Detection of IGF2BP3, HOXB7, and NEK2 mRNA Expression in Brush Cytology Specimens as a New Diagnostic Tool in Patients with Biliary Strictures

Introduction

It is a challenging task to distinguish between benign and malignant lesions in patients with biliary strictures. Here we analyze whether determination of target gene mRNA levels in intraductal brush cytology specimens may be used to improve the diagnosis of bile duct carcinoma.

Materials and Methods

Brush cytology specimens from 119 patients with biliary strictures (malignant: n = 72; benign: n = 47) were analyzed in a retrospective cohort study. mRNA of IGF-II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), Forkhead box M1 (FOXM1), kinesin family member 2C (KIF2C) and serine/threonine kinase NEK2 was determined by semi-quantitative RT-PCR using the ΔCt method.

Results

IGF2BP3 (p<0.0001), HOXB7 (p<0.0001), and NEK2 (p<0.0001) mRNA expression levels were significantly increased in patients with cholangiocarcinoma or pancreatic cancer. Median ΔCt values differed by 3.5 cycles (IGF2BP3), 2.8 cycles (HOXB7) and 1.3 cycles (NEK2) corresponding to 11-fold, 7-fold and 2.5-fold increased mRNA levels in malignant versus benign samples. Sensitivity to detect biliary cancer was 76.4% for IGF2BP3 (80.9% specificity); 72.2% for HOXB7 (78.7% specificity) and 65.3% for NEK2 (72.3% specificity), whereas routine cytology reached only 43.1% sensitivity (85.4% specificity). Diagnostic precision was further improved, when all three molecular markers were assessed in combination (77.8% sensitivity, 87.2% specificity) and achieved 87.5% sensitivity and 87.2% specificity when molecular markers were combined with routine cytology.

Conclusions

Our data suggest that measuring IGF2BP3, HOXB7 and NEK2 mRNA levels by RT-PCR in addition to cytology has the potential to improve detection of malignant biliary disorders from brush cytology specimens.     

Crystal structure of Diedel, a marker of the immune response of Drosophila melanogaster

Background

The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines.

Methodology/Principal Findings

We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 Å resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel β-sheet covered by an α-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families.

Conclusion/Significance

Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small family of proteins present in drosophilids, aphids and DNA viruses infecting lepidopterans. Diedel is an extracellular protein composed of two sub-domains. Two special structural features (hydrophobic surface patch and cis/trans conformation for proline 52) may indicate a putative interaction site, and support an extra cellular signaling function for Diedel, which is in accordance with its proposed role as negative regulator of the JAK/STAT signaling pathway.     

The complete nucleotide sequences of the 5 genetically distinct plastid genomes of Oenothera, subsection Oenothera: II. A microevolutionary view using bioinformatics and formal genetic data

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.     

IRS1-independent defects define major nodes of insulin resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.     

Reduced lung function in a chronic asthma model is associated with prolonged inflammation, but independent of peribronchial fibrosis

Background

In asthma, mechanisms contributing to chronicity remain to be determined. Recent models of sensitisation with prolonged airway allergen challenges reproduce typical features of chronic asthma. However, the interplay between inflammation, structural changes and lung function is poorly understood. This study was performed to delineate functional, structural and immunological airway changes after cessation of long term challenges to elucidate factors contributing to the development of prolonged lung function changes.

Methodology/Principal Findings

Mice sensitised systemically were consecutively challenged intranasally with ovalbumin for two or eight weeks. After the end of challenges, lung function, airway inflammation, features of airway remodelling, local T-cell cytokines and systemic ovalbumin-specific antibodies were monitored. Long term challenges resulted in airway hyperresponsiveness lasting 2 weeks and reduced baseline lung function for 6 weeks after their cessation. In contrast, these changes resolved within one week after short term challenges. Prolonged transforming growth factor beta (TGF-β)1 production and marked peribronchial fibrosis were only induced by long term challenges. Importantly, fibrosis became apparent only after the onset of lung function changes and outlasted them. Further, long term challenges led to prolonged and intense airway inflammation with marked lymphocytosis, but moderate eosinophilia, sustained IL-5 production and ovalbumin-specific IgG2a antibodies, the latter suggesting a Th1 component to the immune response. In contrast, following short term challenges airway inflammation was dominated by eosinophils and associated with a strong, but transient IL-13 response.

Conclusions

Prolonged lung function changes after long term allergen challenges seem to develop and resolve independently of the persistent peribronchial fibrosis. They are more closely associated with intense airway inflammation, marked lymphocytosis, prolonged IL-5 and TGF-β1 production in the airways and a Th1 immune response.     

Left atrial enlargement and clinical considerations in patients with or without a residual interatrial shunt after closure of the left atrial appendage with the WATCHMAN™-device

Background

Interventional closure of the left atrial appendage (LAA) in patients with non-valvular atrial fibrillation, high thromboembolic and bleeding risk or bleeding history is an alternative therapeutic strategy to oral anticoagulation. It is not known if the exclusion of the LAA from the blood circulation affects the left atrial volume (LAV) and consequently its prognostic value or the circulatory performance of the heart in humans.

Methods

We aimed to prospectively assess potential changes in baseline LAV, left ventricular ejection fraction (LVEF), NT-proBNP-level and the covered distance in the 6-min walk-test 6 weeks and 6 months after LAA closure with the WATCHMAN? device. We used serial 3-dimensional transthoracic and transesophageal echocardiography to assess LAV, residual interatrial shunt and device performance in 58 consecutive patients with successful LAA closure.

Results

Accurate 3D–echocardiographic data for LAV measurements were evaluable for 51 (91%) patients. Maximum LAV (LAVmax) at baseline was 102.8?±?30.8 ml and increased significantly to 107.7?±?32.8 ml after 6 weeks (p?<?0.01) and 113.5?±?34.2 ml after 6 months (p?<?0.01). Minimal LAV (LAVmin) increased from 76.9?±?29.5 ml at baseline to 81.8?±?30.2 ml after 45 days (p?<?0.01) and 82.1?±?33.3 ml after 6 months (p?<?0.01). Similarly, their indexes to BSA (LAVImax and LAVImin) increased significantly, as well. Patients without a residual left-to-right interatrial shunt showed a significantly higher increase in LAVmax or LAVmin. Baseline LVEF, NT-proBNP-level or the distance covered at the 6-min walk test did not significantly change 6 weeks or 6 months after LAA closure.

Conclusions

LAVmax and LAVmin increase significantly after interventional LAA closure. LA enlargement does not correlate with clinical progression of heart failure. Persistent left-to-right interatrial shunt counteracts the LA enlargement. A reduced LA compliance after exclusion of the LAA from the blood circulation with consecutive increase in LA pressure may be a potential cause of LA enlargement and warrants further investigation.

Trial registration

German Clinical Trials Register ID: DRKS00010768; Registration Date 07.07.2016.

     

Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: A subcellular correlate to ischemic preconditioning?

Background: There is increasing evidence that mitochondria – owning a high degree of autonomy within the cell – might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K _ATP) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia.Methods and Results: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K _ATP opener diazoxide (30 μmol/l) and was completely inhibited by the K _ATP blocker 5-hydroxydecanoic acid (300 μmol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia.Conclusion: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K_ATP but is independent of ATP preservation.     

Molecular Mechanisms of Inherited Arrhythmias

Inherited arrhythmias and conduction system diseases are known causes of sudden cardiac death and are responsible for significant mortality and morbidity in patients with congenital heart disease and electrical disorders. Knowledge derived from human genetics and studies in animal models have led to the discovery of multiple molecular defects responsible for arrhythmogenesis. This review summarizes the molecular basis of inherited arrhythmias in structurally normal and altered hearts.On the cellular and molecular levels, minor disturbances can provoke severe arrhythmias. Ion channels are responsible for the initiation and propagation of the action potential within the cardiomyocyte. Structural heart diseases, such as hypertrophic or dilated cardiomyopathies, increase the likelihood of cardiac electrical abnormalities. Ion channels can also be up- or down-regulated in congenital heart disease, altering action potential cellular properties and therefore triggering arrhythmias. Conduction velocities may be inhomogeneously altered if connexin function, density or distribution changes. Another important group of electrophysiologic diseases is the heterogeneous category of inherited arrhythmias in the structurally normal heart, with a propensity to sudden cardiac death. There have been many recent relevant discoveries that help explain the molecular and functional mechanisms of long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and other electrical myopathies. Identification of molecular pathways allows the identification of new therapeutic targets, for both disease palliation and cure. As more disease-causing mutations are identified and genotypic-phenotypic correlation is defined, families can be screened prior to symptom-onset and patients may potentially be treated in a genotype-specific manner, opening the doors of cardiac electrophysiology to the emerging field of pharmacogenomics.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.