Gene expression changes in femur bone induced by skeletal muscle unloading
ABSTRACT: Gene expression changes in femur bone induced by acute skeletal muscle unloading, which leads to physiological changes including muscle atrophy, weakness and results in bone remodelling and subsequent loss of bone mass, was investigated by hind-limb suspension (HLS) treatment of Male ICR mice (28–32 g body wt; Harlan, Indianapolis, IN). AgilentTM Whole Mouse Genome Oligo Microarrays were utilised to examine the effects of HLS on mRNA expression profiles of the femur bone in the hindlimbs of freely ambulating control and 24h HLS treated mice. Five independent biological replicates of this experiment were carried out. Five independent biological replicates of this experiment (Control and HLS) were carried out.
Project description:Gene expression changes induced by acute skeletal muscle unloading, which leads to physiological changes including muscle atrophy, fibre-type switching, and loss of ability to transition between lipid and glucose as energy source (metabolic inflexibility), was investigated by hind-limb suspension (HLS) treatment of Male ICR mice (28–32 g body wt; Harlan, Indianapolis, IN). Agilent Whole Mouse Genome Oligo Microarrays were utilised to examine the effects of HLS on mRNA expression profiles of the soleus muscle and the gastrocnemius muscle in the hindlimbs of freely ambulating control and 24h HLS treated mice. Experiment Overall Design: Five independent biological replicates of this experiment (Control and HLS) were carried out.
Project description:Nf-kB activity is associated with the key pathological features of chronic respiratory diseases including epithelial remodelling, excess mucous production, and submucosal gland hyperplasia. However, the role of Nf-kB activity in airway epithelial differentiation remains controversial. In the present study we demonstrate that Nf-kB adaptor protein Myd88 deficiency promotes increased airway submucosal gland abundance and abnormal epithelial differentiation in proximal adult airways. Abnormal airway differentiation was not developmentally determined, became exacerbated following acute lung injury, and did not involve altered epithelial proliferation or apoptosis. Instead, we demonstrate that tracheal Myd88 deficiency promotes upregulation of a unique gene expression profile that includes activation of alternate, Myd88-independent Nf-kB signalling. Finally, we show that these effects are not intrinsically maintained in vitro using an air-liquid interface epithelial culture. This finding indicates that Myd88 deficiency promotes adult airway remodelling by regulating non-epithelial, non-cell autonomous Nf-kB activity. 20 microarray samples of whole trachea RNA in total: 5 samples wildtype control tissue 5 samples Myd88 KO control tissue 5 samples wildtype 3 day polidocanol injury tissue 5 samples Myd88 KO 3 day polidocanol injury tissue
Project description:A major development in obesity research is the recognition that the condition is characterized by chronic mild inflammation. Within adipose tissue, this involves the infiltration of macrophages as well as a direct inflammatory response of adipocytes. This study has used Agilent whole-genome microarrays to examine the effects of macrophage-conditioned medium on the global inflammatory response of human adipocytes. Human adipocytes (SGBS cells), differentiated in culture, were treated with macrophage (U937 cells) conditioned media for 4 or 24 h. Control SGBS cells were treated with unconditioned media (control) or RPMI media alone to control for differences in media used to culture U937 cells. There were 6 replicates per group.
Project description:A major development in pigmentation research was the discovery of the involvement of SLC24A5 in natural variation in skin colour. This study used Agilent whole-genome microarrays to examine the impact of SLC24A5 knockdown on the transcriptome of normal human melanocytes derived from lightly and darkly pigmented donors. 2 melanocyte cell lines were treated for 6h, with 2 separate siRNA duplexes to target SLC24A5. Treatment of the cells with a non-targeting siRNA control duplex, lipofectamine transfection reagent alone and unchallenged cells, cultured alongside the cellular treatments comprised the controls for the experiment. All treatments and controls were conducted in triplicate, in both cell lines.
Project description:A major development in the study of obesity is the recognition that the condition is characterised by chronic mild inflammation. Within adipose tissue, this involves the infiltration of macrophages, as well as the direct inflammatory response of the adipocytes and pre-adipocytes. This study has used Agilent whole-genome microarrays to examine the effects of macrophage-conditioned medium on the global inflammatory response of human pre-adipocytes. Human pre-adipocytes (SGBS cells) were treated with macrophage (U937 cells) conditioned medium for 24 h. Control pre-adipocytes were treated with unconditioned medium (control) or RPMI-1640 media alone to control for differences in media used to culture the U937 cells. There were 5 biological replicates per group.
Project description:A number of chronic, age-related diseases are associated with elevated markers of inflammation such as interleukin-6 (IL-6). In this study, we investigated the hypothesis that sedentary individuals with disparate basal serum IL-6 respond differentially to a structured physical activity programme. Gene expression changes in Peripheral Blood Mononuclear cells (PBMC) induced by physical activity was investigated in sedentary, middle-aged men (mean age 52.6 years and BMI 29.1), with relatively high or low basal serum IL-6 levels (mean of 2.13 and 0.59pg/ml respectively), who undertook a 24-week physical activity programme with blood sampling in the pre-exercise period and at the end of 24-weeks prescribed physical activity. AgilentTM Whole Human Genome Oligo Microarrays were utilised to examine the effects of physical activity on mRNA expression profiles of the Peripheral Blood Mononuclear cells (PBMC) at 2 time points (pre-exercise, and after 24 weeks physical activity. There were 6 individuals per group.
Project description:Obesity is characterised by expansion of white adipose tissue, accompanied by an inflamatory response. It has been proposed that the inflammatory state observed may be linked to relative hypoxia in clusters of adipocytes distant from vasculature. Adipose tissue hypoxia has been observed in both animal models and human studies in obesity, and has been linked to insulin resistance. This study has used Agilent whole-genome microarrays to examine the effects of acute hypoxia on the global gene expression in human adipocytes. Human adipocytes (Zen-bio cells) were incubated in hypoxic conditions (1% O2) for 24 h. Control human adipocytes were incubated under normoxic conditions (21% O2). Eight biological replicates were prepared for each experimental condition, and RNA was pooled from two biological replicates, giving a total of n=4 array replicates.
Project description:Mutations in genes involved in dNTP metabolism can lead to tissue-specific mitochondrial depletion syndromes (MDS), likely because the expression of key enzymes is reduced to critical levels in post mitotic cells. Our goal was to establish an in vitro skeletal muscle cell model to study the muscle specificity of MDS associated with mitochondrial dNTP pool imbalance. We performed a comprehensive analysis at the mRNA level of enzymes and transporters responsible for dNTP pool imbalance in muscle cells in vitro and in vivo. Agilent Mouse Oligo Arrays 4x44K were utilized to examine expression levels in proliferating and differentiated C2C12 cells as well as in the mouse EDL (fast glycolytic) and soleus (slow oxidative) muscles. The comparison of mRNA expression profiles supports the reliability of our in vitro cell system. Proliferating mouse C2C12 myoblasts were collected at about 50 percent confluence. Myoblasts were then induced to differentiate in vitro into myotubes that were harvested after 96 hours and further purified to reduce the contribution of mononucleated cells present in the culture. Gene expression in C2C12 myoblasts and myotubes was compared with fully differentiated muscle fibers in vivo. To this aim, the extensor digitorum longus (EDL) and soleus hind limb muscles were isolated from adult CD1 mice. These muscles were selected because they have different metabolic (glycolytic vs. oxidative) and twitching (fast vs. slow) properties. Triplicate total RNA samples were submitted to gene expression profiling.
Project description:Gene expression changes in Peripheral Blood Mononuclear cells (PBMC) induced by physical activity was investigated in sedentary middle-aged men (mean age 52.6 years and BMI 29.1) who undertook a 24-week physical activity programme with blood sampling in the pre-exercise period , at the end of 24-weeks prescribed physical activity , and following a two-week detraining period. AgilentTM Whole Human Genome Oligo Microarrays were utilised to examine the effects of physical activity on mRNA expression profiles of the Peripheral Blood Mononuclear cells (PBMC) at 3 time points (pre-exercise, after 24 weeks physical activity, and at 26 weeks after 2 weeks detraining. There were 12 participants in this programme.
Project description:To provide insights into the role of Ankrd2 in the pathways controlling myogenic differentiation, the gene networks that are disturbed in Ankrd2 knockout or overexpressing primary cells were explored during differentiation. Ankrd2 plays an important role in myogenic differentiation by modulating the activity of its interacting partners (i.e p53). Since Ankrd2 binds to numerous transcription factors many signaling pathways remained to be explored to determine the functional significance of crosstalk between Ankrd2 and the activation of transcriptional programmes that regulate muscle remodeling. In contrast to previous gene expression studies in which we and others have studied the effect of Ankrd2 silencing in mouse differentiating C2C12 cells (Series GSE7542) and human differentiated CHQ5B muscle cells (Belgrano et al., 2011), we here used an in vivo experimental model in which the expression of Ankrd2 was completely abrogated. Furthermore, since Ankrd2 is known to be strongly upregulated in response to various stress stimuli, we studied the effect of Ankrd2 rescue and acute overexpression in KO and WT cells by adenoviral infection 20 hours prior to collection of cells. The pathways that change significantly are involved in distinct inflammatory response phases having NF-kB as a central node. Based on these results Ankrd2 is emerging for the first time as a repressor of immune and inflammatory responses. Gene expression profiling experiments have been performed using the SurePrint G3 Mouse 8x60K array by Agilent with 8 subarrays, each containing 39,430 60mer oligonucleotide probes for Entrez Gene RNAs, 16,251 for lincRNAs, 96 x 10 control probes, and 32 x 10 spike-in control probes. The Agilent platforms with one-color approach were selected to make possible the all-to all experiments comparison. The animal model, created and characterized in the laboratory of Dr.ML Bang, directed at knocking out Ankrd2 function was used. The adenovirus expressing Ankrd2-HA was produced using the AdEasy strategy (Agilent Technologies). Primary muscle cultures derived from wild type (WT) and Ankrd2-knockout (KO) mice infected with adenovirus expressing Ankrd2 or control GFP were collected at three different stages: proliferating, fusing and differentiated myoblasts. Three biological replicates per condition were included and a total of 36 microarray experiments were thus performed. First, the over-expression of Ankrd2 was checked by Western blot analysis. Then RNA was extracted from each sample and the relative transcriptional profiles were carried out using updated microarrays from Agilent technologies. Information from probe features was extracted from microarray scan data with the Feature Extraction Software (Agilent). Only arrays with at least 8/9 quality control metrics in range were used for the following data analyses. Intra-array normalization was performed with the Feature Extraction Software. Quantile inter-arrays normalization was performed using the Expander software (Sharan et al., 2003).