <p>Results</p> <p>A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently <it>CASP8AP2</it>/<it>FLASH</it>. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of <it>CASP8AP2</it>/<it>FLASH </it>silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.</p> <p>Conclusions</p> <p>We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene <it>NEFH</it>.</p

Anaemia requiring red blood cell transfusion is associated with unfavourable 90-day survival in surgical patients with sepsis

Objective: The mortality associated with sepsis remains unacceptably high, despite modern high-quality intensive care. Based on the results from previous studies, anaemia and its management in patients with sepsis appear to impact outcomes; however, the transfusion policy is still being debated, and the ideal approach may be extremely specific to the individual. This study aimed to investigate the long-term impact of anaemia requiring red blood cell (RBC) transfusion on mortality and disease severity in patients with sepsis. We studied a general surgical intensive care unit (ICU) population, excluding cardiac surgery patients. 435 patients were enrolled in this observational study between 2012 and 2016. Results: Patients who received RBC transfusion between 28 days before and 28 days after the development of sepsis (n = 302) exhibited a significantly higher 90-day mortality rate (34.1% vs 19.6%; P = 0.004, Kaplan–Meier analysis). This association remained significant after adjusting for confounders in the multivariate Cox regression analysis (hazard ratio 1.68; 95% confidence interval 1.03–2.73; P = 0.035). Patients who received transfusions also showed significantly higher morbidity scores, such as SOFA scores, and ICU lengths of stay compared to patients without transfusions (n = 133). Our results indicate that anaemia and RBC transfusion are associated with unfavourable outcomes in patients with sepsis

GOSim – an R-package for computation of information theoretic GO similarities between terms and gene products

<p>Abstract</p> <p>Background</p> <p>With the increased availability of high throughput data, such as DNA microarray data, researchers are capable of producing large amounts of biological data. During the analysis of such data often there is the need to further explore the similarity of genes not only with respect to their expression, but also with respect to their functional annotation which can be obtained from Gene Ontology (GO).</p> <p>Results</p> <p>We present the freely available software package <it>GOSim</it>, which allows to calculate the functional similarity of genes based on various information theoretic similarity concepts for GO terms. <it>GOSim </it>extends existing tools by providing additional lately developed functional similarity measures for genes. These can e.g. be used to cluster genes according to their biological function. Vice versa, they can also be used to evaluate the homogeneity of a given grouping of genes with respect to their GO annotation. <it>GOSim </it>hence provides the researcher with a flexible and powerful tool to combine knowledge stored in GO with experimental data. It can be seen as complementary to other tools that, for instance, search for significantly overrepresented GO terms within a given group of genes.</p> <p>Conclusion</p> <p><it>GOSim </it>is implemented as a package for the statistical computing environment <it>R </it>and is distributed under GPL within the CRAN project.</p

Measurement invariance of six language versions of the post-traumatic stress disorder checklist for DSM-5 in civilians after traumatic brain injury

Publisher Copyright: © 2022, The Author(s).Traumatic brain injury (TBI) is frequently associated with neuropsychiatric impairments such as symptoms of post-traumatic stress disorder (PTSD), which can be screened using self-report instruments such as the Post-Traumatic Stress Disorder Checklist for DSM-5 (PCL-5). The current study aims to inspect the factorial validity and cross-linguistic equivalence of the PCL-5 in individuals after TBI with differential severity. Data for six language groups (n ≥ 200; Dutch, English, Finnish, Italian, Norwegian, Spanish) were extracted from the CENTER-TBI study database. Factorial validity of PTSD was evaluated using confirmatory factor analyses (CFA), and compared between four concurrent structural models. A multi-group CFA approach was utilized to investigate the measurement invariance (MI) of the PCL-5 across languages. All structural models showed satisfactory goodness-of-fit with small between-model variation. The original DSM-5 model for PTSD provided solid evidence of MI across the language groups. The current study underlines the validity of the clinical DSM-5 conceptualization of PTSD and demonstrates the comparability of PCL-5 symptom scores between language versions in individuals after TBI. Future studies should apply MI methods to other sociodemographic (e.g., age, gender) and injury-related (e.g., TBI severity) characteristics to improve the monitoring and clinical care of individuals suffering from PTSD symptoms after TBI.Peer reviewe