Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27

Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis

The Effects of Cholesteryl Ester Transfer Protein Inhibition on High Density Lipoprotein Function

Cardiovascular disease affects about 1.4 million Australians and was responsible for more than 45000 deaths in 2011. Compelling evidence from human population studies has shown a positive correlation between elevated plasma high density lipoprotein cholesterol (HDL-C) levels and a reduced risk of major cardiovascular events. HDLs have several potential cardioprotective functions, the most extensively studied of which is their ability to remove excess cholesterol from macrophages. They also have anti-inflammatory properties, enhance endothelial repair and improve endothelial function. HDLs also have anti-oxidant, anti-apoptotic, anti-thrombotic and anti-diabetic functions. Therapies that increase HDL-C levels reduce atherosclerotic lesion development in animals. These observations have led to the hypothesis that increasing HDL-C levels will reduce cardiovascular events in humans. Although CETP inhibitors have been shown to markedly increase HDL-C levels and reduce major cardiovascular events in a recent clinical outcome trial, there remains doubt about the effects of CETP inhibition on the cardioprotective properties of HDLs. The work in this thesis examines the effects of inhibiting CETP on the cardioprotective functions of HDLs. The studies investigate the effects of a novel CETP inhibitor, AMG-899, on HDL composition, size and several of their cardioprotective properties. Subjects were treated with placebo or AMG-899 (2.5 mg/day or 10 mg/day) over 12 weeks. HDLs were isolated from subjects before and after treatment. AMG-899 treatment increased plasma HDL-C levels and decreased LDL-C levels (Chapter 3). Treatment with AMG-899 also changed the composition of HDLs, increased HDL size (Chapter 3) and increased HDL-mediated ABCA1- and ABCG1-specific cholesterol efflux (Chapter 4). Treatment with 10 mg/day AMG-899 also improved the anti-inflammatory properties of HDLs (Chapter 5), while HDLs isolated from subjects after treatment with 2.5 mg/day and 10 mg/day AMG-899 were equally effective at increasing endothelial proliferation and migration relative to HDLs isolated prior to treatment (Chapter 6). These studies establish that inhibiting CETP activity with AMG-899 does not impair, and in some cases, improves the potentially cardioprotective functions of HDLs. In conclusion, as AMG-899 treatment increases HDL-C levels more effectively than other CETP inhibitors, and does not impair the major cardioprotective functions of HDLs, it is a potential candidate for future clinical development

The Resistance Patterns in <i>E. coli</i> Isolates among Apparently Healthy Adults and Local Drivers of Antimicrobial Resistance: A Mixed-Methods Study in a Suburban Area of Nepal

Evidence-based decision-making to combat antimicrobial resistance (AMR) mandates a well-built community-based surveillance system for assessing resistance patterns among commensals and pathogenic organisms. As there is no such surveillance system in Nepal, we attempted to describe the antimicrobial resistance pattern in E. coli isolated from the fecal samples of apparently healthy individuals in Dhulikhel municipality and also explored the local drivers of AMR. We used a mixed-method design with a cross-sectional quantitative component and a descriptive qualitative component, with focus group discussion and key informant interviews as the data collection method. Fecal samples were collected from 424 individuals randomly selected for the study. E. coli was isolated from 85.9% of human fecal samples, of which 14% were resistant to ≥3 class of antimicrobials (multidrug resistant). Of the 368 isolates, resistance to ampicillin (40.0%), tetracycline (20.7%) and cefotaxime (15.5%) were most prevalent. The major drivers of AMR were: lack of awareness of AMR, weak regulations on sales of antimicrobials, poor adherence to prescribed medications, and incomplete dosage due to financial constraints. These findings indicate the need for strict implementation of a national drug act to limit the over-the-counter sales of antimicrobials. Additionally, awareness campaigns with a multimedia mix are essential for educating people on AMR

Antimicrobial Resistance in E. coli Isolated from Chicken Cecum Samples and Factors Contributing to Antimicrobial Resistance in Nepal

Microorganisms with antimicrobial resistance (AMR) are prevalent among humans and animals, and also found in the environment. Though organisms with AMR can spread to humans via food from animal sources, the burden of AMR in food-producing animals remains largely unknown. Thus, we assessed the resistance pattern among Escherichia coli isolated from chicken cecum samples and explored issues contributing to AMR in animals in the Dhulikhel Municipality of Nepal. We conducted a mixed-methods study, comprising a cross-sectional quantitative component, with collection of chicken cecal samples from slaughter houses/shops. In addition, a descriptive qualitative component was undertaken, with a focus group discussion and key informant interviews among stakeholders involved in animal husbandry. Of the 190 chicken cecum samples collected, 170 (89%) were subjected to culture and drug sensitivity testing, of which E. coli was isolated from 159 (94%) samples. Of the 159 isolates, 113 (71%) had resistance to &ge;3 antimicrobial class. Resistance to tetracycline (86%) and ciprofloxacin (66%) were most prevalent. Overuse of antimicrobials, easy availability of antimicrobials, and lack of awareness among farmers about AMR were major issues contributing to AMR. The high prevalence of resistance among E. coli in chicken cecal samples calls for rational use of antimicrobials, educating farmers, and multi-sectoral coordination

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males.

microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males.HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically.miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size.HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males.

microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males.HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically.miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size.HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss

Changes of HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels over 12 weeks of weight loss diet (n = 47).

<p>Changes of HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels over 12 weeks of weight loss diet (n = 47).</p

Bivariate correlation of changes in HDL-associated miR-223 levels with changes in clinical characteristics over 12 weeks of weight loss diet in the high protein group (n = 20).

<p>Bivariate correlation of changes in HDL-associated miR-223 levels with changes in clinical characteristics over 12 weeks of weight loss diet in the high protein group (n = 20).</p

Clinical characteristics at baseline and changes over 12 weeks of weight loss diets.

<p>Clinical characteristics at baseline and changes over 12 weeks of weight loss diets.</p

Fold-changes of HDL-miRNA levels after 12 weeks of high protein and normal protein diets.

<p>HDL-miRNAs miR-16, miR-17, miR-126, miR-222 and miR-223 were assessed by real-time PCR TaqMan miRNA assays and normalized to both Caenorhabditis elegans (Cel) miR-39 and HDL total protein concentration. Results are expressed as fold change. Values represent the mean±SEM of n = 20 for the high protein (HP) diet group and n = 27 for the normal protein (NP) diet group. *P<0.05 (after multiple testing correction).</p