ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy.

In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.

Acute stress induces long-term metabolic, functional, and structural remodeling of the heart.

Takotsubo cardiomyopathy is a stress-induced cardiovascular disease with symptoms comparable to those of an acute coronary syndrome but without coronary obstruction. Takotsubo was initially considered spontaneously reversible, but epidemiological studies revealed significant long-term morbidity and mortality, the reason for which is unknown. Here, we show in a female rodent model that a single pharmacological challenge creates a stress-induced cardiomyopathy similar to Takotsubo. The acute response involves changes in blood and tissue biomarkers and in cardiac in vivo imaging acquired with ultrasound, magnetic resonance and positron emission tomography. Longitudinal follow up using in vivo imaging, histochemistry, protein and proteomics analyses evidences a continued metabolic reprogramming of the heart towards metabolic malfunction, eventually leading to irreversible damage in cardiac function and structure. The results combat the supposed reversibility of Takotsubo, point to dysregulation of glucose metabolic pathways as a main cause of long-term cardiac disease and support early therapeutic management of Takotsubo.

Implantation of engineered human microvasculature to study human infectious diseases in mouse models.

Animal models for studying human pathogens are crucially lacking. We describe the implantation in mice of engineered human mature microvasculature consisting of endothelial and perivascular cells embedded in collagen hydrogel that allows investigation of pathogen interactions with the endothelium, including in vivo functional studies. Using Neisseria meningitidis as a paradigm of human-restricted infection, we demonstrated the strength and opportunities associated with the use of this approach.

Quality assessment in light microscopy for routine use through simple tools and robust metrics.

Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Cyclic fertilin-derived peptide stimulates in vitro human embryo development.

OBJECTIVE: To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin ß (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor. DESIGN: Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts. SETTING: The study was performed in an academic hospital and research laboratory. PATIENT(S): Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine." INTERVENTION(S): Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action. MAIN OUTCOME MEASURE(S): Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 µM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%. RESULT(S): Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport. CONCLUSION(S): Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.

RhoA within myofibers controls satellite cell microenvironment to allow hypertrophic growth.

Adult skeletal muscle is a plastic tissue that can adapt its size to workload. Here, we show that RhoA within myofibers is needed for overload-induced hypertrophy by controlling satellite cell (SC) fusion to the growing myofibers without affecting protein synthesis. At the molecular level, we demonstrate that RhoA controls in a cell autonomous manner Erk1/2 activation and the expressions of extracellular matrix (ECM) regulators such as Mmp9/Mmp13/Adam8 and macrophage chemo-attractants such as Ccl3/Cx3cl1. Their decreased expression in RhoA mutants is associated with ECM and fibrillar collagen disorganization and lower macrophage infiltration. Moreover, matrix metalloproteinases inhibition and macrophage depletion in controls phenocopied the altered growth of RhoA mutants while having no effect in mutants showing that their action is RhoA-dependent. These findings unravel the implication of RhoA within myofibers, in the building of a permissive microenvironment for muscle hypertrophic growth and for SC accretion through ECM remodeling and inflammatory cell recruitment.

Selective Laser Melted Titanium Alloy for Transgingival Components: Influence of Surface Condition on Fibroblast Cell Behavior.

PURPOSE: To mechanically characterize and assess the biological properties of Ti6Al4V surfaces obtained by Selective Laser Melting in order to determine whether this process is conceivable for production of implant-supported prostheses and particularly trans-gingival components. As-built and polished surfaces were studied in comparison with components obtained by computer numerical control machining technology in order to consider whether the properties are in the same range as the conventional method currently used. MATERIALS AND METHODS: Cylindrical specimens of Ti6Al4V (n = 6) were built with Selective Laser Melting for the characterization of mechanical properties according to ISO 22674 and discs (n = 12) were fabricated in the same conditions for cytotoxicity evaluation. Discs (n = 12) of Ti6Al4V were also obtained by computer numerical control machining as control. Half of the number of discs (n = 6) from each process were polished, to simulate the laboratory protocol for polishing of transmucosal components and half of the discs remained unaltered (as-built). Surface roughness measurements of disc specimens (as-built and polished) were compared with computer numerical control milling specimens (as-built and polished). Proliferation of human gingival fibroblasts on Ti6Al4V surfaces was also assessed for each condition. Viability and cell morphology were then evaluated qualitatively. Ra and Sa data were compared using Student's t-test (α = 0.05) and metabolic activity data were compared using Kruskal-Wallis statistical test (α = 0.05). RESULTS: Selective Laser Melting specimens showed elongation at break greater than 2% and 0.2% yield strength better than 500MPa which complied with ISO 22674 standards. Although Selective Laser Melting samples displayed significantly increased roughness on as-built surfaces compared to computer numerically controlled milling samples (p < 0.05), no statistically significant difference was observed after mechanical polishing (p = 0.279). Regarding metabolic activity, no statistical difference was observed between groups at day 3 (p > 0.05) and fibroblasts showed a viability higher than 97% on all discs. Cell shapes on polished samples suggested moderate adhesion compared to unpolished samples. CONCLUSION: With the manufacturing parameters selected in this study, Selective Laser Melting of Ti6Al4V appeared to be compatible with a prosthetic application type 4 according to ISO 22674. Surfaces obtained, followed by recommended postprocessing provided components with equivalent biological properties compared to computer numerical control machining technology.

Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism.

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor's sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

A neural crest cell isotropic-to-nematic phase transition in the developing mammalian gut.

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

Stiffness increases with myofibroblast content and collagen density in mesenchymal high grade serous ovarian cancer.

Women diagnosed with high-grade serous ovarian cancers (HGSOC) are still likely to exhibit a bad prognosis, particularly when suffering from HGSOC of the Mesenchymal molecular subtype (50% cases). These tumors show a desmoplastic reaction with accumulation of extracellular matrix proteins and high content of cancer-associated fibroblasts. Using patient-derived xenograft mouse models of Mesenchymal and Non-Mesenchymal HGSOC, we show here that HGSOC exhibit distinct stiffness depending on their molecular subtype. Indeed, tumor stiffness strongly correlates with tumor growth in Mesenchymal HGSOC, while Non-Mesenchymal tumors remain soft. Moreover, we observe that tumor stiffening is associated with high stromal content, collagen network remodeling, and MAPK/MEK pathway activation. Furthermore, tumor stiffness accompanies a glycolytic metabolic switch in the epithelial compartment, as expected based on Warburg's effect, but also in stromal cells. This effect is restricted to the central part of stiff Mesenchymal tumors. Indeed, stiff Mesenchymal tumors remain softer at the periphery than at the core, with stromal cells secreting high levels of collagens and showing an OXPHOS metabolism. Thus, our study suggests that tumor stiffness could be at the crossroad of three major processes, i.e. matrix remodeling, MEK activation and stromal metabolic switch that might explain at least in part Mesenchymal HGSOC aggressiveness.

3D-printed protected face shields for health care workers in Covid-19 pandemic.

The coronavirus pandemic resulted in a shortage of protective equipment. To meet the request of eye-protecting devices, an interdisciplinary consortium involving practitioners, researchers, engineers and technicians developed and manufactured thousands of inexpensive 3D-printed face shields, inside hospital setting. This action leads to the concept of "concurrent, agile, and rapid engineering".

Streptococcus pyogenes infects human endometrium by limiting the innate immune response.

Group A Streptococcus (GAS), a Gram-positive human-specific pathogen, yields 517,000 deaths annually worldwide, including 163,000 due to invasive infections and among them puerperal fever. Before efficient prophylactic measures were introduced, the mortality rate for mothers during childbirth was approximately 10%; puerperal fever still accounts for over 75,000 maternal deaths annually. Yet, little is known regarding the factors and mechanisms of GAS invasion and establishment in postpartum infection. We characterized the early steps of infection in an ex vivo infection model of the human decidua, the puerperal fever portal of entry. Coordinate analysis of GAS behavior and the immune response led us to demonstrate that (a) GAS growth was stimulated by tissue products; (b) GAS invaded tissue and killed approximately 50% of host cells within 2 hours, and these processes required SpeB protease and streptolysin O (SLO) activities, respectively; and (c) GAS impaired the tissue immune response. Immune impairment occurred both at the RNA level, with only partial induction of the innate immune response, and protein level, in an SLO- and SpeB-dependent manner. Our study indicates that efficient GAS invasion of the decidua and the restricted host immune response favored its propensity to develop rapid invasive infections in a gynecological-obstetrical context.

Physical plasma therapy accelerates wound re-epithelialisation and enhances extracellular matrix formation in cutaneous skin grafts.

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal-epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF-ß1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extracellular Release of Antigen by Dendritic Cell Regurgitation Promotes B Cell Activation through NF-κB/cRel.

Dendritic cells (DCs) are professional APCs, which sample Ags in the periphery and migrate to the lymph node where they activate T cells. DCs can also present native Ag to B cells through interactions observed both in vitro and in vivo. However, the mechanisms of Ag transfer and B cell activation by DCs remain incompletely understood. In this study, we report that murine DCs are an important cell transporter of Ag from the periphery to the lymph node B cell zone and also potent inducers of B cell activation both in vivo and in vitro. Importantly, we highlight a novel extracellular mechanism of B cell activation by DCs. In this study, we demonstrate that Ag released upon DC regurgitation is sufficient to efficiently induce early B cell activation, which is BCR driven and mechanistically dependent on the nuclear accumulation of the transcription factor NF-κB/cRel. Thus, our study provides new mechanistic insights into Ag delivery and B cell activation modalities by DCs and a promising approach for targeting NF-κB/cRel pathway to modulate the DC-elicited B cell responses.