Divergence of annual and perennial species in the Brassicaceae and the contribution of cis-acting variation at FLC orthologues.

Variation in life history contributes to reproductive success in different environments. Divergence of annual and perennial angiosperm species is an extreme example that has occurred frequently. Perennials survive for several years and restrict the duration of reproduction by cycling between vegetative growth and flowering, whereas annuals live for 1 year and flower once. We used the tribe Arabideae (Brassicaceae) to study the divergence of seasonal flowering behaviour among annual and perennial species. In perennial Brassicaceae, orthologues of FLOWERING LOCUS C (FLC), a floral inhibitor in Arabidopsis thaliana, are repressed by winter cold and reactivated in spring conferring seasonal flowering patterns, whereas in annuals, they are stably repressed by cold. We isolated FLC orthologues from three annual and two perennial Arabis species and found that the duplicated structure of the A. alpina locus is not required for perenniality. The expression patterns of the genes differed between annuals and perennials, as observed among Arabidopsis species, suggesting a broad relevance of these patterns within the Brassicaceae. Also analysis of plants derived from an interspecies cross of A. alpina and annual A. montbretiana demonstrated that cis-regulatory changes in FLC orthologues contribute to their different transcriptional patterns. Sequence comparisons of FLC orthologues from annuals and perennials in the tribes Arabideae and Camelineae identified two regulatory regions in the first intron whose sequence variation correlates with divergence of the annual and perennial expression patterns. Thus, we propose that related cis-acting changes in FLC orthologues occur independently in different tribes of the Brassicaceae during life history evolution.

High-definition endoscopy with iScan and Lugol's solution for the detection of inflammation in patients with nonerosive reflux disease: histologic evaluation in comparison with a control group.

Nonerosive reflux disease (NERD) is commonly diagnosed in patients with symptoms of reflux. The aim of the present study was to determine whether high-definition endoscopy (HD) plus equipped with the iScan function or chromoendoscopy with Lugol's solution might permit the differentiation of NERD patients from those without reflux symptoms, proven by targeted biopsies of endoscopic lesions. A total of 100 patients without regular intake of proton pump inhibitors and with a normal conventional upper endoscopy were prospectively divided into NERD patients and controls. A second upper endoscopy was performed using HD+ with additional iScan function and then Lugol's solution was applied. Biopsy specimens were taken from the gastroesophageal junction in all patients. A total of 65 patients with reflux symptoms and 27 controls were included. HD(+) endoscopy with iScan revealed subtle mucosal breaks in 52 patients; the subsequent biopsies confirmed esophagitis in all cases. After Lugol's solution, 58 patients showed mucosal breaks. Sensitivity for the iScan procedure was 82.5%, whereas that for Lugol's solution was 92.06%. Excellent positive predictive values of 100% and 98.3%, respectively, were noted. The present study suggests that the majority of patients with NERD and typical symptoms of reflux disease can be identified by iScan or Lugol's chromoendoscopy as minimal erosive reflux disease (ERD) patients.

Methylation status of imprinted genes and repetitive elements in sperm DNA from infertile males.

Stochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly higher in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby.

A new algorithm for improving fetal weight estimation from ultrasound data at term.

OBJECTIVE: The purpose of this retrospective study was to find a method of improving the accuracy of fetal birth weight estimation on the basis of traditional ultrasonographic measurements of the head, thorax, and femur at term. In this context, we analyzed a novel regression method comparing to existing algorithms. METHODS: The delivery records of two hospitals were searched for women who delivered macrosomic infants, and the patients' medical records were retrospectively reviewed in order to derive clinical and ultrasonographic data at term. A total of 223 patients with macrosomic infants (birth weight > 4,000 g) were identified. These patients were complemented by data for 212 women who had ultrasound fetal assessments of less than 4,000 g. We used the method of isotonic regression to construct a birth weight prediction function that increases monotonically with each of the input variables and which minimizes the empirical quadratic loss. RESULTS: A suspicion of macrosomia was based on a history of macrosomia, fundal height, and sonographic weight estimation >4,000 g. The mean period between ultrasound weight estimation and delivery was 7.2 days. The ability of the biometric algorithms developed to predict fetal weight at term ranged between a mean absolute error of 312 and 344 g, given a confidence interval of 95%. We demonstrate that predictions of birth weight on the basis of ultrasound data can be improved significantly, if an isotonic regression model is used instead of a linear regression model. CONCLUSIONS: This study demonstrates that ultrasound detection of macrosomia can be improved using the isotonic regression method.

High definition colonoscopy combined with i-Scan is superior in the detection of colorectal neoplasias compared with standard video colonoscopy: a prospective randomized controlled trial.

INTRODUCTION: Colonoscopy is the accepted gold standard for the detection of colorectal cancer. The aim of the current study was to prospectively compare high definition plus (HD+) colonoscopy with I-Scan functionality (electronic staining) vs. standard video colonoscopy. The primary endpoint was the detection of patients having colon cancer or at least one adenoma. METHODS: A total of 220 patients due to undergo screening colonoscopy, postpolypectomy surveillance or with a positive occult blood test were randomized in a 1 : 1 ratio to undergo HD+ colonoscopy in conjunction with I-Scan surface enhancement (90i series, Pentax, Tokyo, Japan) or standard video colonoscopy (EC-3870FZK, Pentax). Detected colorectal lesions were judged according to type, location, and size. Lesions were characterized in the HD+ group by using further I-Scan functionality (p- and v-modes) to analyze pattern and vessel architecture. Histology was predicted and biopsies or resections were performed on all identified lesions. RESULTS: HD+ colonoscopy with I-Scan functionality detected significantly more patients with colorectal neoplasia (38 %) compared with standard resolution endoscopy (13 %) (200 patients finally analyzed; 100 per arm). Significantly more neoplastic (adenomatous and cancerous) lesions and more flat adenomas could be detected using high definition endoscopy with surface enhancement. Final histology could be predicted with high accuracy (98.6 %) within the HD+ group. CONCLUSIONS: HD+ colonoscopy with I-Scan is superior to standard video colonoscopy in detecting patients with colorectal neoplasia based on this prospective, randomized, controlled trial.

Recognition and characterization of small colonic neoplasia with high-definition colonoscopy using i-Scan is as precise as chromoendoscopy.

BACKGROUND: The EPKi system (Pentax, Japan) enables resolution above HDTV. Aim of the study was to test the efficacy of HD+ alone and with the new post-processing digital filter i-Scan or chromoendoscopy (Methylene blue 0.1%) in screening for colorectal cancer. We focused on lesions less than 5 mm as a surrogate marker for the optical possibilities of the EPKi system. METHODS: The last 30 cm of the colon in a screening population were inspected with HD+ alone, in combination with i-Scan (2:1 randomisation) and subsequently with chromoendoscopy. All lesions were characterized and targeted biopsies were performed. RESULTS: i-Scan augmented in 69 patients the identification of lesions from 176 to 335 (p<0.001) and chromoendoscopy to 646 (p<0.001). The additional lesions were mainly flat (type IIb, 74%), which were only recognized using i-Scan or chromoendoscopy. The amount of neoplasias was not significantly different (HD+: 5, i-Scan: 11, Chromoendoscopy: 11), but all could correctly be predicted using i-Scan or chromoendoscopy. CONCLUSIONS: HD+ colonoscopy with and without i-Scan unmask a plethora of small lesions but chromoendoscopy can even advance the number. However, i-Scan was able to predict neoplasia as precisely as chromoendoscopy and might shortly replace chromoendoscopy as a more time efficient tool.

High-definition endoscopy with i-Scan and Lugol's solution for more precise detection of mucosal breaks in patients with reflux symptoms.

BACKGROUND AND STUDY AIMS: Patients with gastroesophageal reflux disease are subdivided into non-erosive (NERD) and erosive reflux disease (ERD). The newly available EPKi processor enables high-definition resolution above HDTV standard (HD+). The aim of the study was to test the efficacy of HD+ esophagogastroduodenoscopy alone and in conjunction with i-Scan (newly developed postprocessing digital filter) and chromoendoscopy (Lugol's solution) for differentiation of reflux patients. METHODS: The distal esophagus of patients with heartburn was inspected with three imaging modalities. HD+ was followed by i-Scan and 15-mL Lugol's solution (1.5 %). The esophagus was evaluated for mucosal breaks (Los Angeles Classification [LA]). Small visible changes were also characterized, and targeted biopsies were performed. End points of the study were the presence and grade of esophagitis and the number of circumscribed changes. RESULTS: A total of 50 patients were included (female 29; mean age 54.7 years). HD+ identified nine patients with mucosal breaks (LA 7A; 2C), i-Scan was able to detect 12 patients (LA 8A; 2B; 2C; 0D) ( P = n. s.) and chromoendoscopy identified 25 patients (LA 16A; 7B; 1C, 1D) ( P < 0.01). Furthermore, a higher grade of esophagitis was recognized by using i-Scan and Lugol's solution in 19 patients. The number of circumscribed lesions could be increased from 21 (HD+) to 58 (i-Scan) ( P < 0.01), and up to 85 after Lugol spraying ( P < 0.01). CONCLUSIONS: Lugol's solution in conjunction with HD+ endoscopy significantly improves the identification of patients with esophagitis and reduces misclassification. The i-Scan filter and chromoendoscopy help to identify reflux-associated lesions.

Cell viability, osteoblast differentiation, and gene expression are altered in human osteoblasts from hypertrophic fracture non-unions.

Recent studies have provided evidence that the number and proliferation capacity of bone marrow-derived mesenchymal stem cells, as well as the number of osteoprogenitor cells are reduced in patients with fracture non-unions. For fracture non-unions that do not heal after appropriate surgical intervention, the question arises as to what extent systemic cellular dysfunctions should be considered as being pathogenetic factors. For this purpose, we have examined the hypothesis that the cell function of osteoblasts isolated from patients with fracture non-unions may differ from those of normal control individuals in an identical and controlled in vitro situation. We analyzed the osteoblast cell viability, formation of alkaline phosphatase-positive (CFU-ALP) and mineralization-positive (CFU-M) colony forming units, as well as global differences of gene expression in osteoblasts from patients with fracture non-unions and from control individuals. We found that cell viability and CFU-M-formation were significantly reduced in non-union osteoblasts. This was accompanied by significant differences in osteoblast gene expression as revealed by Affymetrix-microarray analysis and RT-PCR. We identified a set of significantly down-regulated factors in non-union osteoblasts that are involved in regulation of osteoblast proliferation and differentiation processes (canonical Wnt-, IGF-, TGF-beta-, and FGF-signaling pathways). The results of the present study strongly support the hypothesis that cell viability, differentiation, and gene expression of osteoblasts may be altered in patients who develop recurrent and recalcitrant fracture non-unions. Proteins involved in Wnt-, IGF, TGF-beta-, and FGF-signaling pathways may be of particular interest and may unveil new potential therapies.

Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes--coincident rise of DNA-relaxing activity in nuclear extracts.

High affinity receptors (VDR) for 1,25-dihydroxycholecalciferol (calcitriol) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes (PBL). HL60 cells, expressing some characteristics of promyelocytes, can be induced to monocytoid differentiation by calcitriol. Specific nuclear translocation of [3H]calcitriol/VDR was examined after exposure of whole cells to 10(-9) M/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei. Specific nuclear translocation of [3H]calcitriol/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites/nucleus after 3 h of incubation in HL60 cells, whereas a maximum of approximately 310 binding sites/nucleus was found after 3 h in PBL. Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h. Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse (pulse/chase-experiments). No difference of VDR retention in pulse and pulse/chase-experiments was seen in PBL, where VDR halflife was approximately 30 min. No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in [3H]calcitriol. Radiolabeled hormone/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone--in contrast to identical experiments with intact cells--did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound. The activity of DNA relaxing enzymes (e.g. topoisomerases I and II) in nuclear extracts was measured using a PBR 322-relaxation-assay. Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol (final ethanol concentration 0.0001% v/v) in HL60 and PBL. The enhanced activity disappeared after 2 h in PBL, whereas it was still enhanced by 4 h in HL60. No effect was seen in ethanol treated controls. We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined, most likely due to translocation of receptor proteins after hormone binding. Translocated hormone/receptor complexes compete for a limited number of specific nuclear binding sites. Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment, thus initiating DNA-unwinding, a prerequisite of transcription initiation.