Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.     

A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signalling via interaction with human Act1

Chlamydia pneumoniae is a common respiratory pathogen that has been associated with a variety of chronic diseases including asthma and atherosclerosis. Chlamydiae are obligate intracellular parasites that primarily infect epithelial cells where they develop within a membrane-bound vacuole, termed an inclusion. Interactions between the microorganism and eukaryotic cell can be mediated by chlamydial proteins inserted into the inclusion membrane. We describe here a novel C. pneumoniae -specific inclusion membrane protein (Inc) CP0236, which contains domains exposed to the host cytoplasm. We demonstrate that, in a yeast two-hybrid screen, CP0236 interacts with the NFκB activator 1 (Act1) and this interaction was confirmed in HeLa 229 cells where ectopically expressed CP0236 was co-immunoprecipitated with endogenous Act1. Furthermore, we demonstrate that Act1 displays an altered distribution in the cytoplasm of HeLa cells infected with C. pneumoniae where it associates with the chlamydial inclusion membrane. This sequestration of Act1 by chlamydiae inhibited recruitment of the protein to the interleukin-17 (IL-17) receptor upon stimulation of C. pneumoniae -infected cells with IL-17A. Such inhibition of the IL-17 signalling pathway led to protection of Chlamydia -infected cells from NFκB activation in IL-17-stimulated cells. We describe here a unique strategy employed by C. pneumoniae to achieve inhibition of NFκB activation via interaction of CP0236 with mammalian Act1.     

Obesity and Television Watching in Preschoolers in Greece: The GENESIS Study

The aim of the current work was to evaluate the effect of preschoolers' television (TV) watching time on the prevalence of obesity even after controlling for their total energy intake and their physical activity status. A representative sample of 2,374 Greek children aged 1–5 years was examined (“Growth, Exercise and Nutrition Epidemiological Study in preSchoolers”, GENESIS study). Children's TV watching time on a usual weekday and at a usual weekend was recorded. The overall mean of children's TV viewing time was 1.32 h/day. The majority of participants (74.0%) spent <2 h/day watching TV whereas only 3.1% spent >4 h/day in front of a TV set. Overall, 65.2% of participants were normal weight, 17.2% were overweight, and the rest 17.6% were obese. The prevalence of obesity was significantly higher among those with TV viewing time ≥2 h/day (21.7%) compared to those watching TV <2 h/day (16.1%, P = 0.003). TV viewing time remained significantly associated with the likelihood of being obese even after controlling for potential confounders (i.e., socio demographic and other characteristics and physical activity status) only among children aged 3–5 years. However, further adjusting for children's total energy intake revealed that the association between the TV viewing time and the probability of being obese was no longer statistically significant. On the other hand, physical activity status continued to be an independent factor of being obese. The current findings support the hypothesis that the effect of TV viewing time on childhood obesity is independent of physical activity status and may be attributed to the increased total energy intake during TV watching.     

The canine kallikrein-related peptidase 14: Structural characterization, alternative splicing and differential expression in mammary cancer

Human kallikrein-related peptidases (KLKs) represent a family of 15 serine proteases with diverse roles in many physiological and pathological processes, including carcinogenesis. In the dog, only two KLK genes are known; dKLK1 and canine arginine esterase. Recently, 12 other genes have been predicted using computational methods, but none of them has ever been experimentally validated in canine tissues. In this study we investigated the expression of Canis familiaris KLK14, (CANFA)KLK14, in normal and cancerous mammary tissues. First, it was demonstrated that the in-silico determined canine KLK14 mRNA (GenBank accession no: XM_541464) has been wrongfully predicted on its 5′-end (nucleotides 1–88). The (CANFA)KLK14 mRNA sequence presented here, has high homology to its human counterpart and exhibits all defining-KLK features. In addition to the classical form of the gene, five splice variants were also identified. The splicing events involved 5′-truncation or complete elimination of exon 4 and/or retention of intron I. All encoded protein products of the splice variants were predicted to be truncated and catalytically inactive. The classical form and variant 3 were almost ubiquitously expressed in both normal and neoplastic tissues. Variant 1 was predominantly detected in normal tissues. The classical form and variants 1 and 2 exhibited lower expression levels in tumor compared to normal tissues. Moreover, an Ile155Asn polymorphism was identified. This is the first report on the structural characterization, alternative splicing and tissue expression of canine KLK14 mRNA. These findings may form the basis for the establishment of comparative studies investigating KLK functions in health and disease using the dog as a model.     

Changing social awareness of the illegal killing of migratory birds in the Ionian Islands,western Greece

ABSTRACT

The current study aims at monitoring, measuring and evaluating the ‘Safe havens for wild birds’ campaign, implemented within the framework of the LIFE programme, in Greece and more specifically in the Ionian Islands. The study detects attitude changes that occurred in three target groups (pupils, local hunters and residents) on three islands where the phenomenon of illegal spring killing is more intense. A questionnaire-based survey was conducted in two phases, just before the launch (2013) and after the completion of the campaign (2015), to enable a comparison of answers and data. The results show that pupils and to a lesser extent residents, enhanced their knowledge and awareness of poaching, the migration of avifauna and the consequences of illegal killing on migratory birds, while this aspect of attitude change was not observed in the hunting community which still believes that spring poaching should be treated as a legal activity and part of local culture.     

Characterization of β-amylase alleles in 79 barley varieties with pyrosequencing

β-Amylase is involved in the starch degradation process and therefore influences grain quality. Starch degradation efficiency is dependent on the enzyme thermostability during malting and mashing. Four alleles resulting in different enzyme thermostability are known. These alleles are distinguished by coding single nucleotide polymorphism (cSNP). Pyrosequencing was used for cSNP genotyping of β-amylase alleles in 79 spring barley varieties by using analyser PSQ MA96 System (Pyrosequencing, Biotage). A new cSNP was revealed by means of Pyrosequencing analysis of sequence flanking cSNP⁶⁹⁸, thus recognizing a fifth β-amylase allele. Pyrosequencing is a high-throughput, fast, and precise system for barley SNP genotyping.