The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events.

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a function for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

Supramolecular Assembly of His-Tagged Fluorescent Protein Guests within Coiled-Coil Peptide Crystal Hosts: Three-Dimensional Ordering and Protein Thermal Stability.

The use of biomaterials for the inclusion and stabilization of biopolymers is an ongoing challenge. Herein, we disclose three-dimensional (3D) coiled-coil peptide crystals with metal ions that include and overgrow His-tagged fluorescent proteins within the crystal. The protein guests are found within two symmetry-related growth sectors of the crystalline host that are associated with faces of the growing crystal that display ligands for metal ions. The fluorescent proteins are included within this "hourglass" region of the crystals at a notably high level, display order within the crystal hosts, and demonstrate sufficiently tight packing to enable energy transfer between a donor-acceptor pair. His-tagged fluorescent proteins display remarkable thermal stability to denaturation over extended periods of time (days) at high temperatures when within the crystals. Ultimately, this strategy may prove useful for the prolonged storage of thermally sensitive biopolymer guests within a 3D crystalline matrix.

Carotenoid status in type 2 diabetes patients with and without retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness. Carotenoids are plant-derived pigments required for general health and particularly for vision. In this study, we evaluated the dietary intake and blood carotenoid levels of type 2 diabetes (T2D) patients with and without DR. A cross-sectional case-control study was conducted among 151 age-matched controls and 344 T2D patients, of which 194 had DR and 150 had no DR (NDR). After a complete ophthalmic examination, the demographic, anthropometric and clinical profiles were obtained. Carotenoids in the plasma were measured by HPLC and dietary intakes were obtained using a food frequency questionnaire. The mean plasma levels of carotenoids (except γ-carotene) were significantly lower in the DR group compared to the Control and NDR groups. The dietary intakes of zeaxanthin, lycopene, α-carotene and ß-carotene were significantly lower in the NDR group compared to the Control group, and were further lower in the DR group compared to the NDR group. Plasma carotenoid levels were significantly inversely associated with the duration of diabetes, RBS and HbA1c but positively associated with HDL. This study demonstrated decreased plasma levels and lower dietary intakes of carotenoids in DR subjects.

Where Do International Medical Graduates Matriculate for Internal Medicine Training? A National Longitudinal Study.

INTRODUCTION: In 2020, roughly 25% of applicants who matched into internal medicine (IM) residencies were international medical graduates (IMGs). We examine 12-year trends in distribution of IMGs among IM training programs and explore differences in program perceptions towards IMG recruitment. METHODS: Since 2007, Association of Program Directors in Internal Medicine Annual Surveys have collected data about trainees by medical school graduate type. Sixteen additional questions regarding perceptions of IMGs were included in the 2017 spring survey. RESULTS: The 2017 survey response rate was 63.3% (236/373) and ranged from 61.9 to 70.2% for the 2007-2019 Annual Surveys. During that 12-year period, 55-70% of community programs' and 22-30% of university programs' PGY1 positions were filled by IMGs. In 2017, 45% of community programs' and 15% of university programs' interview and ranking positions were allocated to IMGs. Departmental pressure (university 45.6% [95% CI 43.7-47.5]; community 28.2% [95% CI 26.6-29.7]; p = 0.007), institutional priority (university 64.0% [95% CI 62.1-66.0]; community 41% [95% CI 36.9-44.6]; p = 0.001), and reputational concerns (university 52.8% [95% CI 50.0-55.6]; community 38.5% [95% CI 36.0-40.9]; p = 0.045) were cited as factors influencing recruitment of IMGs. CONCLUSION: Our study was limited to exploring program factors in residency recruitment and did not assess applicant preferences. There is a large, longstanding difference in the recruitment of IMGs to US community-based and university residencies, beginning during the interview and ranking process. Further research in disparities in IMG recruitment is needed, including exploring pressures, preferences, and potential biases associated with the recruitment of IMGs.

Frailty and Nutritional Status among Urban Older Adults in South India.

The purpose of this study was to assess the prevalence of frailty and nutritional status among older adults. This population-based cross-sectional study was conducted in 163 subjects aged 60-88 years, from Hyderabad City, South India. Data were obtained on sociodemographic details and anthropometry and biochemical parameters. Dietary intake was assessed by a three-day 24 h dietary recall, and the probability of adequacy (PA) was calculated using the estimated average requirements. Frailty indicators were as follows: handgrip strength was measured by using a Jamar dynamometer, gait speed was measured by a ten-meter length walk test, and low physical activity level, weight loss, and exhaustion were assessed using a questionnaire. Among the study population, 20% of the participants were frail and 80% were nonfrail. The prevalence of frailty is higher in older (30.1%) than the younger (12.2%) age groups, and it is more so in women (32.4%) than in men (10.1%). The lower educational status and income were associated with frailty. The PA of most of the nutrients was low in the frail group. Noticeably, the mean PA (MPA) across the fourteen micronutrients was significantly higher in nonfrail (38%) compared to the frail group (25%). The prevalence of frailty was higher in the lowest tertile of most of the food groups and nutrient intake compared to the highest tertile. The study revealed a 20% prevalence of frailty among urban older adults and provided evidence that inadequate intake of nutrients is independently associated with frailty.

Membrane Association Transforms an Inert Anti-TCRß Fab' Ligand into a Potent T Cell Receptor Agonist.

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRß H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation.

Separate but Equal? The Sorting of USMDs and Non-USMDs in Internal Medicine Residency Programs.

BACKGROUND: The US internal medicine workforce relies on international and osteopathic medical graduates to fill gaps in residency. Little is known about the distribution and impact of IMGs, DOs, and USMDs concentrating in different types of IM programs. OBJECTIVE: Determining the extent to which USMDs, DOs, and IMGs concentrate in different types of IM programs and comparing Board pass rates by program concentration. DESIGN, SETTINGS, AND PARTICIPANTS: This survey study used data from the AMA's FREIDA database for 476 non-military IM programs in 2017-2018, and 2016-2018 ABIM exam pass rates for 388 accredited programs. MEASUREMENTS: Outcomes were (1) program concentration based on percentage of residents who were USMDs, IMGs, and DOs in 2017-2018 and (2) 2016-2018 program ABIM pass rates as proxies for program quality. Key independent variables were hospital type (community-based, community-based university-affiliated, or university-based) when program concentration was the outcome, and program concentration when Board pass rates were the outcome. RESULTS: Twenty-five percent of programs were "USMD-dominated," 17% were "DO-dominated," 42% were "IMG dominated," and 16% were "integrated." The chances that a university hospital was USMD-dominated were 32 percentage points higher than that for a community hospital (AME = 0.32, baseline probability = 0.11, 95% CI, 0.17-0.46, P < .001). USMD-dominated programs also had significantly higher pass rates by 4.0 percentage points (AME = 0.04, baseline proportion = 0.90, 95% CI, 0.02-0.06, P < .001) than integrated programs, while DO-dominated programs had significantly lower pass rates (AME = - 0.1, baseline proportion = 0.90, 95% CI, - 0.15 to - 0.04, P < .001). CONCLUSION: USMDs and non-USMDs systematically cluster in certain types of residency programs and their training may not be equal, as measured by board pass rates.

Prevalence of vitamin deficiencies in an apparently healthy urban adult population: Assessed by subclinical status and dietary intakes.

OBJECTIVES: Studies in children and pregnant women consistently showed pandemic proportions of micronutrient deficiencies in the Indian subcontinent. However, vitamin deficiencies in apparently healthy adults are seldom recognized, hence the aim of this exploratory study was to assess their subclinical vitamin status and dietary intakes. METHODS: In all, 270 apparently healthy urban adults 30 to 70 y of age, from Hyderabad city, India participated in this study. Blood levels of vitamins (A, B1, B2, B6, total and active B12, D, and folate) and homocysteine were assessed. Anthropometric parameters were measured; dietary intake was obtained by food frequency questionnaire, and probability of adequacy (PA) was calculated by the estimated average requirement. RESULTS: Among the study population, the overall prevalence of deficiency of vitamin B2 was strikingly high (50%) followed by the vitamins B6 (46%), active B12 (46%), total B12 (37%), folate (32%), D (29%), B1 (11%), and A (6%). Hyperhomocysteinemia (HHcys) was widely prevalent (52%) in the study participants. In case of dietary intakes, PA was lowest for vitamin B12 (4%) and folate (9%) followed by vitamins A (22%), B2 (33%), B6 (30%), and B1 (59%). The mean PA of these vitamins was noticeably low (28%). The unadjusted logistic regression analysis found men and those with a deficiency of folate and total and active B12 to be at higher risk for HHcys. In the adjusted model, the risk for active B12 deficiency almost doubled. CONCLUSION: The study demonstrated a high prevalence of multiple subclinical vitamin deficiencies, dietary inadequacies, and HHcys, which are possible risk factors for disease burden among apparently healthy adults.

Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events.

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long-dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

Preliminary Validity Evidence for a Milestones-Based Rating Scale for Chart-Stimulated Recall.

BACKGROUND: Minimally anchored Standard Rating Scales (SRSs), which are widely used in medical education, are hampered by suboptimal interrater reliability. Expert-derived frameworks, such as the Accreditation Council for Graduate Medical Education (ACGME) Milestones, may be helpful in defining level-specific anchors to use on rating scales. OBJECTIVE: We examined validity evidence for a Milestones-Based Rating Scale (MBRS) for scoring chart-stimulated recall (CSR). METHODS: Two 11-item scoring forms with either an MBRS or SRS were developed. Items and anchors for the MBRS were adapted from the ACGME Internal Medicine Milestones. Six CSR standardized videos were developed. Clinical faculty scored videos using either the MBRS or SRS and following a randomized crossover design. Reliability of the MBRS versus the SRS was compared using intraclass correlation. RESULTS: Twenty-two faculty were recruited for instrument testing. Some participants did not complete scoring, leaving a response rate of 15 faculty (7 in the MBRS group and 8 in the SRS group). A total of 529 ratings (number of items × number of scores) using SRSs and 540 using MBRSs were available. Percent agreement was higher for MBRSs for only 2 of 11 items-use of consultants (92 versus 75, P = .019) and unique characteristics of patients (96 versus 79, P = .011)-and the overall score (89 versus 82, P < .001). Interrater agreement was 0.61 for MBRSs and 0.51 for SRSs. CONCLUSIONS: Adding milestones to our rating form resulted in significant, but not substantial, improvement in intraclass correlation coefficient. Improvement was inconsistent across items.

Role Models' Influence on Specialty Choice for Residency Training: A National Longitudinal Study.

BACKGROUND: Role models in medical school may influence students' residency specialty choice. OBJECTIVE: We examined whether medical students who reported clinical exposure to a role model during medical school would have an increased likelihood of selecting the role model's specialty for their residencies. METHODS: We conducted a 5-year prospective, national longitudinal study (2011-2016) of medical students from 24 US allopathic medical schools, starting from the middle of their third year. The primary outcome measure was type of residency specialty choice 4 years after graduation. Main predictors were the clinical specialty of a student's most admired physician and the relative importance of 7 potentially influential factors for specialty choice in the fourth year of medical school. RESULTS: From 919 eligible participants, 564 (61%) responded to the first survey; 474 of the respondents (84%) completed the follow-up survey. We excluded 29 participants who were not in their fourth year by the time of the follow-up survey. Of the follow-up respondents, 427 (96%) had specialty data 4 years after graduation. In our multivariate models, exposure to an admired generalist physician prior to medical school (odds ratio [OR] = 2.21, 95% confidence interval [CI] 1.03-4.73) and during medical school (OR = 2.62, 95% CI 1.69-4.05) had the strongest odds with respect to training in a generalist residency 4 years after graduation. Role model exposure also predicted specialty choice among those training in surgical and radiology, ophthalmology, anesthesiology, and dermatology (ROAD) specialties. CONCLUSIONS: Personal exposure to role models in medical school is an important predictor of residency training in that role model's specialty.

Awareness of Bullying in Residency: Results of a National Survey of Internal Medicine Program Directors.

BACKGROUND: Bullying of medical trainees is believed to occur more frequently in medical education than once thought. OBJECTIVE: We conducted a survey to understand internal medicine program director (PD) perspectives and awareness about bullying in their residency programs. METHODS: The 2015 Association of Program Directors in Internal Medicine (APDIM) annual survey was e-mailed to 368 of 396 PDs with APDIM membership, representing 93% of internal medicine residency programs. Questions about bullying were embedded within the survey. Bivariate analyses were performed on PD and program characteristics. RESULTS: Of a total of 368 PD APDIM members, 227 PDs (62%) responded to the survey. Less than one-third of respondents (71 of 227, 31%) reported being aware of bullying in their residency programs during the previous year. There were no significant differences in program or PD characteristics between respondents who reported bullying in their programs and those who did not (gender, tenure as PD, geographic location, or specialty, all P > .05). Those who acknowledged bullying in their program were more likely to agree it was a problem in graduate medical education (P < .0001), and it had a significant negative impact on the learning environment (P < .0001). The majority of reported events entailed verbal disparagements, directed toward interns and women, and involved attending physicians, other residents, and nurses. CONCLUSIONS: This national survey of internal medicine PDs reveals that a minority of PDs acknowledged recent bullying in their training programs, and reportedly saw it as a problem in the learning environment.

Membrane bending occurs at all stages of clathrin-coat assembly and defines endocytic dynamics.

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question of how coat assembly produces membrane curvature has been studied with molecular and cellular structural biology approaches, without direct visualization of the process in living cells; resulting in two competing models for membrane bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined with electron, atomic force, and super-resolution optical microscopy to measure membrane curvature during CME. Surprisingly, coat assembly accommodates membrane bending concurrent with or after the assembly of the clathrin lattice. Once curvature began, CME proceeded to scission with robust timing. Four color pol-TIRF showed that CALM accumulated at high levels during membrane bending, implicating its auxiliary role in curvature generation. We conclude that clathrin-coat assembly is versatile and that multiple membrane-bending trajectories likely reflect the energetics of coat assembly relative to competing forces.

Geriatrics Curriculum Needs Assessment for Dermatology Residency Programs.

BACKGROUND: Geriatric patients account for a growing proportion of dermatology clinic visits. Although their biopsychosocial needs differ from those of younger adults, there are no geriatrics training requirements for dermatology residency programs. OBJECTIVE: This study explored the state of geriatrics education in dermatology programs in 2016. METHODS: This constructivist study employed cross-sectional, mixed-methods analysis with triangulation of semistructured interviews, surveys, and commonly used curricular materials. We used purposive sampling of 5 US academic allopathic dermatology programs of different sizes, geographic locations, and institutional resources. Participants were interviewed about informal curricula, barriers, and suggestions for improving geriatrics education, and they also completed a survey about the geriatrics topics that should be taught. The constant comparative method with grounded theory was used for qualitative analysis. We identified formal geriatrics curricular content by electronically searching and counting relevant key texts. RESULTS: Fourteen of 17 participants (82%) agreed to be interviewed, and 10 of 14 (71%) responded to the survey. Themes of what should be taught included diagnosing and managing skin diseases common in older adults, holistic treatment, cosmetic dermatology, benign skin aging, and the basic science of aging. Topics currently covered that could be expanded included communication, systems-based challenges, ethical issues, safe prescribing, quality improvement, and elder abuse. Cosmetic dermatology was the most commonly taught formal geriatrics curricular topic. CONCLUSIONS: There were discrepancies among topics participants felt were important to teach about geriatric dermatology and curricular coverage of these areas. We identified challenges for expanding geriatrics curricula and potential solutions.

Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination.

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell-cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation.

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Surveys of Health Professions Trainees: Prevalence, Response Rates, and Predictive Factors to Guide Researchers.

PURPOSE: To establish a baseline overall response rate for surveys of health professions trainees, determine strategies associated with improved response rates, and evaluate for the presence of nonresponse bias. METHOD: The authors performed a comprehensive analysis of all articles published in Academic Medicine, Medical Education, and Advances in Health Sciences Education in 2013, recording response rates. Additionally, they reviewed nonresponse bias analyses and factors suggested in other fields to affect response rate including survey delivery method, prenotification, and incentives. RESULTS: The search yielded 732 total articles; of these, 356 were research articles, and of these, 185 (52.0%) used at least one survey. Of these, 66 articles (35.6%) met inclusion criteria and yielded 73 unique surveys. Of the 73 surveys used, investigators reported a response rate for 63.0% of them; response rates ranged from 26.6% to 100%, mean (standard deviation) 71.3% (19.5%). Investigators reported using incentives for only 16.4% of the 73 surveys. The only survey methodology factor significantly associated with response rate was single- vs. multi-institutional surveys (respectively, 74.6% [21.2%] vs. 62.0% [12.8%], P = .022). Notably, statistical power for all analyses was limited. No articles evaluated for nonresponse bias. CONCLUSIONS: Approximately half of the articles evaluated used a survey as part of their methods. Limited data are available to establish a baseline response rate among health professions trainees and inform researchers which strategies are associated with higher response rates. Journals publishing survey-based health professions education research should improve reporting of response rate, nonresponse bias, and other survey factors.