SNP marker development in Pinus sylvestris L. in stress-responsive genes characterized from Pinus cembra L. transcriptomes.

Massively parallel sequencing of cDNA is an efficient route for generating sequence collections that represent expressed genes under different environmental control. The analysis of their sequence helps in developing molecular markers, such as SNPs, which represent a useful tool in detecting adaptive signals in populations. In this study novel PCR markers, based on stress responsive genes, were designed from the transcriptome of the haploxylon Swiss stone pine (Pinus cembra L.) and tested for SNPs in the diploxylon Scots pine (Pinus sylvestris L.). 84 primers were tested on P. sylvestris DNA samples originating from three different types of habitat. After sequencing and BLAST search of the amplified products, parts of 19 different candidate genes were analysed by considering the polymorphic sites, insertions/deletions as well as synonymous and non-synonymous SNPs. In a total of 3735 sites no indels, eight synonymous and 11 non-synonymous SNPs were found. By providing de novo molecular markers developed in P. cembra and tested for transferability in Scots pine, our results give support for the use of de novo markers targeting conserved regions across different pines. The SNPs detected may have important applications in further studies of adaptive genetic variation, providing tools to study relevant genes important in the long-term adaptation of pine species.

Epicardial fat and coronary artery calcification in patients on long-term hemodialysis.

INTRODUCTION: Recent studies have shown a significant correlation between increased epicardial fat volume (EFV) and mortality, coronary artery disease events, and measures of coronary atherosclerotic burden, for example, coronary calcium. Patients with chronic kidney disease on hemodialysis have an increased prevalence of coronary atherosclerosis and coronary calcium. The mechanisms underlying both may differ from patients with normal kidney function. Only limited data are available on the relationship between epicardial fat and coronary calcium in these patients. METHODS: Ninety-three consecutive patients (62 men and 31 women; mean age, 55 ± 11 years) with chronic kidney failure on regular hemodialysis underwent computed tomography for coronary calcium scoring as well as assessment of cardiovascular risk factors. Calcium scoring was performed using a low-dose, prospectively ECG-triggered high pitch spiral acquisition protocol (dual-source computed tomography, 280-millisecond (ms) rotation, 2 × 128 × 0.6-mm collimation, 120-kV tube voltage, 80-mA·s tube current). Cross-sectional images were reconstructed with 3.0-mm thickness, 1.5-mm increment, and a medium sharp reconstruction kernel (B35f). Agatston score and EVF were analyzed in a semiautomatic fashion using dedicated software. RESULTS: The mean duration of dialysis was 5.7 years. Of all patients, 93% had arterial hypertension, 66% had hyperlipidemia, 30% were diabetic, and 49.5% were current or prior smokers. The mean body mass index (BMI) was 27 ± 4 kg/m. The mean EFV was 162 ± 80 mL, and the mean coronary artery calcification (CAC) was 765 ± 1391 Agatston units (AU). In univariable and multivariable analysis, EFV was significantly correlated to BMI (P < 0.05) and age (P = 0.021), but not to CAC (P = 0.106). In subanalysis for values binned by median, we also found a significant correlation between EFV (binned) and smoking (P = 0.49) as well as a significant correlation between EFV (binned) and CAC for 46 patients younger than 55 years (median age). CONCLUSION: The epicardial fat volume in patients with chronic kidney disease and on hemodialysis is significantly correlated to BMI, age, and smoking but, with the exception of younger patients, not to the coronary calcium score. Our data suggest that in this special patient cohort, other mechanisms might influence the genesis of coronary calcification.

Toward noninvasive follow-up of low-risk bladder cancer - Rationale and concept of the UroFollow trial.

BACKGROUND: Follow-up recommendations for patients with nonmuscle invasive bladder cancer (NMIBC) are largely based upon expert opinion. A growing body of evidence suggests that current follow-up strategies for bladder cancer patients with low and intermediate risk represent overdiagnosis and may lead to overtreatment. The goal of this study is to explore the options of a noninvasive follow-up in patients with pTa G1-2/low-grade NMIBC. METHODS: The risks and options for a urine marker-guided, noninvasive follow-up of patients with pTa G1-2/low-grade NMIBC were defined and the study design for a prospective randomized trial (UroFollow) was developed based upon the current literature. RESULTS: The investigators postulated that follow-up of patients with pTa G1-2/low-grade NMIBC requires a high sensitivity of urinary tumor markers. However, data from prospective studies with prediagnostic urine samples are scarce, even for approved markers, and cross-sectional studies with symptomatic patients overestimate the sensitivity. So far, cell-based markers (e.g., uCyt+ and UroVysion) in urine appeared to have higher sensitivities and specificities in low-grade NMIBC than urine cytology and markers analyzing soluble tumor-associated antigens. Marker panels are more sensitive than single-marker approaches at the expense of a lower specificity. Given a prospective randomized comparison with a marker sensitivity of 80% compared to usual care with cystoscopy, the sample size calculation yielded that 62 to 185 patients under study per arm are needed depending on different recurrence rates. CONCLUSIONS: Based upon these findings the UroFollow trial has been designed as a prospective randomized study comparing a noninvasive marker-based (UroVysion, NMP22, urine cytology, and ultrasound) follow-up with the current standard of care over a period of 3 years.

Urinary transcript quantitation of CK20 and IGF2 for the non-invasive bladder cancer detection.

PURPOSE: Cytokeratin 20 (CK20) and insulin-like growth factor 2 (IGF2) were previously proposed to be elevated in clinical samples from patients with bladder cancer (BCa). A two cohort design validation study was used to assess the relevance for BCa detection by transcript quantitation of both markers in urine samples. Their diagnostic value was assessed in comparison with voided urine cytology (VUC). METHODS: RNA isolation was carried out using cellular sediments of urine samples from 196/103 histologically positive BCa patients, as well as 97/50 control subjects for the test (TC) and validation cohort (VC), respectively. Urinary transcript levels of CK20 and IGF2 were determined by qPCR. RESULTS: Relative transcript levels were significantly elevated 3.4/11-fold for CK20 and 188/64-fold for IGF2 (p < 0.001) in urine sediments of BCa patients compared to controls in the TC and VC, respectively. In a combined analysis, the resulting sensitivity (SN) (SNTC: 77.9; SNVC: 90.3%) and specificity (SP) (SPTC: 88.0; SPVC: 84.0%) were similar to that of VUC. The sensitivity of VUC in combination with CK20 and IGF2 was considerably increased (SNTC: 94.6; SNVC: 93.2%) while specificity was reduced (SPTC: 72.0; SPVC: 82.0%) compared to VUC alone in the test and validation cohort. CONCLUSIONS: Transcript levels of IGF2 and CK20 enabled the detection of BCa with a diagnostic performance similar to VUC. Combined analysis of voided urine cytology together with altered transcript levels of CK20 and IGF2 enhanced sensitivity, but did not improve overall test performance.

Analysis of variation in chloroplast DNA sequences.

This chapter introduces and reviews methods for analyzing variation in chloroplast DNA, mainly by polymerase chain reaction (PCR) and subsequent revelation of polymorphisms. Sources for chloroplast primers are discussed, as well as methods such as Sanger sequencing, PCR followed by restriction fragment length polymorphism (RFLP), gel electrophoresis, fragment analysis on automated DNA sequencers, denaturing high-performance liquid chromatography (dHPLC), and next-generation sequencing (NGS). A special section deals with peculiarities of chloroplast DNA variation, such as tandem repeats and mini- and microsatellites.