Bioinformatic Analysis of Chlamydia trachomatis Polymorphic Membrane Proteins PmpE, PmpF, PmpG and PmpH as Potential Vaccine Antigens

Chlamydia trachomatis is the most important infectious cause of infertility in women with important implications in public health and for which a vaccine is urgently needed. Recent immunoproteomic vaccine studies found that four polymorphic membrane proteins (PmpE, PmpF, PmpG and PmpH) are immunodominant, recognized by various MHC class II haplotypes and protective in mouse models. In the present study, we aimed to evaluate genetic and protein features of Pmps (focusing on the N-terminal 600 amino acids where MHC class II epitopes were mapped) in order to understand antigen variation that may emerge following vaccine induced immune selection. We used several bioinformatics platforms to study: i) Pmps' phylogeny and genetic polymorphism; ii) the location and distribution of protein features (GGA(I, L)/FxxN motifs and cysteine residues) that may impact pathogen-host interactions and protein conformation; and iii) the existence of phase variation mechanisms that may impact Pmps' expression. We used a well-characterized collection of 53 fully-sequenced strains that represent the C. trachomatis serovars associated with the three disease groups: ocular (N=8), epithelial-genital (N=25) and lymphogranuloma venereum (LGV) (N=20). We observed that PmpF and PmpE are highly polymorphic between LGV and epithelial-genital strains, and also within populations of the latter. We also found heterogeneous representation among strains for GGA(I, L)/FxxN motifs and cysteine residues, suggesting possible alterations in adhesion properties, tissue specificity and immunogenicity. PmpG and, to a lesser extent, PmpH revealed low polymorphism and high conservation of protein features among the genital strains (including the LGV group). Uniquely among the four Pmps, pmpG has regulatory sequences suggestive of phase variation. In aggregate, the results suggest that PmpG may be the lead vaccine candidate because of sequence conservation but may need to be paired with another protective antigen (like PmpH) in order to prevent immune selection of phase variants.AN is a recipient of a post-doctoral fellowship (SFRH/BPD/75295/2010) from Fundação para a Ciência e a Tecnologia (FCT)

Chlamydia trachomatis diversity viewed as a tissue-specific coevolutionary arms race

Analysis of 15 serovars of Chlamydia trachomatis reveals an evolutionary arms race in pathogen-host interactions

Fabrication of AZ4562 refractive microlenses array for light enhancement on optical microsystems

This paper presents the fabrication of an array of high aspect ratio photoresist based refractive microlenses (ML) using photolithography and thermal reflow. First, and in order to evaluate and predict the MLs optical properties and physical dimensions, finite element analysis was done. These simulations helped to design the super high resolution chrome on soda lime glass photomask as well as the parameters for the lithographic processes. Then, an array of high aspect ratio structures (length 4.9 mm, width 30 μm and 5 μm spacing between adjacent structures) with 5 μm thickness were fabricated. The thermal reflow technique (using a hotplate) was applied and an array of MLs measuring 5 and 32 μm at the vertex and radius, respectively, was achieved. When the photoresist (PR) is heated up above its glass transition temperature, it melts and the surface tension effect causes the fabricated microstructure to obtain the spherical lens profile. The hotplate thermal reflow is simple and easy to control, thus permitting the fabrication of smooth and homogeneous surfaces essential for good quality refractive microlenses.This work was supported by the Portuguese Foundation for Science and Technology under the projects FCT/PTDC/EEA-ELC/109936/2009 and FCT/MITPT/ EDAM-SI/0025/2008

Adaptive Evolution of the Chlamydia trachomatis Dominant Antigen Reveals Distinct Evolutionary Scenarios for B- and T-cell Epitopes: Worldwide Survey

Background: Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis. Methodology/Principal Findings: Using genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9 % of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P,10 212), specifically within B-cell epitopes (P,10 25), where 25 % of them are at fixation (P,10 25). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhikingeffect

Normalization strategies for real-time expression data in Chlamydia trachomatis

Since Chlamydia trachomatis is a genetically non-tractable pathogen, transcriptomics assumes a fundamental role for the better understanding of its biology. However, the suitability of endogenous controls for normalization of transcriptomic data in this bacterium still needs validation. We aimed to assess the stability of 10 genes for their potential use as endogenous controls in qPCR at both normal and stress (antibiotic treatment) growth conditions throughout the developmental cycle of three strains with different cell-appetence. Normalization was performed using the quantified bacterial genomes. We also tested the applicability of two widely used softwares (geNorm and Normfinder) to our data. For all strains, we found that 16SrRNA was the most stably expressed gene throughout the normal developmental cycle, but it was highly unstable under antibiotic exposure, suggesting prudence when using ribosomal genes as endogenous controls in expression experiments involving stress environments. The geNorm and Normfinder algorithms revealed contrasting results and seem inappropriate for the selected pool of genes. Considering the multiplicity of experimental conditions, there should be an in loco validation of endogenous controls, where 16SrRNA appears to be in the front line. Alternatively, normalization of expression data against genomic DNA, which is less influenced by experimental constraints (especially relevant for intracellular organisms) and stress conditions, likely constitutes a good option. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomati

A Search for Novel Legionella pneumophila Effector Proteins Reveals a Strain Specific Nucleotropic Effector

Legionella pneumophila is an accidental human pathogen that causes the potentially fatal Legionnaires' disease, a severe type of pneumonia. The main virulence mechanism of L. pneumophila is a Type 4B Secretion System (T4SS) named Icm/Dot that transports effector proteins into the host cell cytosol. The concerted action of effectors on several host cell processes leads to the formation of an intracellular Legionella-containing vacuole that is replication competent and avoids phagolysosomal degradation. To date over 300 Icm/Dot substrates have been identified. In this study, we searched the genome of a L. pneumophila strain (Pt/VFX2014) responsible for the second largest L. pneumophila outbreak worldwide (in Vila Franca de Xira, Portugal, in 2014) for genes encoding potential novel Icm/Dot substrates. This strain Pt/VFX2014 belongs to serogroup 1 but phylogenetically segregates from all other serogroup 1 strains previously sequenced, displaying a unique mosaic genetic backbone. The ability of the selected putative effectors to be delivered into host cells by the T4SS was confirmed using the TEM-1 β-lactamase reporter assay. Two previously unknown Icm/Dot effectors were identified, VFX05045 and VFX10045, whose homologs Lpp1450 and Lpp3070 in clinical strain L. pneumophila Paris were also confirmed as T4SS substrates. After delivery into the host cell cytosol, homologs VFX05045/Lpp1450 remained diffused in the cell, similarly to Lpp3070. In contrast, VFX10045 localized to the host cell nucleus. To understand how VFX10045 and Lpp3070 (94% of identity at amino acid level) are directed to distinct sites, we carried out a comprehensive site-directed mutagenesis followed by analyses of the subcellular localization of the mutant proteins. This led to the delineation of region in the C-terminal part (residues 380 to 534) of the 583 amino acid-long VFX10045 as necessary and sufficient for nuclear targeting and highlighted the fundamental function of the VFX10045-specific R440 and I441 residues in this process. These studies revealed a strain-specific nucleotropism for new effector VFX10045/Lpp3070, which anticipates distinct functions between these homologs.This project has been funded by: Research Grant 2016 by the European Society of Clinical Microbiology and lnfectious Diseases (ESCMID) to IF; by National funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB.info:eu-repo/semantics/publishedVersio

Connecting Regional Development to Environmental Education

<p>In this study we investigate how environmental education can be connected with the regional sustainable development We perceive that the dialogues surrounding Environmental Education in formal education have been dividing opinions over the last two decades. This does not happen only in relation to the conceptual precepts, but mainly in relation to the field of activity. Our theoretical support comes from authors engaged with the Critical Environmental Education, signaling for the construction of what we call Reference Framework. Our research is developed from a qualitative perspective, having as a strategy the exploratory case study. For the analysis of the documents we make reference to the methodology of content analysis proposed by Bardin (2011) and reflective application of the Reference Framework. We argue that so far in Brazil, environmental education is divided into two major theoretical trends that reflect the practices of environmental educators. On the one hand, supporting a behaviorist / liberal / conservative tendency of Environmental Education (CARVALHO, 2001; GUIMARÃES, 2000; LOUREIRO, 2008), understand environmental practices from its immediate resolution dimension, focusing actions that situate environmental practices through changes in social behaviors, mostly promoted by environmental activism. On the other hand, we have the opposite, that is, the popular / critical / emancipatory tendency which situates its argumentative and practical content situates in the significant assumption of a new societal posture in relation to the economic models adopted. It establishes the connection between environmental education and local / regional development, delineating a perception of reality, forming a dimension of creating other ways of relating human and non-human, including the emergence of a rationality that impresses ethical socio-environmental values, other forms of understanding of the world and the concept of environmental rationality.</p&gt

Probing deep tissues with laser-induced thermotherapy using near-infrared light

Optically tunable gold nanoparticles have been widely used in research with near-infrared light as a means to enhance laser-induced thermal therapy since it capitalizes on nanoparticles’ plasmonic heating properties. There have been several studies published on numerical models replicating this therapy in such conditions. However, there are several limitations on some of the models which can render the model unfaithful to therapy simulations. In this paper, two techniques of simulating laser-induced thermal therapy with a high-absorbing localized region of interest inside a phantom are compared. To validate these models, we conducted an experiment of an agar-agar phantom with an inclusion reproducing it with both models. The phantom was optically characterized by absorption and total attenuation. The first model is based on the macroperspective solution of the radiative transfer equation given by the diffusion equation, which is then coupled with the Pennes bioheat equation to obtain the temperature. The second is a Monte Carlo model that considers a stochastic solution of the same equation and is also considered as input to the Pennes bioheat transfer equation which is then computed. The Monte Carlo is in good agreement with the experimental data having an average percentage difference of 4.5% and a correlation factor of 0.98, while the diffusion method comparison with experimental data is 61% and 0.95 respectively. The optical characterization of the phantom and its inclusion were also validated indirectly since the Monte Carlo, which used those parameters, was also validated. While knowing the temperature in all points inside a body during photothermal therapy is important, one has to be mindful of the model which fits the conditions and properties. There are several reasons to justify the discrepancy of the diffusion method: low-scattering conditions, absorption, and reduced scattering are comparable. The error bars that are normally associated when characterizing an optical phantom can justify also a part of that uncertainty. For low-size tumors in depth, one may have to increase the light dosage in photothermal therapies to have a more effective treatment.authorsversionpublishe

Influence of the hip joint modeling approaches on the kinematics of human gait

The influence of the hip joint formulation on the kinematic response of the model of human gait is investigated throughout this work. To accomplish this goal, the fundamental issues of the modeling process of a planar hip joint under the framework of multibody systems are revisited. In particular, the formulations for the ideal, dry, and lubricated revolute joints are described and utilized for the interaction of femur head inside acetabulum or the hip bone. In this process, the main kinematic and dynamic aspects of hip joints are analyzed. In a simple manner, the forces that are generated during human gait, for both dry and lubricated hip joint models, are computed in terms of the system’s state variables and subsequently introduced into the dynamics equations of motion of the multibody system as external generalized forces. Moreover, a human multibody model is considered, which incorporates the different approaches for the hip articulation, namely ideal joint, dry, and lubricated models. Finally, several computational simulations based on different approaches are performed, and the main results presented and compared to identify differences among the methodologies and procedures adopted in this work. The input conditions to the models correspond to the experimental data capture from an adult male during normal gait. In general, the obtained results in terms of positions do not differ significantly when the different hip joint models are considered. In sharp contrast, the velocity and acceleration plotted vary significantly. The effect of the hip joint modeling approach is clearly measurable and visible in terms of peaks and oscillations of the velocities and accelerations. In general, with the dry hip model, intra-joint force peaks can be observed, which can be associated with the multiple impacts between the femur head and the cup. In turn, when the lubricant is present, the system’s response tends to be smoother due to the damping effects of the synovial fluid.The first and third authors express their gratitude to the Portuguese Foundation for Science and Technology for the PhD grants SFRH/BD/76573/2011 and SFRH/BD/64477/2009, respectively. The authors would like to thank to the Portuguese Foundation for Science and Technology through the project UID/EEA/04436/2013. The authors are also gratefully acknowledge the financial support from QREN (Quadro de Referência Estratégico Nacional - National Strategic Reference Framework), for this study “INOVSHOES - Padronizar para Customizar Calçado Ortopédico”, project n.º 2010/12032