Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.     

Toll-like receptor and antimicrobial peptide expression in the bovine endometrium

Background  

The endometrium is commonly infected with bacteria leading to severe disease of the uterus in cattle and humans. The endometrial epithelium is the first line of defence for this mucosal surface against bacteria and Toll-like receptors (TLRs) are a critical component of the innate immune system for detection of pathogen associated molecular patterns (PAMPs). Antimicrobial peptides, acute phase proteins and Mucin-1 (MUC-1) also provide non-specific defences against microbes on mucosal surfaces. The present study examined the expression of innate immune defences in the bovine endometrium and tested the hypothesis that endometrial epithelial cells express functional receptors of the TLR family and the non-specific effector molecules for defence against bacteria.     

A Self-Report Risk Index to Predict Occurrence of Dementia in Three Independent Cohorts of Older Adults: The ANU-ADRI

Background and Aims

The Australian National University AD Risk Index (ANU-ADRI, http://anuadri.anu.edu.au) is a self-report risk index developed using an evidence-based medicine approach to measure risk of Alzheimer''s disease (AD). We aimed to evaluate the extent to which the ANU-ADRI can predict the risk of AD in older adults and to compare the ANU-ADRI to the dementia risk index developed from the Cardiovascular Risk Factors, Aging and Dementia (CAIDE) study for middle-aged cohorts.

Methods

This study included three validation cohorts, i.e., the Rush Memory and Aging Study (MAP) (n = 903, age ≥53 years), the Kungsholmen Project (KP) (n = 905, age ≥75 years), and the Cardiovascular Health Cognition Study (CVHS) (n = 2496, age ≥65 years) that were each followed for dementia. Baseline data were collected on exposure to the 15 risk factors included in the ANU-ADRI of which MAP had 10, KP had 8 and CVHS had 9. Risk scores and C-statistics were computed for individual participants for the ANU-ADRI and the CAIDE index.

Results

For the ANU-ADRI using available data, the MAP study c-statistic was 0·637 (95% CI 0·596–0·678), for the KP study it was 0·740 (0·712–0·768) and for the CVHS it was 0·733 (0·691–0·776) for predicting AD. When a common set of risk and protective factors were used c-statistics were 0.689 (95% CI 0.650–0.727), 0.666 (0.628–0.704) and 0.734 (0.707–0.761) for MAP, KP and CVHS respectively. Results for CAIDE ranged from c-statistics of 0.488 (0.427–0.554) to 0.595 (0.565–0.625).

Conclusion

A composite risk score derived from the ANU-ADRI weights including 8–10 risk or protective factors is a valid, self-report tool to identify those at risk of AD and dementia. The accuracy can be further improved in studies including more risk factors and younger cohorts with long-term follow-up.     

Temperature effects on essential oil composition of citronella selections

Four citronella [Cymbopogon nardus (L.) Rendle] selections indigenous to Sri Lanka were grown for 90 days at 27/21° or 32/27°C daylnight temperatures in controlled environments. Leaves were harvested and oil extracted by steam distillation. Analysis for chemical constituents was carried out by gas liquid chromatography. Growing temperatures affected oil composition with the response to temperature differing among selections. The commercially desired constituent. citronellal, was higher at 27/21°C than at 32/27°C in all selections, whereas the commercially undesirable constituent borneol was higher at 32/27°C than at 27/ 21°C. The production of total monoterpene hydrocarbons was enhanced at 27/ 21°C in selections C-4 and C-8 compared to 32/27°C. The level of methyl isoeugenol differed among selections.     

Unraveling the resistance of microbial biofilms: has proteomics been helpful?

Biofilms are surface-attached, matrix-encased, structured microbial communities which display phenotypic features that are dramatically different from those of their free-floating, or planktonic, counterparts. Biofilms seem to be the preferred mode of growth of microorganisms in nature, and at least 65% of all human infections are associated with biofilms. The most notable and clinically relevant property of biofilms is their greater resistance to antimicrobials compared with their planktonic counterparts. Although both bacterial and fungal biofilms display this phenotypic feature, the exact mechanisms underlying their increased drug resistance are yet to be determined. Advances in proteomics techniques during the past decade have facilitated in-depth analysis of the possible mechanisms underpinning increased drug resistance in biofilms. These studies have demonstrated the ability of proteomics techniques to unravel new targets for combating microbial biofilms. In this review, we discuss the putative drug resistance mechanisms of microbial biofilms that have been uncovered by proteomics and critically evaluate the possible contribution of the new knowledge to future development in the field. We also summarize strategic uses of novel proteomics technologies in studies related to drug resistance mechanisms of microbial biofilms.     

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.     

Expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility

Background  

Contamination of the uterine lumen with bacteria is ubiquitous in cattle after parturition. Some animals develop endometritis and have reduced fertility but others have no uterine disease and readily conceive. The present study tested the hypothesis that postpartum cattle that develop persistent endometritis and infertility are unable to limit the inflammatory response to uterine bacterial infection.     

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.     

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.