Time-resolved cryo-EM using Spotiton.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

Kinetics of human brown adipose tissue activation and deactivation.

Brown adipose tissue (BAT) has been identified as a potential target in the treatment and prevention of obesity and metabolic disease. The precise kinetics of BAT activation and the duration of stimulus required to recruit metabolically active BAT, and its subsequent deactivation, are not well-understood. In this clinical trial, 19 healthy adults (BMI: 23.7 ± 0.7 kg/m2, Age: 31.2 ± 2.8 year, 12 female) underwent three different cooling procedures to stimulate BAT glucose uptake, and active BAT volume was determined using 18F-Fluorodeoxyglucose (FDG) PET/CT imaging. We found that 20 min of pre-injection cooling produces activation similar to the standard 60 min (39.9 mL vs. 44.2 mL, p = 0.52), indicating that BAT activity approaches its peak function soon after the initiation of cooling. Furthermore, upon removal of cold exposure, active BAT volume declines (13.6 mL vs. 44.2 mL, p = 0.002), but the deactivation process persists even hours following cessation of cooling. Thus, the kinetics of human BAT thermogenesis are characterized by a rapid increase soon after cold stimulation but a more gradual decline after rewarming. These characteristics reinforce the feasibility of developing mild, short-duration cold exposure to activate BAT and treat obesity and metabolic disease.

Spotiton: New features and applications.

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Šresolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

A student proposal for ethical guidelines in anatomical education: Ethical and policy considerations.

The recent publication of a story regarding anatomical dissection in a medical school has revealed the need for increased attention to the ethical and policy aspects of anatomical education. While most of the attention devoted to these questions thus far has been focused on procedures before and after dissection, from the perspective of medical students, there are important considerations during the process of dissection itself. This proposal was developed by two third-year medical students through a review of the relevant published literature, reflection upon their personal experiences in anatomy courses in two separate institutions, and informal discussion of these topics with peers. The proposal is that basic ethical guidelines should be established and monitored by an independent committee tasked with reviewing them. The proposed guidelines include: First, a clear set of expectations about what the student is expected to learn with respect to anatomical knowledge and dissection technique; second, the establishment by schools or national bodies of minimal ethical standards regarding respectful behavior toward the donor bodies, and the communication of these standards to teachers and students involved in educational dissections; third, the use of materials that encourage students to view their donors with respect and ensure proper treatment of them; and fourth, the establishment of an oversight group (at each medical school and at national level) comprising students, faculty, community members, and staff, who will regularly review the anatomical education program and update these ethical guidelines as appropriate. While many of these proposals are already implemented in some anatomy departments, the establishment of clear guidelines at a national as well as a school-by-school level will permit students the freedom to participate fully in their education, knowing they have met the highest ethical standards as they prepare for a career as a humanistic physician.

Bioethics, Religion, and Public Policy: Intersections, Interactions, and Solutions.

Bioethics in America positions itself as a totalizing discipline, capable of providing guidance to any individual within the boundaries of a health or medical setting. Yet the religiously observant or those driven by spiritual values have not universally accepted decisions made by "secular" bioethics, and as a result, religious bioethical thinkers and adherents have developed frameworks and rich counter-narratives used to fend off encroachment by policies perceived as threatening. This article uses brain death in Jewish law, the case of Jahi McMath, and vaccination refusal to observe how the religious system of ethics is presently excluded from bioethics and its implications.

Differential cardiopulmonary haemodynamic phenotypes in PASC-related exercise intolerance.

Background: Post-acute sequelae of COVID-19 (PASC) affect a significant proportion of patients who have previously contracted SARS-CoV-2, with exertional intolerance being a prominent symptom. This study aimed to characterise the invasive haemodynamic abnormalities of PASC-related exertional intolerance using invasive cardiopulmonary exercise testing (iCPET). Study design and intervention: 55 patients were recruited from the Yale Post-COVID-19 Recovery Program, with most experiencing mild acute illness. Supine right heart catheterisation and iCPET were performed on all participants. Main results: The majority (75%) of PASC patients exhibited impaired peak systemic oxygen extraction (pEO2) during iCPET in conjunction with supranormal cardiac output (CO) (i.e., PASC alone group). On average, the PASC alone group exhibited a "normal" peak exercise capacity, V'O2 (89±18% predicted). ∼25% of patients had evidence of central cardiopulmonary pathology (i.e., 12 with resting and exercise heart failure with preserved ejection fraction (HFpEF) and two with exercise pulmonary hypertension (PH)). PASC patients with HFpEF (i.e., PASC HFpEF group) exhibited similarly impaired pEO2 with well compensated PH (i.e., peak V'O2 and CO >80% respectively) despite aberrant central cardiopulmonary exercise haemodynamics. PASC patients with HFpEF also exhibited increased body mass index of 39±7 kg·m-2. To examine the relative contribution of obesity to exertional impairment in PASC HFpEF, a control group comprising obese non-PASC group (n=61) derived from a historical iCPET cohort was used. The non-PASC obese patients with preserved peak V'O2 (>80% predicted) exhibited a normal peak pulmonary artery wedge pressure (17±14 versus 25±6 mmHg; p=0.03) with similar maximal voluntary ventilation (90±12 versus 86±10% predicted; p=0.53) compared to PASC HFpEF patients. Impaired pEO2 was not significantly different between PASC patients who underwent supervised rehabilitation and those who did not (p=0.19). Conclusions: This study highlights the importance of considering impaired pEO2 in PASC patients with persistent exertional intolerance unexplained by conventional investigative testing. Results of the current study also highlight the prevalence of a distinct high output HFpEF phenotype in PASC with a primary peripheral limitation to exercise.

Acute and subacute neurovascular impact of cryptogenic air emboli.

Background: Cerebral air embolism is a rare cause of acute ischemic stroke that is becoming increasingly well-described in the literature. However, the mechanism and severity of this type of injury can vary, with ischemia typically emerging early in the course of care. To the best of our knowledge, delayed ischemia in this setting has not yet been described. Case Description: A stroke code was called for an unresponsive, hospitalized, 75-year-old man. A computerized tomography (CT) scan of the head revealed air within the right greater than left hemispheric cortical veins with loss of sulcation, concerning for developing ischemia, and CT angiography revealed absent opacification of the distal cortical vessels in the right anterior cerebral artery and middle cerebral artery territories. Magnetic resonance imaging (MRI) of the brain was obtained 5.75 h after the patient's last known well-showed small areas of subtle cortical diffusion restriction. Follow-up CT head within 24 h showed near-complete resolution of the air emboli after treatment with 100% fraction of inspired oxygen on mechanical ventilation. Subsequent MRI, performed 4 days after the initial event, showed extensive cortical diffusion restriction and cerebral edema crossing vascular territories. Conclusion: This case highlights that cerebral air emboli can cause delayed ischemia that may not be appreciated on initial imaging. As such, affected patients may require intensive neurocritical care management, close neurologic monitoring, and repeat imaging irrespective of initial radiographic findings.

Impact of a Medical Clinic Relocation on Travel Time: A Tale of 2 Modes of Transportation.

The aim of this study was to quantify access disparities by examining the impact of a medical clinic relocation on travel time differences for patients using private cars versus public transit. Longitude and latitude of patient home addresses were extracted from electronic medical records for the 4 years before the clinic move. Using offline, open-source, and HIPAA-compliant routing software, roundtrip travel times were computed from each home address to the old and new clinic locations via car and bus. Mean roundtrip travel time by a car changed from 41.3 (IQR 16.1-80.7) to 45.4 (IQR 25.9-78.1) minutes, a 9.9% increase. Mean roundtrip travel time by public transit changed from 67.5 (IQR 51.5-100.2) to 120.8 (100.3-156.1) minutes, a 78.9% increase. Even clinic relocations that minimally impact car travel times can nevertheless yield substantial changes to those traveling by public transit. Clinics and health systems that wish to reduce barriers to accessing health care, especially among those already facing structural inequities, would benefit from utilizing the analytic approach described here.

Relation of Cigarette Smoking and Heart Failure in Adults ≥65 Years of Age (From the Cardiovascular Health Study).

Cigarette smoking is associated with adverse cardiac outcomes, including incident heart failure (HF). However, key components of potential pathways from smoking to HF have not been evaluated in older adults. In a community-based study, we studied cross-sectional associations of smoking with blood and imaging biomarkers reflecting mechanisms of cardiac disease. Serial nested, multivariable Cox models were used to determine associations of smoking with HF, and to assess the influence of biochemical and functional (cardiac strain) phenotypes on these associations. Compared with never smokers, smokers had higher levels of inflammation (C-reactive protein and interleukin-6), cardiomyocyte injury (cardiac troponin T [hscTnT]), myocardial "stress"/fibrosis (soluble suppression of tumorigenicity 2 [sST2], galectin 3), and worse left ventricle systolic and diastolic function. In models adjusting for age, gender, and race (DEMO) and for clinical factors potentially in the causal pathway (CLIN), smoking exposures were associated with C-reactive protein and interleukin-6, sST2, hscTnT, and with N-terminal pro-brain natriuretic protein (in Whites). In DEMO adjusted models, the cumulative burden of smoking was associated with worse left ventricle systolic strain. Current smoking and former smoking were associated with HF in DEMO models (hazard ratio 1.41, 95% confidence interval 1.22 to 1.64 and hazard ratio 1.14, 95% confidence interval 1.03 to 1.25, respectively), and with current smoking after CLIN adjustment. Adjustment for time-varying myocardial infarction, inflammation, cardiac strain, hscTnT, sST2, and galectin 3 did not materially alter the associations. Smoking was associated with HF with preserved and decreased ejection fraction. In conclusion, in older adults, smoking is associated with multiple blood and imaging biomarker measures of pathophysiology previously linked to HF, and to incident HF even after adjustment for clinical intermediates.