Fibroblasts’ secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification

: Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases)

Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection

Expression of apoptosis-related markers and clinical outcome in patients with advanced colorectal cancer

The clinical relevance of bax and bcl-2 protein expression has been investigated in 84 patients with recurrent or metastatic colorectal cancer submitted to a chemotherapy regimen including methotrexate and fluorouracil/leucovorin. Cytoplasmic immunostaining of bax and bcl-2 was present in 65.5% and 38%, respectively, of the tumours. No association was found between bax and bcl-2 or between p53 and bax or bcl-2 protein expression. Moreover, the biomarkers were unrelated to patient and tumour characteristics known to affect the clinical outcome of colorectal cancer patients. In general, the apoptosis-related markers did not appear indicative of short- and long-term clinical response nor of prognosis. Bcl-2-negative lesions were more frequent among patients who reached an objective clinical response, which is in agreement with previously reported data regarding other tumour types. When the interrelationship between p53 and bax expression was examined, a better response rate (40%) was found for patients whose tumours did not express p53 and bax, and a better prognosis (2-year probability of overall survival 75%) for patients with p53-positive and bax-negative tumours. In the present series of patients with advanced colorectal cancer submitted to systemic chemotherapy we did not find a clear association between expression of apoptosis-related markers and clinical outcome, even in the subset of patients in which the apoptotic index as determined by the TUNEL approach was investigated. © 2001 Cancer Research Campaign http://www.bjcancer.co

Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis

SIQUEIRA JR. et al. Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod., v. 104, n. 1, p. 122-130, 2007.Objective. This clinical study was undertaken to compare the effectiveness of 2.5% sodium hypochlorite (NaOCl) and 0.12% chlorhexidine digluconate as irrigants in reducing the cultivable bacterial populations in infected root canals of teeth with apical periodontitis. Study design. According to stringent inclusion/exclusion criteria, 32 teeth with primary intraradicular infections and chronic apical periodontitis were selected and followed in the study. Bacterial samples were taken at the baseline (S1) and after chemomechanical preparation using either NaOCl (n 16) or chlorhexidine (n 16) as irrigants (S2). Cultivable bacteria recovered from infected root canals at the 2 stages were counted. Isolates from S2 samples were identified by means of 16S rRNA gene sequencing analysis. Results. At S1, all canals were positive for bacteria, and the median number of bacteria per canal was 7.32 105 for the NaOCl group and 8.5 105 for the chlorhexidine group. At S2, the median number of bacteria in canals irrigated with NaOCl and chlorhexidine was 2.35 103 and 2 102, respectively. Six of 16 (37.5%) canals from the NaOCl group and 8 of 16 (50%) canals from the chlorhexidine group yielded negative cultures. Chemomechanical preparation using either solution substantially reduced the number of cultivable bacteria in the canals. No significant difference was observed between the NaOCl and chlorhexidine groups with regard to the number of cases yielding negative cultures (P .72) or quantitative bacterial reduction (P .609). The groups irrigated with NaOCl or chlorhexidine showed a mean number of 1.3 and 1.9 cultivable species per canal, respectively. The great majority of isolates in S2 were from gram-positive bacteria, with streptococci as the most prevalent taxa. Conclusions. The present findings revealed no significant difference when comparing the antibacterial effects of 2.5% NaOCl and 0.12% chlorhexidine used as irrigants during the treatment of infected canal

TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCβ3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCβ3 and the TRPV1 channel; 2) serotonin, or a selective agonist, α-methyl-serotonin (α-Me-5-HT), requires the presence of PLCβ3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCβ3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD^+ neurons that represent ≈90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1+ nociceptors led to a significant behavioral deficit for pruritogens, including α-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways

The Higher Spin/Vector Model Duality

This paper is mainly a review of the dualities between Vasiliev's higher spin gauge theories in AdS4 and three dimensional large N vector models, with focus on the holographic calculation of correlation functions of higher spin currents. We also present some new results in the computation of parity odd structures in the three point functions in parity violating Vasiliev theories.Comment: 55 pages, 1 figure. Contribution to J. Phys. A special volume on "Higher Spin Theories and AdS/CFT" edited by M. R. Gaberdiel and M. Vasiliev. v2: references adde

Metabolic profiling and classification of propolis samples from Southern Brazil: an NMR-based platform coupled with machine learning

The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching 90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.Financial support for this investigation by National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Brazilian Biosciences National Laboratory (LNBioCNPEM/MCTI), Foundation for Support of Scientific and Technological Research in the State of Santa Catarina (FAPESC), and Portuguese Foundation for Science and Technology (FCT) is acknowledged. The research fellowship granted by CNPq to the first author is also acknowledged. The work was partially funded by a CNPq and FCT agreement through the PropMine grant

Climate diagnostics of three major drought events in the Amazon and illustrations of their seasonal precipitation predictions

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Structural basis of synaptic vesicle assembly promoted by α-synuclein.

α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this 'double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts

The spectral action and cosmic topology

The spectral action functional, considered as a model of gravity coupled to matter, provides, in its non-perturbative form, a slow-roll potential for inflation, whose form and corresponding slow-roll parameters can be sensitive to the underlying cosmic topology. We explicitly compute the non-perturbative spectral action for some of the main candidates for cosmic topologies, namely the quaternionic space, the Poincare' dodecahedral space, and the flat tori. We compute the corresponding slow-roll parameters and see we check that the resulting inflation model behaves in the same way as for a simply-connected spherical topology in the case of the quaternionic space and the Poincare' homology sphere, while it behaves differently in the case of the flat tori. We add an appendix with a discussion of the case of lens spaces.Comment: 55 pages, LaTe