SA-ODG platform: a semi-automated and PCR-free method to analyse microRNAs in solid tissues.

Over the past two decades, numerous techniques have been developed for analysing microRNAs in body fluids and tissues. However, these techniques still face technical challenges, particularly when compared to well-established techniques for proteins and metabolites. Recently, the ODG platform was introduced, which is an innovative technology that allows for the direct detection and quantification of microRNAs in liquid biopsies without requiring extraction or amplification. This study presents the implementation of the ODG platform within a semi-automated protocol to create the "SA-ODG" platform, enhancing the efficiency and precision of microRNA testing while reducing hands-on time required by laboratory staff. For the first time, the SA-ODG platform has been used to directly quantify microRNAs in solid tissues. The results demonstrate precise analysis of miR-122-5p in mouse liver tissues using SA-ODG. These developments represent a crucial step forward in advancing the field of extraction and amplification-free microRNA detection and quantification.

Telomere length increase after weight loss induced by bariatric surgery: results from a 10 year prospective study.

BACKGROUND/OBJECTIVES: Obesity contributes to telomere attrition. Studies focusing on short-term effects of weight loss have been unable to identify protection of telomere length. This study investigates long-term effects of pronounced weight loss induced by bariatric surgery on telomere length. SUBJECTS/METHODS: One hundred forty-two patients were recruited in a prospective, controlled intervention study, follow-up investigations were done after 10.46±1.48 years. A control group of normal weight participants was recruited and followed from 1995 to 2005 in the Bruneck Study. A total of 110 participants from each study was matched by age and sex to compare changes in telomere length. Quantitative PCR was used to determine telomere length. RESULTS: Telomere length increased significantly by 0.024±0.14 (P=0.047) in 142 bariatric patients within 10 years after surgery. The increase was different from telomere attrition in an age- and sex-matched cohort population of the Bruneck Study (-0.057±0.18; ß=0.08; P=0.003). Significant changes in telomere length disappeared after adjusting for baseline body mass index (BMI) because of general differences in BMI and telomere length between the two study populations (ß=0.07; P=0.06). Age was proportional to telomere length in matched bariatric patients (r=0.188; P=0.049) but inversely correlated with telomere length in participants of the Bruneck Study (r=-0.197; P=0.039). There was no association between percent BMI/excess weight loss and telomere attrition in bariatric patients. Baseline telomere length in bariatric patients was inversely associated with baseline plasma cholesterol and triglyceride concentrations. Telomere shortening was associated with lower high-density lipoprotein cholesterol and higher fasting glucose concentration at baseline in bariatric patients. CONCLUSIONS: Increases in relative telomere length were found after bariatric surgery in the long term, presumably due to amelioration of metabolic traits. This may overrule the influence of age and baseline telomere length and facilitate telomere protection in patients experiencing pronounced weight loss.

A multicentre, randomized, single-blind, parallel-group study comparing the efficacy and tolerability of benzoyl peroxide 3%/clindamycin 1% with azelaic acid 20% in the topical treatment of mild-to-moderate acne vulgaris.

BACKGROUND: Mild-to-moderate acne vulgaris is treated with a range of mono- and combination therapies; however, clinical evidence is still required to optimize treatment recommendations. OBJECTIVE: To compare the efficacy, tolerability and safety of a combination of benzoyl peroxide 3% and clindamycin 1% (BPO + CLN) with azelaic acid 20% (AzA) for the topical treatment of mild-to-moderate acne vulgaris. METHOD: This was a randomized, assessor-blinded, parallel-group, multicentre study conducted in Germany. Patients with a confirmed diagnosis of acne vulgaris, aged 12-45 years, were randomized 1 : 1 to once-daily BPO + CLN gel or twice-daily AzA cream for up to 12 weeks. The primary endpoint was the percentage change in inflammatory lesions from baseline at Week 4. Secondary endpoints included total and inflammatory lesion counts and tolerability assessments. For selected secondary endpoints, inductive statistical analysis was performed post hoc. Patient safety was assessed by adverse event (AE) monitoring. RESULTS: Efficacy was assessed in the modified intent-to-treat (mITT) population [patients using ≥1 dose of study medication (ITT), plus baseline and ≥1 post-baseline lesion count (n = 215)]. There was a statistically significant difference in the primary endpoint, with a median decrease of -52.6% for BPO + CLN (n = 107) vs.-38.8% for AzA (n = 108; P = 0.0004). There was also a greater difference in secondary lesion endpoints at Week 12, with a median decrease in inflammatory lesions of -78.8% and -65.3% and total lesions of -69.0% and -53.9% with BPO + CLN and AzA, respectively (both P < 0.0001). Tolerability was acceptable for both treatments. Overall, 55.6% (BPO + CLN) and 69.7% (AzA) of patients reported treatment-emergent AEs, and 15.7% and 35.8% of patients experienced application site reactions with BPO + CLN (24 events; 17 patients) and AzA (60 events; 39 patients) treatment, respectively (ITT population). CONCLUSION: BPO + CLN demonstrated greater efficacy than AzA in the treatment of mild-to-moderate acne vulgaris and has a positive tolerability and safety profile.

Serum prolactin in advanced chronic liver disease.

Hyperprolactinemia is a frequent endocrine disorder with well known harmful effects on the reproductive system and bone metabolism. Besides prolactinomas several drugs and disorders such as renal failure and hypothyroidism have been shown to cause hyperprolactinemia. Based on former studies, liver cirrhosis has also been suggested to cause hyperprolactinemia, while mechanisms have not been identified yet. In this study, we set out to investigate the prevalence and predictors of hyperprolactinemia in 178 patients with liver cirrhosis of different etio-logies. Eighteen out of 178 patients - 7 females and 11 males - displayed elevated serum pro-lactin levels. When patients were excluded who suffered from co-morbidities or took medication that are discussed to potentially interfere with prolactin metabolism, only 3 males displayed increased serum prolactin levels. Prolactin levels were similar in patients with liver cirrhosis of different etiologies. Our data suggest that hyperprolactinemia is not commonly found in patients with liver cirrhosis, but is mostly associated with intake of drugs or presence of comorbidites which are known to potentially cause hyperprolactinemia. We thus hypothesize that in contrast to former studies liver cirrhosis is not a common cause of hyperprolactinemia and that in the absence of co-morbidities or drugs that are known to potentially increase prolactin levels, marked hyperprolactinemia needs further investigation in patients with liver cirrhosis.

The role of apolipoprotein A5 in non-alcoholic fatty liver disease.

BACKGROUND: Apolipoprotein A5 (apoA5) is a recently described liver-specific protein that has been shown to influence triglyceride (TG) metabolism. ApoA5 transgenic mice display dramatically reduced TG levels, while in contrast apoA5 deficiency in humans was reported to result in marked hypertriglyceridemia. ApoA5 exerts its extracellular effects by increasing lipolysis of TG-rich lipoproteins, while in vitro data suggest additional intrahepatic effects. METHODS: In this study the authors set out to investigate a possible role of apoA5 in non-alcoholic fatty liver disease (NAFLD). We thus determined hepatic apoA5 expression in 15 obese subjects with histologically proven NAFLD undergoing bariatric surgery. In addition, the authors established a hepatic cell culture model of apoA5 knockdown by transfecting human hepatoma cells (HepG2) with apoA5 small interfering (si) RNA, and determined intracellular TG content and expression levels of key enzymes and transcription factors of intrahepatic lipid metabolism in these cells. RESULTS: Pronounced weight loss and associated histologically verified improvement of hepatic steatosis were accompanied by significant reductions of hepatic apoA5 mRNA expression levels. Significant apoA5 knockdown in HepG2 cells resulted in a marked decrease of intracellular TG content. When HepG2 cells were co-transfected with apoA5 and peroxisome proliferator-activated receptor gamma (PPARγ), reductions in hepatic TG accumulation were significantly less pronounced when compared to apoA5 siRNA transfected HepG2 cells. CONCLUSIONS: In obese subjects, hepatic apoA5 mRNA expression decreases after weight loss and improvements in hepatic steatosis. The authors' in vitro data demonstrate that apoA5 influences intrahepatic TG metabolism and that these intracellular effects of apoA5 are accompanied by changes in PPARγ mRNA expression. In summary, the data suggest that as well as several other factors, apoA5 might be involved in the pathogenesis of hepatic steatosis.

Effect of bariatric surgery on circulating chemerin levels.

BACKGROUND: Subclinical inflammation in obesity is critical for development of several obesity-associated disorders. We set out to investigate the effect of pronounced weight loss on circulating chemerin levels, a chemoattractant protein that also influences adipose cell function by paracrine and autocrine mechanisms. MATERIAL AND METHODS: Thirty-two obese patients undergoing bariatric surgery were tested before and on an average of 18 months after gastric banding or gastric bypass surgery. RESULTS: Pronounced weight loss after bariatric surgery was accompanied by improvements in parameters of lipid and glucose metabolism and increased adiponectin levels. Chemoattractant chemerin significantly decreased from 175.91 +/- 24.50 to 145.53 +/- 26.44 ng mL(-1) after bariatric surgery (P < or = 0.01). Concomitantly, hs-CRP as a marker of subclinical inflammation was significantly reduced after weight reduction (P < or = 0.01). CONCLUSIONS: We hypothesize that weight-loss induced reduction in circulating chemerin might in conjunction with other factors be associated with diminished recruitment of macrophages in adipose tissue and reduction of subclinical inflammation, which again could partly explain beneficial long-term effects of weight reduction in obese subjects.

Effect of obesity and insulin sensitivity on adiponectin isoform distribution.

BACKGROUND: Adiponectin is an insulin-sensitizing, antiatherogenic and anti-inflammatory adipocytokine that circulates in three isoforms: a trimer [low-molecular weight (LMW)], a hexamer (trimer-dimer) of medium molecular weight (MMW) and a multimeric high molecular weight (HMW) isoform. Evidence is accumulating that HMW adiponectin is the active isoform of the adipocytokine. We investigated the impact of adipose tissue and insulin sensitivity on adiponectin isoform distribution. MATERIALS AND METHODS: One hundred and eighty-seven normolipidaemic, non-diabetic lean or obese subjects with or without insulin resistance participating in the Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk (SAPHIR) were included in the study. Insulin sensitivity was determined by the short insulin tolerance test and the homeostasis model assessment (HOMA) index. Serum adiponectin isoform distribution was determined by an enzyme immunoassay. RESULTS: Total adiponectin as well as HMW/total adiponectin ratio was significantly increased in female subjects. Circulating total adiponectin levels were lowest in obese patients due to reduced concentrations of HMW adiponectin. As determined by stepwise regression analysis, besides age and high density lipoprotein (HDL) cholesterol, visceral fat area and waist-to-hip ratio predicted concentrations of HMW adiponectin, while insulin sensitivity had no influence on either total adiponectin or its isoforms. CONCLUSIONS: Our results underline that determination of adiponectin isoforms are more useful than measurement of total adiponectin in clinical settings. Our data suggest that adiponectin concentrations are strongly associated with visceral fat area but not with insulin sensitivity. Thus, we hypothesize that insulin resistance is a consequence rather than the cause of hypoadiponectinaemia in obese subjects.

20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn.

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.

Long-term effects of weight loss after bariatric surgery on functional and structural markers of atherosclerosis.

OBJECTIVE: Pronounced weight loss after bariatric surgery was demonstrated to have significant beneficial effects on surrogates of early atherosclerosis. The aim of this prospective examination was to investigate whether these improvements of endothelial function and vascular structure are persistent in the long-term. DESIGN AND METHODS: A total of 52 obese adults were examined before and 5 years after bariatric surgery. Carotid intima media thickness (IMT), brachial flow-mediated dilation (FMD), abdominal fat distribution, and metabolic parameters were determined. Additional 18 months data were available from 27 patients. RESULTS: After 5 years, mean weight loss ± SD of 25% ± 12 in all subjects was accompanied by known improvements in metabolism. Change in IMT was -0.02 mm ± 0.007, whereas FMD improved by +1.5% ± 0.5. In the subgroup IMT decreased by 0.04 mm ± 0.06 within the first 18 months, whereas no significant change was observed between 18 month and 5 years. FMD improved by 3.8% ± 0.6 after 18 months followed by a nonsignificant decrease of -1.4% ± 0.9. CONCLUSIONS: These long-term results demonstrate that bariatric surgery-induced weight loss improves both functional and structural markers of early atherosclerosis providing further evidence for the beneficial effects of weight loss on obesity-associated alterations of the vasculature.