P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma.

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependent kinase inhibitor 2A, isoform INK4a). We further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and in human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis.

Delivering Urgent Care Using Telemedicine: Insights from Experienced Clinicians at Academic Medical Centers.

BACKGROUND: Care delivered using telemedicine has been steadily growing in the USA but represented a small fraction of overall visits before the COVID-19 pandemic as few clinicians had been providing care using telemedicine. Understanding how experienced clinicians have practiced telemedicine can help guide today's exponential adoption of telemedicine. OBJECTIVE: The objective of this study was to explore barriers and facilitators to providing effective, high-quality urgent care using telemedicine ("tele-urgent care") from the perspective of clinicians experienced in telemedicine. APPROACH: We conducted semi-structured interviews between July 2018 and March 2019 of clinicians who had been providing tele-urgent care services to patients as a part of their routine clinical practice. Themes were identified using content analysis with a constant comparative coding approach. KEY RESULTS: Among the 20 clinicians interviewed, the majority were female (90%) and nurse practitioners (65%). We identified four themes related to barriers and facilitators to providing effective, high-quality tele-urgent care. Workplace factors such as a strong information technology (IT) infrastructure, real-time IT support, an electronic health record, and a collegial work environment, often virtual, were necessary standards. Communication and exam techniques from in-person encounters were adapted to tele-urgent care including active listening skills and teaching patients to conduct specific exam maneuvers virtually. The convenience of tele-urgent care should be preserved to support improvements in access to care. Finally, patients and clinicians occasionally had mismatched expectations about what could or would be provided during a tele-urgent care encounter. Managing the added tension that can occur during a telemedicine encounter was important. CONCLUSION: As telemedicine becomes an integral part of the care continuum, incorporating and accounting for these key insights when we train and support clinicians will be necessary to provide effective, high-quality care to patients in the future.

Probing the Impact of the Knob-into-Hole Mutations on the Structure and Function of a Therapeutic Antibody.

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.

Understanding Why Urban, Low-Income Patients Miss Primary Care Appointments: Insights From Qualitative Interviews of West Philadelphians.

Missed primary care appointments occur frequently among urban, low-income patients-some of the costliest and sickest patients. We conducted semi-structured interviews with 43 patients who reside in West Philadelphia (100% insured by Medicaid, 95% were non-Hispanic African Americans, and 47.1 years old on average) to identify why recent primary care appointments were or might have been missed. Existing transportation options, including public transportation, were considered unreliable and alternative options too costly. In addition, we discovered poor health, family obligations, and work requirements prevented appointment attendance. Intervening on the barriers identified may reduce missed appointment rates among disadvantaged populations.

Association of Rideshare-Based Transportation Services and Missed Primary Care Appointments: A Clinical Trial.

Importance: Transportation barriers contribute to missed primary care appointments for patients with Medicaid. Rideshare services have been proposed as alternatives to nonemergency medical transportation programs because of convenience and lower costs. Objective: To evaluate the association between rideshare-based medical transportation and missed primary care appointments among Medicaid patients. Design, Setting, and Participants: In a prospective clinical trial, 786 Medicaid beneficiaries who resided in West Philadelphia and were established primary care patients at 1 of 2 academic internal medicine practices located within the same building were included. Participants were allocated to being offered complimentary ride-sharing services (intervention arm) or usual care (control arm) based on the prescheduled day of their primary care appointment reminder. Those scheduled on even-numbered weekdays were in the intervention arm and on odd-numbered weekdays, the control arm. The primary study outcome was the rate of missed appointments, estimated using an intent-to-treat approach. All individuals receiving a phone call reminder were included in the study sample, regardless of whether they answered their phone. The study was conducted between October 24, 2016, and April 20, 2017. Interventions: A model of providing rideshare-based transportation was designed. As part of usual care, patients assigned to both arms received automated appointment phone call reminders. As part of the study protocol, patients assigned to both arms received up to 3 additional appointment reminder phone calls from research staff 2 days before their scheduled appointment. During these calls, patients in the intervention arm were offered a complimentary ridesharing service. Research staff prescheduled rides for those interested in the service. After their appointment, patients phoned research staff to initiate a return trip home. Main Outcomes and Measures: Missed appointment rate (no shows and same-day cancellations) in the intervention compared with control arm. Results: Of the 786 patients allocated to the intervention or control arm, 566 (72.0%) were women; mean (SD) age was 46.0. (12.5) years. Within the intervention arm, 85 among 288 (26.0%) participants who answered the phone call used ridesharing. The missed appointment rate was 36.5% (144 of 394) for the intervention arm and 36.7% (144 of 392) for the control arm (P = .96). Conclusions and Relevance: The uptake of ridesharing was low and did not decrease missed primary care appointments. Future studies trying to reduce missed appointments should explore alternative delivery models or targeting populations with stronger transportation needs. Trial Registration: clinicaltrials.gov Identifier: NCT02955433.

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye.

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

Next-generation sequencing and novel variant determination in a cohort of 92 familial exudative vitreoretinopathy patients.

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a developmental disease that can cause visual impairment and retinal detachment at a young age. Four genes involved in the Wnt signaling pathway were previously linked to this disease: NDP, FDZ4, LRP5, and TSPAN12. Identification of novel disease-causing alleles allows for a deeper understanding of the disease, better molecular diagnosis, and improved treatment. METHODS: Sequencing libraries from 92 FEVR patients were generated using a custom capture panel to enrich for 163 known retinal disease-causing genes in humans. Samples were processed using next generation sequencing (NGS) techniques followed by data analysis to identify and classify single nucleotide variants and small insertions and deletions. Sanger validation and segregation testing were used to verify suspected variants. RESULTS: Of the cohort of 92, 45 patients were potentially solved (48.9%). Solved cases resulted from the determination of 49 unique mutations, 41 of which are novel. Of the novel variants discovered, 13 were highly likely to cause FEVR due to the nature of these variants (frameshifting indels, splicing mutations, and nonsense variants types). To our knowledge, this is the largest study of a FEVR cohort using NGS. CONCLUSIONS: We were able to determine probable disease-causing variants in a large number of FEVR patients, the majority of which were novel. Knowledge of these variants will help to further characterize and diagnose FEVR.

Design of a phosphorylatable PDZ domain with peptide-specific affinity changes.

Phosphorylation is one of the most common posttranslational modifications controlling cellular protein activity. Here, we describe a combined computational and experimental strategy to design new phosphorylation sites into globular proteins to regulate their functions. We target a peptide recognition protein, the Erbin PDZ domain, to be phosphorylated by cAMP-dependent protein kinase. Comparing the five successful designs to the unsuccessful cases, we find a trade-off between protein stability and the ability to be modified by phosphorylation. In two designs, Erbin's peptide binding function is modified by phosphorylation, where the presence of the phosphate group destabilizes peptide binding. One of these showed an additional switch in specificity by introducing favorable interactions between a designed arginine in the peptide and phosphoserine on the PDZ domain. Because of the diversity of PDZ domains, this opens avenues for the design of related phosphoswitchable domains to create a repertoire of regulatable interaction parts for synthetic biology.