Molecular basis of pigment structural diversity in echinoderms.

The varied pigments found in animals play both ecological and physiological roles. Virtually all echinoderms contain putative pigment biosynthetic enzymes, the polyketide synthases (PKSs). Among these, crinoids have complex pigments found both today and in ancient fossils. Here, we characterize a key pigment biosynthetic enzyme, CrPKS from the crinoid Anneissia japonica. We show that CrPKS produces 14-carbon aromatic pigment precursors. Despite making a compound previously found in fungi, the crinoid enzyme operates by different biochemical principles, helping to explain the diverse animal PKSs found throughout the metazoan (animal) kingdom. Unlike SpPks1 from sea urchins that had strict starter unit selectivity, CrPKS also incorporated starter units butyryl- or ethylmalonyl-CoA to synthesize a crinoid pigment precursor with a saturated side chain. By performing biochemical experiments, we show how changes in the echinoderm pigment biosynthetic enzymes unveil the vast variety of colors found in animals today.

Spontaneously regenerative corticospinal neurons in mice.

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

Matrix metalloproteinases mediate influenza A-associated shedding of the alveolar epithelial glycocalyx.

BACKGROUND: The alveolar epithelium is protected by a heparan sulfate-rich, glycosaminoglycan layer called the epithelial glycocalyx. It is cleaved in patients with acute respiratory distress syndrome (ARDS) and in murine models of influenza A (IAV) infection, shedding fragments into the airspace from the cell surface. Glycocalyx shedding results in increased permeability of the alveolar-capillary barrier, amplifying acute lung injury. The mechanisms underlying alveolar epithelial glycocalyx shedding in IAV infection are unknown. We hypothesized that induction of host sheddases such as matrix metalloproteinases (MMPs) during IAV infection results in glycocalyx shedding and increased lung injury. MATERIALS AND METHODS: We measured glycocalyx shedding and lung injury during IAV infection with and without treatment with the pan-MMP inhibitor Ilomastat (ILO) and in an MMP-7 knock out (MMP-7KO) mouse. C57BL/6 or MMP-7KO male and female mice were given IAV A/PR/8/34 (H1N1) at 30,000 PFU/mouse or PBS intratracheally. For some experiments, C56BL/6 mice were infected in the presence of ILO (100mg/kg) or vehicle given daily by IP injection. Bronchoalveolar lavage (BAL) and lung tissue were collected on day 1, 3, and 7 for analysis of glycocalyx shedding (BAL Syndecan-1) and lung injury (histology, BAL protein, BAL cytokines, BAL immune cell infiltrates, BAL RAGE). Expression and localization of the sheddase MMP-7 and its inhibitor TIMP-1 was examined by RNAScope. For in vitro experiments, MLE-12 mouse lung epithelial cells were cultured and treated with active or heat-inactivated heparinase (2.5 U/mL) prior to infection with IAV (MOI 1) and viral load and MMP-7 and TIMP-1 expression analyzed. RESULTS: IAV infection caused shedding of the epithelial glycocalyx into the BAL. Inhibition of MMPs with ILO reduced glycocalyx shedding by 36% (p = 0.0051) and reduced lung epithelial injury by 40% (p = 0.0404). ILO also reduced viral load by 68% (p = 0.027), despite having no significant effect on lung cytokine production. Both MMP-7 and its inhibitor TIMP-1 were upregulated in IAV infected mice: MMP-7 colocalized with IAV, while TIMP-1 was limited to cells adjacent to infection. However, MMP-7KO mice had similar glycocalyx shedding, epithelial injury, and viral load compared to WT littermates, suggesting redundancy in MMP sheddase function in the lung. In vitro, heparinase treatment before infection led to a 52% increase in viral load (p = 0.0038) without altering MMP-7 or TIMP-1 protein levels. CONCLUSIONS: Glycocalyx shedding and MMPs play key roles in IAV-induced epithelial injury, with significant impact on IAV viral load. Further studies are needed to understand which specific MMPs regulate lung epithelial glycocalyx shedding.

Common and divergent pathways in early stages of glutamate and tau-mediated toxicities in neurodegeneration.

It has been shown that excitotoxicity and tau-mediated toxicities are major contributing factors to neuronal death in Alzheimer's disease (AD). The excitatory amino acid transporter 2 (EAAT2 or GLT-1), the major glutamate transporter in the brain that regulates glutamate levels synaptically and extrasynaptically, has been shown to be deficient in AD brains, leading to excitotoxicity and subsequent cell death. Similarly, buildup of neurofibrillary tangles, which consist of hyperphosphorylated tau protein, correlates with cognitive decline and neuronal atrophy in AD. However, common genes and pathways that are critical in the aforementioned toxicities have not been well elucidated. To investigate the impact of glutamate dyshomeostasis and tau accumulation on translational profiles of affected hippocampal neurons, we used mouse models of excitotoxicity and tau-mediated toxicities (GLT-1-/- and P301S, respectively) in conjunction with BAC-TRAP technology. Our data show that GLT-1 deficiency in CA3 pyramidal neurons leads to translational signatures characterized by dysregulation of pathways associated with synaptic plasticity and neuronal survival, while the P301S mutation induces changes in endocytic pathways and mitochondrial dysfunction. Finally, the commonly dysregulated pathways include impaired ion homeostasis and metabolic pathways. These common pathways may shed light on potential therapeutic targets for ameliorating glutamate and tau-mediated toxicities in AD.

CIRCULATING HEPARAN SULFATE PROFILES IN PEDIATRIC ACUTE RESPIRATORY DISTRESS SYNDROME.

ABSTRACT: Introduction: Sepsis-induced degradation of endothelial glycocalyx heparan sulfate (HS) contributes to the pulmonary microvascular endothelial injury characteristic of acute respiratory distress syndrome (ARDS) pathogenesis. Our objectives were to (1) examine relationships between plasma indices of HS degradation and protein biomarkers of endothelial injury and (2) identify patient subgroups characterized by distinct profiles of HS degradation in children with ARDS. Methods: We analyzed prospectively collected plasma (2018-2020) from a cohort of invasively mechanically ventilated children (aged >1 month to <18 years) with ARDS. Mass spectrometry characterized and quantified patterns of HS disaccharide sulfation. Protein biomarkers reflective of endothelial injury (e.g., angiopoietin-2, vascular cell adhesion molecule-1, soluble thrombomodulin) were measured with a multiplex immunoassay. Pearson correlation coefficients were used to construct a biomarker correlation network. Centrality metrics detected influential biomarkers (i.e., network hubs). K-means clustering identified unique patient subgroups based on HS disaccharide profiles. Results: We evaluated 36 patients with pediatric ARDS. HS disaccharide sulfation patterns, 6S, NS, and NS2S, positively correlated with all biomarkers of endothelial injury (all P < 0.05) and were classified as network hubs. We identified three patient subgroups, with cluster 3 (n = 5) demonstrating elevated levels of 6S and N-sulfated HS disaccharides. In cluster 3, 60% of children were female and nonpulmonary sepsis accounted for 60% of cases. Relative to cluster 1 (n = 12), cluster 3 was associated with higher oxygen saturation index (P = 0.029) and fewer 28-day ventilator-free days (P = 0.016). Conclusions: Circulating highly sulfated HS fragments may represent emerging mechanistic biomarkers of endothelial injury and disease severity in pediatric ARDS.

Studying the Pulmonary Endothelium in Health and Disease: An Official American Thoracic Society Workshop Report.

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

Miniaturized Fab' imaging probe derived from a clinical antibody: Characterization and imaging in CRISPRi-attenuated mammary tumor models.

Clinical imaging-assisted oncosurgical navigation requires cancer-specific miniaturized optical imaging probes. We report a near-infrared (NIR) Fab'-based epidermal growth factor receptor (EGFR)-specific probe carrying 3 NIR fluorophores (Fab'-800CW), which retained high-affinity binding to EGFR ectodomain (equilibrium KD E = 1 nM). Fab'-800CW showed a robust 4-times gain of fluorescence intensity (FI) and a 20% lifetime (FLT) increase under the conditions mimicking intracellular degradation. The probe was tested by using triple-negative breast cancer (TNBC) cell lines obtained by applying CRISPR interference (CRISPRi) effect of EGFR-targeting sgRNA and dCas9-KRAB chimera coexpression in MDA-MB-231 cells (WT cells). FI imaging in cell culture proved a 50% EGFR expression attenuation by CRISPRi. FI imaging in animals harboring attenuated or WT TNBC tumors with ex vivo corroboration identified differences between WT and CRISPRi tumors FI at 30 min post injection. Our results suggest the feasibility of EGFR expression imaging using a Fab'-based probe relevant for imaging-guided cancer surgery.

Differences across race and ethnicity in the quality of antidepressant medication management.

OBJECTIVE: To illustrate the importance of a multidimensional view of disparities in quality of antidepressant medication management (AMM), as well as discriminating "within-facility" disparities from disparities that exist between facilities. DATA SOURCES AND STUDY SETTING: We used data from the Veterans Health Administration's (VA) Corporate Data Warehouse (CDW) which contains clinical and administrative data from VA facilities nationally. STUDY DESIGN: CDW data were used to measure five indicators of AMM quality, including the HEDIS Effective Acute-Phase and Effective Continuation-Phase measures. Mixed effects regression models were used to examine differences in quality indicators between racial/ethnic groups, controlling for other demographic and clinical factors. An adaptation of the Kitagawa-Blinder-Oaxaca (KBO) method was used to decompose mean differences in treatment quality between racial and ethnic groups into within- and between-facility effects. DATA EXTRACTION METHODS: Demographic, clinical, and health service utilization data were extracted for patients in fiscal year 2017 with a diagnosis of depression and a new start of an antidepressant medication. PRINCIPAL FINDINGS: The decomposition of the overall differences between White and Black patients on receiving an initial 90-day prescription (46.7% vs. 32.7%), Effective Acute-Phase (79.7% vs. 66.8%), and Effective Continuation-Phase (64.0% vs. 49.6%) HEDIS measures revealed that most of the overall effects were "within-facility," meaning that Black patients are less likely to meet these measures regardless of where they are treated. Although the overall magnitude of disparities between White and Hispanic patients on these three measures was very similar (46.7% vs. 32.7%; 79.7% vs. 69.2%; 64.0% vs. 53.6%), the differences were more attributable to Hispanic patients being treated in facilities with overall lower performance on these measures. CONCLUSIONS: Discriminating within- and between-facility disparities and taking a multidimensional view of quality are essential to informing efforts to address disparities in AMM quality.

Circulating Biomarkers of Endothelial Dysfunction Associated With Ventilatory Ratio and Mortality in ARDS Resulting From SARS-CoV-2 Infection Treated With Antiinflammatory Therapies.

BACKGROUND: The association of plasma biomarkers and clinical outcomes in ARDS resulting from SARS-CoV-2 infection predate the evidence-based use of immunomodulators. RESEARCH QUESTION: Which plasma biomarkers are associated with clinical outcomes in patients with ARDS resulting from SARS-CoV-2 infection treated routinely with immunomodulators? STUDY DESIGN AND METHODS: We collected plasma from patients with ARDS resulting from SARS-CoV-2 infection within 24 h of admission to the ICU between December 2020 and March 2021 (N = 69). We associated 16 total biomarkers of inflammation (eg, IL-6), coagulation (eg, D-dimer), epithelial injury (eg, surfactant protein D), and endothelial injury (eg, angiopoietin-2) with the primary outcome of in-hospital mortality and secondary outcome of ventilatory ratio (at baseline and day 3). RESULTS: Thirty patients (43.5%) died within 60 days. All patients received corticosteroids and 6% also received tocilizumab. Compared with survivors, nonsurvivors demonstrated a higher baseline modified Sequential Organ Failure Assessment score (median, 8.5 [interquartile range (IQR), 7-9] vs 7 [IQR, 5-8]); P = .004), lower Pao2 to Fio2 ratio (median, 153 [IQR, 118-182] vs 184 [IQR, 142-247]; P = .04), and higher ventilatory ratio (median, 2.0 [IQR, 1.9-2.3] vs 1.5 [IQR, 1.4-1.9]; P < .001). No difference was found in inflammatory, coagulation, or epithelial biomarkers between groups. Nonsurvivors showed higher median neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) levels (median, 8.4 ng/mL [IQR, 7.0-11.2 ng/mL] vs 6.9 ng/mL [IQR, 5.5-8.0 ng/mL]; P = .0025), von Willebrand factor domain A2 levels (8.7 ng/mL [IQR, 7.9-9.7 ng/mL] vs 6.5 ng/mL [IQR, 5.7-8.7 ng/mL]; P = .007), angiopoietin-2 levels (9.0 ng/mL [IQR, 7.9-14.1 ng/mL] vs 7.0 ng/mL [IQR, 5.6-10.6 ng/mL]; P = .01), and syndecan-1 levels (15.9 ng/mL [IQR, 14.5-17.5 ng/mL] vs 12.6 ng/mL [IQR, 10.5-16.1 ng/mL]; P = .01). Only NEDD9 level met the adjusted threshold for significance (P < .003). Plasma NEDD9 level was associated with 60-day mortality (adjusted OR, 9.7; 95% CI, 1.6-60.4; P = .015). Syndecan-1 level correlated with both baseline (ρ = 0.4; P = .001) and day 3 ventilatory ratio (ρ = 0.5; P < .001). INTERPRETATION: Biomarkers of inflammation, coagulation, and epithelial injury were not associated with clinical outcomes in a small cohort of patients with ARDS uniformly treated with immunomodulators. However, endothelial biomarkers, including plasma NEDD9, were associated with 60-day mortality.

Brewing coral terpenes-A yeast based approach to soft coral terpene cyclases.

Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules.

Cerebrospinal fluid proteomic profile of frailty: Results from the PROLIPHYC cohort.

Frailty is a clinical state reflecting a decrease in physiological reserve capacities, known to affect numerous biological pathways and is associated with health issues, including neurodegenerative diseases. However, how global protein expression is affected in the central nervous system in frail subject remains underexplored. In this post hoc cross-sectional biomarker analysis, we included 90 adults (52-85 years) suspected of normal pressure hydrocephalus (NPH) and presenting with markers of neurodegenerative diseases. We investigated the human proteomic profile of cerebrospinal fluid associated with frailty defined by an established cumulated frailty index (FI, average = 0.32), not enriched for neurology clinical features. Using a label-free quantitative proteomic approach, we identified and quantified 999 proteins of which 13 were positively associated with frailty. Pathway analysis with the top positively frailty-associated proteins revealed enrichment for proteins related to inflammation and immune response. Among the 60 proteins negatively associated with frailty, functional pathways enriched included neurogenesis, synaptogenesis and neuronal guidance. We constructed a frailty prediction model using ridge regression with 932 standardized proteins. Our results showed that the "proteomic model" could become an equivalent predictor of FI in order to study chronological age. This study represents the first comprehensive exploration of the proteomic profile of frailty within cerebrospinal fluid. It sheds light on the physiopathology of frailty, particularly highlighting processes of neuroinflammation and inhibition of neurogenesis. Our findings unveil a range of biological mechanisms that are dysregulated in frailty, in NPH subjects at risk of neurodegenerative impairment, offering new perspectives on frailty phenotyping and prediction.

Host-derived protease promotes aggregation of Staphylococcus aureus by cleaving the surface protein SasG.

Staphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections.

Short macrocyclic peptides in sponge genomes.

Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.

Inflammation, endothelial injury, and the acute respiratory distress syndrome after out-of-hospital cardiac arrest.

Background: Acute respiratory distress syndrome (ARDS) is often seen in patients resuscitated from out-of-hospital cardiac arrest (OHCA). We aim to test whether inflammatory or endothelial injury markers are associated with the development of ARDS in patients hospitalized after OHCA. Methods: We conducted a prospective, cohort, pilot study at an urban academic medical center in 2019 that included a convenience sample of adults with non-traumatic OHCA. Blood and pulmonary edema fluid (PEF) were collected within 12 hours of hospital arrival. Samples were assayed for cytokines (interleukin [IL]-1, tumor necrosis factor-α [TNF-α], tumor necrosis factor receptor1 [TNFR1], IL-6), epithelial injury markers (pulmonary surfactant-associated protein D), endothelial injury markers (Angiopoietin-2 [Ang-2] and glycocalyx degradation products), and other proteins (matrix metallopeptidase-9 and myeloperoxidase). Patients were followed for 7 days for development of ARDS, as adjudicated by 3 blinded reviewers, and through hospital discharge for mortality and neurological outcome. We examined associations between biomarker concentrations and ARDS, hospital mortality, and neurological outcome using multivariable logistic regression. Latent phase analysis was used to identify distinct biological classes associated with outcomes. Results: 41 patients were enrolled. Mean age was 58 years, 29% were female, and 22% had a respiratory etiology for cardiac arrest. Seven patients (17%) developed ARDS within 7 days. There were no significant associations between individual biomarkers and development of ARDS in adjusted analyses, nor survival or neurologic status after adjusting for use of targeted temperature management (TTM) and initial cardiac arrest rhythm. Elevated Ang-2 and TNFR-1 were associated with decreased survival (RR = 0.6, 95% CI = 0.3-1.0; RR = 0.5, 95% CI = 0.3-0.9; respectively), and poor neurologic status at discharge (RR = 0.4, 95% CI = 0.2-0.8; RR = 0.4, 95% CI = 0.2-0.9) in unadjusted associations. Conclusion: OHCA patients have markedly elevated plasma and pulmonary edema fluid biomarker concentrations, indicating widespread inflammation, epithelial injury, and endothelial activation. Biomarker concentrations were not associated with ARDS development, though several distinct biological phenotypes warrant further exploration. Latent phase analysis demonstrated that patients with low biomarker levels aside from TNF-α and TNFR-1 (Class 2) fared worse than other patients. Future research may benefit from considering other tools to predict and prevent development of ARDS in this population.

Improving Compliance with Preoperative Nasal Povidone-Iodine to Prevent Surgical Site Infection in Vascular and Neurosurgery Services in a Community Teaching Hospital.

Surgical site infections (SSI) remain a cause of morbidity, prolonged hospitalization, surgical readmission, and death. Nasal colonization with methicillin-resistant Staphylococcal aureus is a frequent cause of device-related SSI and nasal mupirocin has been used for prevention. More recently, povidone-iodine nasal swabs have become an alternative. It is cheaper, ensures compliance and there are no concerns regarding antimicrobial resistance. However, its adoption was suboptimal in a community hospital system in southwestern Ohio, especially in neurosurgery and vascular surgery. Quality improvement techniques, including solicitation of stakeholder input, surgeons and perioperative nurses' education, and the use of reminders to order and administer the povidone-iodine nasal swabs improved physician ordering and nurse administration compliance, leading to fewer infections. The interventions continued after the project was completed, sustaining decreases in neurosurgery and vascular surgery, and fewer SSI through the first years of the pandemic. Despite the complexity of these surgeries, simple interventions were effective in addressing the problem.

A preclinical systematic review and meta-analysis assessing the effect of biological sex in lipopolysaccharide-induced acute lung injury.

It is unclear what effect biological sex has on outcomes of acute lung injury (ALI). Clinical studies are confounded by their observational design. We addressed this knowledge gap with a preclinical systematic review of ALI animal studies. We searched MEDLINE and Embase for studies of intratracheal/intranasal/aerosolized lipopolysaccharide administration (the most common ALI model) that reported sex-stratified data. Screening and data extraction were conducted in duplicate. Our primary outcome was histological tissue injury and secondary outcomes included alveolar-capillary barrier alterations and inflammatory markers. We used a random-effects inverse variance meta-analysis, expressing data as standardized mean difference (SMD) with 95% confidence intervals (CIs). Risk of bias was assessed using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool. We identified six studies involving 132 animals across 11 independent experiments. A total of 41 outcomes were extracted, with the direction of effect suggesting greater severity in males than females in 26/41 outcomes (63%). One study reported on lung histology and found that male mice exhibited greater injury than females (SMD: 1.61, 95% CI: 0.53-2.69). Meta-analysis demonstrated significantly elevated albumin levels (SMD: 2.17, 95% CI: 0.63-3.70) and total cell counts (SMD: 0.80, 95% CI: 0.27-1.33) in bronchoalveolar lavage fluid from male mice compared with female mice. Most studies had an "unclear risk of bias." Our findings suggest sex-related differences in ALI severity. However, these conclusions are drawn from a small number of animals and studies. Further research is required to address the fundamental issue of biological sex differences in LPS-induced ALI.

Methylone is a rapid-acting neuroplastogen with less off-target activity than MDMA.

Background: Post-traumatic stress disorder (PTSD) is a highly prevalent psychiatric disorder that can become chronic and debilitating when left untreated. Available pharmacotherapies are limited, take weeks to show modest benefit and remain ineffective for up to 40% of patients. Methylone is currently in clinical development for the treatment of PTSD. Preclinical studies show rapid, robust and long-lasting antidepressant-like and anxiolytic effects. The mechanism of action underlying these effects is not yet fully understood. This study investigated the downstream gene expression changes and signaling pathways affected by methylone in key brain areas linked to PTSD and MDD. It also sought to determine whether neuroplasticity-related genes were involved. We compared effects of methylone with MDMA to explore similarities and differences in their brain effects because MDMA-assisted psychotherapy has recently shown benefit in clinical trials for PTSD and methylone is a structural analog of MDMA. Methods: Monoamine binding, uptake and release studies were performed and a high-throughput-screen evaluated agonist/antagonist activities at 168 GPCRs in vitro. We used RNA sequencing (RNA-seq) to probe drug-induced gene expression changes in the amygdala and frontal cortex, two brain areas responsible for emotional learning that are affected by PTSD and MDD. Rats were treated with methylone or MDMA (both 10 mg/kg, IP), and their responses were compared with controls. We performed functional enrichment analysis to identify which pathways were regulated by methylone and/or MDMA. We confirmed changes in gene expression using immunohistochemistry. Results: Methylone, a monoamine uptake inhibitor and releaser, demonstrated no off-target effects at 168 GPCRs, unlike MDMA, which showed activity at 5HT2A and 5HT2C receptors. RNA-seq results revealed significant regulation of myelin-related genes in the amygdala, confirmed by immunohistochemistry. In the frontal cortex, methylone significantly upregulated genes implicated in neuroplasticity. Conclusion: Results suggest that (1) methylone is a rapid-acting neuroplastogen that affects key brain substrates for PTSD and MDD and that (2) methylone appears to exhibit higher specificity and fewer off-target effects than MDMA. Together, these results are consistent with the reported clinical experiences of methylone and MDMA and bolster the potential use of methylone in the treatment of PTSD and, potentially, other neuropsychiatric disorders.

RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.