PE is a functional domain responsible for protein translocation and localization on mycobacterial cell wall

The PE family of Mycobacterium tuberculosis includes 98 proteins which share a highly homologous N-terminus sequence of about 110 amino acids (PE domain). Depending on the C-terminal domain, the PE family can be divided in three subfamilies, the largest of which is the PE_PGRS with 61 members. In this study, we determined the cellular localization of three PE proteins by cell fractionation and immunoelectron microscopy by expressing chimeric epitope-tagged recombinant proteins in Mycobacterium smegmatis. We demonstrate that the PE domain of PE_PGRS33 and PE11 (a protein constituted by the only PE domain) contains the information necessary for cell wall localization, and that they can be used as N-terminal fusion partners to deliver a sufficiently long C-terminus-linked protein domain on the mycobacterial cell surface. Indeed, we demonstrate that PE_PGRS33 and Rv3097c (a lipase belonging to the PE family) are surface exposed and localize in the mycobacterial cell wall. Moreover, we found that PE_PGRS33 is easily extractable by detergents suggesting its localization in the mycobacterial outer membrane. Beyond defining the cellular localization of these proteins, and a function for their PE domains, these data open the interesting possibility to construct recombinant mycobacteria expressing heterologous antigens on their surface for vaccine purposes.     

Why pesticides with mutagenic,carcinogenic and reproductive risks are registered in Brazil

Brazil is the biggest market for pesticides in the world. In the registration process, a pesticide must be authorized by the Institute of the Environment, Health Surveillance Agency and Ministry of Agriculture. Evaluations follow a package of toxicological studies submitted by the companies and also based on the Brazilian law regarding pesticides. We confronted data produced by private laboratories, submitted to the Institute of the Environment for registration, with data obtained from scientific databases, corresponding to mutagenicity, carcinogenicity and teratogenicity of pesticides. All studies submitted by the companies were carried out by private laboratories. From 247 pesticide formulations analyzed, none showed positive results for mutagenicity, carcinogenicity or teratogenicity. From 574 articles in the scientific literature, 84% published by public laboratories showed positive results, while 79% of those showing negative results came from private laboratories. There is an ethical concern about a conflict of interest between public/independent laboratories and private laboratories that produce data for registering pesticides. We demonstrated that there is a clear contradiction between public and private laboratories. Brazilian regulatory authorities have approved the registration of pesticides based almost exclusively on the monographs provided by the pesticide industry, because the use of scientific articles or information from the independent literature is strongly belittled by the industry. Pesticide companies argue that scientific articles cannot be trusted. Also, according to the industry, pesticide registration cannot be refused based on results from scientific articles. Thus, the registration of pesticides with mutagenic, carcinogenic and teratogenic risks has been approved in Brazil.     

Folding propensity of anoplin: A molecular dynamics study of the native peptide and four mutated isoforms

Anoplin, a cationic decapeptide amide GLLKRIKTLL‐NH₂ derived from venom sac of the solitary wasp Anoplius samariensis has been investigated through Molecular Dynamics. The wild‐type (WT) and four isoforms were simulated both in water and in the membrane‐mimicking solvent trifluoroethanol (TFE). In water all the investigated species, found to be in rapid equilibrium between different conformational states, can be considered as unfolded. On the other hand, in TFE all the systems enhance their rigidity and, in general, show α‐helix as the main folded conformation. Interestingly, a semi‐quantitative thermodynamic analysis has suggested that the folding driving force is not always the same being in some cases (e.g., the WT Anoplin) of entropic nature and in other cases of energetic nature. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 692–701, 2015.     

Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica)

A new thermophilic spore-forming strain MR3CT was isolated from geothermal soil located on Mount Rittmann in Antarctica. Strain MR3CT was Gram-positive, rod-shaped, occurring in pairs or filamentous. Growth was observed between 45 and 65 degrees C (optimum 61 degrees C) and at pH 5.0-6.5 (optimum pH 5.6). It was capable of utilizing galactose, trehalose, maltose and sucrose. The microorganism produced an exopolysaccharide and synthesized an extracellular constitutive amylolytic activity. The G + C content of DNA was 43.5 mol%. On the basis of 16S rRNA gene sequence similarity, strain MR3CT was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain MR3C1T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MR3CT from the validly published Anoxybacillus species. MR3CT therefore represents a new species, for which the name Anoxybacillus amylolyticus sp. nov., is proposed, with the type strain MR3CT (= ATCC BAA-872T = DSM 15939T = CIP 108338T).     

Evaluating joint-space narrowing and cartilage loss in rheumatoid arthritis by using MRI

Introduction

Magnetic resonance imaging (MRI) has been shown to be superior to radiography (XR) for assessing synovitis, osteitis, and bone erosion in rheumatoid arthritis (RA), particularly in clinical trials. However, relatively little has been reported on the ability of MRI to evaluate articular cartilage loss, or joint-space narrowing (JSN), in the hands and wrists. In a previous study, we adapted the nine-point Genant-modified Sharp XR-JSN score for use with MRI (MRI-JSN). In this study, we compare MRI-JSN with XR-JSN by using images from two multicenter clinical trials.

Methods

Baseline XR and 1.5-Tesla MR images of one hand and wrist from each of 47 subjects with RA enrolled in one of two multicenter clinical trials were evaluated by using the XR-JSN and MRI-JSN methods by a single radiologist experienced in the two methods. Radiographs and MR images were read independently on different occasions.

Results

In total, 575 of 611 joints were compared (one metacarpophalangeal joint of the thumb and 35 proximal interphalangeal joints were outside the MRI field of view and could not be assessed). The 22 (47%) subjects showed JSN with both XR and MRI, and 25 (53%) subjects showed no JSN with either method. No subject showed JSN with only one or the other method. MRI showed high agreement with XR (intraclass correlation coefficient = 0.83). Sensitivity of MRI for JSN, by using XR as the gold standard, was 0.94; specificity was 0.91; accuracy was 0.91; positive predictive value was 0.64; and negative predictive value was 0.99.

Conclusions

This validation exercise suggests that MRI JSN scoring may offer a viable alternative to XR JSN scoring in multicenter clinical trials of RA. However, the relative longitudinal sensitivity of MRI to change and the ability to discriminate therapeutic effect on JSN were not evaluated in this study.     

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.     

High cleavage specificity of a subtilisin-like protease from a hyperthermophilic archaeon under extreme conditions

The cleavage specificity of Pernisine, a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix, was established by mass spectrometry, analysing the peptides generated by digestion of oxidised bovine insulin B chain. The specificity was explored by changing several factors such as substrate/enzyme ratio, temperature and reaction media. Using a S/E ratio of 1000 (w/w) and a temperature of 60 °C, five primary cleavage sites in the insulin B chain were detected suggesting a broad specificity of Pernisine, which is different from that found for other bacterial subtilisin-like proteases. When the S/E ratio and/or temperature were increased, a higher selectivity of Pernisine was observed with a unique cleavage site occurring between Leu15 and Tyr16. In addition, the influence on the enzymatic hydrolysis of different organic solvent concentrations was investigated. The results demonstrated that Pernisine could specifically digest the peptide substrate even in the presence of 80% acetonitrile solution or 30% dimethyl sulfoxyde. Thereby the cleavage specificity of Pernisine can be opportunely modulated by controlling the in vitro digestion conditions, suggesting that this enzyme could be an attractive candidate to use in a variety of biotechnological applications.     

Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand

Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation.     

Epidemiology of dermatophytoses: retrospective analysis from 2005 to 2010 and comparison with previous data from 1975

Dermatophyte infections are extremely frequent worldwide and their epidemiological features vary according to the geographical area and have changed in the last decades. We studied the spectrum of dermatophytoses by means of a retrospective analysis involving 6,133 patients referred to the Mycology Service of the Dermatology Clinic of Policlinico Hospital - University of Bari, Italy during the period 2005-2010. The most frequent clinical forms were tinea unguium (39.2% of the total dermatophytoses), tinea corporis (22.7%) and tinea pedis (20.4%). There was a predominance of women for tinea unguium and corporis and of men for tinea pedis and especially tinea cruris. T. rubrum was the prevalent causative agent, implicated in 64% of total cases, followed by M. canis (14%) and T. mentagrophytes (10%). The retrospective evaluation of epidemiological data collected at our Clinic since 1975 showed a gradual decrease in the frequency of tinea cruris, tinea corporis, and tinea capitis over time. On the contrary, during the past two decades, there has been a progressive increase in the frequency of tinea pedis and especially of tinea unguium. In parallel with this changing pattern, the frequency of isolation of T. rubrum has shown a continuous increase during the last 35 years, whereas a progressive decline of the etiological role of T. violaceum, M. canis and even more of E. floccosum has been noted.     

Structure and orientation of the gH625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system

Glycoprotein H (gH) of the herpes simplex virus type 1 is involved in the complex mechanism of membrane fusion of the viral envelope with host cells. The virus requires four glycoproteins (gB, gD, gH, gL) to execute fusion and the role played by gH remains mysterious. Mutational studies have revealed several regions of gH ectodomain required for fusion and identified the segment from amino acid 625 to 644 as the most fusogenic region. Here, we studied the behavior in a membrane-mimicking DPC micellar environment of a peptide encompassing this region (gH625-644) and determined its NMR solution structure and its orientation within the micelles.