Effect of high or low chronic shear stress on microvascular endothelial cells cultured on native- versus AGE-fibronectin
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ABSTRACT: Pulmonary microvascular endothelial cells were cultured on the inner surface of fibers (each 700 µm inner diameter) coated with either with native- or advanced glycation endproduct (AGE) modified fibronectin in hollow-fiber cartridges (FiberCell Systems, Inc.). Duration of shear stress application was 10 days. Shear stress was gradually increased to 17 dyne/cm2 in 2 samples (GSM32266: native fibronectin high shear stress or GSM45608: AGE-fibronectin) or was kept at constant low level of 1 dyne/cm2 in 1 sample (GSM41248: native fibronectin low shear stress). Keywords: parallel sample
Project description:Pulmonary microvascular endothelial cells were cultured on the inner surface of fibers (each 700 µm inner diameter) coated with either with native- or advanced glycation endproduct (AGE) modified fibronectin in hollow-fiber cartridges (FiberCell Systems, Inc.). Duration of shear stress application was 10 days. Shear stress was gradually increased to 17 dyne/cm2 in 2 samples (GSM32266: native fibronectin high shear stress or GSM45608: AGE-fibronectin) or was kept at constant low level of 1 dyne/cm2 in 1 sample (GSM41248: native fibronectin low shear stress). Keywords: parallel sample
Project description:Chinook salmon (Oncorhynchus tshawytscha) display the greatest variability of return times to freshwater of all Pacific salmon. Populations return to freshwater for spawning at many different times of year, resulting in segregated populations that may use differing molecular pathways for these large behavioral and physiological differences. Using a population of Chinook from California’s Central Valley, we sought to generate novel expressed sequences using Long Serial Analysis of Gene Expression (LongSAGE). We constructed three LongSAGE libraries from brains of samples caught in the spring and fall in freshwater and from the ocean. Using cDNA libraries from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), we were able to assign 59% of putatively differentially expressed tags to genes. Additionally, we tested the expression levels of seven genes, indicated by LongSAGE to be putatively differentially expressed between the fall and spring, and found none significantly differentially expressed. This study is the first to apply LongSAGE to salmon and provides a framework for conducting future research on gene expression differences between Chinook salmon of different populations, as well as underlying mechanisms of differing physiology and behavior. Keywords: seasonal difference Single individuals were used to construct each LongSAGE library. The fall, spring and ocean samples were then compared between each other and examined for differences in the number of tags observed.
Project description:Tissue from macroscopically healthy parts of nephrectomized kidneys. Endothelial cells were purified from the cell outgrowth of glomeruli twice using CD31 magnetic beads and cultured on fibronectin coated surface. Cells were not passaged more than eight times and stained with vWf, CD31, and after stimulation with TNF alpha also with CD62E/P. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.
Project description:Dark-grown maize seedlings with primary roots 12-20 mm in length were transplanted to high (-0.03 MPa) water potential vermiculite. About 1,000 roots were collected. Each root was divided into 4 segments (distances are from the junction of the root apex and root cap): segment 1, 0-3 mm plus the root cap; segment 2, 3-7 mm; segment 3, 7-12 mm; segment 4, 12-20 mm. Details of the conditions and nutrient modifications have been described before (Sharp et al., 1988; Spollen et al, 2000). From each segment, 250 mg of material was used and RNA was isolated form the combined sample containing all four segments. RNA was isolated using the RNeasy Maxi Kit (Qiagen). RNA integrity was verified by denaturing agarose gel electorphoresis and spectrophotometry. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.
Project description:SAGE analysis of fully grown ovarian follicles has been carried out to supply a resource for identification of important molecules involved in vertebrate oogenesis and in early stages of embryonic development. Keywords: SAGE About 250 full-grown ovarian follicles were obtained after gentle squeezing the abdomen of five mature zebrafish females.
Project description:Human cytomegalovirus (HCMV) has been shown to have the potential to alter cellular gene expression early after infection. However, one-gene approaches and the use of closed system gene expression technologies have identified only few cellular genes whose activity changed immediate-early. We therefore used serial analysis of gene expression (SAGE) to investigate the transcriptional program of human fibroblasts in response to HCMV in the immediate-early phase of infection. Differential expression of various cellular genes was monitored. Transcriptional expression changes of genes coding for ribosomal proteins reflected a general cellular response to starvation and stress. But differential regulation of genes coding for transcription factors and proteins associated with cellular metabolism, homeostasis and cell structure may represent transcriptional alterations in response to HCMV infection. Expression kinetics by 5' nuclease fluorigenic real-time PCR of selected genes revealed partial protection of infected cells against initial stress-associated alterations of gene expression and indicated fluctuations of transcriptional levels over time. Additionally, agreement with the quantitative results obtained by SAGE was observed only for genes up-regulated in HCMV-infected cells. This finding pointed to various technical and statistical parameters that all may be critical for quantitative transcriptome studies using global approaches, especially when exploring biological systems in a critical phase of cellular physiology. Keywords: other
Project description:Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.
Project description:Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem structure and function in the ocean. Diatoms are an abundant and widespread functional group of phytoplankton that are responsible for significant amounts of primary production in the ocean, however there has not been a comprehensive study of diatom physiological responses to P deficiency. Here, we coupled deep sequencing of transcript tags and quantitative proteomic analysis from the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. The reads (tags) were mapped to the T. pseudonana genome sequence, confirming expression of 91% of the modeled gene set. A total of 318 genes were differentially regulated with a false discovery rate of p<0.05. A total of 1264 proteins were detected, and of those 136 were differentially expressed with a false discovery rate of p<0.05. Significant changes in the abundance of transcripts and proteins were observed and these changes were coordinated for glycolysis, translation, and multiple biochemical responses to P deficiency. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P (DOP) through increased production of alkaline phosphatase metalloenzymes and a diesterase, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to rapidly respond to variations in environmental P availability. T. pseudonana (Strain 1335 from the Provosoli-Guillard National Center for the Culture of Marine Phytoplankton (CCMP)) was grown in a modified f/2 medium made from Sargasso Sea water. Macronutrients and vitamin B12, biotin, and thiamine solutions were treated with prepared Chelex-100 resin to remove trace metal contaminants followed by trace-metal clean syringe sterilization to yield final nutrient concentrations of 882 μM NaNO3 and 106 μM Na2SiO3, and vitamin concentrations of 75 pM, 400 pM, and 60 nM, respectively. The Fe concentration was also modified from f/2 to 400 nM. All conditions were run in triplicate at 14 ºC, in constant light (120 µmol photons m-2 s-1). Cells were grown with f/2 phosphorus concentrations (P-replete; 36 µM PO4) and with low phosphorus concentrations (P-deficient; 0.4 µM PO4). Growth was monitored daily with cell counts. Duplicate P-replete treatments were pooled and harvested in mid log phase, and triplicate P-deficient treatments were pooled, and also quickly harvested onto 2 µm filters at the onset of P depletion. All RNA samples were snap frozen in liquid nitrogen. Replicate P-deficient cultures were refed to 36 µM phosphate at the onset of P depletion, and subsequently resumed growth. Additional growth studies were performed as described above substituting glycerolphosphate and adenosine monophosphate at 36 µM, for the phosphate in the medium.
Project description:Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Keywords: SAGE; time course; stress response; cold acclimation; freezing tolerance SAGE libraries were generated from A. thaliana leaf tissue collected after exposure to low temperature (4°C) for 0 minutes, 30 minutes, 2 hours, 2 days and one week, the tags were matched to the Arabidopsis genome and statistical analysis was performed to reveal differential gene expression.