Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.

Practice Makes Perfect: Objective Structured Clinical Examinations Across the UME-to-GME Continuum Improve Care of Transgender Simulated Patients.

Background Residents lack confidence in caring for transgender individuals. More exposure and practice throughout training is needed. Objective To explore whether and how prior exposure to transgender health skills during medical school impacted competency with these skills during residency. Methods In 2022, all 101 internal medicine residents at New York University Grossman School of Medicine participated in an objective structured clinical examination (OSCE) station as part of their annual formative assessment where they cared for a standardized patient (SP) who identified as transgender. Three SPs who were members of the transgender community were recruited through online and social media forums. Two resident groups (continuum vs noncontinuum) differed in their prior experiences with transgender OSCEs during medical school. We analyzed SPs' ratings of resident performance using checklist data and SP open-ended feedback to compare performance between groups and resident post-OSCE evaluations to understand residents' perceptions of the educational value of the case. Results Residents with prior experience with transgender SPs (continuum) were more frequently recommended by SPs (88% [21 of 24] vs 70% [54 of 77]) to a family member or friend, were all rated professional (100% [24 of 24] vs 94% [72 of 94]) and scored better in pain information-gathering (92% vs 65%, mean summary score) and gender-affirming care skills (67% vs 52%, mean summary score). Noncontinuum residents lacked experience, missed opportunities to ask about gender identity, and needed work on demonstrating comfort and using proper language. Most residents completing a post-OSCE evaluation (80%, 41 of 51) rated the case as "very valuable." Conclusions Spaced practice and feedback through early exposure to transgender OSCEs were valuable for skill acquisition, giving continuum residents a learning advantage compared to noncontinuum residents.

Implementing an Experiential Telehealth Training and Needs Assessment for Residents and Faculty at a Veterans Affairs Primary Care Clinic.

Background: The transition to telehealth during the COVID-19 pandemic revealed a lack of preexisting telehealth training for clinicians. As a workplace-based simulation methodology designed to improve virtual clinical skills, announced standardized patients (ASPs) may help meet evolving educational needs to sustain quality telehealth care. Objective: We describe the development and implementation of an ASP program to assess and provide feedback to resident and faculty clinicians in virtual practice, and report on performance, feasibility, and acceptability. Methods: From June 2021 to April 2022, resident and faculty clinicians at a VA primary care clinic participated in a video visit in which an ASP portrayed either a 70-year-old man with hearing loss and hypertension or a 60-year-old man with hypertension and financial stress. Following the visit, ASPs provided verbal feedback and completed a behaviorally anchored checklist to rate telehealth and communication skills, chronic disease management, and use of resources. Domain summary scores were calculated as the mean percentage of "well done" items. Participants completed a feedback survey on their experience. Results: Seventy-six televisits (60 primary care residents [postgraduate year 1-3], 16 internal medicine faculty) were conducted from August 2021 to April 2022. Clinicians performed well in communication skills: information gathering (79%, 60 of 76, well done), relationship development (67%, 51 of 76), education and counseling (71%, 54 of 76), and patient satisfaction (86%, 65 of 76). They performed less well in telemedicine skills (38%, 29 of 76). Participants agreed that the experience was a good use of their time (88%, 67 of 76). Conclusions: An ASP-facilitated training for resident and faculty clinicians assessed telehealth skills and clinical practice and identified areas for intervention. Clinicians responded well to the training and feedback.

Unmet needs among people reported with hepatitis C, New York City.

OBJECTIVE: This project sought to describe unmet needs among patients reported with hepatitis C in New York City. DESIGN: From the New York City Health Department's hepatitis C surveillance database, we randomly selected patients whose positive hepatitis C test was in April or May 2005. In 2006, we interviewed patients by telephone and collected information from their clinicians or by medical record review. SETTING: New York City. PARTICIPANTS: We interviewed 180 of the 387 eligible patients and collected information from clinicians for 145 of the 180 patients. MAIN OUTCOME MEASURES: These included whether patients had understood their clinicians' explanation of their hepatitis C diagnosis, if they had been counseled about not drinking alcohol, information about support group attendance, vaccination against hepatitis A and B, health status, treatment, and other factors. RESULTS: Of the 180 patients, 7% stated that they had not understood their clinicians' explanation of their hepatitis C diagnosis, and 26% said that they had not been counseled about avoiding alcohol. Among the 90% of patients who had not attended a hepatitis support group, 31% were interested in attending. Among the 145 patients with information from clinicians, at least 28% were susceptible to hepatitis A and 18% to hepatitis B. CONCLUSIONS: This hepatitis C surveillance project, with information from patients and clinicians, illustrates a valuable use of a chronic hepatitis C surveillance system. The patients described here had several unmet needs, including hepatitis A and B vaccination, basic information about the virus, support groups, and counseling about preventing further liver damage and preventing transmission to others. Relatively simple and affordable health department activities can address these needs, improving quality of life and decreasing the likelihood of liver disease progression.

Protomer alignment modulates specificity of RNA substrate recognition by Ire1.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Autoimmune anti-DNA and anti-phosphatidylserine antibodies predict development of severe COVID-19.

High levels of autoimmune antibodies are observed in COVID-19 patients but their specific contribution to disease severity and clinical manifestations remains poorly understood. We performed a retrospective study of 115 COVID-19 hospitalized patients with different degrees of severity to analyze the generation of autoimmune antibodies to common antigens: a lysate of erythrocytes, the lipid phosphatidylserine (PS) and DNA. High levels of IgG autoantibodies against erythrocyte lysates were observed in a large percentage (up to 36%) of patients. Anti-DNA and anti-PS antibodies determined upon hospital admission correlated strongly with later development of severe disease, showing a positive predictive value of 85.7% and 92.8%, respectively. Patients with positive values for at least one of the two autoantibodies accounted for 24% of total severe cases. Statistical analysis identified strong correlations between anti-DNA antibodies and markers of cell injury, coagulation, neutrophil levels and erythrocyte size. Anti-DNA and anti-PS autoantibodies may play an important role in the pathogenesis of COVID-19 and could be developed as predictive biomarkers for disease severity and specific clinical manifestations.

Different endovascular referral patterns are being learned in medical and surgical residency training programs.

Physicians in residency training will be the referring physicians of tomorrow. We sought to determine the current surgical and medical trainees' perception of vascular surgery's endovascular qualifications and capabilities. An anonymous survey was sent to all general surgery and internal medicine residents at a single academic institution. Respondents answered the question "Which specialty is the most qualified to perform (1) inferior vena cava (IVC) filter insertion; (2) angiograms, angioplasty, and stenting of the carotid arteries; (3) renal arteries; (4) aorta; and (5) lower extremity arteries?" For each question, respondents chose one response, either vascular surgery, interventional radiology, interventional cardiology, or do not know. One hundred respondents completed the survey (general surgery, n=50; internal medicine, n=50). There was a significant difference in the attitudes of surgery and medicine residents when choosing the most qualified endovascular specialist (p<0.05). Surgery residents chose vascular surgery as the most qualified specialty for each listed procedure: carotid (80%, n=40), IVC (56%, n=28), aorta (100%, n=50), extremity (86%, n=43), renal (78%, n=39). Medicine residents chose vascular surgery as the most qualified specialty less frequently: carotid (66%, n=33), IVC (6%, n=3), aorta (88%, n=44), extremity (72%, n=36), renal (16%, n=8). There was no significant difference in specialty selection based on postgraduate year. There is a large discrepancy between surgical and medical trainees' perception of vascular surgery's endovascular abilities, particularly regarding IVC placement and renal artery interventions. If our own institution mirrors the nation, each passing year a significant portion of the 21,722 graduating internal medicine residents go into practice viewing vascular surgeons as second-tier endovascular providers. A concerted campaign should be undertaken to educate medical residents regarding the skills and capabilities of vascular surgeons.