Studies have reported opposing effects of high-fat diet and mechanical stimulation on lineage commitment of the bone marrow stem cells. Yet, how the bone marrow modulates its gene expression in response to the combined effects of mechanical loading and a high-fat diet has not yet been addressed. We investigated whether early-life voluntary physical activity can modulate the effects of a high-fat diet on body composition, bone phenotype and bone marrow gene expression in male Sprague Dawley rats. We show that early-life high-fat diet positively affected body weight, total fat percentage and bone mass indices. In the bone marrow, early-life high-fat diet resulted in adipocyte hypertrophy and a pro-inflammatory and pro-adipogenic gene expression profile. Crucially, the bone marrow of the rats that undertook wheel exercise while on a high-fat diet retained a memory of the early-life exercise. This memory lasted at least 60 days after the cessation of the voluntary exercise and was manifest by: 1) the bone marrow adipocyte size of the exercised rats not exhibiting hypertrophy; and 2) genes associated with mature adipocyte function being down-regulated. Our results are consistent with the marrow adipose tissue having a unique and long-lasting response to high-fat feeding in the presence or absence of exercise. Overall design: Eighty male SD rats were randomised at weaning into : chow-fed group (C-SED) or a high-fat fed group. The high-fat fed group was further divided into three sub-groups: the high-fat sedentary (HF-SED) group, the high-fat late-exercise (HF-LEX) group, and the high-fat early-exercise (HF-EEX) group. At day 120-123, the animals were culled and total RNA was extracted from the bone marrow of the femur. The RNA was sequenced using Illumina Hiseq4000 technology. Differential gene expression analysis was carried out using Tuxedo suite of bioinformatic tools.
A Memory of Early Life Physical Activity Is Retained in Bone Marrow of Male Rats Fed a High-Fat Diet.
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Methods: Triplicate RNA samples from morphologically stage-matched embryos were sequenced to compare expression profiles over time. Strand-specific libraries were prepared using the TruSeq stranded total RNA-ribozero kit (Illumina) and 100-bp paired-end sequencing was performed to depth of 10 million reads per library on an Illumina HiSeq 2000. Methods: On average, 19 million 100 bp paired-end reads per library were generated. These were then adapter and quality trimmed using cutadapt and SolexaQA. Each sequencing data set was independently mapped to the zebrafish genome with a bowtie2 index generated from Danio_rerio.Zv9.70 (Ensembl) downloaded from Illumina's iGenomes collection. Zebrafish genome danRer7was used to provide known transcript annotations from Ensembl using TopHat2 (version 2.0.9) with the following options: “tophat2 --GTF genes.gtf --library-type fr-firststrand -p 24 --mate-inner-dist -8 --mate-std-dev 6 zv9” (on average, 75.38% reads mapped uniquely to the genome). Transcriptomes were assembled with Cufflinks (version 2.2.0) using options: 'cufflinks -p 32 --GTF genes.gtf' and differential expression analysis between control and knockdown embryos was performed using Cuffdiff. A FDR corrected p-value of 0.05 was applied as the cut off to identify differentially regulated transcripts Results: We could show that MO assisted depletion of Rad21 and CTCF affected the transcriptional profiles of embryos in different ways. Overall design: mRNA profiles of (2.5, 3.3, 4.5, 5.3, 10 hpf) wild type (WT) and morpholino depleted Rad21 MO (Rad21) and CTCF MO (CTCF) embryos were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
Cohesin facilitates zygotic genome activation in zebrafish.
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The oncogenic mechanisms and tumour biology underpinning Clear Cell Sarcoma of Kidney (CCSK), the second commonest paediatric renal malignancy, are poorly understood and currently therapy depends heavily on Doxorubicin with cardiotoxic side-effects. Previously, we characterised the balanced t(10;17)(q22;p13) chromosomal translocation, identified at that time as the only recurrent genetic aberration in CCSK. This translocation results in an in-frame fusion of the YWHAE (encoding 14-3-3e) and NUTM2 genes, with a somatic incidence of 12%. Clinico-pathological features of that cohort suggested that this aberration might be associated with higher stage and grade disease. Since no primary CCSK cell line exists, we generated various stably transfected cell lines containing doxycycline-inducible HA-tagged-YWHAE-NUTM2, in order to study the effect of expressing this transcript. 14-3-3e-NUTM2-expressing cells exhibited significantly greater cell migration compared to mock-treated controls. Gene and protein expression studies conducted in parallel on this model system suggested dysregulation of signalling pathways as a basis to the migration changes. Importantly, by blocking these signalling pathways using anti-EGFR, anti-IGF1R and anti-PDGFa neutralising antibodies, the migratory advantage conferred by transcript expression was abrogated. These results support 14-3-3e-NUTM2 expression as a contributor to CCSK tumorigenesis and provide avenues for the exploration of novel therapeutic approaches in CCSK.
Dysregulated mitogen-activated protein kinase signalling as an oncogenic basis for clear cell sarcoma of the kidney.
Disease, Cell lineView Samples
Analysis of CPEB translational regulator target mRNAs
Cytoplasmic polyadenylation element binding protein deficiency stimulates PTEN and Stat3 mRNA translation and induces hepatic insulin resistance.
A zebrafish forward genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in the lysosomal hydrolase Cathepsin L that manifests the hallmarks of human lysosomal storage diseases. In uninfected mutants, macrophages progressively accumulate undigested material in their lysosomes, leading to impaired migration and the accumulation of unengulfed cell debris. During mycobacterial infection, these vacuolated macrophages cannot migrate to phagocytose infected macrophages undergoing apoptosis in the tuberculous granuloma. Consequently, unengulfed apoptotic macrophages undergo secondary necrosis causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal accumulations similarly impair migration to newly infecting mycobacteria. We find that important aspects of this phenotype are recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of alveolar macrophages from smokers exhibit lysosomal accumulations and do not migrate to Mycobacterium tuberculosis. This incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis. Overall design: A forward genetic screen for zebrafish larvae that are hypersusceptible to Mycobacterium marinum infection identified a mutation in the transcription factor snapc1b at 13: 37996163 (T->C). Individuals of wild type (T/T) and mutant (C/C) were genotyped and pooled respectively for RNA isolation and transcriptome analysis.
Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.
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Transplantation of amniotic membrane-expanded limbal epithelium (AMLE) in place of donor tissue grafts results in significantly improved outcomes for patients suffering from severe limbal stem cell deficiency; however the reasons for such superior results are unclear. The purpose of this study was to identify transcriptional gene profiles specific to AMLE and donor central corneal epithelium (CE), which may contribute to the divergent clinical outcomes observed following transplant. Limbal fibroblasts which underlie the epithelium and secrete extracellular matrix proteins following injury/surgery were also profiled. Using cell culture, immunofluorescence, microarray gene expression profiling and qRT-PCR validation; this study aims to identify enriched biological processes and pathways which characterise AMLE and CE tissues. We hope the study outcomes will shed light onto the factors which contribute to provide the improved clinical outcomes associated with AMLE transplantation.
Comparative transcriptomic analysis of cultivated limbal epithelium and donor corneal tissue reveals altered wound healing gene expression.
Specimen partView Samples
We have sequenced using single end and paired end sequencing GMPs, CMPs, EoPs, SiglecF+IL5ra- GMPs and eosinophils to be able to characterise this new subset of GMPs and to be able to give it some context within a lineage trajectory analysis Overall design: RNA-seq was performed on GMPs (n=2), CMPs (n=2), EoPs (n=2), Eosinophils (n=3) and SiglecF+IL5ra- GMPs isolated from C57BL/6 (n=5) and Myb hypomorphic Plt4/Plt4 mice (n=4).
Identification of a Siglec-F+ granulocyte-macrophage progenitor.
Specimen part, Cell line, SubjectView Samples
Macrophages polarize to divergent functional phenotypes depending on their microenvironment in a highly coordinated process of metabolic and transcriptional rewiring that is still poorly understood. We developed an Integrated Metabolomics and Gene Expression (IMAGE) profiling and analysis pipeline and applied it to extensively characterize global metabolic programs of macrophage polarization. IMAGE analysis identified 7 major (novel and known) regulatory modules responsible for metabolic rewiring during polarization, which we validated through extensive carbon and nitrogen labeling experiments. M1-specific modules included: inflammatory variant of the aspartate-arginosuccinate shunt; TCA cycle break at Idh expression accompanied by citrate accumulation and production of itaconate and fatty acid synthesis. In M2 macrophages we discovered significant role of glutamine in polarization, providing nitrogen for UDP-GlcNAc synthesis. Consistently, glutamine deprivation results in significant M2-specific defect in polarization. Our data provide, for the first time, a global view of the integrated transcriptional and metabolic changes that result in M1 and M2 polarization. Overall design: Bone-marrow derived macrophages were generated from C57BL/6 mice were plated at ~100k cells per well in 96-well plate and stimulated with either Il4 or combination of LPS&IFNg or left unstimulated for 24 h mRNA was derived from lysates using Invitrogen oligo-dT beads
Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.
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To determine the regulatory pathways necessary for astrocytoma formation within complex adult brain microenvironments, we engineered mice for adult astrocyte-specific disruption of key regulators (pRb, Kras and Pten). Drivers of all astrocytoma grades were identified using CreERTM-inducible alleles. Inactivation of pRb was necessary to initiate grade II disease, and was the only lesion to do so. Additional activation of Kras progressed disease to grade III, while further Pten inactivation facilitated grade IV (glioblastoma) progression. These outcomes were elicited whether somatic events were induced broadly or focally. In vivo inactivation of pRb, which induced astrocyte proliferation and apoptosis, activated the MAPK pathway, while Kras activation and Pten loss triggered PI3K pathways.
Evolutionary etiology of high-grade astrocytomas.
Sex, TimeView Samples