Transcriptomic analysis of plasmid and plasmid-related chromosomal ORFs in C. trachomatis strains with different cell-appetence

Despite the undergoing chromosomal size-reduction of Chlamydia trachomatis, almost all strains maintain the conserved 7,5kb plasmid. It has been recently considered a virulence factor, as plasmid-bearing strains evidenced a higher ability to successfully colonize epithelial cells and sustain infection than plasmidless strains. However, the biological role of the eight plasmid ORFs remains poorly characterized. Thus, we aim to correlate the relative expression of each plasmid ORFs with the number of plasmids per genome as well as with the relative expression of chromosomal genes that seem to be regulated by the plasmid, during the whole Chlamydia developmental cycle. Both the transcriptomic analysis of all the selected ORFs and the determination of the plasmid copy number were performed by qPCR in prototype and currently circulating strains with distinct cell-appetence and ecological success, throughout Chlamydia developmental cycle. So far, our results indicate: 1) huge differences (up to 100-fold) in the expression levels between plasmid ORFs; 2) for the same ORF, different expression levels and profiles among strains; 3) variation in the number of plasmids per genome during the infectious cycle; 4) the higher expression level of the plasmid ORFs do not seem to correlate with a higher number of plasmids per genome. These results, together with our ongoing transcriptomic survey of the plasmid-related chromosomal genes, will contribute to shed some light on the molecular function of the plasmid ORFs in chlamydial biology

Molecular features underlying the higher ecological success of C. trachomatis E and F genotypes

In the light of the >98% genomic similarity among Chlamydia trachomatis serovars, the higher worldwide ecological success of E and F is enigmatic. We intend to provide a quick overview of the molecular data that distinguish these from the remaining strains. Examples are: - E and F possess a similar chromosomal genetic make-up distinct from the remaining genotypes. Some loci linked to this independent co-segregation comprehend membrane proteins, hypothetical virulence factors, and regulatory regions (published data). - Some loci reveal nonrandom mutational patterns, where mutations exclusive of E and F are clustered in specific protein domains, likely promoting strains functional and/or structural attributes (published data). - Based on data from a worldwide survey, MOMP of E and F exhibit the lowest mutation rate (22.3-fold lower), implying more fitted antigenic profiles to deal with host immunity (published data). - The likelihood of E and F strains to undergo genetic recombination is about 12-fold lower than that of the other genotypes (P<10-2), suggesting a putative clonal evolution, where superimposed favorable clones may be strongly maintained in vivo (preliminary data from our lab). - Strains E and F do not seem to originate higher infectious load in vivo, when compared with other genital genotypes (published data). Full-genomic data from multiple and diverse clinical isolates will be essential to decipher the secret behind the higher ecological success of E and F strains

Inactivation of Listeria monocytogenes by pulsed light in packaged and sliced salpicão, a ready-to-eat traditional cured smoked meat sausage

The efficacy of pulsed light (PL) for the surface decontamination of a sliced ready-to-eat cured meat product, salpicão, was studied. The surface of the slices was inoculated with Listeria monocytogenes (107 cfu/g), and then PL treatment was applied. Microbial analyses (L. monocytogenes, coagulase-negative Staphylococci (CNS), Lactic acid bacteria (LAB) and Enterobacteriaceae) were performed immediately after the treatment. pH, aw, colour and volatile profile were also evaluated. Response Surface experimental Design was applied regarding factors voltage (ranging from 1828 to 3000 V) and distance (ranging from 2.6 to 5 cm) to the light source, and a maximum of 16.11 J/cm2 energy dose was achieved. A reduction of 1.58 log cfu/g in L. monocytogenes count was obtained when a fluence of 5.31 J/cm2 was applied. Enterobacteriaceae, LAB and CNS endogenous microbial populations were not influenced by PL treatment. PL application reduced a* values and influenced b* values without impacting the sample ΔE or L* values. The potential oxidative effect of this technology was evaluated by solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS), and no significant changes were verified after its application in this sliced cured meat product.info:eu-repo/semantics/publishedVersio

Caloric Curves in two and three-dimensional Lennard-Jones-like systems including Long-range forces

We present a systematic study of the thermodynamics of two and three-dimensional generalized Lennard-Jones ($LJ$) systems focusing on the relationship between the range of the potential, the system density and its dimension. We found that the existence of negative specific heats depends on these three factors and not only on the potential range and the density of the system as stated in recent contributions.Comment: LaTex, 12 pages, 7 figure

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

In silico scrutiny of genes revealing phylogenetic congruence with clinical prevalence or tropism properties of Chlamydia trachomatis strains

Microbes possess a multiplicity of virulence factors that confer them the ability to specifically infect distinct biological niches. Contrary to what is known for other bacteria, for the obligate intracellular human pathogen Chlamydia trachomatis, the knowledge of the molecular basis underlying serovars’ tissue specificity is scarce. We examined all ~900 genes to evaluate the association between individual phylogenies and cell-appetence or ecological success of C. trachomatis strains. Only ~1% of the genes presented a tree topology showing the segregation of all three disease groups (ocular, urogenital, and lymphatic) into three wellsupported clades. Approximately 28% of the genes, which include the majority of the genes encoding putative type III secretion system effectors and Inc proteins, present a phylogenetic tree where only lymphogranuloma venereum strains form a clade. Similarly, an exclusive phylogenetic segregation of the most prevalent genital serovars was observed for 61 proteins. Curiously, these serovars are phylogenetically cosegregated with the lymphogranuloma venereum serovars for ~20% of the genes. Some clade-specific pseudogenes were identified (novel findings include the conserved hypothetical protein CT037 and the predicted a-hemolysin CT473), suggesting their putative expendability for the infection of particular niches. Approximately 3.5% of the genes revealed a significant overrepresentation of nonsynonymous mutations, and the majority encode proteins that directly interact with the host. Overall, this in silico scrutiny of genes whose phylogeny is congruent with clinical prevalence or tissue specificity of C. trachomatis strains may constitute an important database of putative targets for future functional studies to evaluate their biological role in chlamydial infections.This work was supported by a grant, ERA-PTG/0004/2010, from Fundação para a Ciência e a Tecnologia (FCT) (to J.P.G.), in the frame of ERA-NET PathoGenoMics. A.N. is recipient of a FCT post-doctoral fellowship (SFRH/BPD/75295/2010), V.B. and R.F. are recipients of Ph.D. fellowships (SFRH/BD/68527/2010 and SFRH/BD/68532/2010, respectively) from FCT, and V.D. is a recipient of fellowship on behalf of the grant ERA-PTG/0004/2010

Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8996265/Background and objectives: To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV- 2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. Methodology: We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. Results: From the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 10 6 6 0.2 10 6 per-base per-infection cycle (mean across both lineages of SARS-CoV-262SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. Conclusions and implications: These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates—an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators—viral populations where mutations occur at a significantly faster rate.M.A. was supported by ‘Fundação para a Ciência e Tecnologia’ (FCT), fellowships PD/BD/138735/2018. Research was supported by FCT Project PTDC/BIA-EVL/31528/2017 to I.G. and by funds from Portuguese NIH.info:eu-repo/semantics/publishedVersio

Is type of work associated with physical activity and sedentary behaviour in women with fibromyalgia A cross-sectional study from the al-Ándalus project

Objectives To analyse the association between the type of work (productive vs reproductive work) and the levels of physical activity and sedentary behaviour in women with fibromyalgia. Method This cross-sectional study involved 258 women with fibromyalgia from southern Spain. Of them, 55% performed reproductive work (unpaid, associated with caregiving and domestic roles) exclusively, while 45% had productive job (remunerated, that results in goods or services). Physical activity of light, moderate and vigorous intensity in the leisure time, at home, at work, and totally were measured through the leisure time physical activity instrument and with the physical activity at home and work instrument, respectively. Sedentary behaviour was measured by the Sedentary Behaviour Questionnaire. Results After adjusting for age, fat percentage, education level and marital status, the multivariate analysis of covariance model informed the existence of significant differences between type of work groups (p<0.001). Women with productive work engaged in more light physical activity at work (mean difference =448.52 min; 95 % CI 179.66 to 717.38; p=0.001), and total physical activity of light (809.72 min; 535.91 to 1085.53; p<0.001) and moderate (29