Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.     

Polymorphism rs11085226 in the Gene Encoding Polypyrimidine Tract-Binding Protein 1 Negatively Affects Glucose-Stimulated Insulin Secretion

Objective

Polypyrimidine tract-binding protein 1 (PTBP1) promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in β-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion.

Methods

We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs) within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698) covering 100% of genetic variation with an r²≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT.

Results

PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04). The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103). Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108). Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018), but the rs351974 was not.

Conclusions

We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo.     

MALDI-TOF mass spectrometry screening of cholelithiasis risk markers in the gene of HNF1alpha

In recent years MALDI-TOF MS gained importance for high-throughput DNA analysis. In the present study this technique was used for the pathogenetic analysis of gallstone disease. The intestinal apical sodium-dependent bile acid transporter (ASBT) shows a genetic association with gallstone disease. ASBT has 3 binding sites in its 5'UTR for hepatocyte nuclear factor 1alpha (HNF1alpha). We hypothesized that genetic alterations in the HNF1alpha gene could influence ASBT expression. The gene HNF1alpha was sequenced in 46 Stuttgart random samples, composed of 16 controls and 30 gallstone patients. Subsequently, two independent cohorts (Stuttgart: 67 gallstones carriers, 109 controls, Leutkirch: 112 gallstone carriers, 99 controls) were screened by MALDI-TOF MS. The subjects were further divided by gender and weight. 24 known polymorphisms and two novel SNPs in the 3'UTR of HNF1alpha were detected (c.*220G>A and c.*1151G>A). After gender-specific sub-division of the pooled cohorts, 4 SNPs resulted in significant differences between male gallstone carriers and male controls (Stuttgart/Leutkirch: rs2255531 OR=2.78; p=0.006, rs1169288 OR=2.13; p=0.032, rs7310409 OR=2.34; p=0.025 and rs1169294 OR=2.13; p=0.031). Two novel variants in the 3'UTR of HNF1alpha were detected and four SNPs of HNF1alpha show a significant association to cholelithiasis in male gallstone patients. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Combined Micro-PET/Micro-CT Imaging of Lung Tumours in SPC-raf and SPC-myc Transgenic Mice

Introduction

SPC-raf and SPC-myc transgenic mice develop disseminated and circumscribed lung adenocarcinoma respectively, allowing for assessment of carcinogenesis and treatment strategies. The purpose of this study was to investigate the technical feasibility, the correlation of initial findings to histology and the administered radiation dose of combined micro-PET/micro-CT in these animal models.

Material and Methods

14 C57BL/6 mice (4 nontransgenic, 4 SPC-raf transgenic, 6 SPC-myc transgenic) were examined using micro-CT and ¹⁸F-Fluoro-deoxyglucose micro-PET in-vivo. Micro-PET data was corrected for random events and scatter prior to reconstruction with a 3D-FORE/2D-OSEM iterative algorithm. Rigid micro-PET/micro-CT registration was performed. Tumour-to-non-tumour ratios were calculated for different lung regions and focal lesions. Diffuse tumour growth was quantified using a semiautomated micro-CT segmentation routine reported earlier. Regional histologic tumour load was assessed using a 4-point rating scale. Gamma radiation dose was determined using thermoluminescence dosimeters.

Results

Micro-CT allowed visualisation of diffuse and circumscribed tumours in SPC-raf and SPC-myc transgenic animals along with morphology, while micro-PET provided information on metabolism, but lacked morphologic detail. Mean tumour-to-non-tumour ratio was 2.47 for circumscribed lesions. No significant correlation could be shown between histological tumour load and tumour-to-nontumour ratio for diffuse tumours in SPC-raf transgenic animals. Calculation of the expected dose based on gamma dosimetry yielded approximately 140 mGy/micro-PET examination additional to approximately 200 mGy due to micro-CT.

Conclusions

Combined micro-PET/micro-CT imaging allows for in-vivo assessment of lung tumours in SPC-raf and SPC-myc transgenic mice. The technique has potential for the evaluation of carcinogenesis and treatment strategies in circumscribed lung tumours.     

The Return of the Intruder: Immediate and Later Effects of Different Approach Distances in a Territorial Songbird

Male songbirds often maintain territories throughout the breeding season, and one of the main functions of song is to deter invaders. Therefore, the distance of an unknown singing rival should play a crucial role within territorial singing interactions of males. This distance is expected to be assessed as more threatening the closer the rival approaches. Here, we tested this assumption by conducting nocturnal playbacks from two different distances in territorial Common nightingales (Luscinia megarhynchos). Immediate vocal responses of birds were examined by analysing changes in song structure as well as temporal response features. The next morning, follow‐up playbacks from an intermediate distance allowed us to investigate longer‐lasting effects of nocturnal playbacks. We found that the distance of a simulated rival had an effect on both immediate and later vocal responses of territorial male nightingales with different song parameters being affected during nocturnal and diurnal singing. This indicates that birds perceive intruder distances and adjust their response behaviour both immediately and in later interactions.     

A chemical genetic approach for covalent inhibition of analogue-sensitive aurora kinase

The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast.     

Alpha-synuclein aggregation involves a bafilomycin A 1-sensitive autophagy pathway

Synucleinopathies like Parkinson disease and dementia with Lewy bodies (DLB) are characterized by α-synuclein aggregates within neurons (Lewy bodies) and their processes (Lewy neurites). Whereas α-synuclein has been genetically linked to the disease process, the pathological relevance of α-synuclein aggregates is still debated. Impaired degradation is considered to result in aggregation of α-synuclein. In addition to the ubiquitin-proteasome degradation, the autophagy-lysosomal pathway (ALP) is involved in intracellular degradation processes for α-synuclein. Here, we asked if modulation of ALP affects α-synuclein aggregation and toxicity. We have identified an induction of the ALP markers LAMP-2A and LC3-II in human brain tissue from DLB patients, in a transgenic mouse model of synucleinopathy, and in a cell culture model for α-synuclein aggregation. ALP inhibition using bafilomycin A 1 (BafA1) significantly potentiates toxicity of aggregated α-synuclein species in transgenic mice and in cell culture. Surprisingly, increased toxicity is paralleled by reduced aggregation in both in vivo and in vitro models. The dichotomy of effects on aggregating and nonaggregating species of α-synuclein was specifically sensitive to BafA1 and could not be reproduced by other ALP inhibitors. The present study expands on the accumulating evidence regarding the function of ALP for α-synuclein degradation by isolating an aggregation specific, BafA1-sensitive, ALP-related pathway. Our data also suggest that protein aggregation may represent a detoxifying event rather than being causal for cellular toxicity.     

Hitchhiking with forests: population genetics of the epiphytic lichen Lobaria pulmonaria in primeval and managed forests in southeastern Europe

Availability of suitable trees is a primary determinant of range contractions and expansions of epiphytic species. However, switches between carrier tree species may blur co‐phylogeographic patterns. We identified glacial refugia in southeastern Europe for the tree‐colonizing lichen Lobaria pulmonaria, studied the importance of primeval forest reserves for the conservation of genetically diverse populations and analyzed differences in spatial genetic structure between primeval and managed forests with fungus‐specific microsatellite markers. Populations belonged to either of two genepools or were admixed. Gene diversity was higher in primeval than in managed forests. At small distances up to 170 m, genotype diversity was lower in managed compared with primeval forests. We found significant associations between groups of tree species and two L. pulmonaria genepools, which may indicate “hitchhiking” of L. pulmonaria on forest communities during postglacial migration. Genepool B of L. pulmonaria was associated with European Beech (Fagus sylvatica) and we can hypothesize that genepool B survived the last glaciation associated within the refuge of European Beech on the Coastal and Central Dinarides. The allelic richness of genepool A was highest in the Alps, which is the evidence for a northern refuge of L. pulmonaria. Vicariant altitudinal distributions of the two genepools suggest intraspecific ecological differentiation.