Browsing by Author "Ganapathy, Sivakumar"
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Item A Genomic and Functional Analysis of Bacterial Diversity in Agricultural Soil for Chlorpyrifos Biodegradation(2018-12) Islam, Nelufa Yesmin; Iyer, Rupa; Ganapathy, Sivakumar; Cai, Chengzhi; Shireen, Wajiha; Zouridakis, GeorgeChlorpyrifos (CP) is a widely used organophosphate (OP) insecticide and a potent environemntal neurotoxin. This research project focuses on the potential of bacteria, both native to agricultural soil and part of a designed consortium composed of Iyer laboratory strains, to completely degrade CP and its toxic byproducts in different types of agricultural soil. Sequence data from isolated agricultural microorganisms was analyzed using the RAST (Rapid Annotation using Subsystem Technology) server to identify putative CP degradation biomarkers. Metabolite production and degradation kinetics analysis gas chromatography mass spectrophotometry (GCMS) analysis was conducted on each soil sample and compared to soil spiked with different combinations of bacterial consortia over a period of 7 days to determine the effectiveness of CP degradation in non-augmented and augmented soil. Genomic analysis of ranch, garden and crop soil microorganisms revealed multiple CP degradation biomarkers including a family of diverse OPHC2-like metallo-β-lactamase (MBL) enzymes, and 3-oxoadipate enol-lactonases. GCMS analysis of these soil samples inoculated with CP support putative microbial degradation activity show that 4 CP metabolites are consistently released including 3,5,6-trichloropyridinol (TCP), phosphorothioic acid, fumaric acid and ethanol. Non-augmented ranch soil and crop field soil display a greater degradation capacity than garden soil possibly due to greater CP pesticide exposure at these sites. Overall, degradation kinetics for augmented and non-augmented soil samples was 0.79d-1 and 0.19d-1 and half-life 1.03 and 5.45 days respectively. CP inoculated soil spiked with a bacterial consortium consisting of all 3 strains exhibited the highest degradation rate with 78.55% of CP degraded after 48 hours. The outcome of this study suggests that while native agricultural populations are capable of low-level CP degradation, supplementing contaminated soil with a bacterial consortium consisting of Pseudomonas putida, Ochrobactrum anthropi and Rhizobium radiobacter could be a highly effective and safe biological approach to facilitating rapid CP degradation.Item A GIS-Based Comparative Study of Superfund Contaminants and the Threat They Represent to Eastern Harris County Communities(2017-12) Hosseinzadeh, Hemen; Iyer, Rupa; Ganapathy, Sivakumar; Khan, Shuhab D.There are six Texas Superfund sites that can be found in Eastern Harris County along the San Jacinto River. Three sites north of the I-10 overpass include the French Limited (FLTD), Sikes Disposal Pits (SDP) and Highlands Acid Pits (HAP), all of which contain benzene, metals, Methyl Tert-Butyl Ether, and Volatile Organic Compounds above the Maximum Contaminants Level (MCL) and lie in close proximity to Channelview, Highlands, and Crosby. Three sites to the south of the 1-10 overpass include the San Jacinto River Waste Pits (SJRWP), Patrick’s Bayou (PB) Superfund, and US Oil Recovery (USOR) Superfund site, which contain benzene as well as polychlorinated dioxins, furans, and biphenyls Polychlorinated Dibenzo-p-dioxins, Polychlorinated dibenzofurans, and Polychlorinated Biphenyls above the MCL and lie near communities in Deer Park, Pasadena, Galena Park, and Jacinto City. Therefore, each Superfund site presents a significant health risk to the nearby urban population centers due to possibility of leakage of contaminants into the San Jacinto River. There are 226,496 people who live and/or work in the aforementioned study area within five miles of at least one Superfund site. Data pooled from each Superfund site was compiled into tables and maps to determine where and which demographics are most at risk of exposure to these toxins. As of 2010, the study area has an approximately 76% Hispanic population with 42% percent of the total population having less than a high school level education, and 38% earning less than $50K annually. These demographic statistics suggest possible communication barriers for the population to respond adequately to a potential health threat. In particular, 17 cancer clusters, which include incidences of brain, melanoma, breast, cervical, kidney, liver, leukemia, and myeloma, were found throughout the study area, excluding only urban areas around USOR (Galena Park, Pasadena, Jacinto City). Bacterial collection in these neighborhoods would indicate the presence of degradation activities and show possible contamination necessitating treatment. Current remediation efforts at the Superfund sites is ongoing, but flooding remains a major threat to urban areas including drinking water wells, residential neighborhoods and parks, as well as elementary schools around the SJRWP and SDP.Item Bacterial Consortium and Effect of Different Conditions on Cell Growth Using Naphthalene as a Sole Carbon Source by the Bacteria Isolated from Soil Samples Collected from Oil Refineries in Texas(2017-05) Jagtap, Dipti; Iyer, Rupa; Ganapathy, Sivakumar; Robles Hernandez, Francisco C.Petrochemical refineries and human activities are responsible for release of carcinogenic compounds known as Polycyclic Aromatic Hydrocarbons (PAHs). Adverse effects of these compounds on human health and ecosystem resulted in an increased awareness, and lead to dramatic increase in research intended to remove PAHs from the ecosystem. In the present study naphthalene was used as a model compound. It was hypothesized that the microorganisms isolated from naphthalene contaminated soil collected from petrochemical refineries in Texas could be utilizing naphthalene as a carbon source. The naphthalene degradation capability of microorganisms could be an indication of their ability to degrade PAHs. In this study the isolated strains were identified as Enterobacter cloacae and Cellulosimicrobium cellulans based on results from 16S rDNA gene sequencing analysis. Bacterial consortium and effect of different conditions on cell growth using naphthalene as a sole carbon source were studied.Item Bioimaging of Flow Dynamics in BTB(2017) Raghunandan, Santhanakrishnan; Alba, Kamran; Ganapathy, SivakumarGloriosa superba is a well-known source of the bioactive colchicine. It is one of the primary sources of treatment for gout and under drug development for cancer as well as cardiovascular diseases. The balloon type bubble bioreactor (BTBR) has been successfully used for the production of biopharmaceuticals. The biomanufacturing of colchicine can be increased by understanding the flow dynamics, mixing intensity inside the BTBR. The preliminary results illustrate the homogenous and heterogeneous flow patterns, the direction of flow inside the reactor at low and high gas injection rates. We will present the mean bubble diameter, the maximum and the minimum vorticity, density of the fluid, viscosity, and surface tension bioimaging as well as quantitative data, which could provide better understanding of the fluid dynamics for the biomanufacturing of biorhizome for therapeutic colchicine production.Item Gloriosa superba and Colchicum autumnale Trascriptomes: Computational Mapping and Identification of Rhizome-specific Genes(2017) Bass, John Samuel; Ganapathy, SivakumarGloriosa superba and Colchicum autumnale are rhizome-producing plants from the Colchicaceae family, which is known for their production of colchicine, a FDA approved gout drug that also has anticancer properties. In order to improve the biomanufacturing of colchicine, understanding and identifying the biorhizome-specific and developmental genes are necessary. RNA-Seq was used to identify the rhizome-specific and developmental genes from G. superba and C. autumnale. The transcriptome of both species were compared against NCBI protein databases. Computational mapping revealed 32312 assembled multiple-tissue transcripts of G. superba represented 15089 unigenes and C. autumnale has 60927, which represents 21948 unigenes in known plant specific gene ontology (GO). Further GO analysis was used to identify known rhizome-specific and developmental genes in G. superba and C. autumnale, utilizing Blast2GO and CLC Genomics Workbench. We will present the identified rhizome-specific and developmental genes, which could help to improve the biomanufacturing of colchicine.Item In Vivo Gloriosa Superba and Colchicum Autumnale Multi-Tissues Transcriptome Analysis for Colchicine Pathway and Rhizome Development Candidate Genes Identification(2018-12) Bass, John Samuel; Ganapathy, Sivakumar; Iyer, Rupa; Cirino, Patrick C.; Shireen, Wajiha; Zouridakis, GeorgeBackground: The continued emergence of side-effects caused by synthetic drugs underscores the need for plant-based drugs in human medicine. Medicinal rhizomatous crops are “the goldmine for modern drugs”, and include such species as Gloriosa superba L., and Colchicum autumnale L., the producer of colchicine, a plant-based medicine. The natural isomer of bioactive colchicine is used to effectively treat major diseases such as cancer, cardiovascular disease, and gout. The medicinal properties of colchicine are well characterized, however, almost nothing is known about its biosynthetic mechanism and colchicine pathway has not been elucidated that are significant barriers in biomanufacturing of biomedicine. The comparative transcriptomes study of G. superba and C. autumnale can serve as sequence resource and synthetic biology toolbox components for identifying biomedicine pathway and rhizome development genes, which could aid colchicine pathway metabolic engineering or synthetic biotechnology to improve colchicine biomanufacturing. Result: Predominantly colchicine synthesizing two monocots such as G. superba and C. autumnale transcriptomes were used to identify putative protein involved in the colchicine biosynthetic pathway and rhizome development along with transcription factors. Mining of the transcriptomes using Blast2GO, 20 and 29 candidate genes [3 and 1 candidate N-methyltransferase (NMT); 10 and 16 candidate 3-O-methyltransferase (3-OMT); cytochrome P450s, a class that could catalyze several steps in the pathway namely, 2 and 5 candidate CYP96T1, 1 and 4 candidate CYP82E10; 4 and 3 candidate N-acetyltransferase (NAT)] were identified in colchicine pathway for G. superba and C. autumnale, respectively. Similarly, 19 and 15 candidate rhizome developmental genes [2 and 1 candidate GIGANTEA (GI), 5 and 4 candidate CONSTANS (CO), 2 and 1 candidate Phytochrome B (PHYB), 2 and 5 candidate Sucrose Synthase (SuSy), 5 and 2 candidate Flowering Locus T (FT), and 3 and 2 candidate REVOLUTA (REV)] were identified in G. superba and C. autumnale, respectively. While 16 and 12 transcription factors in rhizome development and regulating secondary metabolic pathways in rhizomes [3 and 1 candidate MADS-box, 6 and 2 candidate AP2-EREBP, 2 and 2 candidate bHLH, 1 and 2 candidate MYB, 2 and 2 candidate NAC, and 2 and 3 WRKY] were screened in G. superba and C. autumnale, respectively. These genes could represent potential leads for metabolic engineering of G. superba or synthetic biotechnology of colchicine metabolism for enhanced colchicine and biorhizome biomass in biomanufacturing. Conclusion: The study of G. superba and C. autumnale genes predicated to encode colchicine pathway enzymes are highly significant for fundamental information on plant-based biomedicine biosynthesis, which could facilitate engineered production in biorhizomes, a potentially important area of synthetic biotechnology. Additionally, increasing our understanding of rhizome genomics could improve colchicine production in G. superba, and generate important knowledge that could be applied to many other medicinal plant species, and could allow engineered production of additional biomedicines in biorhizomes, a potentially important area of expansion for synthetic biotechnology to solve overarching biomanufacturing challenges.Item Isolation and Identification of Biphenyl and Polychlorinated Biphenyl Degrading Microbes(2017-12) Ahmed, Hasan Hasnaeen; Iyer, Rupa; Ganapathy, Sivakumar; Cai, ChengzhiThe San Jacinto River system south of the I-10 overpass is heavily contaminated with biphenyl and polychlorinated biphenyls (PCBs), carcinogenic aromatic hydrocarbons that have a deleterious impact on the environment. While typically recalcitrant to most forms of environmental degradation, biphenyl and PCBs compounds are known to share a common microbial degradation pathway with other chlorinated aromatics such as polychlorinated dioxin (PCDD) and dibenzofuran (PCDF). As such the purpose of this research is to isolate and test bacterial strains collected from contaminated San Jacinto River sediment and soil for the capacity to degrade or fully metabolize biphenyl and its derivatives. As conventional clean-up is expensive, such strains can be used in the field of bioremediation as a more cost-effective way of treating environmental contamination. The samples were screened for six weeks in minimal media with putative positive samples isolated for further bioinformatics analysis. Microorganisms identified as Pseudomonas pseudoalcaligenes, Pseudomonas nitroreducens, and Comamonas sp. were noted to harbor a putative 2,3 biphenyl dioxygenase enzyme and were further assessed for possible degradative capacity through a combination of growth kinetics and GC-MS analysis to determine the quality and quantity of the intermediate metabolites. Sample growth results suggest that each of the three isolated study strains are unable to utilize biphenyl or 3-chlorobiphenyl as a sole carbon source. In addition, expected metabolites of biphenyl degradation were not observed in either the supernatant or cell lysate indicating that expression of these putative 2,3 biphenyl dioxygenase enzymes and/or their catalytic activity against biphenyl is likely to be minimal.Item Micro-Patterned Substrates for Differentiating Mesenchymal Stem Cells into Insulin Producing Cells(2017-12) Friguglietti, Jefferson; Merchant, Fatima Aziz; Zagozdzon-Wosik, Wanda; Balan, Venkatesh; Ganapathy, Sivakumar; Flavier, Albert B.; Zouridakis, GeorgeConventional insulin therapy for Type 1 diabetes mellitus is often accompanied by long-term complications such as heart disease and kidney damage, if patients do not follow a very strict and controlled regime of taking insulin shots. Transplantation of pancreatic islets is a therapeutic option available for Type 1 Diabetes, where in donor islets are transplanted into patients for controlling glucose levels without the need of insulin shots. Although the current islet transplantation Edmonton protocol has made progress in successfully treating diabetic patients, a lack of viable donor cells and side effects associated with immunosuppressant drugs make alternative therapeutic options critical. Cell replacement therapy via differentiation of adult stem cells into glucose-responsive insulin producing cells (IPCs) has recently provided hope for Type I diabetes. However, inadequate functional performance of the differentiated cells with poor long-term insulin production has slowed further progress. Thus, there is a critical need for improving the total yield of differentiated cells and their functional performance. In this study we investigated the potential of a novel substrate of micro-patterned Titanium diboride (TiB2) on Silicon (Si) wafers for culturing adult human bone marrow mesenchymal stem cells (hBM-MSCs) and differentiating them into insulin producing cells (IPCs). The hypothesis is that these substrates enable formation of aggregates, thereby enabling a 3D micro-environment for differentiation. Stereomicroscopy showed MSCs preference for TiB2 patterns over Si and the formation of uniform aggregates only on the TiB2 after the differentiation protocol. Moreover, MSCs not only remained at 80% or more viable when aggregated, but phenotyping analysis for the presence of biomarker CD105 demonstrated conserved multi-lineage potential throughout the 9 day pre-differentiation incubation period. More importantly, our results suggest a 2-3 fold increase of insulin secretion from MSCs differentiated on the micro-patterned substrates when compared against differentiation in conventional tissue culture flasks.Item Multiphase Fluid Mechanics in Biomanufacturing(2018-12) Raghunandan, Santhanakrishnan; Ganapathy, Sivakumar; Alba, Kamran; Iyer, Rupa; Balan, Venkatesh; Shireen, Wajiha; Zouridakis, GeorgeThe aim of this fluid mechanics work was to study the fundamental hydrodynamics mechanism in a balloon type bubble bioreactor (BTBB), which could provide a key guidance to improve biomanufacturing of biopharmaceuticals. A multiphase (liquid and air) computational fluid dynamics was performed with particle tracking velocimetry to observe flow patterns inside BTBB. The control experiment was investigated in 5 and 20L BTBB with water observed an increase in the bubble diameter (0.15 to 0.24 cm for 2L working volume in 5L; 0.27 to 0.36 cm for 16L working volume in 20L) with increasing volumetric flow rate (1 cc/min to 5.5 cc/min) and working volume (2L, 4L, 8L and 16L). However, the bubble diameter reduced with increasing interface forces such as viscosity, density and surface tension when compared with water (0.14 cm at 5.5 cc/min in salt solution; while it was 0.24 cm in water). The decrease in density and viscous fluid could be due to low detachment time and increasing density of the bubble or prevention of bubble coalescence. The reduction in surface tension (30 dynes/cm) resulted in the activation of sparger pores leading to the formation of numerous small bubbles. A homogeneous or laminar flow was observed in higher flow rates (3 cc/min) with increasing viscosity (5 mPa.s) and the flow was turbulent or heterogeneous with flow rates higher than 3 cc/min (4, 5, 5.5 cc/min). Moreover, the dual effect of increasing viscosity and density decreased the bubble sizes in case of CO2 and had laminar flow for higher flow rates (3 cc/min) when compared with the individual effect of density and viscosity. The flow data shows that the differential fluid mechanics pattern in non-uniform geometry BTBB, which can be used to design a flow sensor that could accurately controlling the mixing rate or mass transfer in large-scale biomanufacturing.Item Transcriptome Analysis of Colchicine Producing Plant Species: Gloriosa superba and Colchicum autumnale(2017) Frenk, Annette; Ganapathy, SivakumarColchicine is an alkaloid product of plant biosynthesis which is FDA approved and commonly used to treat gout. It has also been proven to be beneficial in heart attack recovery and its antimitotic properties make it a promising cancer treatment. Traditionally, the natural isomer of colchicine is exacted from Gloriosa superba after harvesting the plant and the compound itself was initially isolated from Colchicum autumnale. The biosynthetic pathway of colchicine is not yet characterized, so in order to improve colchicine production in manufacturing, the pathway genes and mechanisms must be researched and clarified. To elucidate this biosynthetic pathway G. superba and C. autumnale transcriptomes were analyzed and compared against NCBI and Swissprot protein databases. Afterwards, annotation data of these transcriptomes revealed that there were 60927 assembled multi-tissue transcripts of C. autumnale which represented 21948 unigenes and G. superba has 32312 assembled multi-tissue transcripts which represented 15089 unigenes in known plantspecific Gene Ontology (GO). In order to further identify colchicine pathways and gene, further GO term analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) for both organisms. The results obtained represents comparative GO term and KEGG pathways, which could aid in identifying colchicine pathway genes.