Project description:Background & aimsGlutamine plays a protective role in intestinal cells during physiologic stress; however, the protection mechanisms are not fully understood. Autophagy functions in bulk degradation of cellular components, but has been recognized recently as an important mechanism for cell survival under conditions of stress. We therefore sought to see if glutamine's actions involve the induction of autophagy in intestinal cells and, if so, the mechanisms that underlie this action.MethodsFormation of microtubule-associated protein light chain 3 (LC3)-phospholipid conjugates (LC3-II) in rat intestinal epithelial IEC-18 cells and human colonic epithelial Caco-2(BBE) cells was determined by Western blotting and localized by confocal microscopy. Activation of mammalian target of rapamycin (mTOR) pathway, mitogen-activated protein (MAP) kinases, caspase-3, and poly (ADP-ribose) polymerase were monitored by Western blotting.ResultsGlutamine increased LC3-II as well as the number of autophagosomes. Glutamine-induced LC3-II formation was paralleled by inactivation of mTOR and p38 MAP kinase pathways, and inhibition of mTOR and p38 MAP kinase allowed LC3-II induction in glutamine-deprived cells. Under glutamine starvation, LC3-II recovery after heat stress or the increase under oxidative stress was blunted significantly. Glutamine depletion increased caspase-3 and poly (ADP-ribose) polymerase activity after heat stress, which was inhibited by treatment with inhibitors of mTOR and p38 MAP kinase.ConclusionsGlutamine induces autophagy under basal and stressed conditions, and prevents apoptosis under heat stress through its regulation of the mTOR and p38 MAP kinase pathways. We propose that glutamine contributes to cell survival during physiologic stress by induction of autophagy.
Project description:Inflammatory conditions as they occur during periodontal disease often result in decreased alveolar bone levels and a loss of connective tissue homeostasis. Here we have focused on the effect of microRNA-138 (miR-138) as a potential regulator of periodontal stem cells as they affect homeostasis during inflammatory conditions. Our data indicate that miR-138 was significantly upregulated in our periodontal disease animal model. Interaction of miR-138 with a predicted targeting site on the osteocalcin (OC) promoter resulted in a 3.7-fold reduction of luciferase activity in promoter assays compared with controls; and miR-138 overexpression in periodontal progenitors significantly inhibited OC (3.4-fold), Runx2 (2.8-fold), and collagen I (2.6-fold). Moreover, treatment with inflammatory modulators such as interleukin (IL)-6 and lipopolysaccharide (LPS) resulted in a significant 2.2-fold (IL-6) or 1.9-fold (LPS) increase in miR-138 expression, while OC and Runx2 expression was significantly decreased as a result of treatment with each inflammatory mediator. Further defining the role of miR-138 in the OC-mediated control of mineralization, we demonstrated that the LPS-induced downregulation of OC expression was partially reversed after miR-138 knockdown. LPS, miR-138 mimic, and OC small interfering RNA inhibited osteoblast differentiation marker alkaline phosphatase activity, while miR-138 inhibitor and OC protein addition enhanced alkaline phosphatase activity. Supporting the role of OC as an essential modulator of osteoblast differentiation, knockdown of miR-138 or addition of OC protein partially rescued alkaline phosphatase activity in periodontal ligament (PDL) cells subjected to LPS treatment. Our data establish miR-138 inhibitor as a potential therapeutic agent for the prevention of the bone loss associated with advanced periodontal disease.
Project description:To better understand the effect of hypoxia, RNA-Seq technology was used to profile the Aspergillus fumigatus during adaptation to hypoxia at 12, 24 and 36 h time points. Four samples examined: from the fungus grown under normoxia and hypoxia conditions
Project description:To realistically evaluate the effects of the environment in space, it is necessary to understand the effects of external factors during sample transport from Earth to space. The present study focused on temperature, profiling the altered gene expression that develops under low cultivation temperatures in C. elegans, used as a space life science model. The 7903 genes were selected as differentially expressed genes, and divided into five sets with similar expression patterns using k-means clustering. Results from Gene Ontology analysis are significantly indicated that the cell cycle related genes, and the TGFβ/insulin-like signal pathway related genes changed. The TGFβ/insulin-like signal pathway is expected to be activated due to low temperatures, as well as by other stressors. To determine the genes whose expression changed four thermal conditions (10, 15, 20, and 25 °C), DNA microarray analysis was performed. The data consisted of 12 samples, consisting of three biological replicates at each temperature.
Project description:To understand the biological variability that exists between different individuals, the resting (untreated) transcriptome from 15 different individuals was examined. Transcripts expressed in a bimodal manor across the 15 biological replicates were identified for further examination.
Project description:To understand the biological variability that exists between different individuals, the resting (untreated) transcriptome from 15 different individuals was examined. Transcripts expressed in a bimodal manor across the 15 biological replicates were identified for further examination. In the study presented here, HUVEC isolates from 15 different individuals were cultured until passage 4 in fully supplemented growth conditions. RNA was extracted and hybridised onto CodeLink microarrays. The expression profiles in the untreated condition were examined along side a subset of the same biological replicates treated with a cocktail of inflammatory mediators (10ng/ml TNF-α, Il-1β, Il-8) for a period of 24 hours prior to RNA extraction (also submitted).
Project description:To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.