Project description:Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Experiment Overall Design: Exposure of mouse ears to sulfur mustard alone, sulfur mustard preceded by drug treatment, or vehicle compounds. Naive controls were also included. Biological replicates of at least n=3 were examined for each exposure condition.
Project description:Sulfur mustard is a vesicant chemical warfare agent, which has been used during Iraq-Iran-war. Many veterans and civilians still suffer from long-term complications of sulfur mustard exposure, especially in their lung. Although the lung lesions of these patients are similar to Chronic Obstructive Pulmonary Disease (COPD), there are some differences due to different etiology and clinical care. Less is known on the molecular mechanism of sulfur mustard patients and specific treatment options. microRNAs are master regulators of many biological pathways and proofed to be stable surrogate markers in body fluids. Based on that microRNA expression for serum samples of sulfur mustard patients were examined, to establish specific microRNA patterns as a basis for diagnostic use and insight into affected molecular pathways. Patients were categorized based on their long-term complications into three groups and microRNA serum levels were measured. The differentially regulated microRNAs and their corresponding gene targets were identified. Cell cycle arrest, ageing and TGF-beta signaling pathways showed up to be the most deregulated pathways. The candidate microRNA miR-143-3p could be validated on all individual patients. In a ROC analysis miR-143-3p turned out to be a suitable diagnostic biomarker in the mild and severe categories of patients. Further microRNAs which might own a link to the biology of the sulfur mustard patients are miR-365a-3p, miR-200a-3p, miR-663a. miR-148a-3p, which showed up only in a validation study, might be linked to the airway complications of the sulfur mustard patients. All the other candidate microRNAs do not directly link to COPD phenotype or lung complications. In summary the microRNA screening study characterizes several molecular difference in-between the clinical categories of the sulfur mustard exposure groups and established some useful microRNA biomarkers.
Project description:Inoculation of endophyte-free (E-) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant foliar fungal endophyte in healthy T. cacao, induced significant changes in the expression of hundreds of host genes. Further, E+ leaves exhibit enhanced pathogen resistance, increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes that all correspond to the changes in expression of specific functional genes in related pathways. Moreover, a cacao gene highly up-regulated in E+ leaves increases pathogen resistance apart from any direct endophyte effects. Thus, benefits of increased pathogen resistance in E+ plants are partially due to enhanced induction of intrinsic host defense pathways, and potential costs include reduced photosynthetic capacity and endophyte metabolism of host tissues. Similar effects are likely to be properties of most plant-endophyte interactions, suggesting general relevance to the design and interpretation of studies of genetic and phenotypic expression in plants. The objective of this experiment was to identify Theobroma cacao genes that are differentially expressed between leaves inoculated with fungal endophyte Colletotrichum tropicale (E+ leaves) and control un-inoculated leaves (E- leaves) 3 days post endophyte inoculation. The experiment was conducted in a Percival growth chamber (model I35LL, 115 volts, 1/4 Hp, series: 8503122.16, Percival Scientific, Inc., Perry IA) with 12/12 h light/dark photoperiod and temperatures of 30M-BM-:C and 26M-BM-:C respectively. Inoculation was done by aspersion of endophyte spores (2X10^6 spore/ml) to a group of T. cacao seedlings and a second group of seedlings were maintained as control un-inoculated (E- leaves). Then three biological replicates (each one consisting of one leaf from different plants) per treatment E+ and four leaves per treatment E- leaves) were collected and processed for a two color oligo microarray analysis.
Project description:Inoculation of endophyte-free (E-) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant foliar fungal endophyte in healthy T. cacao, induced significant changes in the expression of hundreds of host genes. Further, E+ leaves exhibit enhanced pathogen resistance, increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes that all correspond to the changes in expression of specific functional genes in related pathways. Moreover, a cacao gene highly up-regulated in E+ leaves increases pathogen resistance apart from any direct endophyte effects. Thus, benefits of increased pathogen resistance in E+ plants are partially due to enhanced induction of intrinsic host defense pathways, and potential costs include reduced photosynthetic capacity and endophyte metabolism of host tissues. Similar effects are likely to be properties of most plant-endophyte interactions, suggesting general relevance to the design and interpretation of studies of genetic and phenotypic expression in plants.