The effects of stress induced by cortisol administration on the repeatability of swimming performance tests in the European sea bass (Dicentrarchus labrax L.)

The impact of stress induced by cortisol administration on the swimming performance of the European sea bass was tested measuring the fish recovery capability using a respirometer. The recovery test consisted of two exhaustive swimming exercises (U _crit) separated by a recovery period. Thirty-eight fish were subjected to this trial preceded or not (control group) by a cortisol (cortisol group) or saline (placebo) injection. For validation, the classical stress parameters (cortisol, glucose, lysozyme, Hct, Hb and RBCC) were monitored. During the recovery tests, only the cortisol group showed a significant reduction in the recovery capability that was interpreted as a sign of possible physiological impairment. Hct, Hb and RBCC did not show significant inter and intra-group differences, while cortisol and glucose increased as a response to fatigue in all the groups at the end of the trials. No immune (lysozyme) response was observed in control and placebo groups, while a reduction was evidenced in the cortisol group.     

Glycoform-Selective Prion Formation in Sporadic and Familial Forms of Prion Disease

The four glycoforms of the cellular prion protein (PrP^C) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrP^Sc) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrP^Sc in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJD^V180I) or from Thr to Ala at residue 183 (fCJD^T183A). Here we report that fCJD^V180I, but not fCJD^T183A, exhibits a proteinase K (PK)-resistant PrP (PrP^res) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrP^res species in both fCJD^V180I and VPSPr is likewise attributable to the absence of PrP^res glycosylated at the first N-linked glycosylation site at residue 181, as in fCJD^T183A. In contrast to fCJD^T183A, both VPSPr and fCJD^V180I exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrP^V180I with a typical glycoform profile from cultured cells generates detectable PrP^res that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJD^V180I share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrP^C to PrP^Sc is inhibited, probably by a dominant-negative effect, or by other co-factors.     

Whole Exome Sequencing Identifies a Troponin T Mutation Hot Spot in Familial Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) commonly causes heart failure and shows extensive genetic heterogeneity that may be amenable to newly developed next-generation DNA sequencing of the exome. In this study we report the successful use of exome sequencing to identify a pathogenic variant in the TNNT2 gene using segregation analysis in a large DCM family. Exome sequencing was performed on three distant relatives from a large family with a clear DCM phenotype. Missense, nonsense, and splice variants were analyzed for segregation among the three affected family members and confirmed in other relatives by direct sequencing. A c.517T C>T, Arg173Trp TNNT2 variant segregated with all affected family members and was also detected in one additional DCM family in our registry. The inclusion of segregation analysis using distant family members markedly improved the bioinformatics filtering process by removing from consideration variants that were not shared by all affected subjects. Haplotype analysis confirmed that the variant found in both DCM families was located on two distinct haplotypes, supporting the notion of independent mutational events in each family. In conclusion, an exome sequencing strategy that includes segregation analysis using distant affected relatives within a family represents a viable diagnostic strategy in a genetically heterogeneous disease like DCM.     

Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Str?ussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy

Prion diseases are classically characterized by the accumulation of pathological prion protein (PrP^Sc) with the protease resistant C-terminal fragment (PrP^res) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrP^res) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrP^Sc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrP^res production and its cleavage sites after PK digestion were analyzed, along with the PrP^Sc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrP^Sc components. All these PrP^Sc types shared common and distinctive biochemical features compared to PrP^Sc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrP^res cleavage sites and PrP^Sc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrP^Sc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.     

Ips pini (Curculionidae: Scolytinae) is a vector of the fungal pathogen, Sphaeropsis sapinea (Coelomycetes), to Austrian pines, Pinus nigra (Pinaceae)

Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton, is among the most common and widely distributed pathogens of conifers worldwide. S. sapinea is disseminated over short distances by rain splash and moist wind, but significant knowledge gaps regarding long-range dispersal remain. Our objective was to determine whether or not the pine engraver beetle, Ips pini Say, is a vector of the pathogen onto Austrian pines (Pinus nigra Arnold). In 2004 and 2005, individuals of I. pini were collected with pheromone traps at two locations in central Ohio (197 and 1,017 individuals for 2004 and 2005, respectively) and screened for the presence of S. sapinea. In the field, fresh logs of Austrian pine were baited with pheromone lures, mechanically wounded, or left undisturbed. After 2 mo, logs were evaluated for insect feeding and the presence of S. sapinea along beetle galleries. Fresh logs were also inoculated in the greenhouse with adult I. pini that were either artificially infested or uninfested with S. sapinea spores to determine vectoring potential. Phoresy rates for individual collections ranged from 0 to 4.1%; average rates were 1.5 and 2.0% for 2004 and 2005, respectively. Isolation frequencies of S. sapinea from baited (15 +/- 5%) and unbaited logs (3 +/- 1%) differed significantly (P=0.009). I. pini was also capable of transmitting the pathogen under controlled conditions. Based on phoresy rates, association, and artificial inoculation studies, we conclude that I. pini is able to transmit S. sapinea to Austrian pine stems.     

Regional mapping of myocardial hibernation phenotype in idiopathic end‐stage dilated cardiomyopathy

Myocardial hibernation (MH) is a well‐known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle‐ejection fraction (LV‐EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter‐ventricular septum (IVS) and antero‐lateral free wall (FW) were transmurally (i.e. sub‐epicardial, mesocardial and sub‐endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U‐shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub‐endocardial and sub‐epicardial layers of DCM as compared with ICM. HIF1‐α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM‐LV and ICM‐LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end‐stage ICM as well as in end‐stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.     

Topical KGF treatment as a therapeutic strategy for vaginal atrophy in a model of ovariectomized mice

One of the most frequent complaints for post‐menopausal women is vaginal atrophy, because of reduction in circulating oestrogens. Treatments based on local oestrogen administration have been questioned as topic oestrogens can reach the bloodstream, thus leading to consider their safety as controversial, especially for patients with a history of breast or endometrial cancers. Recently, growth factors have been shown to interact with the oestrogen pathway, but the mechanisms still need to be fully clarified. In this study, we investigated the effect of keratinocyte growth factor (KGF), a known mitogen for epithelial cells, on human vaginal mucosa cells, and its potential crosstalk with oestrogen pathways. We also tested the in vivo efficacy of KGF local administration on vaginal atrophy in a murine model. We demonstrated that KGF is able to induce proliferation of vaginal mucosa, and we gained insight on its mechanism of action by highlighting its contribution to switch ERα signalling towards non‐genomic pathway. Moreover, we demonstrated that KGF restores vaginal trophism in vivo similarly to intravaginal oestrogenic preparations, without systemic effects. Therefore, we suggest a possible alternative therapy for vaginal atrophy devoid of the risks related to oestrogen‐based treatments, and a patent (no. RM2012A000404) has been applied for this study.     

Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.     

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X

Background  

Classical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae.