Crystal ribcage: a platform for probing real-time lung function at cellular resolution.

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies.

Baseline oxygen consumption decreases with cortical depth.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase-the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

Unveiling the Adoption and Barriers of Telemedicine in US Hospitals: A Comprehensive Analysis (2017-2022).

BACKGROUND: Telemedicine has emerged as a vital healthcare delivery model, especially pronounced during the COVID-19 pandemic. Our study uniquely focuses on an institutional lens, examining US hospitals to offer targeted policy implications. OBJECTIVE: To investigate the trend in telemedicine adoption across US hospitals from 2017 to 2022 and analyze the institutional challenges they encounter, particularly in the realm of electronic health information exchange. DESIGN: Cross-sectional study leveraging data from the American Hospital Association's (AHA) annual surveys for the years 2017 to 2021 and the 2022 AHA IT Supplement Survey. SETTING: The study includes a national sample of US hospitals, covering a diverse range of hospital types including large, nonprofit, teaching, and system-affiliated institutions. PARTICIPANTS: US hospitals form the study's participants, with a substantial response rate to the surveys. MAIN MEASURES: Key metrics include the number of telemedicine patient encounters, percentage of hospitals offering telemedicine services, and institutional challenges to electronic health information exchange. KEY RESULTS: Telemedicine encounters saw a 75% increase, growing from approximately 111.4 million in 2020 to nearly 194.4 million in 2021. The percentage of hospitals offering at least one form of telemedicine service went from 46% in 2017 to 72% in 2021. Larger, nonprofit, and teaching hospitals were more prone to telehealth adoption, without notable urban-rural disparities. While over 90% of hospitals allow patients to view and download medical records, only 41% permit online data submission. Importantly, 25% of hospitals identified Certified Health IT Developers such as EHR vendor as frequent culprits in information blocking, with cost being the primary obstacle. CONCLUSIONS: The findings underscore the rapid yet uneven adoption of telemedicine services in U.S. hospitals. The results point to the need for comprehensive policy interventions to address the challenges identified and realize telemedicine's full potential in healthcare delivery and resilience.

Factors Associated with Compliance to the Hospital Price Transparency Final Rule: a National Landscape Study.

BACKGROUND: The Hospital Price Transparency Final Rule, effective January 1, 2021, requires hospitals to post online a machine-readable file that includes payer-specific negotiated commercial prices for all services. The regulation aims to improve the affordability of hospital care by promoting price competition. However, a low compliance level among hospitals would compromise the operational effectiveness of this regulation. Understanding hospitals' compliance status to the regulation has important implications for its enforcement effort and effectiveness assessment. OBJECTIVE: To analyze nationwide hospitals' compliance status to the Hospital Price Transparency Rule. DESIGN: Cross-sectional observational study. PARTICIPANTS: A total of 3558 Medicare-certified general acute-care hospitals were examined. MAIN MEASURES: A binary compliance rating was generated by using data collected by Turquoise Health. "Noncompliance" means that no machine-readable file was posted or the posted file contains no commercial negotiated prices. "Compliance" means that a machine-readable file was posted with commercial negotiated prices for at least one insurance plan. KEY RESULTS: As of June 1, 2021, 55% of the 3558 Medicare-certified general acute-care hospitals we examined had not posted a machine-readable file containing commercial negotiated prices. Wide variations of compliance existed across states and hospital referral regions. A hospital's compliance status is strongly associated with the average compliance status of peer hospitals in the same market. Hospitals with greater IT preparedness, for-profit hospitals, system-affiliated hospitals, large hospitals, and non-urban hospitals had greater compliance. More concentrated hospital markets had greater average compliance. CONCLUSIONS: Hospitals take into consideration the behavior of their peers in the same market when making price disclosure decisions. Compliant hospitals are likely to have better IT preparedness, more financial resources and personnel expertise to mitigate the cost required for the implementation of the Price Transparency Rule. The compliance cost, therefore, might be a barrier for some hospitals.

Quantitative muscle analysis in facioscapulohumeral muscular dystrophy using whole-body fat-referenced MRI: Protocol development, multicenter feasibility, and repeatability.

INTRODUCTION/AIMS: Functional performance tests are the gold standard to assess disease progression and treatment effects in neuromuscular disorders. These tests can be confounded by motivation, pain, fatigue, and learning effects, increasing variability and decreasing sensitivity to disease progression, limiting efficacy assessment in clinical trials with small sample sizes. We aimed to develop and validate a quantitative and objective method to measure skeletal muscle volume and fat content based on whole-body fat-referenced magnetic resonance imaging (MRI) for use in multisite clinical trials. METHODS: Subjects aged 18 to 65 years, genetically confirmed facioscapulohumeral muscular dystrophy 1 (FSHD1), clinical severity 2 to 4 (Ricci's scale, range 0-5), were enrolled at six sites and imaged twice 4-12 weeks apart with T1-weighted two-point Dixon MRI covering the torso and upper and lower extremities. Thirty-six muscles were volumetrically segmented using semi-automatic multi-atlas-based segmentation. Muscle fat fraction (MFF), muscle fat infiltration (MFI), and lean muscle volume (LMV) were quantified for each muscle using fat-referenced quantification. RESULTS: Seventeen patients (mean age ± SD, 49.4 years ±13.02; 12 men) were enrolled. Within-patient SD ranged from 1.00% to 3.51% for MFF and 0.40% to 1.48% for MFI in individual muscles. For LMV, coefficients of variation ranged from 2.7% to 11.7%. For the composite score average of all muscles, observed SDs were 0.70% and 0.32% for MFF and MFI, respectively; composite LMV coefficient of variation was 2.0%. DISCUSSION: We developed and validated a method for measuring skeletal muscle volume and fat content for use in multisite clinical trials of neuromuscular disorders.

Plasma-based longitudinal mutation monitoring as a potential predictor of disease progression in subjects with adenocarcinoma in advanced non-small cell lung cancer.

BACKGROUND: Identifying and tracking somatic mutations in cell-free DNA (cfDNA) by next-generation sequencing (NGS) has the potential to transform the clinical management of subjects with advanced non-small cell lung cancer (NSCLC). METHODS: Baseline tumor tissue (n = 47) and longitudinal plasma (n = 445) were collected from 71 NSCLC subjects treated with chemotherapy. cfDNA was enriched using a targeted-capture NGS kit containing 197 genes. Clinical responses to treatment were determined using RECIST v1.1 and correlations between changes in plasma somatic variant allele frequencies and disease progression were assessed. RESULTS: Somatic variants were detected in 89.4% (42/47) of tissue and 91.5% (407/445) of plasma samples. The most commonly mutated genes in tissue were TP53 (42.6%), KRAS (25.5%), and KEAP1 (19.1%). In some subjects, the allele frequencies of mutations detected in plasma increased 3-5 months prior to disease progression. In other cases, the allele frequencies of detected mutations declined or decreased to undetectable levels, indicating clinical response. Subjects with circulating tumor DNA (ctDNA) levels above background had significantly shorter progression-free survival (median: 5.6 vs 8.9 months, respectively; log-rank p = 0.0183). CONCLUSION: Longitudinal monitoring of mutational changes in plasma has the potential to predict disease progression early. The presence of ctDNA mutations during first-line treatment is a risk factor for earlier disease progression in advanced NSCLC.

Comparison of BEAMing and Droplet Digital PCR for Circulating Tumor DNA Analysis.

BACKGROUND: Circulating tumor DNA (ctDNA) assays are increasingly used for clinical decision-making, but it is unknown how well different assays agree. We aimed to assess the agreement in ctDNA mutation calling between BEAMing (beads, emulsion, amplification, and magnetics) and droplet digital PCR (ddPCR), 2 of the most commonly used digital PCR techniques for detecting mutations in ctDNA. METHODS: Baseline plasma samples from patients with advanced breast cancer enrolled in the phase 3 PALOMA-3 trial were assessed for ESR1 and PIK3CA mutations in ctDNA with both BEAMing and ddPCR. Concordance between the 2 approaches was assessed, with exploratory analyses to estimate the importance of sampling effects. RESULTS: Of the 521 patients enrolled, 363 had paired baseline ctDNA analysis. ESR1 mutation detection was 24.2% (88/363) for BEAMing and 25.3% (92/363) for ddPCR, with good agreement between the 2 techniques (κ = 0.9l; 95% CI, 0.85-0.95). PIK3CA mutation detection rates were 26.2% (95/363) for BEAMing and 22.9% (83/363) for ddPCR, with good agreement (κ = 0.87; 95% CI, 0.81-0.93). Discordancy was observed for 3.9% patients with ESR1 mutations and 5.0% with PIK3CA mutations. Assessment of individual mutations suggested higher rates of discordancy for less common mutations (P = 0.019). The majority of discordant calls occurred at allele frequency <1%, predominantly resulting from stochastic sampling effects. CONCLUSIONS: This large, clinically relevant comparison showed good agreement between BEAMing and ddPCR, suggesting sufficient reproducibility for clinical use. Much of the observed discordancy may be related to sampling effects, potentially explaining many of the differences in the currently available ctDNA literature.

Discovery of N-substituted 7-azaindoles as Pan-PIM kinases inhibitors - Lead optimization - Part III.

N-substituted azaindoles were discovered as promising pan-PIM inhibitors. Lead optimization is described en route toward the identification of a clinical candidate. Modulation of physico-chemical properties allowed to solve inherent hERG and permeability liabilities. Compound 17 showed tumor growth inhibition in a KG1 tumor-bearing mouse model.

Prospective for remediation and purification of wastes from Xikuangshan mine by using Si-based substances.

Heavy metal mining includes several procedures producing water and solid wastes. These wastes may have high content of heavy metals and other pollutants. Usually, traditional technologies for purification of solid and liquid wastes are expensive and require a lot of special constructions. Recent investigations have shown that some Si-rich substances enable to regulate the mobility of pollutants in soil and water and enhance the plant resistance to its toxicity. Based on these findings, new way for purification of waste-waters and detoxification of pollutants can be elaborated. Laboratory test was conducted with contaminated solid and liquid wastes from Xikuangshan mine. In column and incubation tests, the contents and mobility of the following pollutants were evaluated in Si-treated and untreated samples: As, Cd, Co, Cr, Cu, Hg, Pb, Ni and Zn. The investigations have shown that the Si-rich substances can be used for filtration of contaminated waste-water. The concentrations of soluble pollutants were reduced by 5-10 times and more. The incubation tests with solid wastes and Si-rich compounds have demonstrated that some Si-based substances reduced the contaminant mobility by 2-4 times. The efficiency of tested substances depended on their solubility on Si. The data has demonstrated that some types of local materials including industrial wastes can be used for purification of waste-waters and detoxification of solid wastes.

Challenges and dynamics of public health reporting and data exchange during COVID-19: insights from US hospitals.

The US health care response during the early stages of the COVID-19 pandemic unveiled challenges in public health reporting systems and electronic clinical data exchange. Using data from the 2020 and 2022 American Hospital Association information technology supplement surveys, this study examined US hospitals' experiences in public health reporting, accessing clinical data from external providers for COVID-19 patient care, and their success in reporting vaccine-related adverse events to relevant state and federal agencies. Results showcase significant disparities in reporting practices across government levels due to inconsistent requirements. Although many hospitals leaned toward automated data transmission, a substantial portion continued to depend on manual processes. Pertaining to electronic clinical data, while entities like large commercial laboratories outperformed others, a considerable number were sluggish in delivering critical information. Moreover, a small percentage of hospitals reported challenges in recording vaccine-related adverse events, emphasizing the need for transparent reporting systems. The study underscores the necessity for standardized reporting protocols, explicit directives, and a pivot from manual to automated processes. Tackling these challenges is pivotal for ensuring prompt and reliable data, bolstering future public health responses, and rejuvenating public trust in health institutions.

Time-Lagged Functional Ultrasound for Multi-Parametric Cerebral Hemodynamic Imaging.

We introduce an ultrasound speckle decorrelation-based time-lagged functional ultrasound technique (tl-fUS) for the quantification of the relative changes in cerebral blood flow speed (rCBF [Formula: see text]), cerebral blood volume (rCBV) and cerebral blood flow (rCBF) during functional stimulations. Numerical simulations, phantom validations, and in vivo mouse brain experiments were performed to test the capability of tl-fUS to parse out and quantify the ratio change of these hemodynamic parameters. The blood volume change was found to be more prominent in arterioles compared to venules and the peak blood flow changes were around 2.5 times the peak blood volume change during brain activation, agreeing with previous observations in the literature. The tl-fUS shows the ability of distinguishing the relative changes of rCBFspeed, rCBV, and rCBF, which can inform specific physiological interpretations of the fUS measurements.

An open-label pilot study of losmapimod to evaluate the safety, tolerability, and changes in biomarker and clinical outcome assessments in participants with facioscapulohumeral muscular dystrophy type 1.

INTRODUCTION: Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease caused by aberrant DUX4 expression, leading to progressive muscle weakness. No effective pharmaceutical treatment is available. Losmapimod, a small molecule selective inhibitor of p38 α/ß MAPK, showed promising results in a phase 1 trial for the treatment of FSHD, prompting additional studies. We report the findings of an open-label phase 2 trial (NCT04004000) investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and exploratory efficacy of losmapimod in participants with FSHD1. METHODS: This study was conducted at a single site in the Netherlands from August 2019 to March 2021, with an optional, ongoing open-label extension. Participants aged 18 to 65 years with FSHD1 took 15 mg of losmapimod twice daily for 52 weeks. Primary endpoints were measures of losmapimod safety and tolerability. Secondary endpoints were assessments of losmapimod pharmacokinetics and pharmacodynamics. RESULTS: Fourteen participants were enrolled. No deaths, serious treatment-emergent adverse events (TEAEs), or discontinuations due to TEAEs were reported. Losmapimod achieved blood concentrations and target engagements that were previously associated with decreased DUX4 expression in vitro. Clinical outcome measures showed a trend toward stabilization or improvement. CONCLUSIONS: Losmapimod was well tolerated and may be a promising new treatment for FSHD; a larger phase 3 study is ongoing.

Visual stimulation drives retinotopic acetylcholine release in the mouse visual cortex.

Cholinergic signaling is involved with a variety of brain functions including learning and memory, attention, and behavioral state modulation. The spatiotemporal characteristics of neocortical acetylcholine (ACh) release in response to sensory inputs are poorly understood, but a lack of intra-region topographic organization of cholinergic projections from the basal forebrain has suggested diffuse release patterns and volume transmission. Here, we use mesoscopic imaging of fluorescent ACh sensors to show that visual stimulation results in ACh release patterns that conform to a retinotopic map of visual space in the mouse primary visual cortex, suggesting new modes of functional cholinergic signaling in cortical circuits.x.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

SIGNIFICANCE: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (mesoscopic) imaging of neuronal activity with fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AIM: Develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. APPROACH: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. RESULTS: We demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved crystal skull window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. CONCLUSIONS: Our widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Significance: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. Aim: We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Approach: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. Results: We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRAB ACh 3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Conclusions: Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Safety and efficacy of losmapimod in facioscapulohumeral muscular dystrophy (ReDUX4): a randomised, double-blind, placebo-controlled phase 2b trial.

BACKGROUND: Facioscapulohumeral muscular dystrophy is a hereditary progressive myopathy caused by aberrant expression of the transcription factor DUX4 in skeletal muscle. No approved disease-modifying treatments are available for this disorder. We aimed to assess the safety and efficacy of losmapimod (a small molecule that inhibits p38α MAPK, a regulator of DUX4 expression, and p38ß MAPK) for the treatment of facioscapulohumeral muscular dystrophy. METHODS: We did a randomised, double-blind, placebo-controlled phase 2b trial at 17 neurology centres in Canada, France, Spain, and the USA. We included adults aged 18-65 years with type 1 facioscapulohumeral muscular dystrophy (ie, with loss of repression of DUX4 expression, as ascertained by genotyping), a Ricci clinical severity score of 2-4, and at least one skeletal muscle judged using MRI to be suitable for biopsy. Participants were randomly allocated (1:1) to either oral losmapimod (15 mg twice a day) or matching placebo for 48 weeks, via an interactive response technology system. The investigator, study staff, participants, sponsor, primary outcome assessors, and study monitor were masked to the treatment allocation until study closure. The primary endpoint was change from baseline to either week 16 or 36 in DUX4-driven gene expression in skeletal muscle biopsy samples, as measured by quantitative RT-PCR. The primary efficacy analysis was done in all participants who were randomly assigned and who had available data for assessment, according to the modified intention-to-treat principle. Safety and tolerability were assessed as secondary endpoints. This study is registered at ClinicalTrials.gov, number NCT04003974. The phase 2b trial is complete; an open-label extension is ongoing. FINDINGS: Between Aug 27, 2019, and Feb 27, 2020, 80 people were enrolled. 40 were randomly allocated to losmapimod and 40 to placebo. 54 (68%) participants were male and 26 (33%) were female, 70 (88%) were White, and mean age was 45·7 (SD 12·5) years. Least squares mean changes from baseline in DUX4-driven gene expression did not differ significantly between the losmapimod (0·83 [SE 0·61]) and placebo (0·40 [0·65]) groups (difference 0·43 [SE 0·56; 95% CI -1·04 to 1·89]; p=0·56). Losmapimod was well tolerated. 29 treatment-emergent adverse events (nine drug-related) were reported in the losmapimod group compared with 23 (two drug-related) in the placebo group. Two participants in the losmapimod group had serious adverse events that were deemed unrelated to losmapimod by the investigators (alcohol poisoning and suicide attempt; postoperative wound infection) compared with none in the placebo group. No treatment discontinuations due to adverse events occurred and no participants died during the study. INTERPRETATION: Although losmapimod did not significantly change DUX4-driven gene expression, it was associated with potential improvements in prespecified structural outcomes (muscle fat infiltration), functional outcomes (reachable workspace, a measure of shoulder girdle function), and patient-reported global impression of change compared with placebo. These findings have informed the design and choice of efficacy endpoints for a phase 3 study of losmapimod in adults with facioscapulohumeral muscular dystrophy. FUNDING: Fulcrum Therapeutics.