Showing papers by "Martin J. J. Ronis published in 2019"

Improved method for isolating high-quality RNA from mouse bone with RNAlater at room temperature.

Abstract: Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR.

Liver tumorigenesis is promoted by a high saturated fat diet specifically in male mice and is associated with hepatic expression of the proto-oncogene Agap2 and enrichment of the intestinal microbiome with Coprococcus

Abstract: Liver cancer results in a high degree of mortality, especially among men. As fatty liver disease is a risk factor for development of hepatocellular carcinoma, we investigated the role of dietary fat type in tumor promotion by high-fat diets in mice after initiation with the chemical carcinogen diethyl nitrosamine. Tumor incidence and multiplicity were significantly greater in males than those in females. In males, fat type had complex effects on tumorigenesis. Preneoplastic foci were most prevalent in mice fed a polyunsaturated fat diet enriched in docosahexaenoic acid, whereas carcinomas and large visible liver tumors were significantly greater in mice fed a saturated fat diet made with cocoa butter relative to mice fed mono- or polyunsaturated fats. Different mechanisms thus seemed involved in early and late tumor promotion. The hepatic transcriptome and gut microbiome were assessed for traits associated with tumorigenesis. Hepatic expression of more than 20% of all genes was affected by sex, whereas fat type affected fewer genes. In males, the saturated fat diet induced expression of the proto-oncogene Agap2 and affected the expression of several cytochrome P450 genes, and genes involved in lipid, bile acid and fatty acid metabolism. The gut microbiome had a higher level of genus Akkermansia and a lower level of Firmicutes in females than in males. Males fed saturated fat had an altered microbiome, including an enrichment of the genus Coprococcus. In conclusion, sex and the dietary fat type affect the gut microbiome, the hepatic transcriptome and ultimately hepatic tumor growth.

Reduced Serum Osteocalcin in High-Risk Alcohol Using People Living With HIV Does Not Correlate With Systemic Oxidative Stress or Inflammation: Data From the New Orleans Alcohol Use in HIV Study.

Abstract: Background HIV infection is now largely a chronic condition as a result of the success of antiretroviral therapy. However, several comorbidities have emerged in people living with HIV (PLWH), including alcohol use disorders and musculoskeletal disorders. Alcohol use has been associated with lower bone mineral density, alterations to circulating bone turnover markers, and hypocalcemia. The pathophysiological basis of bone loss in the PLWH population is unclear but has been suggested to be linked to oxidative stress and inflammation. To test the hypothesis that PLWH consuming excessive alcohol have altered markers of bone turnover and/or calcium homeostasis in association with oxidative stress, we correlated measurements of alcohol consumption with markers of oxidative stress and inflammation, serum calcium concentrations, and measurements of bone turnover, including c-terminal telopeptide cross-links (CTX-1) and osteocalcin. Methods Data were drawn from cross-sectional baseline data from the ongoing New Orleans Alcohol Use in HIV (NOAH) study, comprised of 365 in care PLWH. Alcohol consumption measures (Alcohol Use Disorders Test, 30-day timeline follow-back calendar, and phosphatidylethanol [PEth]) were measured in a subcohort of 40 subjects selected based on highest and lowest PEth measurements. Multivariate linear regression was performed to test the relationships between alcohol consumption and systemic oxidative stress (4-hydroxynonenal; 4-HNE) and inflammation (c-reactive protein; CRP). Results Serum calcium and CTX-1 did not differ significantly between the high and low-PEth groups. Individuals in the high-PEth group had significantly lower serum osteocalcin (median low-PEth group: 13.42 ng/ml, inter-quartile range [IQR] 9.26 to 14.99 ng/ml; median high-PEth group 7.39 ng/ml, IQR 5.02 to 11.25 ng/ml; p = 0.0005, Wilcoxon rank-sum test). Osteocalcin negatively correlated with PEth (Spearman r = -0.45, p = 0.05) and self-reported measures after adjusting for covariates. Alcohol consumption showed mild, but significant, positive associations with serum 4-HNE, but not with CRP. Osteocalcin did not correlate with either 4-HNE or CRP. Conclusions In this subcohort of PLWH, we detected significant associations between at-risk alcohol use and osteocalcin, and at-risk alcohol use and serum 4-HNE, suggesting suppression of bone formation independent of increased systemic oxidative stress with increasing alcohol consumption.

Estradiol and NADPH oxidase crosstalk regulates responses to high fat feeding in female mice.

Abstract: We previously demonstrated protection against high fat-induced obesity in female but not male p47phox−/− mice lacking NADPH oxidase NOX1/2 activity. To test the role of estradiol (E2)-NOX crosstalk...