Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed inEscherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced inE. coliby influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in anE. colisystem that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368and Lys370play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions

Microbiological and 16S rRNA analysis of sulphite-reducing clostridia from river sediments in central Italy

<p>Abstract</p> <p>Background</p> <p>Microbiological indicators are commonly used in the assessment of public health risks associated with fecal contamination of freshwater ecosystems. Sediments are a reservoir of microorganisms, and can thus provide information on past pollution events, not obtainable through the testing of surface water. Moreover, pathogens present in sediment may represent future threats to human health. <it>Clostridium perfringens</it>, a typical colonizer of sediments, has been suggested as an alternative indicator of fecal pollution. In order to be suitable for such purpose, the microorganism should be widely distributed in contaminated environments. The objective of this study was thus to determine the composition of the anaerobic community in sediment samples of the lower Tiber basin, in central Italy, through a combined approach involving granulometric analysis of sediment samples, as well as a microbiological and molecular (16S rRNA) analysis of strains.</p> <p>Results</p> <p>Granulometry showed a similar, clayey sediment composition, in most sampling sites. The microbiological method, employing, an adaptation of the standard method, proved to be effective in isolating anaerobic bacteria from the environmental matrix for the purpose of genetic analysis. Eighty-three strains of bacteria were isolated and the partial 16S rRNA gene sequenced. While biochemical analysis detected only <it>C. perfringens </it>strains, phylogenetic analysis indicated the presence of three clusters: <it>C. perfringens, C. bifermentans </it>and <it>B. cereus</it>, comprising eight taxa. <it>C. perfringens</it>, the commonest in almost all sediment sampling sites, was present in all sites, and in both seasons (seasonal sampling was carried out only along the Tiber and Aniene rivers). None of the described genetic profiles showed complete similarity with GenBank sequences.</p> <p>Conclusion</p> <p>The study underlines the value of <it>C. perfringens </it>as an alternative microbial indicator of fecal contamination in river sediments. This is supported by the bacterium's presence in all sampling sites, and in both seasons, coupled with its detectability using commercial diagnostic kits.</p> <p>The study also illustrates the presence of an anaerobic community of considerable biodiversity in the lower Tiber basin, with <it>C. perfringens </it>as its main component. The 16S rRNA analysis, while confirming the phylogenetic relationships among isolated species, also showed haplotype patterns different from those present in the NCBI database.</p

Biochemical and neuropathological findings in a Creutzfeldt-Jakob Disease patient with the rare Val180Ile-129Val haplotype in the prion protein gene

Genetic Creutzfeldt-Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrPSc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians

Ongoing outbreak of hepatitis A in Italy: preliminary report as of 31 May 2013

Since January 2013, an unusual increase in hepatitis A cases has been detected in northern Italy. A total number of 352 cases were reported to the integrated surveillance system between January and the end of May 2013 and this represents a 70% increase compared to the same period of the previous year. The outbreak is ongoing and the public health authorities are continuing their investigations to establish the transmission vehicle and to control the outbreak

An exploration of the influence of diagonal dissociation and moderate changes in speed on locomotor parameters in trotting horses

Background. Although the trot is described as a diagonal gait, contacts of the diagonal pairs of hooves are not usually perfectly synchronized. Although subtle, the timing dissociation between contacts of each diagonal pair could have consequences on gait dynamics and provide insight into the functional strategies employed. This study explores the mechanical effects of different diagonal dissociation patterns when speed was matched between individuals and how these effects link to moderate, natural changes in trotting speed. We anticipate that hind-first diagonal dissociation at contact increases with speed, diagonal dissociation at contact can reduce collision-based energy losses and predominant dissociation patterns will be evident within individuals. Methods. The study was performed in two parts: in the first 17 horses performed speedmatched trotting trials and in the second, five horses each performed 10 trotting trials that represented a range of individually preferred speeds. Standard motion capture provided kinematic data that were synchronized with ground reaction force (GRF) data from a series of force plates. The data were analyzed further to determine temporal, speed, GRF, postural, mass distribution, moment, and collision dynamics parameters. Results. Fore-first, synchronous, and hind-first dissociations were found in horses trotting at (3.3 m/s � 10%). In these speed-matched trials, mean centre of pressure (COP) cranio-caudal location differed significantly between the three dissociation categories. The COP moved systematically and significantly (P D :001) from being more caudally located in hind-first dissociation (mean locationD0.41�0.04) through synchronous (0.36 � 0.02) to a more cranial location in fore-first dissociation (0.32 � 0.02). Dissociation patterns were found to influence function, posture, and balance parameters. Over a moderate speed range, peak vertical forelimb GRF had a strong relationship with dissociation time (RD:594; P 0:05) or speed (RD:223; P D:023). Discussion. The results indicate that at moderate speeds individual horses use dissociation patterns that allow them to maintain trunk pitch stability through management of the cranio-caudal location of the COP. During the hoof-ground collisions, reduced mechanical energy losses were found in hind-first dissociations compared to fully synchronous contacts. As speed increased, only forelimb vertical peak force increased

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt–jakob disease:An international study

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt\u2013Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160\u2013165

Influence of timing on CSF tests value for Creutzfeldt-Jakob disease diagnosis

Background: The analysis of markers in the cerebrospinal fluid (CSF) is useful in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, the time at which the study of these markers is most sensitive remains controversal. Objective: To assess the influence of time of sampling on the value of CSF tests in the diagnosis of sCJD. Method: In the framework of a multinational European study, we studied the results of 14-3-3, S100b, neurone specific enolase (NSE) and tau protein in 833 CSF samples from sCJD patients at different stages of disease and in 66 sequentially repeated lumbar punctures (LP). Results: 14-3-3 and tau protein tended to increase in sensitivity from onset (88%, 81%) to the advanced stage (91%, 90%). This was significant only in the methionine-valine (MV) heterozygous group of patients at codon 129. The absolute levels of S100b (p < 0.05), NSE and tau protein increased in the last stage of disease. High levels of tau protein, NSE and S100b were associated with shorter survival times (p < 0.01). Sixty-six sCJD patients underwent repeated LP. These sCJD patients were younger, had longer disease durations and were more frequently MV at codon 129 (p < 0.001) than the whole group. 14-3-3 sensitivity increased from 64% to 82% in the second LP (p = 0.025) and 88% sCJD patients had at least one positive result. Conclusions: Sensitivity and absolute levels of CJD markers increased with disease progression and were modulated by the codon 129 genotype. Early negative results should be inter-preted with caution, especially in young patients or those who are MV at codon 129

hepatitis a outbreak in italy 2013 a matched case control study

Between January and May 2013 a hepatitis A (HA) incidence increase was detected in Italy, signalling an outbreak. A retrospective matched case-control study was conducted to identify the source of infection. A case was defined as a resident of any of five regions (Apulia, autonomous province of Bolzano, Emilia-Romagna, Friuli-Venezia-Giulia and autonomous province of Trento), who had symptom onset between 1 January and 31 May2013 as well a positive test for anti-HA virus IgM. We compared each case with four age-and neighbourhood-matched controls. Overall 119 cases and 419 controls were enrolled. Berries were found as the main risk factor for HA (adjusted odds ratio (ORadj): 4.2; 95% confidence interval (CI): 2.5-7.0) followed by raw seafood (ORadj: 3.8; 95% CI: 2.2-6.8; PAF: 26%). Sequencing the virion protein (VP)1-2a region from 24 cases yielded a common sequence (GenBank number: KF182323). The same sequence was amplified from frozen mixed berries consumed by some cases as well as from isolates from Dutch and German HA patients, who had visited some of the affected Italian provinces during the outbreak. These findings suggested berries as the main source of the Italian outbreak. Control measures included voluntary recall of the confirmed frozen mixed berry batches and a trace-back investigation was initiated. The Ministry of Health website recommends frozen berries to be cooked for two minutes before eating.

Collisional mechanics of the diagonal gaits of horses over a range of speeds

One of the goals of the neuromotor control system is to minimize the cost of locomotion by reducing mechanical energy losses. Collisional mechanics, which studies the redirection of the downwards motion of the center of mass (COM) by ground reaction forces (GRF) generated by the limbs, represents an important source of energy loss. The primary objective of this study was to compare collisional mechanics and the associated mechanical energy losses in horses performing diagonally-synchronized gaits over a range of speeds. It is to be expected that collisional energy losses will be high when the COM velocity vector is closely aligned with the GRF vector. This condition is achieved in piaffe, an artificial gait performed in dressage competitions that has a diagonal limb coordination pattern similar to trot but performed with little or no forward velocity. Therefore, we hypothesized that collisional energy losses would be higher in piaffe than in trot. Synchronized kinematic and GRF data were collected from three highly-trained horses performing piaffe, passage and trot at a range of speeds. Derived variables were vertical excursion and velocity of the trunk COM, fore and hind limb compression expressed as percentage reduction of standing limb lengths, range of limb pro-retraction, GRF vector magnitude and vector angle, collision angle (Φ), and mechanical cost of motion (CoMotmech). Linear regression was used to investigate the relationship between CoMotmech and speed for each gait. Partial correlation was used to seek relationships between COM excursion and limb mechanics for each gait. Piaffe, passage and trot were clearly separated on the basis of speed. In all gaits the trunk was high at contact and lift off and descended to its lowest point in midstance following the pattern typical of spring mass mechanics. Mechanical cost was significantly (p &lt; .05) and inversely related to speed in trot and piaffe with the value increasing steeply as speed approached zero due to a near vertical orientation of both the COM velocity vector and the GRF vector. Limb compression during stance was significantly (p &lt; .05) linked to trunk COM vertical excursion in all gaits, with a stronger relationship in the forelimb. Hindlimb compression was, however, large in piaffe where the force magnitudes are notably smaller. The study illustrates the potential value of studying artificial gaits to provide data encompassing the entire range of locomotor capabilities. The results supported the experimental hypothesis by showing a threefold increase in collisional energy losses in piaffe compared with trot. In all gaits, dissociation between diagonal limb contacts and lift offs was thought to be an important strategy in reducing in collisional losses. Piaffe, the most costly gait, has similar characteristics to hopping on the spot. It appears that greater hindlimb compliance and a lower step frequency are important energy conservation strategies for piaffe