Complete Genome Sequence of Escherichia coli Strain M8, Isolated from ob/ob Mice.
ABSTRACT: Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb.
Project description:OBJECTIVE:Obesity increases morbidity and resource utilization in sepsis patients. Sepsis transitions from early/hyper-inflammatory to late/hypo-inflammatory phase. Majority of sepsis-mortality occurs during the late sepsis; no therapies exist to treat late sepsis. In lean mice, we have shown that sirtuins (SIRTs) modulate this transition. Here, we investigated the role of sirtuins, especially the adipose-tissue abundant SIRT-2 on transition from early to late sepsis in obese with sepsis. METHODS:Sepsis was induced using cecal ligation and puncture (CLP) in ob/ob mice. We measured microvascular inflammation in response to lipopolysaccharide/normal saline re-stimulation as a "second-hit" (marker of immune function) at different time points to track phases of sepsis in ob/ob mice. We determined SIRT-2 expression during different phases of sepsis. We studied the effect of SIRT-2 inhibition during the hypo-inflammatory phase on immune function and 7-day survival. We used a RAW264.7 (RAW) cell model of sepsis for mechanistic studies. We confirmed key findings in diet induced obese (DIO) mice with sepsis. RESULTS:We observed that the ob/ob-septic mice showed an enhanced early inflammation and a persistent and prolonged hypo-inflammatory phase when compared to WT mice. Unlike WT mice that showed increased SIRT1 expression, we found that SIRT2 levels were increased in ob/ob mice during hypo-inflammation. SIRT-2 inhibition in ob/ob mice during the hypo-inflammatory phase of sepsis reversed the repressed microvascular inflammation in vivo via activation of endothelial cells and circulating leukocytes and significantly improved survival. We confirmed the key finding of the role of SIRT2 during hypo-inflammatory phase of sepsis in this project in DIO-sepsis mice. Mechanistically, in the sepsis cell model, SIRT-2 expression modulated inflammatory response by deacetylation of NF?Bp65. CONCLUSION:SIRT-2 regulates microvascular inflammation in obese mice with sepsis and may provide a novel treatment target for obesity with sepsis.
Project description:The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.
Project description:GWAT store most of the TAG in mice, ob/ob mice is an obese mice. Ob/ob/Fsp27-/- mice are lean when compared with ob/ob mice. The GWAT weight was dramatically reduced in ob/ob/Fsp27-/- mice. We next extract the total RNA from the GWAT of ob/ob and ob/ob/Fsp27-/- mice, to perform microaary analysis using Mouse Genome 430 2.0 arrays, Affymetrix. We then analysised the up-regulated and down regulated pathways. We extract the RNA of GWAT from 4 month old mice and hybridization on Affymetrix microarrays. We then analysis the data.
Project description:ob/ob mice is an obese mice. CIDE family proteins including Cidea, Cideb and Cidec play important role in lipid metabolism. Cidea is mainly expressed in the brown adipose tissue (BAT). Cidec is mainly expressed in the BAT and white adipose tissue (WAT). We generated ob/ob/Cidea-/-/Cidec-/- mice to investigate the phenotype of fat tissue. ob/ob/Cidea-/-/Cidec-/- mice are lean when compared with ob/ob mice. The tissue weight and TAG content of BAT and WAT was extreamly decreased in ob/ob/Cidea-/-/Cidec-/- mice compared with that in ob/ob mice. We next extract the total RNA from the BAT and WAT of ob/ob and ob/ob/Cidea-/-/Cidec-/- mice, to perform microarray analysis using Mouse Gene 1.0 ST array system, Affymetrix. We then analysised the up-regulated and down regulated pathways. Overall design: We extract the RNA of BAT and WAT from 4 month old mice and hybridization on Affymetrix microarrays. We then analysis the data.
Project description:OBJECTIVES:Obesity is an independent risk factor for cardiovascular disease. Reverse cholesterol transport (RCT) is an important cardioprotective mechanism. This study aimed to investigate RCT changes in a murine model of obesity. METHODS:Ob/ob and control mice were injected with [3H]-cholesterol-labelled macrophages and cholesterol accumulation quantified after 48 h. Ex vivo, cholesterol efflux and uptake were determined in hepatic and adipose tissues. RESULTS:Ob/ob mice had more labelled cholesterol in their plasma (86%, p<0.001), suggesting impaired RCT. SR-BI-mediated cholesterol efflux was elevated from ob/ob mice (serum, 33%; apoB-depleted plasma, 14%, p<0.01) and HDL-c were also higher (60%, p<0.01). Ex vivo it was found that cholesterol uptake was significantly lower into the livers and adipose tissue of ob/ob mice, compared to non-obese wildtype controls. Furthermore, ex vivo cholesterol efflux was reduced in ob/ob liver and adipose tissue towards apoA-I and HDL. Consistent with this, protein levels of SR-BI and ABCG1 were significantly lower in ob/ob hepatic and adipose tissue than in wildtype mice. Finally, labelled cholesterol concentrations were lower in ob/ob bile (67%, p<0.01) and faeces (76%, p<0.0001). CONCLUSION:Obesity causes impairment in RCT due to reduced plasma cholesterol uptake and efflux by hepatocytes and adipocytes. A reduction in the capacity for plasma cholesterol clearance may partly account for increased CVD risk with obesity.
Project description:To identify a female mouse model of diabetic peripheral neuropathy (DPN), we characterized DPN in female BTBR ob/ob mice and compared their phenotype to non-diabetic and gender-matched controls. We also identified dysregulated genes and pathways in sciatic nerve (SCN) and dorsal root ganglia (DRG) of female BTBR ob/ob mice to determine potential DPN mechanisms.Terminal neuropathy phenotyping consisted of examining latency to heat stimuli, sciatic motor and sural sensory nerve conduction velocities (NCV), and intraepidermal nerve fiber (IENF) density. For gene expression profiling, DRG and SCN were dissected, RNA was isolated and processed using microarray technology and differentially expressed genes were identified.Similar motor and sensory NCV deficits were observed in male and female BTBR ob/ob mice at study termination; however, IENF density was greater in female ob/ob mice than their male counterparts. Male and female ob/ob mice exhibited similar weight gain, hyperglycemia, and hyperinsulinemia compared to non-diabetic controls, although triglycerides were elevated more so in males than in females. Transcriptional profiling of nerve tissue from female mice identified dysregulation of pathways related to inflammation.Similar to males, female BTBR ob/ob mice display robust DPN, and pathways related to inflammation are dysregulated in peripheral nerve.
Project description:Particles generated from wear of prosthesis joint bearing surfaces induce inflammation-mediated periprosthetic bone resorption (osteolysis). Morbidly obese leptin-deficient ob/ob mice are resistant to polyethylene particle-induced bone loss, suggesting that leptin, a hormone produced by adipocytes that circulates in concentrations proportional to total body adiposity, increases osteolysis. To confirm that particles induce less osteolysis in leptin-deficient mice after controlling for cold stress (room temperature)-induced bone loss, ob/ob mice on a C57BL/6 (B6) background and colony B6 wildtype (WT) mice housed at thermoneutral temperature were randomized to control or particle treatment groups (N?=?5/group). Polyethylene particles were implanted over calvaria and mice sacrificed 2 weeks later. Compared to particle-treated WT mice, particle-treated ob/ob mice had lower osteolysis score, less infiltration of immune cells, and less woven bone formation. To determine the role of leptin in particle-induced osteolysis, ob/ob mice were randomized into one of 4 groups (n?=?6-8/group): (1) control, (2) particles, (3) particles?+?continuous leptin (osmotic pump, 6??g/d), or (4) particles?+?intermittent leptin (daily injection, 40??g/d). Leptin treatment increased particle-induced osteolysis in ob/ob mice, providing evidence that the adpiokine may play a role in inflammation-driven bone loss. Additional research is required to determine whether altering leptin levels within the physiological range results in corresponding changes in polyethylene-particle-induced osteolysis.
Project description:OBJECTIVE: Direct health care costs of obesity continue to grow throughout the world and research on obesity disease models are on the rise. The ob/ob mouse is a well-characterized model of obesity and associated risk factors. Successful breeding and backcrossing onto different backgrounds are essential to create knockout models. Ob/ob mice are sterile and heterozygotes must be identified by genotyping to maintain breeding colonies. Several methods are employed to detect the ob mutant allele, a single nucleotide polymorphism (SNP). Gel based methods are time consuming and inconsistent, and non-gel based assays rely upon expensive and complex reagents or instruments. A fast, high-throughput, cost effective, and consistent method to identify Lep(ob) mutation is much needed. DESIGN AND METHODS: Primers to produce an amplicon for High Resolution Melting Analysis (HRM) of the Lep(ob) SNP were designed and validated. RESULTS: Fluorescence normalized high resolution melting curve plots delineated ob/+, ob/ob, and WT genotypes. Genotypes were also confirmed phenotypically. CONCLUSIONS: HRM of the Lep(ob) SNP allows closed-tube identification of the Lep(ob) mutation using a real-time PCR machine now common to most labs/departments. Advantages of this method include assay sensitivity/accuracy, low cost dyes, less optimization, and cost effectiveness as compared to other genotyping techniques.
Project description:Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.