Published molecular profiling studies in patients with lymphoma suggested the influence of hypoxia inducible factor-1 alpha (HIF1) targets in prognosis of DLBCL. Yet, the role of hypoxia in hematological malignancies remains unclear. We observed that activation of HIF1 resulted in global translation repression during hypoxic stress in DLBCL. Protein translation efficiency as measured using 35S-labeled methionine incorporation revealed a 50% reduction in translation upon activation of HIF1. Importantly, translation was not completely inhibited and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with a decrease in mitochondrial function. Screening of primary DLBCL patient samples revealed that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies demonstrated that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide strong support for the direct contribution of HK2 in B-cell lymphoma development and suggest that HK2 is a key metabolic driver of the DLBCL phenotype.ne incorporation revealed a 50% reduction in translation upon activation of HIF1. Importantly, translation was not completely blunted and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with decrease in mitochondrial function. Screening of DLBCL patient samples identified that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies show that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide more definitive proof of direct contribution of HK2 in development of B-cell lymphoma and suggest that HK2 is a key metabolic driver of DLBCL phenotype.
Role of hypoxia in Diffuse Large B-cell Lymphoma: Metabolic repression and selective translation of HK2 facilitates development of DLBCL.
Cell line, Treatment
View SamplesBiofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions.
Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.
Specimen part, Disease, Time
View SamplesBiofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions.
Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.
Specimen part, Time
View SamplesNumerous studies have established a critical role for BMP signaling in skeletal development. In the developing axial skeleton, sequential SHH and BMP signals are required for specification of a chondrogenic fate in somitic tissue. A similar paradigm is thought to operate in the limb, but the signals involved are unclear. To investigate the nature of these signals we examined BMP action in mesenchymal populations derived from the early murine limb bud (~ E10.5). These populations exhibited a graded response to BMPs, in which early limb mesenchymal (EL) cells (from the distal hind limb) displayed an anti-chondrogenic response, whereas BMPs promoted chondrogenesis in older cell populations. To better understand the molecular basis of disparate BMP action in these various populations, gene expression profiling with Affymetrix microarrays was employed to identify BMP-regulated genes. These analyses showed that BMPs induced a distinct gene expression pattern in the EL cultures versus later mesenchymal limb populations (IM and LT).
Regulation of BMP-dependent chondrogenesis in early limb mesenchyme by TGFbeta signals.
Specimen part
View SamplesInnate immune pattern recognition receptors play critical roles in pathogen detection and initiation of antimicrobial responses. We and others have previously demonstrated the importance of the beta-glucan receptor Dectin-1 in the recognition of pathogenic fungi by macrophages and dendritic cells, and have elucidated some of the mechanisms by which Dectin-1 signals to coordinate the antifungal response. While Dectin-1 signals alone are sufficient to trigger phagocytosis and Src-Syk-mediated induction of antimicrobial reactive oxygen species, collaboration with Toll-like receptor (TLR)2 signaling enhances NF-kB activation and regulates cytokine production. In this study we demonstrate that Dectin-1 signaling can also directly modulate gene expression via activation of nuclear transcription of activated T cells (NFAT) transcription factors. Dectin-1 ligation by zymosan particles or live Candida albicans yeast triggers NFAT activation in macrophages and dendritic cells. Dectin-1-triggered NFAT activation plays a role in the induction of Egr2 and Egr3 transcription factors, and cyclooxygenase 2 (Cox-2). Furthermore, we show that NFAT activation regulates IL-2, IL-10 and IL-12 p70 production by zymosan-stimulated dendritic cells. These data establish NFAT activation in myeloid cells as a novel mechanism of regulation of the innate antimicrobial response.
Dectin-1 stimulation by Candida albicans yeast or zymosan triggers NFAT activation in macrophages and dendritic cells.
No sample metadata fields
View SamplesPrimary micromass cultures derived from 11.5 day old mouse embryo limb buds were cultured for 15 days in differentiating conditions (beta-glycerophosphate and ascorbic acid). Total RNA from differentiating chondrocytes was isolated every three days i.e. days 3,6,9,12 and 15 and hybridized to MOE430A chips. Objective: Gain a view of the temporal gene expression changes occuring during chondrocyte differentiation.
Microarray analyses of gene expression during chondrocyte differentiation identifies novel regulators of hypertrophy.
No sample metadata fields
View SamplesBackground: Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated.
Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development.
No sample metadata fields
View SamplesCell body and pseudopod RNA are differentially regulated during the migration of the metastatic cancer cells.We wanted to identify the RNA which are upregulated in the pseudopodial (PS) fraction as compared to cell body fraction (CB).
Pseudopodial actin dynamics control epithelial-mesenchymal transition in metastatic cancer cells.
Cell line
View SamplesA variety of cell cultures models and in vivo approaches have been used to study gene expression during chondrocyte differentiation. The extent to which the in vitro models reflect bona fide gene regulation in the growth plate has not been quantified. In addition, studies that evaluate global gene expression changes among different growth plate zones are limited. To address these issues, we completed a microarray screen of three growth plate zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective growth plate zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs confirmed documented data and pointed to novel aspects of chondrocyte differentiation. Parallel comparisons of the microdissected tibiae data set to our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the growth plate. These studies will allow us to better understand zone-specific gene expression patterns in the growth plate. Ultimately, this work will help define both the genomic context in which genes are expressed in the long bones and the extent to which the micromass culture system is able to recapitulate chondrocyte development in endochondral ossification.
Genome-wide analyses of gene expression during mouse endochondral ossification.
No sample metadata fields
View SamplesMicroarray gene expression experiments to identify differentially expressed genes and pathways in Jag1 conditional/null livers reveal up-regulation of many genes related to fibrosis and ECM interactions.
Microarray data reveal relationship between Jag1 and Ddr1 in mouse liver.
Age, Specimen part
View Samples