Pro-inflammatory macrophage activation does not require inhibition of mitochondrial respiration.

Pro-inflammatory macrophage activation is a hallmark example of how mitochondria serve as signaling organelles. Upon classical macrophage activation, oxidative phosphorylation sharply decreases and mitochondria are repurposed to accumulate signals that amplify effector function. However, evidence is conflicting as to whether this collapse in respiration is essential or largely dispensable. Here we systematically examine this question and show that reduced oxidative phosphorylation is not required for pro-inflammatory macrophage activation. Only stimuli that engage both MyD88- and TRIF-linked pathways decrease mitochondrial respiration, and different pro-inflammatory stimuli have varying effects on other bioenergetic parameters. Additionally, pharmacologic and genetic models of electron transport chain inhibition show no direct link between respiration and pro-inflammatory activation. Studies in mouse and human macrophages also reveal accumulation of the signaling metabolites succinate and itaconate can occur independently of characteristic breaks in the TCA cycle. Finally, in vivo activation of peritoneal macrophages further demonstrates that a pro-inflammatory response can be elicited without reductions to oxidative phosphorylation. Taken together, the results suggest the conventional model of mitochondrial reprogramming upon macrophage activation is incomplete.

MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

The metabolic cofactor Coenzyme A enhances alternative macrophage activation via MyD88-linked signaling.

Metabolites and metabolic co-factors can shape the innate immune response, though the pathways by which these molecules adjust inflammation remain incompletely understood. Here we show that the metabolic cofactor Coenzyme A (CoA) enhances IL-4 driven alternative macrophage activation [m(IL-4)] in vitro and in vivo. Unexpectedly, we found that perturbations in intracellular CoA metabolism did not influence m(IL-4) differentiation. Rather, we discovered that exogenous CoA provides a weak TLR4 signal which primes macrophages for increased receptivity to IL-4 signals and resolution of inflammation via MyD88. Mechanistic studies revealed MyD88-linked signals prime for IL-4 responsiveness, in part, by reshaping chromatin accessibility to enhance transcription of IL-4-linked genes. The results identify CoA as a host metabolic co-factor that influences macrophage function through an extrinsic TLR4-dependent mechanism, and suggests that damage-associated molecular patterns (DAMPs) can prime macrophages for alternative activation and resolution of inflammation.

Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome.

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome.

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

The Association Between Distance Learning, Stress Level, and Perceived Quality of Education in Medical Students After Transitioning to a Fully Online Platform.

BACKGROUND: Before COVID-19, preclinical medical students traditionally attended didactic lectures in in-person settings. Due to social distancing, students were required to switch to online meeting platforms, such as Zoom. For medical students accustomed to in-person interactions, these changes may add more stress to the already stressful medical school experience. Furthermore, it was unclear if students' stress levels were related to their preference for one learning modality over another. The purpose of this study was thus to explore associations between lecture modality (synchronous Zoom lectures versus live, in-person lectures) and stress in second-year medical students after they transitioned from a face-to-face learning experience to a fully online lecture platform. METHODOLOGY: Cross-sectional data were collected from 112 second-year medical students enrolled in a large U.S. medical school using an anonymous questionnaire delivered electronically via social media and emails. The survey contained items pertaining to students' attitudes towards different types of lecture modalities and how they relate to personal stress. Descriptive data and Spearman's rank correlation tests were conducted using IBM Corp. Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp. RESULTS: This study examined correlations between preclinical medical school lecture delivery and personality type, stress levels, attendance, and burnout. Overall, no significance was found between mode of delivery and personality type. On the other hand, the mode of delivery significantly affected stress levels, attendance, and burnout. Moderate to strong correlations were found between the item "Zoom lectures have reduced stress compared to in-person lectures" and preference for Zoom, quality of education using Zoom compared to the in-person lectures, belief that Zoom lectures should continue as part of the curriculum delivery method, staying motivated with lectures fully online with Zoom, and liking that Zoom lectures save commute time to campus. CONCLUSIONS: Findings suggest that a fully online curriculum may play a role in reducing stress in medical students without compromising the quality of education.

Macrophage COX2 Mediates Efferocytosis, Resolution Reprogramming, and Intestinal Epithelial Repair.

BACKGROUND AND AIMS: Phagocytosis (efferocytosis) of apoptotic neutrophils by macrophages anchors the resolution of intestinal inflammation. Efferocytosis prevents secondary necrosis and inhibits further inflammation, and also reprograms macrophages to facilitate tissue repair and promote resolution function. Macrophage efferocytosis and efferocytosis-dependent reprogramming are implicated in the pathogenesis of inflammatory bowel disease. We previously reported that absence of macrophage cyclooxygenase 2 (COX2) exacerbates inflammatory bowel disease-like intestinal inflammation. To elucidate the underlying pathogenic mechanism, we investigated here whether COX2 mediates macrophage efferocytosis and efferocytosis-dependent reprogramming, including intestinal epithelial repair capacity. METHODS: Using apoptotic neutrophils and synthetic apoptotic targets, we determined the effects of macrophage specific Cox2 knockout and pharmacological COX2 inhibition on the efferocytosis capacity of mouse primary macrophages. COX2-mediated efferocytosis-dependent eicosanoid lipidomics was determined by liquid chromatography tandem mass spectrometry. Small intestinal epithelial organoids were employed to assay the effects of COX2 on efferocytosis-dependent intestinal epithelial repair. RESULTS: Loss of COX2 impaired efferocytosis in mouse primary macrophages, in part, by affecting the binding capacity of macrophages for apoptotic cells. This effect was comparable to that of high-dose lipopolysaccharide and was accompanied by both dysregulation of macrophage polarization and the inhibited expression of genes involved in apoptotic cell binding. COX2 modulated the production of efferocytosis-dependent lipid inflammatory mediators that include the eicosanoids prostaglandin I2, prostaglandin E2, lipoxin A4, and 15d-PGJ2; and further affected secondary efferocytosis. Finally, macrophage efferocytosis induced, in a macrophage COX2-dependent manner, a tissue restitution and repair phenotype in intestinal epithelial organoids. CONCLUSIONS: Macrophage COX2 potentiates efferocytosis capacity and efferocytosis-dependent reprogramming, facilitating macrophage intestinal epithelial repair capacity.

A systematic review of screening for perinatal depression and anxiety in community-based settings.

Screening for perinatal depression and anxiety in community-based maternal and child health settings may help close the detection and treatment gap among women at higher risk for these conditions. We aim to review perinatal depression and anxiety screening tools, timing, and follow-up processes for positive screens in community-based settings. We conducted a systematic review of the literature to identify papers describing screening and interventions for perinatal depression and anxiety in community-based settings. We identified 49 papers describing 47 studies of perinatal depression or anxiety screening in community-based settings. The Edinburgh Postnatal Depression Scale (EPDS) was the most frequently used screening tool. Referral and referral tracking for those who screened positive for symptoms were inadequately described. Types of training and technical assistance provided for screening varied widely. It is feasible and acceptable to screen for perinatal depression in community settings, but there is a need for systematic research examining which screening tools to use, the ideal frequency of screening, and referral completion rates. There is a lack of information regarding perinatal anxiety screening and a lack of uniformity in training regarding screening in community-based settings. Future studies should compare the efficacy of screening in community-based settings to screening in healthcare settings.

Exercise Training in Patients With Heart Failure With Preserved Ejection Fraction: A Community Hospital Pilot Study.

BACKGROUND: Patients with heart failure with preserved ejection fraction (HFpEF) experience poor exercise tolerance and quality of life. Little is known about the feasibility or effects of HFpEF exercise training (ET) in a community hospital setting. OBJECTIVE: The aim of this study was to examine the feasibility and pilot data of a community-based HFpEF ET intervention. METHODS: This was a single-group (n = 16), pretest-posttest, 9-week ET intervention. The Minnesota Living With Heart Failure Questionnaire, Patient Health Questionnaire-9, cardiopulmonary exercise test (peak VO2), and 6-minute walk test were used for evaluation. RESULTS: Participants (n = 16) attended 88% of prescribed ET sessions and 94% completed all pretest-posttest assessments. Significant improvements in Minnesota Living With Heart Failure Questionnaire (P = .01), Patient Health Questionnaire-9 (P ≤ .01), exercise test time (P = .01) and 6-minute walk test (P = .001), but not in peak VO2 (P = .16), were found. CONCLUSIONS: The ET intervention was feasible and safe, and findings support improved quality of life, depressive symptoms, and exercise tolerance. Larger controlled trials are warranted.

Parenthood During Graduate Medical Education: A Scoping Review.

PURPOSE: To conduct a scoping review of the literature on parenthood during graduate medical education (GME) and to develop a conceptual framework to inform policy and guide research. METHOD: The authors searched PubMed and Embase for articles published from January 1993 through August 7, 2017, using a query framework that combined the concepts of "person" (e.g., "trainee") and "parenthood" (e.g., "breastfeeding"). They included studies describing parenthood or pregnancy of trainees in U.S. GME training programs. Two authors independently screened citations and abstracts and performed kappa coefficient tests to evaluate interreviewer reliability. Two authors performed a full-text review of and extracted data from each included article, and 4 authors coded data for all articles. The authors used descriptive statistics and qualitative synthesis to analyze data. RESULTS: Ninety articles met inclusion criteria, and nearly half (43/90; 48%) were published between 2010 and 2017. The authors developed 6 themes that surround resident parenthood: well-being, maternal health, others' perceptions, relationships, program preparation, and policy. They mapped these themes by relationship of stakeholders (e.g., infant and family, institutions) to the resident-parent to create a conceptual framework describing parenthood during GME. CONCLUSIONS: The findings from this scoping review have implications for policy and research. Those authoring parental leave policies could collaborate with national board leaders to develop consistent standards and include nontraditional families. Gaps in the literature include the effect of resident parenthood on patient care, postpartum health, and policy execution. Research in these areas would advance the literature on parenthood during residency.

Reduction of Pain during Flexible Cystoscopy: A Systematic Review and Meta-Analysis.

PURPOSE: The BCAN (Bladder Cancer Advocacy Network) Patient Survey Network identified pain during intravesical procedures as a research priority for patients. Although intraurethral lidocaine is the standard of care in this setting, evidence of its use is equivocal. We systematically reviewed studies of interventions to reduce discomfort during cystoscopy and intravesical therapy of bladder cancer. We performed a meta-analysis of interventions using available randomized, controlled trials. MATERIALS AND METHODS: Search terms derived from the key questions were incorporated into the literature search constructed by a research librarian and the English medical literature from 1990 to 2017 was accessed. The initial search yielded 626 potential studies and the final review incorporated 62. We combined 12 trials into a meta-analysis with a random effects model of the efficacy of intraurethral lidocaine vs plain lubricant to reduce pain during flexible cystoscopy as measured on a 10-point visual analogue scale. RESULTS: Data from 12 randomized controlled trials in a total of 1,549 patients were included in the final intraurethral lidocaine meta-analysis. The standardized mean difference between visual analogue scale pain scores in patients who underwent flexible cystoscopy with intraurethral lidocaine and plain lubricant was -0.22 (95% CI -0.39--0.05). Evidence was insufficient to evaluate other interventions to mitigate the discomfort of invasive bladder procedures. CONCLUSIONS: Intraurethral lidocaine provides statistically significant pain reduction in men who undergo flexible cystoscopy, particularly with a longer dwell time. The evidence was insufficient for other tested interventions. A prospective study is needed to further clarify interventions to decrease patient discomfort during cystoscopy and other intravesical procedures in a diverse population.

Paraneoplastic vertigo as the presenting symptom of a testicular seminoma.

Vertigo is a common presenting symptom, but rarely may be caused by a malignancy. We present a case of a 44-year-old man who presented with nystagmus and vertigo precipitated by movement, with accompanying nausea and weight loss. Diagnostic workup revealed a right testicular mass that was resected and found to be a seminoma. The patient's symptoms resolved after surgical resection and treatment with corticosteroids.

A cluster of amyotrophic lateral sclerosis in New Hampshire: a possible role for toxic cyanobacteria blooms.

Cyanobacteria produce many neurotoxins including beta-methylamino-L-alanine (BMAA) that has been liked to amyotrophic lateral sclerosis (ALS) and neurodegenerative disease. A number of ALS cases have been diagnosed among residents of Enfield, NH, a town encompassing a lake with a history of cyanobacteria algal blooms. To investigate an association between toxic cyanobacterial blooms in New Hampshire and development of ALS, we reviewed records from our institution and other community databases to obtain demographic information on patients diagnosed with ALS within New England. We identified nine ALS patients who lived near Lake Mascoma in Enfield, NH, an incidence of sporadic ALS that is 10 to 25 times the expected incidence of 2/100,000/year. We suggest that the high incidence of ALS in this potential cluster could be directly related to chronic exposure to cyanobacterial neurotoxins such as BMAA. Possible routes of toxin exposure include inhalation of aerosolized toxins, consuming fish, or ingestion of lake water. Further investigation, including analysis of brain tissue for cyanobacterial toxins, will be helpful to test for an association between BMAA and ALS.

A microtitre plate assay for measuring glycosidase activity.

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.

Caring for older people who have a hearing disability.

Approximately a quarter of the residents in our nursing home use hearing aids, and the information given in this article on supporting people who wear hearing aids was particularly useful.