Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Glucocorticoid signaling in pancreatic islets modulates gene regulatory programs and genetic risk of type 2 diabetes

Glucocorticoids are key regulators of glucose homeostasis and pancreatic islet function, but the gene regulatory programs driving responses to glucocorticoid signaling in islets and the contribution of these programs to diabetes risk are unknown. In this study we used ATAC-seq and RNA-seq to map chromatin accessibility and gene expression from eleven primary human islet samples cultured in vitro with the glucocorticoid dexamethasone at multiple doses and durations. We identified thousands of accessible chromatin sites and genes with significant changes in activity in response to glucocorticoids. Chromatin sites up-regulated in glucocorticoid signaling were prominently enriched for glucocorticoid receptor binding sites and up-regulated genes were enriched for ion transport and lipid metabolism, whereas down-regulated chromatin sites and genes were enriched for inflammatory, stress response and proliferative processes. Genetic variants associated with glucose levels and T2D risk were enriched in glucocorticoid-responsive chromatin sites, including fine-mapped variants at 51 known signals. Among fine-mapped variants in glucocorticoid-responsive chromatin, a likely casual variant at the 2p21 locus had glucocorticoid-dependent allelic effects on beta cell enhancer activity and affected SIX2 and SIX3 expression. Our results provide a comprehensive map of islet regulatory programs in response to glucocorticoids through which we uncover a role for islet glucocorticoid signaling in mediating genetic risk of T2D.     

Serial Expression Analysis of Liver Regeneration-Related Genes in Rat Regenerating Liver

We investigated the gene expression changes in rat hepatic restoration with Rat Genome 230 2.0 chip containing 11,789 known genes and 13,231 unknown genes (taking up 90 percent of rat whole genome) following a 2/3 hepatectomy. The expression profiles and roles of these genes in rat liver regeneration (LR) were assayed using bioinformatics and systems biology method. Among the above genes, 1,004 known genes and 857 unknown genes were found to be associated with rat LR. The numbers of the known genes up-regulated, down-regulated, and up/down-regulated were 622, 443, and 15, respectively; that of the unknown genes were 367, 400, and 14, respectively. Out of the above two groups of genes, the ones up- and down-regulated 20 times or more were 62 and 38, 8, and 14, respectively. Notably, The highest expression level of dehydrogenase/reductase member 7 (DHRS7) was more than 968-fold compared to control, and alpha-1-B glycoprotein (A1BG), the product of gene with the lowest expression abundance, was 58 times lower than control. During rat liver regeneration, 467 up–regulated, 282 down–regulated, 10 up/down-regulated genes, and 1,031 undetected genes in our study interacted with each other and formed a network with a total of 4,014 connectivities. Among them, the genes for the regulation, synthesis, transport, signal transduction, protein modification, and physiological response formed 630, 290, 691, 373, 2010, and 20 connectivities, respectively; and the genes jun, fos, myc, ptgs2, ccna2, ccl2 had relatively higher degree of connectivity. The results indicated that cell apoptosis and inflammatory response were enhanced in the initial phase and the early part of progressive phase in LR.     

Molecular characterisation of TNF,AIF, dermatopontin and VAMP genes of the flat oyster Ostrea edulis and analysis of their modulation by diseases

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes.