Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.     

Endothelin-B receptors and ventricular arrhythmogenesis in the rat model of acute myocardial infarction

The arrhythmogenic effects of endothelin-1 (ET-1) are mediated via ETA-receptors, but the role of ETB-receptors is unclear. We examined the pathophysiologic role of ETB-receptors on ventricular tachyarrhythmias (VT/VF) during myocardial infarction (MI). MI was induced by coronary ligation in two animal groups, namely in wild-type (n = 63) and in ETB-receptor-deficient (n = 61) rats. Using a telemetry recorder, VT/VF episodes were evaluated during phase I (the 1st hour) and phase II (2–24 h) post-MI, with and without prior β-blockade. Action potential duration at 90% repolarization (APD90) was measured from monophasic epicardial recordings and indices of sympathetic activation were assessed using fast-Fourier analysis of heart rate variability. Serum epinephrine and norepinephrine were measured with radioimmunoassay. MI size was similar in the two groups. There was a marked temporal variation in VT/VF duration; during phase I, it was higher (p = 0.0087) in ETB-deficient (1,519 ± 421 s) than in wild-type (190 ± 34 s) rats, but tended (p = 0.086) to be lower in ETB-deficient (4.2 ± 2.0 s) than in wild-type (27.7 ± 8.0 s) rats during phase II. Overall, the severity of VT/VF was greater in ETB-deficient rats, evidenced by higher (p = 0.0058) mortality (72.0% vs. 32.1%). There was a temporal variation in heart rate and in the ratio of low- to high-frequency spectra, being higher (<0.001) during phase I, but lower (p < 0.05) during phase II in ETB-deficient rats. Likewise, 1 h post-MI, serum epinephrine (p = 0.025) and norepinephrine (p < 0.0001) were higher in ETB-deficient (4.20 ± 0.54, 14.24 ± 1.39 ng/ml) than in wild-type (2.30 ± 0.59, 5.26 ± 0.67 ng/ml) rats, respectively. After β-blockade, VT/VF episodes and mortality were similar in the two groups. The ETB-receptor decreases sympathetic activation and arrhythmogenesis during the early phase of MI, but these effects diminish during evolving MI.     

Assessment of PCBs exposure in human hair using double focusing high resolution mass spectrometry and single quadrupole mass spectrometry

Polychlorinated biphenyls (PCBs) levels were assessed in human hair samples, originating from two main agricultural regions of Greece. The analysis was performed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-double focusing high resolution mass spectrometry (GC-DFHRMS). The main analytical procedure involved hair decontamination, solid-liquid extraction and cleanup steps. The recoveries of PCBs ranged from 71.2% to 101.6%, with accuracies greater than 87.5% and the between-run precisions (%RSD) lower than 25% for all analytes. Differences in the frequencies of detection and the median values of PCBs were detected between the examined regions and between the applied analytical techniques. All Peloponnesus' hair examined samples were found positive for each examined PCB, while the percentage of the total positive samples ranged from 86.1% (for PCB 138) to 94.4% (for PCB 28 and 153 congeners) using GC-DFHRMS. The Cretan hair samples were less contaminated (SUM PCBs=0.61 and 1.47pg/mg) unlike the Peloponnesus' samples (SUM PCBs=24.68 and 38.74pg/mg) measured by GC-DFHRMS and GC-MS, respectively. PCBs with high chlorination gave lower concentration values compared to low chlorination PCBs in both populations. No significant differences were observed between women and men. The GC-DFHRMS technique provided higher percentage of positive samples and low PCBs median values, due to higher sensitivity and interferences from isobaric ions in the GC-MS technique and is therefore considered as a powerful tool for such assessments in hair specimens.     

Necrotizing acute pancreatitis current status - emerging new strategies in surgical management

Despite that pancreatic necrosis complicates only 15 % of cases of acute pancreatitis (AP), it is associated with high morbidity and considerable mortality. In an attempt to improve prognosis, many surgical strategies have been described during the last few decades. Currently, necrosectomy remains the cornerstone in the surgical treatment of infected pancreatic necrosis and in selected cases of sterile necrotizing pancreatitis. Following necrosectomy, continuous closed lavage is recommended by many authors, while closed abdominal packing /drainage and repeated planned necrosectomies- commonly using the zipper technique-are also acceptable alternative strategies. Open abdomen (laparostomy) is rarely indicated in carefully selected cases (typically in abdominal compartment syndrome associated with necrotizing AP). During the last decade, minimally invasive techniques (including percutaneous drainage, retroperitoneal endoscopic approach, transgastric endoscopic approach etc) have been extensively studied by some groups not only in the management of pancreatic abscesses and / or pseudocysts, but also as primary methods of treatment of necrotizing AP. Results have been impressive, but experience currently is limited to only a few centers around the world.     

Social determinants of self-reported emotional and behavioral problems in Greek adolescents

OBJECTIVE: This study aimed to assess the social factors associated with self-reported emotional and behavioral problems among Greek adolescents. METHODS: At age 18, a population-based sample of 3373 Greek adolescents completed the Youth Self-Report (YSR) questionnaire. RESULTS: The results from the multivariate analysis indicated that both lack of intimate friendships and not having parental monitoring were independently associated with problem behavior among both male and female adolescents. Engagement in sexual activities was significantly associated with higher YSR scores among both genders. Absence of academic motivation was associated with higher YSR scores in girls only. CONCLUSIONS: Our results confirm that social factors, including negative family functions, lack of academic motivation and not having close friends, were associated with emotional and behavioral problems among adolescents and imply the need to develop a supportive social network for adolescents.     

Parallel manifestation of Crohn’s disease and acute pericarditis: a report of two cases

Pericarditis is an uncommon extraintestinal manifestation of inflammatory bowel disease (IBD), which may occur at any time during its natural course. Moreover, it may be associated with the medications used to treat IBD, especially mesalamine. We report on two patients with acute pericarditis who were subsequently diagnosed with Crohn’s disease. It is likely that mild, longstanding, virtually asymptomatic intestinal disease preceded the onset of pericarditis in both cases.     

Lamivudine/pegylated interferon alfa-2b sequential combination therapy compared with lamivudine monotherapy in HBeAg-negative chronic hepatitis B

BACKGROUND AND AIM: Monotherapy has been proven insufficient in achieving sustained control of chronic hepatitis B. We aimed to assess the efficacy of combined sequential administration of lamivudine and pegylated interferon alfa-2b in patients with hepatitis Be antigen (HBeAg)-negative chronic hepatitis B. METHODS: Eighteen patients were given sequential combination treatment starting with 3 months of lamivudine monotherapy followed by 9 months of pegylated interferon alfa-2b (after a 3-month period of concomitant administration of the two drugs) and 24 patients received lamivudine monotherapy. RESULTS: At the end of treatment, 88.9% of the patients who received sequential combination treatment and 70.8% of those who received lamivudine monotherapy had hepatitis B virus (HBV) DNA levels below 400 copies/mL (P = not significant). At the end of treatment, 72.2% of the patients who received sequential combination treatment and 70.8% of those who received lamivudine monotherapy achieved alanine aminotransferase normalization (P = not significant). After 12 months of follow up, 33.3% of the patients who received sequential combination treatment and 16.7% of those who received lamivudine monotherapy had HBV-DNA levels below 400 copies/mL (P = 0.4). After 12 months of follow up, 72.2% of the patients who received sequential combination treatment and 25.0% of those who received lamivudine monotherapy had normal alanine aminotransferase levels (P < 0.01). Twenty-five percent of the patients in the lamivudine monotherapy group had virological breakthrough compared to none in the sequential combination treatment group (P = 0.06). CONCLUSIONS: Sequential combination treatment is able to improve sustained biochemical response rates and prevent the emergence of lamivudine-resistant mutants in patients with HBeAg-negative chronic hepatitis B.