Mycoplasma Contamination in The 1000 Genomes Project

Background: In silco Biology is increasingly important and is often based on public datasets. While the problem of contamination is well recognised in microbiology labs the corresponding problem of database corruption has received less attention. Results: Mapping 50 billion next generation DNA sequences from The Thousand Genome Project against published genomes reveals many that match one or more Mycoplasma but are not included in the reference human genome GRCh37.p5. Many of these are of low quality but NCBI BLAST searches confirm some high quality, high entropy sequences match Mycoplasma but no human sequences. Conclusions: It appears at least 7percent of 1000G samples are contaminated

Prediction, diagnosis and treatment of experimental graft-versus-host disease : an investigation of genomic, molecular and cellular factors in graft-versus-host disease and mesenchymal stromal cell therapy in a rat model of allogeneic stem cell transplantation

Patients who suffer from leukemia or lymphoma can be cured by the transplantation of bone marrow stem cells from a volunteer donor (allogeneic hematopoietic cell transplantation). Recipients frequently acquire an immune disorder known as graft-versus-host disease (GvHD) which represents the main hindrance of this therapeutic procedure at present. Graft-borne donor T cells are activated by disparate antigens in the host leading to a cascade of adverse immune reactions and damage of host tissues that give rise to clinical GvHD. The objectives of this thesis were twofold: firstly, to develop diagnostic tools and identify reliable biomarkers of GvHD; secondly, to improve GvHD treatment by mesenchymal stromal cell (MSC) therapy. In a collaborative effort, we helped develop the rat skin explant, an ex-vivo assay of GvHD, as well as a DNA microarray for the study of genetic factors that regulate GvHD. Analysis of gene expression in rat skin explants revealed MHC and innate immune receptor genes that were associated with graft-versus-host reactions and are thus of potential use as diagnostic biomarkers of GvHD. In a model of MHC-mismatched bone marrow transplantation that caused acute GvHD in rats, we isolated natural killer cell and T cell subsets including regulatory T cells that were differentially distributed in disease. Using this animal model, we also tested whether MSC injections could prevent GvHD. We concluded that repeated interventions failed to alleviate disease or improve survival in transplanted rats. In contrast, MSC potently suppressed T cell proliferation and cytokine secretion in vitro. Proliferation was inhibited through the enzymatic synthesis of nitric oxide. In summary, this work resulted in the characterization of genetic and cellular markers of GvHD in the skin ex vivo and after transplantation in vivo as well as the isolation of nitric oxide synthesis as a key pathway used by rat MSC to inhibit allogeneic T cell responses in vitro

Impact of the innate immune response on mammary epithelia

Lipocalin 2 (Lcn2) is a member of the lipocalin family, members of which are usually small extracellular proteins with the abilities of binding and transporting small hydrophobic molecules. Lcn2 was named neutrophil gelatinase-associated lipocalin (NGAL) in human, neu related lipocalin (NRL) in rat and SIP24, 24p3, uterocalin, and siderocalin in mouse. The expression level of Lcn2 in the mammary gland is very high during involution. Results in this thesis show that the expression of rat Lcn2 (NRL) is higher in primiparous rat mammary glands after 28 days of involution than its expression in age matched virgin (AMV) mammary glands. Parity is associated with a lower incidence of breast cancer. The population size of macrophages in primiparous mammary glands is also higher in parous glands. More immune surveillance provided by the larger number of macrophages in the mammary gland after involution may be one reason for the observed parity induced protection against breast cancer. The expression levels of Lcn2 and other inflammatory genes are induced in HC11 cells, a mammary gland epithelial cell line, by mycoplasma infection or the mycoplasma membrane lipopeptide macrophage-activating lipopeptide-2 (MALP-2). Using the Lcn2 promoter linked to a luciferase reporter plasmid, we investigated the mechanism by which MALP-2 regulates gene expression and demonstrated that it activates NFkappaB and C/EBP and induces IkappaBzeta;. Reduction in IkappaBzeta by RNAi reduced Lcn2 promoter activation by MALP-2

Drug resistance, and the role of p53, in lung cancer cell lines

This thesis sets out to increase our knowledge of mechanisms by which lung cancer cells develop resistance to chemotherapeutic agents. The involvement of the tumour suppressor p53 in the development of drug resistance in lung cancer cell lines was investigated. p53 is a tumour suppressor gene, which is mutated in more than half of all tumours. Most chemotherapeutic drugs cause DNA damage that is sensed by p53, which either arrests the cell cycle to allow DNA repair or induces apoptosis. Wildtype p53 was transfected into several cell lines; A549, which expresses wild-type p53, DLKP-SQ, which expresses mutant p53 and HI299 which is p53 null. Clones were isolated from these cell lines and tested for changes in sensitivity to the chemotherapeutic agents adriamycin, taxol and carboplatin. A549 transfected clones showed an increase in p53 protein after transfection, however, these cells did not display substantial changes in sensitivity to the chemotherapeutic agents tested. The cell line H1299, which is p53 null, expressed p53 protein after stable transfection, and was chosen for further analysis. This p53-expressing cell line displayed only small changes in sensitivity to chemotherapeutic agents compared to control-transfected cells. No correlation was observed between the p53 status of the cell lines and the ease of developing resistant variants. To further investigate the molecular basis of drug resistance in lung cancer, a panel of four cell lines were chosen for pulse-selection with chemotherapeutic agents. The cell lines include two adenocarcinoma (AC) and two large cell carcinoma (LCC) cell lines. They were pulse-selected with taxol and carboplatin, both of which are clinically relevant drugs for treatment of this type of cancer. The eight novel cell lines obtained were tested for sensitivity to a cross-section of chemotherapeutic agents and for expression of the multidrug resistance (MDR) efflux pump proteins, Pgp and MRP-1. The taxol-selected variants were chosen for further analysis because the resistance profile was stable and the concentration of drug used for selection was at a clinically achievable level. Microarray analysis was used to identify genes associated with the development of taxol resistance. Ten differentially expressed genes with a possible role in taxol resistance were chosen from this analysis. These genes were further investigated by siRNA transfection, in order to determine the functional relevance of these genes in drug resistance. The taxol-selected variants analysed by microarray analysis provide a unique opportunity to study less well characterised mechanisms of taxol resistance, since these variants do not display classical MDR

Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease.

Design of a variant surface antigen-supplemented microarray chip for whole transcriptome analysis of multiple Plasmodium falciparum cytoadherent strains, and identification of strain-transcendent rif and stevor genes

<p>Abstract</p> <p>Background</p> <p>The cytoadherence of <it>Plasmodium falciparum </it>is thought to be mediated by variant surface antigens (VSA), encoded by <it>var</it>, <it>rif</it>, <it>stevor </it>and <it>pfmc-2tm </it>genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different <it>P. falciparum </it>strains. However, many <it>P. falciparum </it>genomes have recently been sequenced, allowing the development of specific microarray probes for each VSA gene.</p> <p>Methods</p> <p>All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer software. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray.</p> <p>Results</p> <p>From each parasite strain 50-56 <it>var </it>genes, 125-132 <it>rif </it>genes, 26-33 <it>stevor </it>genes and 3-8 <it>pfmc-2tm </it>genes were identified. Bioinformatic analysis of the new VSA sequences showed that 13 <it>rif genes </it>and five <it>stevor </it>genes were well-conserved across at least three strains (83-100% amino acid identity). The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using <it>P. falciparum </it>clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by <it>ITvar9</it>. Whole transcriptome analysis showed that the most highly up-regulated gene in rosetting parasites was <it>ITvar9 </it>(19 to 429-fold up-regulated over six time points). Only one <it>rif </it>gene (<it>IT4rifA_042</it>) was up-regulated by more than four fold (five fold at 12 hours post-invasion), and no <it>stevor </it>or <it>pfmc-2tm </it>genes were up-regulated by more than two fold. 377 non-VSA genes were differentially expressed by three fold or more in rosetting parasites, although none was as markedly or consistently up-regulated as <it>ITvar9</it>.</p> <p>Conclusions</p> <p>Probes for the VSA of newly sequenced <it>P. falciparum </it>strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of <it>ITvar9 </it>was confirmed, and the data did not support the involvement of other VSA families in rosette formation.</p

Identification and characterisation of micrornas involved in the pathogenesis of HIV–associated non-Hodgkin's lymphoma

Background: Since its discovery about three decades ago, the Human Immunodeficiency Virus (HIV) has claimed over millions of lives globally. Although our understanding of the mode of transmission and action of this causative agent for the Acquired Immune Deficiency Syndrome (AIDS) has increased through research, and treatment regimens developed and improved, in certain parts of the world the pandemic continues to expand. Sub-Saharan Africa, which is the epicentre of this global health concern, accounts for approximately 66% of the total number of individuals affected, with South Africa enduring the heaviest burden. South Africa has the world's largest antiretroviral therapy (ART) programme and as such, HIV infected people are living longer, and consequently the incidence of HIV co-morbidities has increased dramatically. HIV/AIDS defining cancers are such co-morbidities with Non- Hodgkin's lymphomas (NHL) being the second most common HIV-associated cancer. Diffuse Large B-cell lymphoma (DLBCL) and Burkitt's lymphoma (BL) are the main subtypes and both present aggressively in HIV positive patients with rapid progression. The use of highly active antiretroviral therapy (HAART) has decreased the incidence of DLBCL in HIV positive patients, however the prevalence of these cancers still remain high in some settings. It has been suggested that the pathogenesis of these cancers in HIV infected individuals is complex and different to that in HIV uninfected individuals, with the possibility that the virus may have an oncogenic role. This has already been demonstrated in the case of the HIV/AIDSdefining cancer Kaposi Sarcoma. However, the same has not been unequivocally demonstrated in HIV-associated NHL. In light of this, the mechanisms through which viruses and viral components promote cellular transformation is an area of active research. One of these mechanisms manipulated by viruses is through the dysregulation of cellular microRNAs (miRNAs) which are small non-coding RNA molecules that are key regulators of gene expression. While they are essential for normal cellular functioning, their expression has been found to be deregulated in diseases including cancer. Several studies have described specific miRNA signatures for NHLs including for DLBCL and BL but none have been described for the HIV-association of these cancers. Aim: The aim of this project was to identify and characterise miRNAs involved in the pathogenesis of HIV-associated NHLs. This thesis reports on the changes in expression of miRNAs in B-cells exposed to an attenuated form (structurally intact but non-infectious) of HIV. Methods: We designed a custom miRNA microarray to identify deregulated miRNAs in the BL cell line Ramos that were exposed to HIV compared to microvesicle treated cells. It was initially planned to use both normal B-cells (L1439A) and BL cells for analysis but Ramos was selected due to technical reasons for this step. Thereafter we validated selected miRNAs by quantitative real-time PCR (qPCR) using single-tube TaqMan® Assays which was predominantly performed in the lymphoblastoid cell line L1439A, which is derived from a healthy donor. We then focused on further characterising the role of one miRNA in the development of HIV-associated NHL by using prediction programmes to predict its putative gene targets and then confirmed its target by using qPCR and western blot analyses. Results: Extensive and comprehensive analysis of the array data led to the identification of a large number of miRNAs which were differentially expressed, with 32 being selected for further studies. These 32 miRNAs include 16 upregulated and 16 downregulated miRNAs, and were selected because they displayed changes in expression by two or more folds. Thereafter, four miRNAs, namely miR-363-3p, miR-222-3p, miR-200c-3p and miR-575, were chosen for validation based on their reported involvement in cancer for validation. The results of two miRNAs (miR-575 (upregulated) (p<0.05) and miR-200c-3p (downregulated) (p<0.05)) were found to be consistent with the results obtained from the miRNA microarray whilst the other two were opposite to that result (both downregulated) (p<0.05). Using online tools as well as the published literature, several potential target genes of miR-575 were identified, namely DENND5A, CDK1, CSTA and ATAD5. One particular target, the BH3- like motif containing inducer of cell death (BLID), which is involved in apoptosis, has previously been confirmed as a gene target in non small cell lung cancer. Using qPCR, we found that BLID messenger RNA (mRNA) was downregulated in normal B-cells when exposed to HIV-1 AT-2. Unfortunately, the BLID protein could not be detected using western blot analysis despite several attempts at detecting varying concentrations of the protein and using two different positive control cell lines. Conclusion: The reverse correlation, between miR-575 and BLID mRNA expression in the same cell line and under the same treatment conditions, supports the notion that the downregulation of miR-575 may be physiologically relevant. However, this could not be further verified as the BLID protein could not be detected in the L1439A cells, even in the microvesicle treated control cells. Future studies should look at further characterisation of miR- 575 in the pathogenesis of HIV-associated NHLs by investigating other predicted gene targets of the miRNA. This will then be followed by loss and gain of function assays to confirm the miRNA:mRNA relationship. Furthermore, functional analyses, such as measure of apoptosis, expression of key regulators of the cell cycle, and other cellular events characteristic of cancer should be carried out to define the role of the miR-575 in the development of HIV-associated lymphoma