Therapeutic implications of the warburg effect assessing the survival of MRC5 and a549 cell lines upon exposure to honey and d glucose - biomed 2013.

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. 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 lung alveolar epithelial cancer cell line A549 were used in this study as models for normal lung and lung cancer respectively. 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 (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), and cell counting (T4 Cellometer; automated cell counting system) assays as well as phase-contrast photo-imaging. Our results indicate that exposure of both cell lines to these organics lead to 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. These findings show a promising role for honey and D-glucose as biotherapeutic metabolites of interest for selective management of cancerous cells.

Assessing the survival of MRC5 and a549 cell lines upon exposure to pyruvic Acid, sodium citrate and sodium bicarbonate - biomed 2013.

Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate 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 for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (p<0.05) differential negative effects on the A549 cell line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line, presenting a potential promise for their use as cancer biotherapeutics.

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.

Role of fructose diphosphate (fdp) and glycerol on the differential survival of mrc-5 and a549 cell lines.

Lung cancer is a one of the most prevalent and deadly cancers in United States. Research has shown that cancer cells exhibit higher glycolytic rates than normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would have a negative impact on their survival and will alter their growth and viability due to a vast decrease in their essential glycolytic ATP production with the resultant energetic collapse and that 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 in this study as models for normal lung and lung cancer in vitro. Using standard methods, both cell lines were maintained and exposed to FDP and glycerol reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments were 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 organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. FDP and glycerol 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, presenting promising indicators for their cancer therapeutic potential.

Therapeutic implications of the warburg effect: role of oxalates and acetates on the differential survival of mrc-5 and a549 cell lines.

Lung cancer is a one of the most prevalent and deadly cancers in United States. Research has shown that cancer cells exhibit higher glycolytic rates than normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon (Warburg effect), we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would have a negative impact on their survival and will alter their growth and viability due to a vast decrease in their essential glycolytic ATP production with the resultant energetic collapse and that 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 in this study as models for normal lung and lung cancer in vitro. Using standard methods, both cell lines were maintained and exposed to oxalic acid and zinc acetate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments were 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 organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Oxalic acid and zinc acetate 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, presenting promising indicators for their cancer therapeutic potential.

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.

Assessing the survival of mrc-5 and a549 cell lines upon exposure to ascorbic Acid and sodium ascorbate - biomed 2011.

Lung cancer is among the highly prevalent and deadly cancers in the United States and worldwide. Cells that are cancerous exhibit higher rates of glycolysis as compared to normal cells. In an attempt to exploit this uniquely enhanced glucose-dependent ATP generation phenomenon, the authors hypothesize that exposure of cancer cells to normal organic metabolites that are capable of inhibiting glycolysis would have a negative impact on survival by altering growth and viability characteristics vastly through decline in ATP build up essentially leading to collapse in energy supply; normal lung cells will not demonstrate such changes. The human lung fibroblast cell line MRC-5 and the cancerous human lung alveolar epithelial cell line A549 were utilized in this study as in vitro models of normal and cancerous lung cell lines respectively. Using standard methods, both cell lines were maintained in culture and exposed to ascorbic acid and sodium ascorbate reagents at concentration levels ranging from 31.3-2,000 µg/ml. Cell survival measurements using MTT andT4 Cellometric assays monitored with phase-contrast photo-imaging were carried out in quadruplicates. Results indicate that exposure characteristics to these metabolites followed concentration-dependent cell mortality/survival curves by the cancerous versus normal cell lines respectively. Ascorbic acid and sodium ascorbate showed statistically significant (p<0.05) differential negative effects on the cancerous A549 cell line in comparison to unexposed controls as well as to effects measured with the normal lung MRC-5 cell line; this is highly indicative of a promising therapeutic potential.

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.

Comorbidity of bipolar disorder with substance abuse: selection of prioritized genes for translational research.

Bipolar disorder is a highly heritable mental illness. The global burden of bipolar disorder is complicated by its comorbidity with substance abuse. Several genome-wide linkage/association studies on bipolar disorder as well as substance abuse have focused on the identification and/or prioritization of candidate disease genes. A useful step for translational research of these identified/prioritized genes is to identify sets of genes that have particular kinds of publicly available data. Therefore, we have leveraged the availability of links to related resources in the Entrez Gene database to develop a web-based resource for selecting genes based on presence or absence in particular biological data resources. The utility of our approach is demonstrated using a set of 3,399 genes from multiple eukaryotes that have been studied in the context of bipolar disorder and/or substance abuse. A web resource to automate the selection of genes that contain certain database links is available at http://compbio.jsums.edu/bpd.

Microarray analysis of mercury-induced changes in gene expression in human liver carcinoma (HepG2) cells: importance in immune responses.

Mercury is widely distributed in the biosphere, and its toxic effects have been associated with human death and several ailments that include cardiovascular diseases, anemia, kidney and liver damage, developmental abnormalities, neurobehavioral disorders, autoimmune diseases, and cancers in experimental animals. At the cellular level, mercury has been shown to interact with sulphydryl groups of proteins and enzymes, to damage DNA, and to modulate cell cycle progression and/or apoptosis. However, the underlying molecular mechanisms of mercury toxicity remain to be elucidated. Our laboratory has demonstrated that mercury exposure induces cytotoxicity and apoptosis, modulates cell cycle, and transcriptionally activates specific stress genes in human liver carcinoma cells. The liver is one of the few organs capable of regeneration from injury. Dormant genes in the liver are therefore capable of reactivation. In this research, we hypothesize that mercury-induced hepatotoxicity is associated with the modulation of specific gene expressions in liver cells that can lead to several disease states involving immune system dysfunctions. In testing this hypothesis, we used an Affymetrix oligonucleotide microarray with probe sets complementary to more than 20,000 genes to determine whether patterns of gene expressions differ between controls and mercury (1-3 microg/mL) treated cells. There was a clear separation in gene expression profiles between controls and mercury-treated cells. Hierarchical cluster analysis identified 2,211 target genes that were affected. One hundred and eighty-eight of these genes were up-regulated, among which forty eight were significant (p = 0.001) with greater than a two-fold change difference in the concentration range (1-3 microg/mL) of mercury-treated cells; twelve genes were moderately over-expressed with an increase of more than one fold (p = 0.004). 2,023 genes were down-regulated with only forty of them reaching statistically significant decline at p = 0.05 according to the Welch's ANOVA/Welch's t-test. Further analyses of affected genes identified genes located on all human chromosomes with higher than normal effects on genes found on chromosomes 1-14, 17-20 (sex-determining region Y)-box18SRY, 21 (splicing factor, arginine/serine-rich 15 and ATP-binding), and X (including BCL6-co-repressor). These genes are categorized as control and regulatory genes for metabolic pathways involving the cell cycle (cyclin-dependent kinases), apoptosis, cytokine expression, Na+/K+ ATPase, stress responses, G-protein signal transduction, transcription factors, DNA repair as well as metal-regulatory transcription factor 1, MTF1 HGNC, chondroitin sulfate proteoglycan 5 (neuroglycan C), ATP-binding cassette, sub-family G (WHITE), cytochrome b-561 family protein, CDC-like kinase 1 (CLK1 HGNC) (protein tyrosine kinase STY), Na+/H+ exchanger regulatory factor (NHERF HGNC), potassium voltage-gated channel subfamily H member 2 (KCNH2), putative MAPK activating protein (PM20, PM21), ras homolog gene family, polymerase (DNA directed), delta regulatory subunit (50 kDa), leptin receptor involved in hematopoietin/interferon-class (D200- domain) cytokine receptor activity and thymidine kinase 2, mitochondrial TK2 HGNC and related genes. Significant alterations in these specific genes provide new directions for deeper mechanistic investigations that would lead to a better understanding of the molecular basis of mercury-induced toxicity and human diseases that may result from disturbances in the immune system.

Molecular and cellular mechanisms associated with autoimmune diseases.

Evidence points to increases in the incidence and prevalence of several autoimmune diseases in the United States. As a result, the cost to public health from clinical management of autoimmune conditions is on the rise. The initiation and progression of autoimmune disturbances involves both genetic and environmental factors. Deficiencies in important proteins that normally participate in maintaining checks and balances within the internal milieu may render an individual prone to developing autoantibodies. Structural abnormalities or decline in normal levels of the pentraxins (serum amylase-P protein, the acute phase proteins, complement, and C-reactive proteins) have been shown to induce autoimmunity. Irregular transmission of information arising from multiple signal transduction pathways typically associated with the serine/threonine cascade routes of mitogen activating phosphorylation kinases, has also been found to induce autoimmunity. The kind of ligand/receptor interactions drives physical recruitment of different signals within the lymphocyte; these links define the quality and quantity of subsequent immune responses. CD95 or the Fas/Apo-1 and its ligand CD95L participate in regulating lymphocyte populations and therefore influence various aspects of immune responses. Mutational abnormalities resulting from synthesis of proteins by the CD95 and/or its ligand CD95L may result in alterations in the apoptotic pathways. Apoptosis may be completely inhibited, activated or partially stimulated. Modulation of apoptosis may lead to accumulation of self-antigens. Subsequently the immune system may be stimulated to react against self-molecules through lymphatic hyperplasia. This process may end up in proliferative disorders and enhanced susceptibility to autoimmune syndromes. This paper deals with mechanisms of autoimmunopathogenesis at the cellular and molecular levels. Emphasis is laid on the role of T and B cell receptor/ligand interactions, functions and malfunctions due to structural and quantitative alterations in T- B- cell clusterof antigen determinants. Genetically susceptible patients who develop spontaneous autoimmune diseases are examined and the etiological factors implicated in the initiation and subsequent dissemination of autoimmune diseases is discussed.

Environmental exposure to mercury and its toxicopathologic implications for public health.

Mercury is a toxic and hazardous metal that occurs naturally in the earth's crust. Natural phenomena such as erosion and volcanic eruptions, and anthropogenic activities like metal smelting and industrial production and use may lead to substantial contamination of the environment with mercury. Through consumption of mercury in food, the populations of many areas, particularly in the developing world, have been confronted with catastrophic outbreaks of mercury-induced diseases and mortality. Countries such as Japan, Iraq, Ghana, the Seychelles, and the Faroe Islands have faced such epidemics, which have unraveled the insidious and debilitating nature of mercury poisoning. Its creeping neurotoxicity is highly devastating, particularly in the central and peripheral nervous systems of children. Central nervous system defects and erethism as well as arrythmias, cardiomyopathies, and kidney damage have been associated with mercury exposure. Necrotizing bronchitis and pneumonitis arising from inhalation of mercury vapor can result in respiratory failure. Mercury is also considered a potent immunostimulant and -suppressant, depending on exposure dose and individual susceptibility, producing a number of pathologic sequelae including lymphoproliferation, hypergammaglobulinemia, and total systemic hyper- and hyporeactivities. In this review we discuss the sources of mercury and the potential for human exposure; its biogeochemical cycling in the environment; its systemic, immunotoxic, genotoxic/carcinogenic, and teratogenic health effects; and the dietary influences on its toxicity; as well as the important considerations in risk assessment and management of mercury poisoning.