Genomic and Transcriptomic Investigation of Endemic Burkitt Lymphoma and Epstein Barr Virus

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein-Barr virus (EBV), unlike sporadic Burkitt Lymphoma (sBL) that occurs with a lower incidence in developed countries. Despite this increased burden the study of eBL has lagged. Additionally, while EBV was isolated from an African Burkitt lymphoma tumor 50 years ago, however, the impact of viral variation in oncogenesis is just beginning to be fully explored. In my thesis research, I focused on investigating molecular genetics of the endemic form of this lymphoma with a particular emphasis on the role of the virus and its variation in pathogenesis using novel sequencing and bioinformatic strategies. First, we sought to understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from 30 primary eBL tumors and compared to sBL tumors. BL tumor samples were prospectively obtained from 2009 until 2012 in Kenya. Within eBL tumors, minimal expression differences were found based on anatomical presentation site, in-hospital survival rates, and EBV genome type; suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex in eBL tumors carrying type 2 EBV compared to type 1 EBV. Secondly, in comparison to previously published pediatric sBL specimens, the majority of the expression and pathway differences were related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than the geographic designation. Moreover, the common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, we identified a set of new genes mutated in BL. Overall, these suggested that EBV, particularly EBV type 1, supports BL oncogenesis alleviating the need for particular driver mutations in the human genome. Second, we sought to comprehensively define sequence variations of EBV across the viral genome in eBL tumor cells and normal infections, and correlate variations with clinical phenotypes and disease risk. We investigated the whole genome sequence of EBV from primary tumors (N=41) and plasma from eBL patients (N=21) as well as EBV in the blood of healthy children (N=29) within the same malaria endemic region. We conducted a genome wide association analysis study with viral genomes of healthy kids and BL kids. Furthermore, we found that the frequencies of EBV types among healthy kids were at equal levels while they were skewed in favor of type 1 (70%) among eBL kids. To pinpoint the fundamental divergence between viral genome subtypes, type 1 and type 2, we constructed phylogenetic trees comparing to all public EBV genomes. The pattern of variation defined the substructures correlated with the subtypes. This investigation not only deciphers the puzzling pathogenic differences between subtypes but also helps to understand how these two EBV types persist in the population at the same time. Overall, this research provides insight into the molecular underpinning of eBL and the role of EBV. It further provides the groundwork and means to unravel the complexity of EBV population structure and provide insight into the viral variation that may influence oncogenesis and outcomes in eBL and other EBV-associated diseases. In addition, genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions

Biosorption kinetics of a direct azo dye Sirius Blue K-CFN by Trametes versicolor

In this study, lyophilized Trametes versicolor biomass is used as a sorbent for biosorption of a textile dye, Sirius Blue K-CFN, from an aqueous solution. The batch sorption was studied with respect to dye concentration, adsorbent dose and equilibrium time. The effect of pH and temperature on dye uptake was also investigated and kinetic parameters were determined. Optimal initial pH (3.0), equilibrium time (2 hrs), initial dye concentration ( 100 mg l-1) and biomass concentration (1.2 mg l-1) were determined at 26\ubaC. The maximum biosorption capacity (qmax) of Sirius Blue K-CFN dye on lyophilized T. versicolor biomass is 62.62 mg/g. The kinetic and isotherm studies indicated that the biosorption process obeys to a pseudo-second order model and Langmuir isotherm model. In addition, the biosorption capacities of fungal biomass compared to other well known adsorbents such as activated carbon and Amberlite, fungal biomass biosorptions capacities were found to be more efficient

Utilization of Molecular Inversion Probes in Malaria Sequencing

While massively parallel sequencing of whole genomes shed light on many previously puzzling genetic questions, the high costs associated with this approach makes its use impractical when large cohorts need to be sequenced at high coverage. Available capture technologies reduces the sequencing costs by enriching template material for the regions of interest. However, these technologies are also prohibitively costly at high sample numbers. Capture methods utilizing molecular inversion probes (MIPs) offer a flexible alternative to enrich template material that multiplex well for thousands of samples and require minimal resources. Here, for our work in malaria, we extend the utility of MIPs, improving the capture length and efficiency. We have also dramatically decreased the capture time from 24-48 h to 1 h. Combined, these improvements allow the potential for rapid and reliable application of MIP captures in research and, importantly, clinical settings

Biodegradation kinetics of o-cresol by Pseudomonas putida DSM 548 (pJP4) and o-cresol removal in a batch-recirculation bioreactor system

The biodegradation kinetics of o-cresol was examined by acclimatized P. putida DSM 548 (pJP4) in batch experiments at varying initial o-cresol concentrations (from 50 to 500 mg/L). The kinetic parameters of o-cresol aerobic biodegradation were estimated by using the Haldane substrate inhibition equation. The biodegradation kinetics of o-cresol was investigated. In batch culture reactors, the Maximum specific growth rate (\u3bcmax), Monod constant (Ks) and the inhibition constant (Ki) were established as 0.519 h-1, 223.84 mg/L and 130.883 mg/L, respectively. o-cresol biodegradation in a batch-recirculation bioreactor system by immobilized P. putida was also studied. The recycled packed bed reactor system, which was composed of Ca-alginate beads and pumice on which cells immobilized, has been performed to determine possible stability for further developments

Differential Gene Expression Analysis and Clinical Correlations within Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in equatorial Africa and is associated with malaria and Epstein-Barr virus co-infections. Molecular alterations within the eBL tumor genome and transcriptome have not been adequately investigated or compared to sporadic Burkitt lymphoma (sBL). Given that eBL has distinct clinical presentations in the jaw as opposed to the abdomen which are associated with survival, we hypothesize that transcriptome sequencing (RNA-seq) and potentially underlying genetic alterations will enhance our understanding of pathogenesis. Our results compare genome-wide RNA transcript abundances between eBL tumors from children (ages 6-7 yrs) with Stage I (Jaw tumor, n=14) and Stage II (abdominal, n=24) disease from Western Kenya to previously published work analyzing sBL which present in older children residing in developed countries and that tend not to be associated with EBV. Our initial analysis confirms mutational changes with likely functional alterations in the genes ID3 and TCF3, the key regulators of oncogenic pathways implicated in BL. However, the specific mutations observed in sBL are at lower frequency within eBL cases. This work represents the first comprehensive gene expression profile analysis of different eBL tumors. Hierarchical clustering, gene ontology and pathway analysis will provide insight into pathogenesis and new targets for chemotherapy

Epstein Barr virus genomes reveal population structure and type 1 association with endemic Burkitt lymphoma [preprint]

Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is associated with malaria and Epstein Barr virus (EBV). In order to better understand the role of EBV in eBL, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (N=58) and healthy controls (N=40) residing in the same geographic region in Kenya. Comparing cases and controls, we found that EBV type 1 was significantly associated with eBL with 74.5% of patients (41/55) versus 47.5% of healthy children (19/40) carrying type 1 (OR=3.24, 95% CI=1.36 - 7.71, P=0.007). Controlling for EBV type, we also performed a genome-wide association study identifying 6 nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. Additionally, we observed that viruses isolated from plasma of eBL patients were identical to their tumor counterpart consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions as well as one novel genome with a 20 kb deletion resulting in the loss of multiple lytic and virion genes. Comparing EBV types, genes show differential variation rates as type 1 appears to be more divergent. Besides, type 2 demonstrates novel substructures. Overall, our findings address the complexities of EBV population structure and provide new insight into viral variation, which has the potential to influence eBL oncogenesis

Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma

BACKGROUND: Burkitt lymphoma (BL) is characterized by overexpression of the c-myc oncogene, which in the vast majority of cases is a consequence of an IGH/MYC translocation. While myc is the seminal event, BL is a complex amalgam of genetic and epigenetic changes causing dysregulation of both coding and non-coding transcripts. Emerging evidence suggest that abnormal modulation of mRNA transcription via miRNAs might be a significant factor in lymphomagenesis. However, the alterations in these miRNAs and their correlations to their putative mRNA targets have not been extensively studied relative to normal germinal center (GC) B cells. METHODS: Using more sensitive and specific transcriptome deep sequencing, we compared previously published small miRNA and long mRNA of a set of GC B cells and eBL tumors. MiRWalk2.0 was used to identify the validated target genes for the deregulated miRNAs, which would be important for understanding the regulatory networks associated with eBL development. RESULTS: We found 211 differentially expressed (DE) genes (79 upregulated and 132 downregulated) and 49 DE miRNAs (22 up-regulated and 27 down-regulated). Gene Set enrichment analysis identified the enrichment of a set of MYC regulated genes. Network propagation-based method and correlated miRNA-mRNA expression analysis identified dysregulated miRNAs, including miR-17~95 cluster members and their target genes, which have diverse oncogenic properties to be critical to eBL lymphomagenesis. Central to all these findings, we observed the downregulation of ATM and NLK genes, which represent important regulators in response to DNA damage in eBL tumor cells. These tumor suppressors were targeted by multiple upregulated miRNAs (miR-19b-3p, miR-26a-5p, miR-30b-5p, miR-92a-5p and miR-27b-3p) which could account for their aberrant expression in eBL. CONCLUSION: Combined loss of p53 induction and function due to miRNA-mediated regulation of ATM and NLK, together with the upregulation of TFAP4, may be a central role for human miRNAs in eBL oncogenesis. This facilitates survival of eBL tumor cells with the IGH/MYC chromosomal translocation and promotes MYC-induced cell cycle progression, initiating eBL lymphomagenesis. This characterization of miRNA-mRNA interactions in eBL relative to GC B cells provides new insights on miRNA-mediated transcript regulation in eBL, which are potentially useful for new improved therapeutic strategies

FLT1 and transcriptome-wide polyadenylation site (PAS) analysis in preeclampsia

Maternal symptoms of preeclampsia (PE) are primarily driven by excess anti-angiogenic factors originating from the placenta. Chief among these are soluble Flt1 proteins (sFlt1s) produced from alternatively polyadenylated mRNA isoforms. Here we used polyadenylation site sequencing (PAS-Seq) of RNA from normal and PE human placentae to interrogate transcriptome-wide gene expression and alternative polyadenylation signatures associated with early-onset PE (EO-PE; symptom onset 34 weeks) cohorts. While we observed no general shift in alternative polyadenylation associated with PE, the EO-PE and LO-PE cohorts do exhibit gene expression profiles distinct from both each other and from normal placentae. The only two genes upregulated across all transcriptome-wide PE analyses to date (microarray, RNA-Seq and PAS-Seq) are NRIP1 (RIP140), a transcriptional co-regulator linked to metabolic syndromes associated with obesity, and Flt1. Consistent with sFlt1 overproduction being a significant driver of clinical symptoms, placental Flt1 mRNA levels strongly correlate with maternal blood pressure. For Flt1, just three mRNA isoforms account for > 94% of all transcripts, with increased transcription of the entire locus driving Flt1 upregulation in both EO-PE and LO-PE. These three isoforms thus represent potential targets for therapeutic RNA interference (RNAi) in both early and late presentations

Biosorption kinetics of a direct azo dye Sirius Blue K-CFN by Trametes versicolor

In this study, lyophilized Trametes versicolor biomass is used as a sorbent for biosorption of a textile dye, Sirius Blue K-CFN, from an aqueous solution. The batch sorption was studied with respect to dye concentration, adsorbent dose and equilibrium time. The effect of pH and temperature on dye uptake was also investigated and kinetic parameters were determined. Optimal initial pH (3.0), equilibrium time (2 hrs), initial dye concentration ( 100 mg l-1) and biomass concentration (1.2 mg l-1) were determined at 26�C. The maximum biosorption capacity (q max) of Sirius Blue K-CFN dye on lyophilized T. versicolor biomass is 62.62 mg/g. The kinetic and isotherm studies indicated that the biosorption process obeys to a pseudo-second order model and Langmuir isotherm model. In addition, the biosorption capacities of fungal biomass compared to other well known adsorbents such as activated carbon and Amberlite, fungal biomass biosorptions capacities were found to be more efficient

Biosorption kinetics of a direct azo dye Sirius Blue K-CFN by Trametes versicolor

WOS: 000293631500003In this study, lyophilized Trametes versicolor biomass is used as a sorbent for biosorption of a textile dye, Sirius Blue K-CFN, from an aqueous solution. The batch sorption was studied with respect to dye concentration, adsorbent dose and equilibrium time. The effect of pH and temperature on dye uptake was also investigated and kinetic parameters were determined. Optimal initial pH (3.0), equilibrium time (2 hrs), initial dye concentration (100 mg l(-1)) and biomass concentration (1.2 mg l(-1)) were determined at 26 degrees C. The maximum biosorption capacity (q(max)) of Sirius Blue K-CFN dye on lyophilized T. versicolor biomass is 62.62 mg/g. The kinetic and isotherm studies indicated that the biosorption process obeys to a pseudo-second order model and Langmuir isotherm model. In addition, the biosorption capacities of fungal biomass compared to other well known adsorbents such as activated carbon and Amberlite, fungal biomass biosorptions capacities were found to be more efficient