Project description:ngs2012_02_microd-ngs microd-part2-Effect of dilution on RNA seq sensitivity-One sample of total RNA extracted from whole embryo by laser assisted microdissection was diluted from 5ng to 10pg to check sensitivity of the library construction and subsequent sensitivity of transcript identification by RNA seq. Overall design: 6 samples with biological replicats. null
Project description:This study, using a growth hormone (GH)-deficient dwarf animal model and peripheral GH replacement, investigated the effects of circulating IGF-1 during adolescence on IGF-1 levels in the brain. Our results demonstrated that hippocampal IGF-1 protein concentrations during adolescence are highly regulated by circulating IGF-1, which were reduced by GH deficiency and restored by systematic GH replacement. In contrast, IGF-1 levels in the CSF were decreased by GH deficiency but not restored by GH replacement. Furthermore, analysis of gene expression using microarrays and RT-PCR indicated that circulating IGF-1 levels did not modify the transcription of IGF-1 or its receptor in the hippocampus but did regulate genes that are involved in microvascular structure and function, brain development, and synaptic plasticity, which potentially support brain structures involved in cognitive function during this important developmental period. GH-deficient dwarf (dw/dw) and heterozygous (HZ) rats were identified at postnatal day 33-34. Starting from d35, dw/dw rats received subcutaneous injection of either 300µg GH (dw/dw+GH) or saline (dw/dw+sal) for 7d or 30d. HZ animals received saline for the same periods as controls. Animals (n=4/group for each time point) were sacrificed, the hippocampi were dissected, and total RNA were isolated for subsequent transcriptomic profiling.
Project description:Current methods for analysing chromosome conformation in mammalian cells are either insensitive and low resolution or low throughput. Since the methods are expensive and relatively difficult to perform and analyse they are not widely used outside of specialised laboratories. Here we have re-designed the Capture-C method producing a new approach, called next generation (NG) Capture-C, which solves most of the current setbacks in analysing chromosome conformation. NG Capture-C produces unprecedented levels of sensitivity and reproducibility, which can be used to analyse any number of genetic loci and/or many samples in a single experiment. NG Capture is straightforward to perform, requiring only standard reagents and access to basic next generation sequencing platforms. The complete and detailed protocol presented here, with new publically available tools for library design and data analysis, will allow most laboratories to analyse chromatin conformation at levels of sensitivity and throughput that were previously impossible. Overall design: NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments, in this case the promoters of genes were exclusively used. The capture step has been optimised to markedly increase the sensitivity of the method (by introducing a double capture step), providing unprecedented levels of sensitivity and reliability for high resolution analysis of the chromatin landscape.
Project description:Genomic analysis of expression in brain, kidney, stomach, liver, and salivary tissues of perinatal ERRγ null and wild type mice. Experiment Overall Design: Total RNA was extracted from brain, kidney, stomach, liver, and salivary tissues of perinatal ERRγ null and wild type mice and hybridized to Affymetrix MOE430A gene chips in triplicate.
Project description:Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by re-implantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor (PR), epidermal growth factor receptor (EGFR), HER2 and the basal cell marker cytokeratin 5 (CK5). Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in MRI-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8 to 12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings. Overall design: RNA-seq from three patient-derived breast cancer brain metastases.
Project description:Genomic analysis of expression in brain, kidney, stomach, liver, and salivary tissues of perinatal ERRγ null and wild type mice. Overall design: Total RNA was extracted from brain, kidney, stomach, liver, and salivary tissues of perinatal ERRγ null and wild type mice and hybridized to Affymetrix MOE430A gene chips in triplicate.
Project description:Cloutier2009 - Brain Energy Metabolism
This model was taken from the
automatically converted to SBML. Following the
submission the parameters are manually encoded and annotated as
spices and global quantities by BioModels curators.
original model was:
Cloutier M, Bolger FB, Lowry JP, Wellstead P. (2009) -
original CellML model was created by:
University of Auckland
This model is described in the article:
An integrative dynamic model
of brain energy metabolism using in vivo neurochemical
Cloutier M, Bolger FB, Lowry JP,
J Comput Neurosci 2009 Dec; 27(3):
An integrative, systems approach to the modelling of brain
energy metabolism is presented. Mechanisms such as glutamate
cycling between neurons and astrocytes and glycogen storage in
astrocytes have been implemented. A unique feature of the model
is its calibration using in vivo data of brain glucose and
lactate from freely moving rats under various stimuli. The
model has been used to perform simulated perturbation
experiments that show that glycogen breakdown in astrocytes is
significantly activated during sensory (tail pinch)
stimulation. This mechanism provides an additional input of
energy substrate during high consumption phases. By way of
validation, data from the perfusion of 50 microM propranolol in
the rat brain was compared with the model outputs. Propranolol
affects the glucose dynamics during stimulation, and this was
accurately reproduced in the model by a reduction in the
glycogen breakdown in astrocytes. The model's predictive
capacity was verified by using data from a sensory stimulation
(restraint) that was not used for model calibration. Finally, a
sensitivity analysis was conducted on the model parameters,
this showed that the control of energy metabolism and transport
processes are critical in the metabolic behaviour of cerebral
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Project description:Two polypyrimidine tract RNA-binding proteins (PTBs), one near-ubiquitously expressed (Ptbp1) and another highly tissue-restricted (Ptbp2), regulate RNA in interrelated but incompletely understood ways. Ptbp1, a splicing regulator, is replaced in the brain and differentiated neuronal cell lines by Ptbp2. To define the roles of Ptbp2 in the nervous system, we generated two independent Ptbp2-null strains, unexpectedly revealing that Ptbp2 is expressed in neuronal progenitors and is essential for postnatal survival. A HITS-CLIP (high-throughput sequencing crosslinking immunoprecipitation)-generated map of reproducible Ptbp2–RNA interactions in the developing mouse neocortex, combined with results from splicing-sensitive microarrays, demonstrated that the major action of Ptbp2 is to inhibit adult-specific alternative exons by binding pyrimidine-rich sequences upstream of and/or within them. These regulated exons are present in mRNAs encoding proteins associated with control of cell fate, proliferation, and the actin cytoskeleton, suggesting a role for Ptbp2 in neurogenesis. Indeed, neuronal progenitors in the Ptbp2-null brain exhibited an aberrant polarity and were associated with regions of premature neurogenesis and reduced progenitor pools. Thus, Ptbp2 inhibition of a discrete set of adult neuronal exons underlies early brain development prior to neuronal differentiation and is essential for postnatal survival. Eight Ptbp2 HITS-CLIP libraries generated from mouse embryonic brain (four libraries from each of two biologic replicates).
Project description:ARN sample of brain from mouse were obtened at 6 times points: 1h, 1h30, 3h, 4h, 18h, 24h, after injection of oleic acid or physiological serum Brain total RNAs from mouse injected with acid oleic or physiological serum were profiled after hybridization with Agilent SurePrint G3 Mouse GE 8x60K Microarray to identify genes with differential expression Overall design: Brain from 31 C57BL6 mice are divided into 6 groups . Each group included tree or two mices given physiological serum (Phi) and 3 mices given oleic acid (OA). Each group was identified according to the incubation time: 1h, 1h30, 3h, 4h, 18h and 24h.