The different types of muscle fibres respond in a specific way to hypertrophy or atrophy. The mechanisms underlying these heterogeneous adaptations remain poorly understood. Using single-nucleus RNA sequencing, we propose that fast glycolytic fibres show genetic limitations to hypertrophy induced by mechanical overload. We show that a prior fibre transition, achieved by reducing SIX1 protein expression (hypomorphism), enhances and accelerates overload-induced hypertrophy, bypassing the genetic limitations of fast glycolytic fibres. In contrast and unexpectedly, Six1 knockout in myofibers abolished overload-induced hypertrophy and instead caused atrophy of IIb/IIx fibers, despite the induction of a strong slow oxidative phenotype. In particular, Six1 deletion leads to metabolic defects caused by inhibition of glycolysis, AMPK and mitochondrial biogenesis. Our findings highlight the critical role of SIX1/AMPK/glycolysis-dependent aerobic metabolism in muscle growth and suggest that fibre type transitions, coupled with preserved metabolic function, may optimise hypertrophic responses. ### Competing Interest Statement The authors have declared no competing interest.

Incomplete reporting of microscopy methods undermines transparency, reproducibility, and data reuse. Despite recent initiatives, comprehensive, broadly endorsed, and accessible reporting guidelines are still lacking. Here, we present a bare minimal microscopy reporting requirements checklist that integrates human- and machine-readable input to provide clear, actionable guidance for researchers, reviewers, and publishers and to advance community standards in microscopy.

BACKGROUND AND AIMS:Immunotherapy has continued to revolutionize cancer treatment, achieving remarkable progress over the past decade. However, in most solid tumors, its effectiveness is often impaired because of immune resistance driven by the tumor microenvironment (TME). In fibrotic cancers, such as cholangiocarcinoma (CCA), the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) form a dense, rigid stroma that hinders immune cell infiltration and promotes immunosuppression. Overcoming these physical and biological barriers is crucial for fully unleashing the potential of immunotherapeutic approaches. APPROACH RESULTS:We developed a dual-action strategy combining photothermal therapy (PTT) using gold-decorated iron oxide nanoflowers (GIONFs) and PD-1 immune checkpoint blockade. This approach was evaluated in preclinical models of CCA to assess its impact on ECM remodeling, CAF modulation, immune cell activation, and tumor growth.The combinatorial strategy effectively reduced ECM stiffness, reprogrammed CAF subsets, and enhanced cytotoxic T-cell infiltration. In preclinical CCA models, treated tumors shift from immune-cold to immune-hot states. This combination amplifies anti-tumor immune responses, decreases immunosuppressive stromal signatures, and improves tumor control. CONCLUSIONS:Our findings emphasize the therapeutic potential of combining nanotechnology and immunotherapy to reshape the TME and overcome immunotherapy resistance in fibrotic tumors, such as CCA. Targeting both ECM and immune checkpoints has emerged as a promising and versatile strategy to enhance the efficacy of immunotherapy against desmoplastic cancers.

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

Neural tissues of the central nervous system are among the softest and most fragile in the human body, protected from mechanical perturbation by the skull and the spine. In contrast, the enteric nervous system is embedded in a compliant, contractile tissue and subject to chronic, high-magnitude mechanical stress. Do neurons and glia of the enteric nervous system display specific mechanical properties to withstand these forces? Using nano-indentation combined with immunohistochemistry and second harmonic generation imaging of collagen, we discovered that enteric ganglia in adult mice are an order of magnitude more resistant to deformation than brain tissue. We found that glia-rich regions in ganglia have a similar stiffness to neuron-rich regions and to the surrounding smooth muscle: ∼3 kPa at a 3 μm indentation depth and ∼7 kPa at an 8 μm depth. Differences in the adhesion strength of the different tissue layers to the glass indenter were scarce. The collagen shell surrounding ganglia and inter-ganglionic fibers may play a key role in strengthening the enteric nervous system to resist the manifold mechanical challenges it faces.

Hintergrund Minimizing lung function impairment is a goal to managing paediatric asthma. Dupilumab (DPL), a fully human monoclonal antibody, blocks the shared receptor component of IL-4/IL-13, key drivers of type 2 asthma. Long-term DPL use in EXCURSION (NCT03560466) sustained lung function improvement in 6–11-year-old patients (pts) with uncontrolled type 2 asthma who had completed VOYAGE (NCT02948959); rapid improvement in lung function was observed in patients who switched from placebo (PBO) to DPL. This analysis assessed numerically higher lung function improvements seen consistently in the DPL/DPL vs PBO/DPL arms in EXCURSION.

L'amélioration de la fonction respiratoire est un élément essentiel de la prise en charge de l'asthme pédiatrique. Le dupilumab (DPL), un anticorps monoclonal entièrement humain, bloque la composante commune des récepteurs de l'IL-4/IL-13, qui jouent un rôle clé dans l'asthme de type 2. L'utilisation à long terme du DPL dans l'étude EXCURSION (NCT03560466) a maintenu l'amélioration de la fonction respiratoire chez des patients de 6 à 11 ans souffrant d'asthme de type 2 non contrôlé parvenus au terme de l'étude VOYAGE (NCT02948959) ; on a observé une amélioration rapide de la fonction respiratoire chez les patients passés du placebo (PBO) au DPL. Cette analyse vise à analyser l'amélioration numériquement plus importante de la fonction respiratoire systématiquement observée pour le groupe DPL/DPL vs PBO/DPL dans EXCURSION. Les patients ont reçu un traitement de fond additionnel par le DPL 100/200 mg ou un PBO toutes les 2 semaines (1×/2 s) pendant 52 semaines au cours de l'étude VOYAGE et par le DPL 100/200 mg 1×/2 s/300 mg 1×/4s pendant 52 semaines au cours de l'étude EXCURSION. Critère d'évaluation: modification par rapport à la valeur à l'inclusion dans l'étude initiale (VIEI) du pourcentage du VEMS prédit (pVEMSp) selon la durée de l'asthme, l'âge à l'apparition de l'asthme ou l'âge à l'instauration du DPL. À la semaine 0, 106 patients ont été inclus dans le groupe PBO/DPL et 209 dans le groupe DPL/DPL. À la semaine 52, la variation par rapport à la VIEI du pVEMSp pour DPL/DPL vs PBO/DPL était numériquement plus importante dans les sous-groupes avec une durée moindre de l'asthme (valeur de p pour l'interaction : p = 0,33), un âge plus avancé lors de l'apparition (p = 0,73) et un âge moins avancé lors de l'instauration du DPL (p = 0,84) ; aucune de ces différences n'atteignaient le seuil de significativité statistique. La réponse au DPL en termes d'amélioration de la fonction respiratoire après 1 à 2 ans de traitement ne diffère pas en fonction de la durée de l'asthme ni de l'âge lors de son apparition ou de l'âge à l'instauration du DPL, chez des patients de 6 à 11 ans souffrant d'asthme de type 2 non contrôlé.

To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on different microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and may, therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time or even between two microscopes of the same model. In addition to illumination stability, working within the linear range of the illumination power allows to adjust accurately the illumination power absolute value (in mW) using a fraction (or %) of its maximal value through the imaging software. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is intended for confocal systems (raster scanning and spinning disks), Multi-photon systems (used for 2P- or 3P-imaging, SHG-imaging, etc.), and widefield systems. It represents the collective experience of over 50 imaging scientists.

In Streptococcus pyogenes, the type II fatty acid (FA) synthesis pathway FASII is feedback-controlled by the FabT repressor bound to an acyl-Acyl carrier protein. Although FabT defects confer reduced virulence in animal models, spontaneous fabT mutants arise in vivo. We resolved this paradox by characterizing the conditions and mechanisms requiring FabT activity, and those promoting fabT mutant emergence. The fabT defect leads to energy dissipation, limiting mutant growth on human tissue products, which explains the FabT requirement during infection. Conversely, emerging fabT mutants show superior growth in biotopes rich in saturated FAs, where continued FASII activity limits their incorporation. We propose that membrane alterations and continued FASII synthesis are the primary causes for increased fabT mutant mortality in nutrient-limited biotopes, by failing to stop metabolic consumption. Our findings elucidate the rationale for emerging fabT mutants that improve bacterial survival in lipid-rich biotopes, but lead to a genetic impasse for infection.

Regeneration of orofacial tissues is hampered by the lack of adequate vascular supply. Implantation of in vitro engineered, prevascularized constructs has emerged as a strategy to allow the rapid vascularization of the entire graft. Given the angiogenic properties of dental pulp stem cells, we hereby established a preclinical model of prevascularized constructs loaded with stem cells from human exfoliating deciduous teeth (SHED) in a 3-dimensional-printed material and provided a functional analysis of their in vivo angiogenesis, vascular perfusion, and permeability. Three different cell-loaded collagen hydrogels (SHED-human umbilical vein endothelial cell [HUVEC], HUVEC with SHED-conditioned medium, and SHED alone) were cast in polylactic acid (PLA) grids and ectopically implanted in athymic mice. At day 10, in vivo positron emission tomography (PETscan) revealed a significantly increased uptake of radiotracer targeting activated endothelial cells in the SHED-HUVEC group compared to the other groups. At day 30, ex vivo micro-computed tomography imaging confirmed that SHED-HUVEC constructs had a significantly increased vascular volume compared to the other ones. Injection of species-specific lectins analyzed by 2-photon microscopy demonstrated blood perfusion of the engineered human vessels in both prevascularized groups. However, in vivo quantification showed increased vessel density in the SHED-HUVEC group. In addition, coinjection of fluorescent lectin and dextran revealed that prevascularization with SHED prevented vascular leakage, demonstrating the active role of SHED in the maturation of human-engineered microvascular networks. This preclinical study introduces a novel PLA prevascularized and implantable construct, along with an array of imaging techniques, to validate the ability of SHED to promote functional human-engineered vessels, further highlighting the interest of SHED for orofacial tissue engineering. Furthermore, this study validates the use of PETscan for the early detection of in vivo angiogenesis, which may be applied in the clinic to monitor the performance of prevascularized grafts.

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.

The protocols in this collection describe how to measure and analyze the photon conversation factor (PCF photo-electrons/count), readnoise, and dynamic range of a light microscopy detection system; which can either be a point detector or an area detector, using an in-homogeneous detector illumination scheme (as opposed to uniform illumination). The collection includes protocols on how to prepare a suitable sample for the acquisition, how to acquire data, as well as a respective analysis protocol. There are three aims a detection system can be characterized for, briefly: Aim 1 - experiment QC: Characterize the microscope performance using detection settings that match the experiment. Aim 2 - instrument QC.: Monitoring of microscope performance over time for service purposes, to maintain image quality constant and at high level. Aim 3 - system characterization: Full characterization of detection path performance under the range of settings applied by users. For a more detailed description refer to protocol 1. Introduction - Background and Aims. This protocol collection represents the collective experience of over 100 imaging scientists and industry experts. Measurements made by our working group with these protocols will be available in a public database. Please note, that this is an evolving document, to be versioned and updated, based on community feedback and new data.

AbstractInStreptococcus pyogenes, the type II fatty acid (FA) synthesis pathway FASII is feedback- controlled by the FabT repressor bound to an acyl-Acyl carrier protein. Despite FabT defects being linked to reduced virulence in animal models, spontaneousfabTmutants arisein vivo. To resolve this paradox, we characterized the conditions and mechanisms that require FabT activity, and those that promotefabTmutant emergence. The primaryfabTmutant defect is energy dissipation: specific nutrients are consumed, but the mutant fails to grow on human tissue, cells, or cell filtrates where nutrients are limited. These features explain the FabT requirement during infection. Conversely,fabTmutants exhibited a marked growth advantage over the wild-type in biotopes rich in saturated FAs.fabTmutants emerge in this context, where continued FASII activity prevented environmental FA incorporation. Anex vivomuscle model demonstrated that wild-typeS. pyogeneswas inhibited, whilefabTmutant growth was stimulated, but conditional to FASII activity. Our findings elucidate the rationale for emergingfabTmutants that improve survival in lipid-rich biotopes, but lead to a genetic impasse for infection.

Background: Patients with mild asthma are believed to represent the majority of patients with asthma. Disease-associated risks such as exacerbations, lung function decline, and death have been understudied in this patient population. There have been no prior efforts from major societies to describe research needs in mild asthma. Methods: A multidisciplinary, diverse group of 24 international experts reviewed the literature, identified knowledge gaps, and provided research recommendations relating to mild asthma definition, pathophysiology, and management across all age groups. Research needs were also investigated from a patient perspective, generated in conjunction with patients with asthma, caregivers, and stakeholders. Of note, this project is not a systematic review of the evidence and is not a clinical practice guideline. Results: There are multiple unmet needs in research on mild asthma driven by large knowledge gaps in all areas. Specifically, there is an immediate need for a robust mild asthma definition and an improved understanding of its pathophysiology and management strategies across all age groups. Future research must factor in patient perspectives. Conclusions: Despite significant advances in severe asthma, there remain innumerable research areas requiring urgent attention in mild asthma. An important first step is to determine a better definition that will accurately reflect the heterogeneity and risks noted in this group. This research statement highlights the topics of research that are of the highest priority. Furthermore, it firmly advocates the need for engagement with patient groups and for more support for research in this field.

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.

BACKGROUND:Dupilumab efficacy and safety in children aged 6-11 years with uncontrolled, moderate-to-severe asthma were shown in the VOYAGE study-a 52-week, multinational, multicentre, phase 3 randomised, double-blind, placebo-controlled trial. We aimed to evaluate the long-term safety and efficacy of dupilumab in children with moderate-to-severe asthma who previously participated in the VOYAGE study. METHODS:365 of 408 children with moderate-to-severe asthma from VOYAGE enrolled in EXCURSION, a 52 week, open-label extension study conducted at 70 centres across 17 countries. 240 children continued with add-on dupilumab (dosed according to bodyweight: 100 mg for those weighing ≤30 kg and 200 mg for those weighing more than 30 kg at EXCURSION baseline) once every 2 weeks administered by subcutaneous injection (dupilumab/dupilumab group) and 125 children on placebo during VOYAGE initiated dupilumab (100 or 200 mg, according to bodyweight), once every 2 weeks administered by subcutaneous injection (placebo/dupilumab group). Following a protocol amendment, for a subset of children weighing 30 kg or less, the dose was changed to 300 mg once every 4 weeks. The primary endpoint for the open-label extension study was the number and proportion of patients with any treatment-emergent adverse event (TEAE) during the 52-week study period in the overall population (defined as children aged 6-11 years old with moderate-to-severe asthma who previously completed VOYAGE). Statistical analyses were descriptive. This study is registered with ClinicalTrials.gov (NCT03560466; EXCURSION). FINDINGS:Children who completed VOYAGE were eligible to enrol in EXCURSION between June 21, 2018 and Aug 18, 2020. During EXCURSION, the safety profile and proportion of patients reporting TEAEs were consistent with those observed during the parent study (VOYAGE). In the overall population, 232 (63·6%) of 365 patients experienced at least one TEAE (dupilumab/dupilumab: 147 [61·3%]; placebo/dupilumab: 85 [68·0%]). The most frequently reported TEAEs were nasopharyngitis, pharyngitis, and upper respiratory tract infections. INTERPRETATION:In EXCURSION, long-term treatment with dupilumab was well tolerated with an acceptable safety profile. FUNDING:Sanofi and Regeneron Pharmaceuticals.

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.

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.

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 β (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.

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.