Wellington K. Ayensu

Identification of Drought-Responsive Universal Stress Proteins in Viridiplantae

Genes encoding proteins that contain the universal stress protein (USP) domain are known to provide bacteria, archaea, fungi, protozoa, and plants with the ability to respond to a plethora of environmental stresses. Specifically in plants, drought tolerance is a desirable phenotype. However, limited focused and organized functional genomic datasets exist on drought-responsive plant USP genes to facilitate their characterization. The overall objective of the investigation was to identify diverse plant universal stress proteins and Expressed Sequence Tags (ESTs) responsive to water-deficit stress. We hypothesize that cross-database mining of functional annotations in protein and gene transcript bioinformatics resources would help identify candidate drought-responsive universal stress proteins and transcripts from multiple plant species. Our bioinformatics approach retrieved, mined and integrated comprehensive functional annotation data on 511 protein and 1561 ESTs sequences from 161 viridiplantae taxa. A total of 32 drought-responsive ESTs from 7 plant genera Glycine, Hordeum, Manihot, Medicago, Oryza, Pinus and Triticum were identified. Two Arabidopsis USP genes At3g62550 and At3g53990 that encode ATP-binding motif were up-regulated in a drought microarray dataset. Further, a dataset of 80 simple sequence repeats (SSRs) linked to 20 singletons and 47 transcript assembles was constructed. Integrating the datasets on SSRs and drought-responsive ESTs identified three drought-responsive ESTs from bread wheat (BE604157), soybean (BM887317) and maritime pine (BX682209). The SSR sequence types were CAG, ATA and AT respectively. The datasets from cross-database mining provide organized resources for the characterization of USP genes as useful targets for engineering plant varieties tolerant to unfavorable environmental conditions.

Developmental Regulation of Genes encoding Universal stress proteins in Schistosoma mansoni

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

Functional Annotation Analytics of Rhodopseudomonas palustris Genomes

Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

Functional Annotation Analytics of Bacillus Genomes Reveals Stress Responsive Acetate Utilization and Sulfate Uptake in the Biotechnologically Relevant Bacillus megaterium

Bacillus species form an heterogeneous group of Gram-positive bacteria that include members that are disease-causing, biotechnologically-relevant, and can serve as biological research tools. A common feature of Bacillus species is their ability to survive in harsh environmental conditions by formation of resistant endospores. Genes encoding the universal stress protein (USP) domain confer cellular and organismal survival during unfavorable conditions such as nutrient depletion. As of February 2012, the genome sequences and a variety of functional annotations for at least 123 Bacillus isolates including 45 Bacillus cereus isolates were available in public domain bioinformatics resources. Additionally, the genome sequencing status of 10 of the B. cereus isolates were annotated as finished with each genome encoded 3 USP genes. The conservation of gene neighborhood of the 140 aa universal stress protein in the B. cereus genomes led to the identification of a predicted plasmid-encoded transcriptional unit that includes a USP gene and a sulfate uptake gene in the soil-inhabiting Bacillus megaterium. Gene neighborhood analysis combined with visual analytics of chemical ligand binding sites data provided knowledge-building biological insights on possible cellular functions of B. megaterium universal stress proteins. These functions include sulfate and potassium uptake, acid extrusion, cellular energy-level sensing, survival in high oxygen conditions and acetate utilization. Of particular interest was a two-gene transcriptional unit that consisted of genes for a universal stress protein and a sirtuin Sir2 (deacetylase enzyme for NAD+-dependent acetate utilization). The predicted transcriptional units for stress responsive inorganic sulfate uptake and acetate utilization could explain biological mechanisms for survival of soil-inhabiting Bacillus species in sulfate and acetate limiting conditions. Considering the key role of sirtuins in mammalian physiology additional research on the USP-Sir2 transcriptional unit of B. megaterium could help explain mammalian acetate metabolism in glucose-limiting conditions such as caloric restriction. Finally, the deep-rooted position of B. megaterium in the phylogeny of Bacillus species makes the investigation of the functional coupling acetate utilization and stress response compelling.

Abstract 3369: Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is a one of the most prevalent and deadly cancers in United States. Experimental evidence support that cancer cells do exhibit higher glycolytic rates than normal cells (Warburg effect). To exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would negatively impact their survival and alter their growth and viability resulting from the vast decrease in their essential glycolytic ATP production; no negative consequences will be seen on normal lung cells. The human lung fibroblast cell line MRC-5 and the human alveolar epithelial cell line A549 were used as models for normal lung and lung cancer in vitro in this study. Using standard methods, both cell lines were maintained and exposed to honey and D-glucose reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging. Our results indicate that exposure of both cell lines to these natural nutraceutic organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Honey and D-glucose showed statistically significant (p<0.05) differential negative effects on the A549 line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line. Results show a promising role of honey and D-glucose as metabolites of interest for selective management of cancerous cells. Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu, Joseph A. Cameron. Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2014-3369

Abstract 1565: Significance of elemental content and distribution profiles in assessing established cell lines as surrogates for cancer tissues from two different organs.

Studies have shown that defective metal homeostasis is very essential to the development of the cancer phenotype in many cancers including lung and liver carcinomas. Low Zn levels as well as overexpression of metalothioneins were implicated in the development and progression of various cancers. Wider elemental profiles that relate cancer and normal phenotypes with regards to metal homeostasis were not well elucidated in the literature. To this end, established cell lines are currently used as accepted models for studying cancer but the level of their representation of actual cancer tissues was not clear. This study is attempting to assess the relevance of cell lines currently is use as surrogates for cancer and establish their relationship to actual normal and cancer tissues from humans. We hypothesize that elemental content and distribution profiles in cancer and normal tissue are significantly different from those possessed by established cell lines. We used Inductivity Coupled Plasma (ICP-Mass Spectroscopy) for lung and liver samples obtained from normal and cancer human cadaver tissues (US Biomax) and cell models of lung and liver carcinomas; the A549 and HepG2 cell lines (ATCC), to analyze for elements and test the hypothesis. Samples prepared using standardized digestion procedures were loaded onto the ICP-MS equipment to test for a profile consisting of 12 elements, namely, Zn, Cu, Fe, Mg, Mn, Ca, Al, Co, Ba, Cr, Ni and Se. Analysis of data showed significant variations in elemental content and distribution profiles between the cell models and actual tissues consistent with the hypothesis. It is concluded that elemental homeostasis is essential for normal tissue function and that shifts in their distribution and content are essential in determining the use of cell models as surrogates for studying cancer. These results are promising and warrant further studies to confirm the relevance of cell line models in relation to their use as screening tools for examining targeted cancer therapeutics. Citation Format: Ibrahim O. Farah, Zikri Arslan, Wellington K. Ayensu. Significance of elemental content and distribution profiles in assessing established cell lines as surrogates for cancer tissues from two different organs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1565. doi:10.1158/1538-7445.AM2013-1565

Autoimmune Diseases: The Role of Environment and Gene Interactions

Wellington K. Ayensu1,3, Emmanuel O. Keku2, Raphael D. Isokpehi1,3, Ibrahim O. Farah1, Chris A. Arthur4 and Sophia S. Leggett4 1College of Science, Engineering & Technology, Jackson State University, Jackson, 2Department of Public Health and Preventive Medicine, School of Medicine, St. George’s University, St. George, Grenada, 3Bioinformatics Section; Jackson State University, Jackson, 4School of Health Sciences, College of Public Service, Jackson State University, Jackson, 1,3,4USA 2West Indies

Altered GABAA receptor expression as biomarker of mercury toxicity in embryonic neurogenesis

Mouse brain microarray dataset (GDS2702) on embryonic days 14.5 (E14.5), 16.5 (E16.5 and 18.5 (E18.5) was investigated for gene expression patterns of GABA receptors. Gabbr1, Gabra2, Gabrb3 and Gabrg2 were the only four GABA receptor genes expressed at stage E14.5 and E16.5. However, at stage E18.5, four additional genes (Gabra1, Gabra3, Gabrg1 and Gabrg3) were expressed. Gabbr1 had the highest intensity in all the three embryonic days. Gabra6, Gabrb2, Gabrd, Gabrr1 and Gabrr2 were not expressed in all the 3 samples analyzed. It is proposed that Gabra6 receptor is a potential biomarker for neuronal toxicity that can arise from maternal exposures to low doses of mercury through diet, water and air pollutions.