Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway
ABSTRACT: In fungal species, differentiation to the filamentous/hyphal cell type is critical for entry into host cells and virulence. Comparative RNA sequencing was used to explore the pathways that regulate differentiation to the filamentous cell type in yeast. This approach uncovered a role for the stress-response MAPK pathway, HOG, during the increased metabolic respiration that induces filamentous growth. In this context, the AMPK Snf1p and ER stress kinase Ire1p regulated the HOG pathway. Cross-modulation between the HOG and filamentous growth (ERK-type) MAPK pathways optimized the differentiation response. The regulatory circuit described here may extend to behaviors in metazoans. Comparison of expression patterns of wild-type and mutant yeast cells grown in salt, tunicamycin or galactose by comparative RNA sequencing analysis.
Project description:Large scale RNA-Seq analysis was performed to investigate the transcriptomic response to osteoarthritis in cartilage and investigate potential subgroups of patients. Data were collected from intact knee cartilage (posterior lateral condyle) from at total of 60 patients with osteoarthritis (OA) following total knee replacement and 10 control non-OA patients following amputation.
Project description:The role of shear stress, the frictional force of blood flow, on the endothelium has been well documented. Differences in shear stress can have profound effects on endothelial and blood vessel biology. Endothelial cells (ECs), termed endocardial ECs, line the heart chambers and are exposed to complex shear stress patterns. While it has been demonstrated that shear stress is important for heart development, little has been shown on the role of shear stress on adult ECs. 4D-MRI studies demonstrate regional differences in blood residence time. We sought to determine the effect of regional differences in endocardial shear stress on the endocardial transcriptome using RNA sequencing (RNA-seq) on 3 different regions (apex, mid-ventricle, outflow tract) from 8 adult pigs, for a total of 24 RNA-seq assays.
Project description:Atherosclerosis is a progressive disease characterized by the accumulation of lipids in the large and medium sized arteries. Lipoproteins and the endothelium play critical roles in the onset of atherosclerosis through the regulation of trans-endothelial lipoprotein flux in the subintima, the expression of adhesion molecules and proinflamatory cytokine, and the recruitment of monocytic precursors to intimal macrophage foam cells. Although it has been greatly studied in animal models, including rabbits, pigs, non-human primates and rodents, little is known about the regression of the disease. There are beneficial effects seen clinically in modification of diet and blood lipid. To determine the effect of withdrawl of risk factors (diet) on endothelial gene expression, in this study we characterized the temporal changes of carotid endothelial transcriptome in hyperlipidemic pigs using next-generation RNA sequencing. Ten litters of castrated barrows swine (Yorkshire x Landrace) were raised on a normal diet. The swine (~250 lbs) were then fed with a diet high in fat and cholesterol (HFHC). One cohort (5 animals) was on HFHC for 20 weeks prior to tissue collections. Another cohort (5 animals) was on HFHC for 12 weeks and then on a normal diet (ND) for 8 weeks. The normal diet consisted of a standard commercial corn/soybean meal diet (18% crude protein) at 100% ad libitum intake. The isocaloric HFHC diet consisted of 16.5% crude protein, 15% fat and 1.5% cholesterol at 80% of the ad libitum feed rate (by weight) such that the caloric intake/kg BW were approximately the same as the control diet. Diets were adjusted biweekly according to body weight. Food was withheld for 24 hours prior to endothelium collection in order to permit the collection of blood for fasting lipid levels.
Project description:The development of autonomic nerve fibres in the tumour microenvironment is a pivotal event that regulates prostate cancer initiation and dissemination, but how nerves emerge in tumours is presently unknown. Here we show that Doublecortine-expressing (DCX+) neural precursors from the central nervous system (CNS) infiltrate prostate tumours and differentiate into neo-neurons that contribute to tumour development. In human primary prostate tumours and transgenic mouse cancer tissues, the density of DCX+ neural progenitors is strongly associated with tumour aggressiveness, invasion and recurrence. We found that DCX+ neural precursors egress from the subventricular zone, a neurogenic area of the CNS, and circulate in the blood to reach the tumour where they initiate neurogenesis. Hence, the DCX+ cells in prostate tumour can differentiate into neurons ex vivo and build up a tumour-associated neural network in vivo. Selective genetic depletion of DCX+ cells in mice significantly inhibits the early phases of prostate cancer development, whereas orthotopic transplantation of DCX+ cells purified from prostate tumour or brain tissues promotes tumour growth and cancer cell dissemination. These results unveil a unique crosstalk between the CNS and the tumour that drives a process of neurogenesis necessary for prostate cancer development, and indicate a novel neural element of the tumour microenvironment as a potential target for cancer treatment.
Project description:Atherosclerosis is a progressive disease characterized by the accumulation of lipids in the large and medium sized arteries. Lipoproteins and the endothelium play critical roles in the onset of atherosclerosis through the regulation of trans-endothelial lipoprotein flux in the subintima, the expression of adhesion molecules and proinflamatory cytokine, and the recruitment of monocytic precursors to intimal macrophage foam cells. Although it has been greatly studied in animal models, including rabbits, pigs, non-human primates and rodents, the early development of atherosclerosis is unknown. In this study, we characterized the temporal changes of carotid endothelial transcriptome in hyperlipidemic pigs using next-generation RNA sequencing. Twenty litters of castrated barrows swine (Yorkshire x Landrace) were raised on a normal diet. The swine (~250 lbs) were then fed with a diet high in fat and cholesterol (HFHC) for 0, 2, 4, 8, or 12 weeks (n=4 animals for each time point) prior to tissue collections. The normal diet consisted of a standard commercial corn/soybean meal diet (18% crude protein) at 100% ad libitum intake. The isocaloric high fat/high cholesterol diet consisted of 16.5% crude protein, 15% fat and 1.5% cholesterol at 80% of the ad libitum feed rate (by weight) such that the caloric intake/kg BW were approximately the same as the control diet. Diets were adjusted biweekly according to body weight. Food was withheld for 24 hours prior to endothelium collection in order to permits the collection of blood for fasting lipid levels.
Project description:This experiment captures the expression of genes between two sites of human cartilage within the same patients to allow investigation of genomic responses to damage during osteoarthritis. Eight patients with symptomatic OA undergoing total knee replacement (n=8, age range 65-79 years, mean age 70.3) were used in this study. Cartilage from paired osteochondral samples were isolated from the intact PLC (posterior lateral condyle) and the damaged DMC (distal medial condyle) for RNA-seq analysis.
Project description:Purpose: Next-generation sequencing (NGS) was used to select genes potentially associated with exercise adaptation in Arabian horses. Methods: Whole transcriptome profiling of blood was performed for untrained horses and horses from which samples were collected during at 3 different periods of training procedure (T1-during intense training period - March, T2- before starts - May and T3 -after flat racing season - October). The muscle transcriptome sequencing was performed for 37 blood samples using Illumina HiScan SQ in 75 single-end cycles. The quantifying transcript abundances was made using the RSEM supported by STAR aligner. The raw reads were aligned to the Equus caballus reference genome. Differentially expressed genes in blood tissue were detected by DESeq2. The RNA-seq results were validated using by qPCR. Results: The increase of the number of DEGs between subsequent training periods has been observed and the highest amount of DEGs was detected between untrained horses (T0) and horses at the end of the racing season (T3) – 440. The comparison of transcriptome of T2 vs T3 and T0 vs T3 showed a significant advantage of up-regulated genes during long-term exercise (up-regulation of 266 and 389 DEGs in T3 period compared T2 and T0; respectively). Our results showed that the largest number of identified genes encoded transcription factors, nucleic acid binding proteins and G-protein modulators, which mainly were transcriptional activated at the last training phase (T3) . Moreover, in the T3 period the identified DEGs represented genes coded for cytoskeletal proteins including actin cytoskeletal proteins and kinases. The most abundant exercise-upregulated genes were involved in pathways important in regulating the cell cycle (PI3K-Akt signaling pathway), cell communication (cAMP-dependent pathway), proliferation, differentiation and apoptosis as well as immunity processes (Jak-STAT signaling pathway). We also observed exercise induced expression of genes related in regulation of actin cytoskeleton, gluconeogenesis (FoxO signaling pathway; Insulin signaling pathway), glycerophospholipid metabolism and calcium signaling. Conclusions: TOur results allow to identify changes in genes expression profile following training schedule in Arabian horses. Based on comparison analysis of blood transcriptomes, several exercise-regulated pathways and genes most affected by exercise were detected. We pinpointed overrepresented molecular pathways and genes essential for exercise adaptive response via maintaining of body homeostasis. The observed transcriptional activation of such gene as LPGAT1, AGPAT5, PIK3CG, GPD2, FOXN2, FOXO3, ACVR1B and ACVR2A can be a base for further research in order to identify genes potentially associated with race performance in Arabian horses. Such markers will be essential to choice the training type, and could result in differences in racing performance specific to various breeds. The blood transcriptome sequencing was performed for 37 samples collected form Arabian horses using Illumina HiScan SQ in75 single-end cycles and in 3-4 technical repetitions.repetitions.
Project description:The gain-of-function mutation in the pleckstrin homology domain of AKT1 (AKT1E17K) occurs in lung and breast cancer. By use of human cellular models and of a AKT1E17K transgenic Cre-inducible murine strain (R26-AKT1E17K mice) we have demonstrated that AKT1E17K is a bona-fide oncogene for lung epithelial cells. However, the role of AKT1E17K in breast cancer remains to be determined. Here, we report the generation and the characterization of a MMTV-CRE;R26-AKT1E17K mouse strain that expresses the mutant AKT1E17K allele in the mammary epithelium. We observed that R26AKT1E17K;MMTV-Cre mice presented a variety of proliferative alterations of the mammary epithelium that were classified as adenosis with low-to-high grade dysplasia. In addition, AKT1E17K stimulates the development of mammary tumors with incidence of 43%. Tumors were morphologically classified as ductal adenocarcinoma of medium-high grade though subsequent immunoistochemical characterization suggested they were of basal-like origin, being PR-/HER2-/ERα+ and CK8-/CK10-/CK5+/CK14+. We also observed that tumors expressing mutant AKT1E17K presented activation of the downstream signaling axis GSK3/cyclin D1 in mammary epithelium, in parallel with increased proliferation rate as demonstrated by cell number count and measurement of Ki67. In conclusion, AKT1E17K is a bona fidae oncogene that is able to initiate tumors at high efficiency in murine mammary epithelium in vivo.
Project description:Glucocorticoids are critical regulators of energy metabolism and immunity and major drug targets in inflammatory disease. It is also known that the circadian clock regulates both metabolism and immunity. This experiment is part of wider study to understand the coupling between circadian clock components and Glucocorticoid receptor in multiple mouse tissues. Here, RNA-seq analysis was performed in vehicle and dexamethasone (a synthetic Glucocorticoid) treated WT mouse liver and lung tissues at two circadian times, to understand time of the day variation in Glucocorticoid regulated genes. Mice were treated at ZT6 (6 hours after lights on, 1:30pm) or at ZT18 (6 hours after lights off, 1:30am) with dexamethasone (1 mg/kg intraperitoneal) or vehicle (methylcyclodextrin 1mg/kg intraperitoneal) for 2 hours before sacrifice by cervical dislocation. Lung and liver tissue were lysed and total RNA prepared using SV Total RNA Isolation System (Promega). Quality and integrity of total RNA samples were assessed by 2100 Bioanalyzer or a 2200 TapeStation (Agilent Technologies) according to the manufacturer’s instructions. RNA sequencing (RNA-seq) libraries were generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer’s protocol. Then paired-end sequenced (101 + 101 cycles, plus indices) on an Illumina HiSeq2500 instrument.
Project description:Intestinal tumours from genetically engeenierd mouse modesl (GEMMs), of various gentopyes, were isolated at endpoint and RNA was isolated. In an additional experiment, epithelial tumour organoids were isolated at end point and grown in vitro. These organoids were harvested 72 hours after seeding and RNA was isolated.