Profibrotic monocyte-derived alveolar macrophages are expanded in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19.

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

The H3.3K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation.

Histone H3.3 is frequently mutated in tumors, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the epigenetic landscape, its effects on gene expression dynamics remain unclear. Here, we use a synthetic reporter to measure the effects of H3.3K36M on silencing and epigenetic memory after recruitment of the ZNF10 Krüppel-associated box (KRAB) domain, part of the largest class of human repressors and associated with H3K9me3 deposition. We find that H3.3K36M, which decreases H3K36 methylation and increases histone acetylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a model for establishment and maintenance of epigenetic memory, where the H3K36 methylation pathway is necessary to maintain histone deacetylation and convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

Recent Developments in Machine Learning for Mass Spectrometry.

Statistical analysis and modeling of mass spectrometry (MS) data have a long and rich history with several modern MS-based applications using statistical and chemometric methods. Recently, machine learning (ML) has experienced a renaissance due to advents in computational hardware and the development of new algorithms for artificial neural networks (ANN) and deep learning architectures. Moreover, recent successes of new ANN and deep learning architectures in several areas of science, engineering, and society have further strengthened the ML field. Importantly, modern ML methods and architectures have enabled new approaches for tasks related to MS that are now widely adopted in several popular MS-based subdisciplines, such as mass spectrometry imaging and proteomics. Herein, we aim to provide an introductory summary of the practical aspects of ML methodology relevant to MS. Additionally, we seek to provide an up-to-date review of the most recent developments in ML integration with MS-based techniques while also providing critical insights into the future direction of the field.

Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

The Lunar Environment Heliophysics X-ray Imager (LEXI) Mission.

The Lunar Environment heliospheric X-ray Imager (LEXI) is a wide field-of-view soft X-ray telescope developed to study solar wind-magnetosphere coupling. LEXI is part of the Blue Ghost 1 mission comprised of 10 payloads to be deployed on the lunar surface. LEXI monitors the dayside magnetopause position and shape as a function of time by observing soft X-rays (0.1-2 keV) emitted from solar wind charge-exchange between exospheric neutrals and high charge-state solar wind plasma in the dayside magnetosheath. Measurements of the shape and position of the magnetopause are used to test temporal models of meso- and macro-scale magnetic reconnection. To image the boundary, LEXI employs lobster-eye optics to focus X-rays to a microchannel plate detector with a 9.1×∘9.1∘ field of view.

PLASMA DYNAMICS OF NEUTROPHIL EXTRACELLULAR TRAPS AND CELL-FREE DNA IN SEPTIC AND NONSEPTIC VASOPLEGIC SHOCK: A PROSPECTIVE COMPARATIVE OBSERVATIONAL COHORT STUDY.

ABSTRACT: Background: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 h after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or noninfectious (following cardiac surgery, CARDIAC) origin. Methods: This is a prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H, and 48H in SEPSIS and CARDIAC patients. The vasopressor inotropic score (VIS), the Sequential Organ Failure Assessment (SOFA) score, and time spent with invasive ventilation, in ICU and in hospital, were recorded. Associations between NETs/cfDNA and VIS and SOFA were analyzed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalization times by generalized linear regression. Results: Both NETs and cfDNA remained elevated over 48 h in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time-weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], P = 0.005; cfDNA median difference 0.48 [0.20-1.02], P < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, P < 0.01, rho = 0.36-0.57 in CARDIAC, P ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, P < 0.01, rho = 0.38-0.47 in CARDIAC, P < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalization times. Conclusion: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 h in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or noninfectious etiology.

Lesion-remote astrocytes govern microglia-mediated white matter repair.

Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.

Targeted therapy, immunotherapy, and small molecules and peptidomimetics as emerging immunoregulatory agents for melanoma.

Primary cutaneous melanoma is the most lethal of all skin neoplasms and its incidence is increasing. Clinical management of advanced melanoma in the last decade has been revolutionised by the availability of immunotherapies and targeted therapies, used alone and in combination. This article summarizes advances in the treatment of late-stage melanoma including use of protein kinase inhibitors, antibody-based immune checkpoint inhibitors, adoptive immunotherapy, vaccines and more recently, small molecules and peptidomimetics as emerging immunoregulatory agents.

The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation.

Histone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the cellular epigenetic landscape, it remains unclear how it affects the dynamics of gene expression. Here, we use a synthetic reporter to measure the effect of H3.3K36M on silencing and epigenetic memory after recruitment of KRAB: a member of the largest class of human repressors, commonly used in synthetic biology, and associated with H3K9me3. We find that H3.3K36M, which decreases H3K36 methylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a new model for establishment and maintenance of epigenetic memory, where H3K36 methylation is necessary to convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

Novel mesh suture may resist bone cutting seen with wire-based sternal closures.

Objective: Sternal dehiscence is frequently associated with wire-based closures cutting through fragile bone, allowing sternal motion, separation, and infection. We investigated whether bone cutting could be limited by using a newly available mesh suture with improved force distribution. Methods: Five sternal models were closed using 8 interrupted single sternal wires, double sternal wires, braided poly(ethylene terephthalate) sutures, single-wrapped mesh sutures, or double-wrapped mesh sutures. To simulate chest-wall forces, closed sternal models were pulled apart using 1020 N of axial force applied incrementally. Double sternal wire and double-wrapped mesh suture were further compared by closing 3 new models with each material and subjecting these models to cyclic loading cycles, simulating breathing and coughing. Image analysis of needle hole size measured "bone cutting" by each closure material and sternal distraction as a function of force. Results: All models exhibited maximal separation at the xiphoid. During axial loading, needle hole size increased 7.2% in the double-wrapped mesh suture model and 9.2% in the double-wire model. Single-wrapped mesh suture, single wires, and braided poly(ethylene terephthalate) extended needle hole size by 6.7%, 47.0%, and 168.3% of original size, respectively. The double-wire model resisted sternal distraction best, separating 0.285 cm at the xiphoid. During cyclic loading, mesh suture exhibited significantly less bone cutting (P = .02) than double wire, with comparable levels of sternal separation (P = .07). Conclusions: Mesh suture may resist bone cutting seen in sternal wire closure in bone models with comparable distraction to currently used sternal closure methods.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

Immune Checkpoint Inhibitors, Small-Molecule Immunotherapies and the Emerging Role of Neutrophil Extracellular Traps in Therapeutic Strategies for Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of many cancer types, including head and neck cancers (HNC). When checkpoint and partner proteins bind, these send an "off" signal to T cells, which prevents the immune system from destroying tumor cells. However, in HNC, and indeed many other cancers, more people do not respond and/or suffer from toxic effects than those who do respond. Hence, newer, more effective approaches are needed. The challenge to durable therapy lies in a deeper understanding of the complex interactions between immune cells, tumor cells and the tumor microenvironment. This will help develop therapies that promote lasting tumorlysis by overcoming T-cell exhaustion. Here we explore the strengths and limitations of current ICI therapy in head and neck squamous cell carcinoma (HNSCC). We also review emerging small-molecule immunotherapies and the growing promise of neutrophil extracellular traps in controlling tumor progression and metastasis.

High-throughput discovery and characterization of viral transcriptional effectors in human cells.

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across ∼1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in viral interferon regulatory factors (VIRFs), and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in Kaposi's sarcoma-associated herpesvirus (KSHV). For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

Perioperative outcomes of open versus robot-assisted radical cystectomy in octogenarians: a population based analysis.

Octogenarians undergoing cystectomy experience higher morbidity and mortality compared to younger patients. Though the non-inferiority of robot-assisted radical cystectomy (RARC) compared to open radical cystectomy (ORC) has been established in a generalized population, the benefits of the robotic approach have not been well studied in an aged population. The National Cancer Database (NCDB) was queried for all patients who underwent cystectomy for bladder cancer from 2010 to 2016. Of these, 2527 were performed in patients age 80 or older; 1988 and 539 underwent ORC and RARC, respectively. On Cox regression analysis, RARC was associated with significantly reduced odds for both 30- and 90-day mortality (HR 0.404, p = 0.004; HR 0.694, p = 0.031, respectively), though the association with overall mortality was not significant (HR 0.877, p = 0.061). The robotic group had a significantly shorter length of stay (LOS) compared to open surgery (10.3 days ORC vs. 9.3 days RARC, p = 0.028). The proportion of cases performed robotically increased over the study period from 12.2% in 2010 to 28.4% in 2016 (p = 0.009, R2 = 0.774). The study is limited by a retrospective design and a section bias, which was not completely control for in the analysis. In conclusion, RARC provides improved perioperative outcomes in aged patients compared to ORC and a trend toward greater utilization of this technique was observed.

Tensile strength and knot security of five suture materials exposed to natural summer conditions of a temperate lake.

OBJECTIVE: Wild fish and other aquatic ectotherms are often subjected to procedures during field research that require wound closure using sutures. A variety of absorbable sutures are available for such purposes, yet degradation processes are highly dependent on temperature, and the environments in which wild ectotherms are released are almost always colder than the conditions for which absorbable sutures are typically designed (i.e., ~37°C). We therefore studied the degradation of various suture materials under a set of biologically relevant conditions for temperate freshwater fish. METHODS: Using a force gauge, we tested the tensile strengths and knot securities of loops tied with five different absorbable suture materials (PDS-II, dyed coated Vicryl, undyed coated Vicryl, plain gut, and chromic gut) prior to and during submersion in a temperate lake over an 8-week period. RESULT: The naturally derived collagen-based suture materials (i.e., plain gut and chromic gut) exhibited major decreases in tensile strength within 2 weeks of submersion but maintained relatively high knot security throughout the study period. The synthetic suture loops had poorer initial knot securities that increased following submersion and showed little to no evidence of degradation after 8 weeks. CONCLUSION: Variable rates of absorbable suture degradation, or lack thereof, were observed. We discuss the implications of these trends for fish welfare considerations such as suture retention, wound healing, inflammation, and infection under natural conditions.

IBRAP: integrated benchmarking single-cell RNA-sequencing analytical pipeline.

Single-cell ribonucleic acid (RNA)-sequencing (scRNA-seq) is a powerful tool to study cellular heterogeneity. The high dimensional data generated from this technology are complex and require specialized expertise for analysis and interpretation. The core of scRNA-seq data analysis contains several key analytical steps, which include pre-processing, quality control, normalization, dimensionality reduction, integration and clustering. Each step often has many algorithms developed with varied underlying assumptions and implications. With such a diverse choice of tools available, benchmarking analyses have compared their performances and demonstrated that tools operate differentially according to the data types and complexity. Here, we present Integrated Benchmarking scRNA-seq Analytical Pipeline (IBRAP), which contains a suite of analytical components that can be interchanged throughout the pipeline alongside multiple benchmarking metrics that enable users to compare results and determine the optimal pipeline combinations for their data. We apply IBRAP to single- and multi-sample integration analysis using primary pancreatic tissue, cancer cell line and simulated data accompanied with ground truth cell labels, demonstrating the interchangeable and benchmarking functionality of IBRAP. Our results confirm that the optimal pipelines are dependent on individual samples and studies, further supporting the rationale and necessity of our tool. We then compare reference-based cell annotation with unsupervised analysis, both included in IBRAP, and demonstrate the superiority of the reference-based method in identifying robust major and minor cell types. Thus, IBRAP presents a valuable tool to integrate multiple samples and studies to create reference maps of normal and diseased tissues, facilitating novel biological discovery using the vast volume of scRNA-seq data available.