The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available

Wogonin Improves Histological and Functional Outcomes, and Reduces Activation of TLR4/NF-κB Signaling after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. This study was undertaken to investigate the effects of wogonin, a flavonoid with potent anti-inflammatory properties, on functional and histological outcomes, brain edema, and toll-like receptor 4 (TLR4)- and nuclear factor kappa B (NF-κB)-related signaling pathways in mice following TBI.Mice subjected to controlled cortical impact injury were injected with wogonin (20, 40, or 50 mg·kg(-1)) or vehicle 10 min after injury. Behavioral studies, histology analysis, and measurement of blood-brain barrier (BBB) permeability and brain water content were carried out to assess the effects of wogonin. Levels of TLR4/NF-κB-related inflammatory mediators were also examined. Treatment with 40 mg·kg(-1) wogonin significantly improved functional recovery and reduced contusion volumes up to post-injury day 28. Wogonin also significantly reduced neuronal death, BBB permeability, and brain edema beginning at day 1. These changes were associated with a marked reduction in leukocyte infiltration, microglial activation, TLR4 expression, NF-κB translocation to nucleus and its DNA binding activity, matrix metalloproteinase-9 activity, and expression of inflammatory mediators, including interleukin-1β, interleukin-6, macrophage inflammatory protein-2, and cyclooxygenase-2.Our results show that post-injury wogonin treatment improved long-term functional and histological outcomes, reduced brain edema, and attenuated the TLR4/NF-κB-mediated inflammatory response in mouse TBI. The neuroprotective effects of wogonin may be related to modulation of the TLR4/NF-κB signaling pathway

Microbial Diversity of a Brazilian Coastal Region Influenced by an Upwelling System and Anthropogenic Activity

BACKGROUND: Upwelling systems are characterised by an intense primary biomass production in the surface (warmest) water after the outcrop of the bottom (coldest) water, which is rich in nutrients. Although it is known that the microbial assemblage plays an important role in the food chain of marine systems and that the upwelling systems that occur in southwest Brazil drive the complex dynamics of the food chain, little is known about the microbial composition present in this region. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a molecular survey based on SSU rRNA gene from the three domains of the phylogenetic tree of life present in a tropical upwelling region (Arraial do Cabo, Rio de Janeiro, Brazil). The aim was to analyse the horizontal and vertical variations of the microbial composition in two geographically close areas influenced by anthropogenic activity (sewage disposal/port activity) and upwelling phenomena, respectively. A lower estimated diversity of microorganisms of the three domains of the phylogenetic tree of life was found in the water of the area influenced by anthropogenic activity compared to the area influenced by upwelling phenomena. We observed a heterogenic distribution of the relative abundance of taxonomic groups, especially in the Archaea and Eukarya domains. The bacterial community was dominated by Proteobacteria, Cyanobacteria and Bacteroidetes phyla, whereas the microeukaryotic community was dominated by Metazoa, Fungi, Alveolata and Stramenopile. The estimated archaeal diversity was the lowest of the three domains and was dominated by uncharacterised marine Crenarchaeota that were most closely related to Marine Group I. CONCLUSIONS/SIGNIFICANCE: The variety of conditions and the presence of different microbial assemblages indicated that the area of Arraial do Cabo can be used as a model for detailed studies that contemplate the correlation between pollution-indicating parameters and the depletion of microbial diversity in areas close to anthropogenic activity; functional roles and geochemical processes; phylogeny of the uncharacterised diversity; and seasonal variations of the microbial assemblages

Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

International audienceBACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader

A Mathematical Model for Interpretable Clinical Decision Support with Applications in Gynecology

Over time, methods for the development of clinical decision support (CDS) systems have evolved from interpretable and easy-to-use scoring systems to very complex and non-interpretable mathematical models. In order to accomplish effective decision support, CDS systems should provide information on how the model arrives at a certain decision. To address the issue of incompatibility between performance, interpretability and applicability of CDS systems, this paper proposes an innovative model structure, automatically leading to interpretable and easily applicable models. The resulting models can be used to guide clinicians when deciding upon the appropriate treatment, estimating patient-specific risks and to improve communication with patients.We propose the interval coded scoring (ICS) system, which imposes that the effect of each variable on the estimated risk is constant within consecutive intervals. The number and position of the intervals are automatically obtained by solving an optimization problem, which additionally performs variable selection. The resulting model can be visualised by means of appealing scoring tables and color bars. ICS models can be used within software packages, in smartphone applications, or on paper, which is particularly useful for bedside medicine and home-monitoring. The ICS approach is illustrated on two gynecological problems: diagnosis of malignancy of ovarian tumors using a dataset containing 3,511 patients, and prediction of first trimester viability of pregnancies using a dataset of 1,435 women. Comparison of the performance of the ICS approach with a range of prediction models proposed in the literature illustrates the ability of ICS to combine optimal performance with the interpretability of simple scoring systems.The ICS approach can improve patient-clinician communication and will provide additional insights in the importance and influence of available variables. Future challenges include extensions of the proposed methodology towards automated detection of interaction effects, multi-class decision support systems, prognosis and high-dimensional data

Early markers for myocardial ischemia and sudden cardiac death.

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB

Molecular evolution of cyclin proteins in animals and fungi

<p>Abstract</p> <p>Background</p> <p>The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi.</p> <p>Results</p> <p>We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution.</p> <p>Conclusions</p> <p>The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events.</p