Project description:A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.
Project description:Leishmania infantum (Kinetoplastida:Trypanosomatidae) is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The motile promastigote stage infects the hematophagous sand fly vector host and amastigotes survives and multiplies within phagocytes of the mammalian host. Promastigotes are routinely cultured in liquid undefined media and are considered to mimic the environment within the sand fly gut. We have put this to the test by high-throughput gene expression profiling by shotgun DNA microarrays generated in our laboratory. This has been possible thanks to RNA amplification.
Project description:Spingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. The objectives of this study were to characterize how strain RW1 responses to changes in different components of the total water potential (solute and matric potential) and to then connect these responses to more realistic scenarios of soil desiccation. To accomplish this task, transcriptome profiling was used to investigate the effects of decreasing the solute potential with sodium chloride (solute stress), decreasing the matric potential with high-molecular weight polyethylene glycol (matric stress), or inoculating cells directly into unsaturated sand (sand desiccation stress). Transcriptome profiling revealed a general response to solute, matric, and sand desiccation stress that involved synthesizing trehalose and modifying the composition of exopolysaccarides. Transcriptome profiling also revealed responses that were unique to each stress. Only solute and matric stress triggered the down-regulation of flagella genes. Only solute and sand desiccation stress triggered the up-regulation of two RNA polymerase ECF-type sigma factors along with several membrane proteins, mechanosensitive channels, and solute transporters. Finally, only matric stress triggered the up-regulation of the RNA polymerase sigma-32 factor along with several molecular chaperones. Together, this study revealed a general response to solute, matric and sand desiccation stress but also unique responses to only a subset of these stresses, suggesting that each stress affects strain RW1 in a fundamentally different way.
Project description:Spingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. The objectives of this study were to characterize how strain RW1 responses to changes in different components of the total water potential (solute and matric potential) and to then connect these responses to more realistic scenarios of soil desiccation. To accomplish this task, transcriptome profiling was used to investigate the effects of decreasing the solute potential with sodium chloride (solute stress), decreasing the matric potential with high-molecular weight polyethylene glycol (matric stress), or inoculating cells directly into unsaturated sand (sand desiccation stress). Transcriptome profiling revealed a general response to solute, matric, and sand desiccation stress that involved synthesizing trehalose and modifying the composition of exopolysaccarides. Transcriptome profiling also revealed responses that were unique to each stress. Only solute and matric stress triggered the down-regulation of flagella genes. Only solute and sand desiccation stress triggered the up-regulation of two RNA polymerase ECF-type sigma factors along with several membrane proteins, mechanosensitive channels, and solute transporters. Finally, only matric stress triggered the up-regulation of the RNA polymerase sigma-32 factor along with several molecular chaperones. Together, this study revealed a general response to solute, matric and sand desiccation stress but also unique responses to only a subset of these stresses, suggesting that each stress affects strain RW1 in a fundamentally different way. Comparative transcriptome profiling was performed to assess the effects of acute (30 min) solute and matric stress (3 samples for acute solute stress, 3 samples for acute matric stress, 3 controls), the effects of chronic (24 hours) solute and matric stress (3 samples for chronic solute stress, 3 samples for chronic matric stress, 3 controls), and the effects of sand desiccation stress (4 samples for sand desiccation treatment, 3 controls).
Project description:We sought to evaluate the brain gene expression profiles of male courtship display. To assess male display and courtship behavior, we designed a courtship preference assay. We evaluated social interactions between males and females using a 40 gallon tank design with a ‘rock’ habitat at one end and ‘sand’ at the other, separated by glass bottom. When parental rock species (Petrotilapia nigra (TaxId 526958), Maylandia zebra (TaxId 106582), Labeotropheus feulliborni) are placed in this tank paradigm, males court females over the rocks. Males of sand species (Mchenga conophorus, Aulonocara baenschi (TaxId 143496), Tramitichromis intermedius (TaxId 323801)) court females over sand and construct species appropriate bowers. When single rock x sand F1 males were placed in this set up with F1 females, males invariably courted females over the ‘rock’ habitat, suggesting genetic dominance. When two rock x sand F1 males were allowed to compete for F1 females in this tank paradigm, something interesting happened. One male, typically the larger, courted females over the rock habitat, and the other simultaneously constructed bowers to court females in the sand. We detected no difference in GSI (gonadal somatic index) between F1 males behaving as ‘socially rock’ vs. ‘socially sand.’ This observation of divergent behavior among interacting F1 brothers suggests an interaction between the genome and the social environment in these males.
Project description:Synergism of Iraqi Sand/Cigarette Smoke Co-Exposure in Rats. 24 samples are used. A total of 102 rats will be separated into six exposure groups: each group consisting of 17 male CD Sprague-Dawley rats, 10 to 15 weeks old. The six exposure groups were each exposed via two routes: 1. nose-only inhalation exposures --- air or cigarette smoke; 2. whole-body inhalation exposures -- air or manufactured silica sand or Iraqi sand. The nose only exposures were conducted 3 hours per day, 5 days per week for 6 weeks. During the last two weeks of the nose - only exposures, whole-body exposure were also conducted 18-19 hours per day, 7 days per week.