A role for Stat4 in IL-12-induced up-regulation of selectin ligands on Th1 cells was explored. Th1 cells generated from Stat4−/− mice exhibited no IL-12-inducible P-selectin ligands, no up-regulation of core 2 β1,6-glucosaminyltransferase I (C2GlcNAcT-I), and low levels of the Th1 transcription factor T-bet. In contrast, Stat4−/− Th1 cells exhibited only a partial defect in expression of IL-12-inducible E-selectin ligands and expressed equivalently high levels of α1,3-fucosyltransferase VII (FucT-VII) as wild-type Th1 cells. FucT-VII expression was induced by T cell activation, and was enhanced by IL-12 independently of Stat4, whereas C2GlcNAcT-I up-regulation was mediated exclusively by IL-12, acting through Stat4. These data show that FucT-VII and C2GlcNAcT-I are controlled through distinct pathways and imply the existence of at least one other IL-12-inducible glycosyltransferase required for E-selectin and possibly P-selectin ligand formation in Th1 cells.

AbstractEpigenetic modification, specifically DNA methylation, is one possible mechanism for intergenerational plasticity. Before inheritance of methylation patterns can be characterized, we need a better understanding of how environmental change modifies the parental epigenome. To examine the influence of experimental ocean acidification on eastern oyster (Crassostrea virginica) gonad tissue, oysters were cultured in the laboratory under control (491 ± 49 μatm) or high (2550 ± 211 μatm) pCO2 conditions for four weeks. DNA from reproductive tissue was isolated from five oysters per treatment, then subjected to bisulfite treatment and DNA sequencing. Irrespective of treatment, DNA methylation was primarily found in gene bodies with approximately 22% of CpGs (2.7% of total cytosines) in the C. virginica genome predicted to be methylated. In response to elevated pCO2, we found 598 differentially methylated loci primarily overlapping with gene bodies. A majority of differentially methylated loci were in exons (61.5%) with less intron overlap (31.9%). While there was no evidence of a significant tendency for the genes with differentially methylated loci to be associated with distinct biological processes, the concentration of these loci in gene bodies, including genes involved in protein ubiquitination and biomineralization suggests DNA methylation may be important for transcriptional control in response to ocean acidification. Changes in gonad methylation also indicate potential for these methylation patterns to be inherited by offspring. Understanding how experimental ocean acidification conditions modify the oyster epigenome, and if these modifications are inherited, allows for a better understanding of how ecosystems will respond to environmental change.

Epigenetic modification, specifically DNA methylation, is one possible mechanism for intergenerational plasticity. Before inheritance of methylation patterns can be characterized, we need a better understanding of how environmental change modifies the parental epigenome. To examine the influence of experimental ocean acidification on eastern oyster (Crassostrea virginica) gonad tissue, oysters were cultured in the laboratory under control (491 ± 49 µatm) or high (2550 ± 211 µatm) pCO 2 conditions for 4 weeks. DNA from reproductive tissue was isolated from five oysters per treatment, then subjected to bisulfite treatment and DNA sequencing. Irrespective of treatment, DNA methylation was primarily found in gene bodies with approximately 22% of CpGs (2.7% of total cytosines) in the C. virginica genome predicted to be methylated. In response to elevated pCO 2 , we found 598 differentially methylated loci primarily overlapping with gene bodies. A majority of differentially methylated loci were in exons (61.5%) with less intron overlap (31.9%). While there was no evidence of a significant tendency for the genes with differentially methylated loci to be associated with distinct biological processes, the concentration of these loci in gene bodies, including genes involved in protein ubiquitination and biomineralization, suggests DNA methylation may be important for transcriptional control in response to ocean acidification. Changes in gonad methylation also indicate potential for these methylation patterns to be inherited by offspring. Understanding how experimental ocean acidification conditions modify the oyster epigenome, and if these modifications are inherited, allows for a better understanding of how ecosystems will respond to environmental change.

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments.