Reconstructing the free-energy landscape of Met-enkephalin using dihedral Principal Component Analysis and Well-tempered Metadynamics

Well-Tempered Metadynamics (WTmetaD) is an efficient method to enhance the reconstruction of the free-energy surface of proteins. WTmetaD guarantees a faster convergence in the long time limit in comparison with the standard metadynamics. It still suffers however from the same limitation, i.e. the non trivial choice of pertinent collective variables (CVs). To circumvent this problem, we couple WTmetaD with a set of CVs generated from a dihedral Principal Component Analysis (dPCA) on the Ramachadran dihedral angles describing the backbone structure of the protein. The dPCA provides a generic method to extract relevant CVs built from internal coordinates. We illustrate the robustness of this method in the case of the small and very diffusive Metenkephalin pentapeptide, and highlight a criterion to limit the number of CVs necessary to biased the metadynamics simulation. The free-energy landscape (FEL) of Met-enkephalin built on CVs generated from dPCA is found rugged compared with the FEL built on CVs extracted from PCA of the Cartesian coordinates of the atoms.Comment: 17 pages, 9 figures (4 in color

Interactive Training System for Interventional Electrocardiology Procedures

International audienceRecent progress in cardiac catheterization and devices al-lowed to develop new therapies for severe cardiac diseases like arrhyth-mias and heart failure. The skills required for such interventions are still very challenging to learn, and typically acquired over several years. Vir-tual reality simulators can reduce this burden by allowing to practice such procedures without consequences on patients. In this paper, we propose the first training system dedicated to cardiac electrophysiology, includ-ing pacing and ablation procedures. Our framework involves an efficient GPU-based electrophysiological model. Thanks to an innovative mul-tithreading approach, we reach high computational performances that allow to account for user interactions in real-time. Based on a scenario of cardiac arrhythmia, we demonstrate the ability of the user-guided simulator to navigate inside vessels and cardiac cavities with a catheter and to reproduce an ablation procedure involving: extra-cellular poten-tial measurements, endocardial surface reconstruction, electrophysiology mapping, radio-frequency (RF) ablation, as well as electrical stimulation. This works is a step towards computerized medical learning curriculum

Mechanisms of Antibody-based defense against Pneumocystis

Pneumocystis (PC) pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. This dissertation evaluates functions of antibodies and conserved fungal cell wall carbohydrate antigens in host defense against PC. We demonstrate that a novel recombinant protein consisting of the extracellular domain of the beta-glucan receptor dectin-1 fused to the constant portion of murine IgG1, binds beta-glucan and recognizes Fc-γ receptors; and functionally, impairs growth of PC in the lungs of immunocompromised mice. As an antibody-like molecule targeting a conserved fungal cell wall carbohydrate enhanced host defense against PC, we questioned whether the host produces similar antibodies. We identified natural IgM antibodies, conserved across species and not requiring microbial stimulation for production, that recognize fungal cell wall carbohydrates beta-glucan and chitosan/chitin. In mice, naïve serum containing natural antibodies impairs the growth of PC organisms in the lungs at intermediate stages of infection, while at earliest stages limits pulmonary neutrophil recruitment. Mice unable to secrete IgM, sIgM(-/-), manifest similar impairments in pathogen clearance at intermediate stages of infection. Additionally, sIgM(-/-) mice demonstrate diminished trafficking of fungal cell wall carbohydrate antigen by CD11c+ cells to draining lymph nodes, impaired production of Th2 and Th17 cytokines in lymph nodes after PC challenge, and altered adaptive antibody responses, with diminished anti-PC IgG1 (Th2 associated) while enhanced IgG2a (Th1 associated) adaptive antibody responses. Thus, sIgM, of which a significant component is natural antibody, influences PC Ag presentation at earliest stages of infection, enhancing host defense and biasing the host towards Th2 adaptive responses. Additionaly, we observe that beta-glucan and chitosan/chitin are targets of induced antibody responses. PC challenge leads to the induction of specific serum IgG, and increased quantities of multiple isotypes at the lung mucosa against these carbohydrates. Mucosal responses against beta-glucan and chitosan/chitin after PC challenge are regulated by CD4+ T cells, and we provide evidence that functional memory B cell responses against fungal wall carbohydrates are generated as a consequence of PC challenge. Collectively, these studies demonstrate the importance of conserved fungal cell wall carbohydrate antigens, and primitive antibody isotypes, in host defense responses against PC

The Beta-Glucan Receptor Dectin-1 Recognizes Specific Morphologies of Aspergillus fumigatus

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3–glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1α (MIP-1α), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte–CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan–initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense

Human Salmonella Typhi exposure generates differential multifunctional cross‐reactive T‐cell memory responses against Salmonella Paratyphi and invasive nontyphoidal Salmonella

Objective There are no vaccines for most of the major invasive Salmonella strains causing severe infection in humans. We evaluated the specificity of adaptive T memory cell responses generated after Salmonella Typhi exposure in humans against other major invasive Salmonella strains sharing capacity for dissemination. Methods T memory cells from eleven volunteers who underwent controlled oral challenge with wt S. Typhi were characterised by flow cytometry for cross‐reactive cellular cytokine/chemokine effector responses or evidence of degranulation upon stimulation with autologous B‐lymphoblastoid cells infected with either S. Typhi, Salmonella Paratyphi A (PA), S. Paratyphi B (PB) or an invasive nontyphoidal Salmonella strain of the S. Typhimurium serovar (iNTSTy). Results Blood T‐cell effector memory (TEM) responses after exposure to S. Typhi in humans evolve late, peaking weeks after infection in most volunteers. Induced multifunctional CD4+ Th1 and CD8+ TEM cells elicited after S. Typhi challenge were cross‐reactive with PA, PB and iNTSTy. The magnitude of multifunctional CD4+ TEM cell responses to S. Typhi correlated with induction of cross‐reactive multifunctional CD8+ TEM cells against PA, PB and iNTSTy. Highly multifunctional subsets and T central memory and T effector memory cells that re‐express CD45 (TEMRA) demonstrated less heterologous T‐cell cross‐reactivity, and multifunctional Th17 elicited after S. Typhi challenge was not cross‐reactive against other invasive Salmonella. Conclusion Gaps in cross‐reactive immune effector functions in human T‐cell memory compartments were highly dependent on invasive Salmonella strain, underscoring the importance of strain‐dependent vaccination in the design of T‐cell‐based vaccines for invasive Salmonella

The Beta-Glucan Receptor Dectin-1 Recognizes Specific Morphologies of Aspergillus Fumigatus

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3-glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1α (MIP-1α), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte-CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan-initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense

Automatic coronary calcium scoring in chest CT using a deep neural network in direct comparison with non-contrast cardiac CT:A validation study

Purpose: To evaluate deep-learning based calcium quantification on Chest CT scans compared with manual evaluation, and to enable interpretation in terms of the traditional Agatston score on dedicated Cardiac CT. Methods: Automated calcium quantification was performed using a combination of deep-learning convolution neural networks with a ResNet-architecture for image features and a fully connected neural network for spatial coordinate features. Calcifications were identified automatically, after which the algorithm automatically excluded all non-coronary calcifications using coronary probability maps and aortic segmentation. The algorithm was first trained on cardiac-CTs and refined on non-triggered chest-CTs. This study used on 95 patients (cohort 1), who underwent both dedicated calcium scoring and chest-CT acquisitions using the Agatston score as reference standard and 168 patients (cohort 2) who underwent chest-CT only using qualitative expert assessment for external validation. Results from the deep-learning model were compared to Agatston-scores(cardiac-CTs) and manually determined calcium volumes(chest-CTs) and risk classifications. Results: In cohort 1, the Agatston score and AI determined calcium volume shows high correlation with a correlation coefficient of 0.921(p < 0.001) and R-2 of 0.91. According to the Agatston categories, a total of 67(70 %) were correctly classified with a sensitivity of 91 % and specificity of 92 % in detecting presence of coronary calcifications. Manual determined calcium volume on chest-CT showed excellent correlation with the AI volumes with a correlation coefficient of 0.923(p < 0.001) and R-2 of 0.96, no significant difference was found (p = 0.247). According to qualitative risk classifications in cohort 2, 138(82 %) cases were correctly classified with a k-coefficient of 0.74, representing good agreement. All wrongly classified scans (30(18 %)) were attributed to an adjacent category. Conclusion: Artificial intelligence based calcium quantification on chest-CTs shows good correlation compared to reference standards. Fully automating this process may reduce evaluation time and potentially optimize clinical calcium scoring without additional acquisitions

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi (S. Typhi) infection and disease remains incomplete. T follicular helper cells (TFH), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional TFH cells reside in germinal centers, circulating TFH (cTFH) (a memory subset of TFH) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S. Typhi vaccine and then challenged with virulent S. Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cTFH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cTFH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cTFH2 and cTFH17, but not cTFH1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cTFH subsets produced higher levels of S. Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cTFH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cTFH subsets and anti-S. Typhi antibodies. Taken together, our results suggest that circulating TFH2 and TFH17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses

Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina

Natural IgM antibodies in diverse species recognize conserved carbohydrates in fungal cell walls and influence early host defense against Pneumocystis in mice

SARS-CoV-2 RNA and Nucleocapsid Antigen Are Blood Biomarkers Associated With Severe Disease Outcomes That Improve in Response to Remdesivir.

BACKGROUND: Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter Adaptive COVID-19 Treatment Trial 1, which randomized patients to remdesivir or placebo. METHODS: Longitudinal specimens collected during hospitalization from a substudy of 642 patients with COVID-19 were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS: Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95% CI, 1.40-2.71) for levels &gt;245 pg/mL vs 1.04 (95% CI, .76-1.42) for levels &lt;245 pg/mL. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS: Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy. CLINICAL TRIAL REGISTRATION: NCT04280705 (ClinicalTrials.gov)