Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia

Background: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb2+, Al+3, SeO2, Cu2+, AsO4, and Zn2+. With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. Results: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. Conclusions: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils

Impact of Manganese, Copper and Zinc Ions on the Transcriptome of the Nosocomial Pathogen Enterococcus faecalis V583

Mechanisms that enable Enterococcus to cope with different environmental stresses and their contribution to the switch from commensalism to pathogenicity of this organism are still poorly understood. Maintenance of intracellular homeostasis of metal ions is crucial for survival of these bacteria. In particular Zn2+, Mn2+ and Cu2+ are very important metal ions as they are co-factors of many enzymes, are involved in oxidative stress defense and have a role in the immune system of the host. Their concentrations inside the human body vary hugely, which makes it imperative for Enterococcus to fine-tune metal ion homeostasis in order to survive inside the host and colonize it. Little is known about metal regulation in Enterococcus faecalis. Here we present the first genome-wide description of gene expression of E. faecalis V583 growing in the presence of high concentrations of zinc, manganese or copper ions. The DNA microarray experiments revealed that mostly transporters are involved in the responses of E. faecalis to prolonged exposure to high metal concentrations although genes involved in cellular processes, in energy and amino acid metabolisms and genes related to the cell envelope also seem to play important roles

Lithium alters expression of RNAs in a type-specific manner in differentiated human neuroblastoma neuronal cultures, including specific genes involved in Alzheimer's disease.

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer's disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all-trans retinoic acid (ATRA). We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium's modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD

Arsenic phytoremediation: Engineering of an arsenic-specific phytosensor and molecular insights of arsenate metabolism through investigations of \u3cem\u3eArabidopsis thaliana, Pteris cretica,\u3c/em\u3e and \u3cem\u3ePteris vittata\u3c/em\u3e

This dissertation is a compilation of four studies that were conducted in the laboratory of Dr. C. Neal Stewart, Jr. at the University of Tennessee, Knoxville. The first study describes an investigation into arsenate metabolism in Arabidopsis thaliana using microarray technology. The second study summarizes progress made to date towards the development of an As-specific phytosensor, or a plant genetically engineered to detect the presence of As in the environment. The third study describes efforts towards genetic transformation of Pteris cretica and Pteris vittata, both As-hyperaccumulating ferns that have been recently demonstrated as effective in the removal of As from contaminated areas. This paper demonstrates the development of a modified tissue culture protocol that was effective in callus generation from both Pteris vittata and Pteris cretica gametophytes as well as regeneration of plantlets from that callus. Attempts towards genetic transformation were made via biolistic bombardment and Agrobacterium-mediated transient expression using leaf infiltration. Optimization of the Pteris tissue culture protocol will facilitate continued efforts towards the genetic transformation of this unique plant, thereby enabling means of more effectively exploring the underlying mechanisms of As hyperaccumulation. The final study reports a field-scale investigation of plant metal uptake at a local contaminated site in Knoxville, TN. The Smokey Mountain Smelters Site is an abandoned secondary aluminum smelter where waste product from the smelting process (slag) was illegally dumped in large piles over much of the property. Interestingly, wild vegetation was found growing on the slag piles without any obvious symptoms of toxicity. Therefore, a study was conducted to quantify the v metal uptake of these plants, characterize the metal profile of the slag material, and investigate the capacity of Pteris cretica in extracting arsenic from slag on-site. As a result, these studies have provided new insights into arsenate metabolism in plants, and generated many testable hypotheses to enhance our understanding of plant genetic responses to metal stress. The following introduction serves to provide a background on phytoremediation, arsenic, and plant responses to the toxic metalloid

Screening of Mutant Arabidopsis Thaliana and Chlamydomonas Reinhardtii for their Potential Use as Phytosensors in 2,4,6 Trinitrotoluene (TNT) Contaminated Environments

Plant biotechnology is a diverse field that is expanding from agricultural research towards environmental applications. The focus of this project was to exploit vegetative effects, such as photosynthesis and growth in genomic model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii to 2,4,6-trinitrotoluene (TNT) with a goal to develop biomonitoring systems. Plants and algae have evolved with various biochemical pathways that have the potential to be exploited for the use of sensing explosives and chemical warfare agents in soil, water and air. The first part of the project involved characterizing the effects of TNT on germination and early seedling development of wild-type Arabidopsis thaliana. It was determined that 10 mM TNT was the tolerance level for Arabidopsis and was used to screen fast neutron irradiated mutant Arabidopsis to evaluate the phenotypic stress responses in the seedlings. TNT responsive mutant lines (lines 1, 2, 3, and 4) were selected on a basis of a leaf color change from dark green to pale green. The second part of the project was to determine the growth response of wild-type and mutant Chlamydomonas reinhardtii to TNT. Growth response studies of wild-type Chlamydomonas revealed that 3 mg/ml of TNT was the maximum TNT concentration that allowed growth. Insertional mutant lines were screened on 3 mg/ml TNT where one mutant (CL48) was selected on the basis of a color change from green to white. Growth response of CL48 in TNT indicated that this mutant line was hypersensitive to TNT compared with transformation recipient line and wild-type Chlamydomonas. The third part of the project involved using microarray technology to determine the differential gene expression of Chlamydomonas in response to TNT. Approximately 158 responsive genes were differentially expressed. Genes involved in photosynthesis and energy metabolism were up-regulated in the presence of TNT. TNT may cause oxidative stress since many oxidative stress related genes were up-regulated. Among the down-regulated genes, the expression of cell wall-related genes was repressed. Several unidentified genes were also induced or repressed. The overall study promotes future work involving the identification of the genes that are involved in TNT response

Schizophrenia is a TH2 dominant autoimmune disease possibly against acetylcholine receptors of CNS

Schizophrenia is a very common psychiatric disorder. However, its etiology and pathogenesis is still unknown. Current theory saying that neurotransmitter imbalance such as serotonin or dopamine only provides limited effectiveness in schizophrenia treatment by drugs changing serotonin and dopamine concentration. Despite of such treatment, majority of schizophrenia patients still have very poor prognosis. Thus, the neurotransmitter imbalance theory is not correct. Here, I propose that schizophrenia is actually a TH2 dominant autoimmune disorder. The candidate of autoantigen could be acetylcholine receptors of CNS. My theory can explain the positive as well as negative symptoms of schizophrenia. By microarray analysis of PBMCS, one-tenth of the total 519 significantly expressed genes are immune-related genes. Among them, TH2 related genes are significantly up-regulated including IL-4, histidine decarboxylase, aldehyde dehydrogenase, CCR9, IgE Fc receptor, GATA2, serotonin receptor, phospholipase A2, and prostaglandin D2 synthase. Besides, TH1 and TH17 related genes are down-regulated including CXCL5, cathepsin C, and neutrophil related S100 binding proteins. The new theory sheds a light to better control this detrimental illness. Anti-inflammatory agents could be used to manage schizophrenia in the near future

Mechanism (S) of Metal-Induced Apoptosis in Saccharomyces Cerevisiae

Heavy metals, such as copper and cadmium have been linked to a number of cellular dysfunctions in single and multicellular organisms that are associated with apoptosis. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cu and Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in YCA1∆ mutant. Other apoptotic markers, including sub-G1 DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. We also show that low levels of copper can induce a similar apoptotic response in yeast within the first hour of exposure. Such cellular responses were verified by analyzing mitochondrial perturbation, generation of superoxide ions, activation of the yeast caspase1, and the eventual fragmentation of nuclear DNA (through TUNEL). In analyzing the response of yeast to the different metals, we also demonstrated that the metal-induced PCD is instigated through the sequential activity of at least two caspase-like proteins (i.e., Yca1 and Atg4), both of which appear to be in involved in the process of inducing mitochondrial stress. The additional caspase-like activity is shown to be derived from an enzyme involved in the latter stages of autophagy (Atg4), and provides an intriguing association of apoptosis with autophagy. Here we also demonstrate that metals such as copper and cadmium causes oxidative damage to mitochondrial proteins. Such oxidative attack is targeted and we show that oxidation of certain crucial proteins is required for apoptosis upon metal exposure. By showing that such targeted protein oxidation is dependent on YCA1 and ATG, we also confirm the finding that in yeast that have been exposed to a heavy metal, YCA1 and ATG are essential for damaging mitochondria and to initiate apoptosis. These novel findings highlight several new perspectives about the mechanism of metal-dependent apoptosis, while opening up future analyses to the power of the yeast model system

Precious transition metals: the importance of Zn2+, Mn2+ and Cu2+ in the human pathogen Enterococcus faecalis

Dissertation presented to obtain the Ph.D degree in BiologyEnterococcus faecalis is a commensal bacterium able to colonize different sites in the human host, such as the gastrointestinal tract, the genito-urinary tract and the oral cavity. It can also be found in numerous other environments, including soil, sand, water, food products and plants. These bacteria show a dual behavior: they can behave quite harmlessly as commensals, but are able to become opportunistic pathogens and cause serious infections, such as urinary tract infections and endocarditis, in hospital settings. The question as to how these bacteria are able to change from commensalism to pathogenicity has directed many recent studies to focus on the environmental host conditions that may trigger this transition as well as on the underlying molecular mechanisms. Metals are very important elements in the host environment, as they are key components of many proteins and are involved in numerous cell processes in both the host and the invading pathogen. The maintenance of metal homeostasis is fundamental to both to ensure that metabolism and cell functions are functioning properly. Variations in this homeostasis must be tightly regulated. In several Gram positive pathogens, metal homeostasis and regulation has been linked to their pathogenicity. The lack of knowledge on this subject in E. faecalis motivated the work presented in this thesis.(...)Apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de apoio BD nº SFRH/BD/30362/200

An Analysis of Global Gene Expression Resulting from Exposure to Energetic Materials

AN ANALYSIS OF GLOBAL GENE EXPRESSION RESULTING FROM EXPOSURE TO ENERGETIC MATERIALS A Dissertation Presented for the Doctor of Philosophy Degree University of Tennessee, Knoxville VERNON LASHAWN MCINTOSH JR. August 2010 Dedication This dissertation is dedicated to my family. My mother and father Debra and Vernon McIntosh instilled in me the respect for academic excellence and the drive maximize my potential. Early on, my younger brother Kyle started showing signs of a shared interest in biology thus my desire to be a positive role model for him kept me motivated. Last but certainly not least, my loving wife and best friend Nichole has been there to offer love and support throughout my entire undergraduate and graduate degrees. It’s difficult to imagine making it this far without her (and that’s not just because she paid the bills). Abstract Characteristic transcriptional biomarkers have been identified for microbial cultures exposed to 2, 4, 6-trinitrotoluene (TNT), 2, 6-dinitrotoluene (DNT), or triacetone-triperoxide (TATP). This study describes the generation of expression profiles for exposure to each compound, the functional significance of each response, and the identification of the characteristic alterations in gene expression associated with exposure to each compound. Expression profiles were generated from a total of three different candidate organisms: Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida. Common to all three organisms, TNT exposure resulted in increased expression of genes involved in toxin resistance and drug efflux systems. The S.cerevisiae and E.coli expression profiles were both characterized by increased expression of genes involved in iron-sulfur cluster assembly, sulfur containing amino acids, sulfate transport and assimilation and the metabolism of nitrogen compounds. Only E.coli and Saccharomyces were used to generate DNT induced expression profiles; both profiles exhibited high degrees of similarity with each organism’s respective TNT profiles. This was especially true of the E.coli profile where 25 of the 30 alterations were also observed after exposure to TNT. A computational discriminant functional analysis was performed to identify characteristic biomarkers for each exposure. For each compound a set of transcriptional biomarkers (10 or less) was developed. An additional set of biomarkers was developed encompassing both TNT and DNT exposure. These sets of genes serve as a transcriptional fingerprint for exposure to each respective compound. The sensitivity and specificity of each transcriptional fingerprint is sufficient to correctly identify exposure to energetic materials against a background of non-energetic compound exposures. This study makes several novel contributions to the greater body of scientific knowledge: • This is the first documented study of the interactions of TATP in any biological system. • This is the first comprehensive gene expression study of the TNT response by P. putida, E.coli or E.coli. • This is the first application of computational class prediction in the development of biomarkers for exposure to energetic material