Rad Path in a Flash: Creation of the First AMSER Sponsored Digital Flashcard Deck and a Review of Digital Flashcard Use among? Medical Students.

RATIONALE AND OBJECTIVES: Digital flashcards are an increasingly popular study method for medical students today. The purpose of this study is to assess students' opinions on digital flashcards and to evaluate the need for radiology-focused digital flashcards. We created the first official Radiology-Pathology (Rad-Path) Correlation course digital flashcard deck sponsored by the Alliance of Medical Student Educators in Radiology (AMSER), and evaluated its effectiveness in increasing medical students' confidence levels of understanding Rad-Path concepts. MATERIALS AND METHODS: A 16-question survey was developed and publicly shared with medical students to assess the need for a high-quality radiology deck. In addition, students who trialed the AMSER Rad-Path Anki deck were assessed on their confidence of radiology pathology concepts prior to and after using these digital flashcards. RESULTS: 185 survey responses were received for the first survey about digital flashcard use. Overall, 87.6% of medical student respondents had used digital flashcards during medical school, and 33.5% had used digital flashcards to study radiology topics specifically. 67.6% of respondents stated they would use a digital flashcard deck if sponsored by an academic radiology society. Regarding the survey of the students who used the AMSER Rad-Path flashcards, 31 responses were received in the pre- and follow-up survey. Mean confidence in understanding the radiology pathology learning objectives increased when the Rad-Path Anki deck was utilized, and students rated the flashcards favorably. CONCLUSION: Students reported a high use of digital flashcards for studying topics in medical school. We identified a perceived need in students for a series of high-quality digital flashcards covering radiology topics. We created the first deck of AMSER Rad-Path flashcards which was well received by the medical students.

Continuous Fluorescence Assay for In Vitro Translation Compatible with Noncanonical Amino Acids.

The tolerance of the translation apparatus toward noncanonical amino acids (ncAAs) has enabled the creation of diverse natural-product-like peptide libraries using mRNA display for use in drug discovery. Typical experiments testing for ribosomal ncAA incorporation involve radioactive end point assays to measure yield alongside mass spectrometry experiments to validate incorporation. These end point assays require significant postexperimental manipulation for analysis and prevent higher throughput analysis and optimization experiments. Continuous assays for in vitro translation involve the synthesis of fluorescent proteins which require the full complement of canonical AAs for function and are therefore of limited utility for testing of ncAAs. Here, we describe a new, continuous fluorescence assay for in vitro translation based on detection of a short peptide tag using an affinity clamp protein, which exhibits changes in its fluorescent properties upon binding. Using this assay in a 384-well format, we were able to validate the incorporation of a variety of ncAAs and also quickly test for the codon reading specificities of a variety of Escherichia coli tRNAs. This assay enables rapid assessment of ncAAs and optimization of translation components and is therefore expected to advance the engineering of the translation apparatus for drug discovery and synthetic biology.

Extensive breaking of genetic code degeneracy with non-canonical amino acids.

Genetic code expansion (GCE) offers many exciting opportunities for the creation of synthetic organisms and for drug discovery methods that utilize in vitro translation. One type of GCE, sense codon reassignment (SCR), focuses on breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. SCR has great potential for genetic code expansion, but extensive SCR is limited by the post-transcriptional modifications on tRNAs and wobble reading of these tRNAs by the ribosome. To better understand codon-tRNA pairing, here we develop an assay to evaluate the ability of aminoacyl-tRNAs to compete with each other for a given codon. We then show that hyperaccurate ribosome mutants demonstrate reduced wobble reading, and when paired with unmodified tRNAs lead to extensive and predictable SCR. Together, we encode seven distinct amino acids across nine codons spanning just two codon boxes, thereby demonstrating that the genetic code hosts far more re-assignable space than previously expected, opening the door to extensive genetic code engineering.

p-Chloropropynyl Phenylalanine, a Versatile Non-Canonical Amino Acid for Co-Translational Peptide Macrocyclization and Side Chain Diversification.

Macrocyclization has proven to be a beneficial strategy to improve upon some of the disadvantages of peptides as therapeutics. Nevertheless, many peptide cyclization strategies are not compatible with in vitro display technologies like mRNA display. Here we describe the novel amino acid p-chloropropynyl phenylalanine (pCPF). pCPF is a substrate for a mutant phenylalanyl-tRNA synthetase and its introduction into peptides via in vitro translation leads to spontaneous peptide macrocyclization in the presence of peptides containing cysteine. Macrocyclization occurs efficiently with a wide variety of ring sizes. Moreover, pCPF can be reacted with thiols after charging onto tRNA, enabling the testing of diverse ncAAs in translation. The versatility of pCPF should facilitate downstream studies of translation and enable the creation of novel macrocyclic peptide libraries.

Development of a Cyclic, Cell Penetrating Peptide Compatible with In Vitro Selection Strategies.

A key limitation for the development of peptides as therapeutics is their lack of cell permeability. Recent work has shown that short, arginine-rich macrocyclic peptides containing hydrophobic amino acids are able to penetrate cells and reach the cytosol. Here, we have developed a new strategy for developing cyclic cell penetrating peptides (CPPs) that shifts some of the hydrophobic character to the peptide cyclization linker, allowing us to do a linker screen to find cyclic CPPs with improved cellular uptake. We demonstrate that both hydrophobicity and position of the alkylation points on the linker affect uptake of macrocyclic cell penetrating peptides (CPPs). Our best peptide, 4i, is on par with or better than prototypical CPPs Arg9 (R9) and CPP12 under assays measuring total cellular uptake and cytosolic delivery. 4i was also able to carry a peptide previously discovered from an in vitro selection, 8.6, and a cytotoxic peptide into the cytosol. A bicyclic variant of 4i showed even better cytosolic entry than 4i, highlighting the plasticity of this class of peptides toward modifications. Since our CPPs are cyclized via their side chains (as opposed to head-to-tail cyclization), they are compatible with powerful technologies for peptide ligand discovery including phage display and mRNA display. Access to diverse libraries with inherent cell permeability will afford the ability to find cell permeable hits to many challenging intracellular targets.

Comparison of the Modified Jones Tube Technique and the DMEK EndoGlide Technique With and Without Viscoelastic Material for DMEK Tissue Preparation.

PURPOSE: The aim of this study was to compare endothelial cell loss for DMEK (Descemet membrane endothelial keratoplasty) tissue preparation techniques using the modified Jones tube and the DMEK EndoGlide with and without viscoelastic material to protect the endothelium. METHODS: This ex vivo study included 10 DMEK grafts prepared using each of the 3 abovementioned techniques. After tissue preparation, transport conditions were simulated for a minimum of 45 hours before deployment of the DMEK tissue and quantification of endothelial cell loss. Comparisons between preparation technique groups were made using the Wilcoxon rank-sum test. RESULTS: The Jones tube group had a mean endothelial cell loss of 11.0 ± 4.8% compared with the EndoGlide group with 12.9 ± 6.7% and the EndoGlide with viscoelastic group with 25.7 ± 15.0%. The differences between the EndoGlide with viscoelastic group and the other 2 were statistically significant both before (P < 0.01 and P = 0.01) and after (P = 0.01 and P = 0.02) adjusting for baseline characteristics. The difference between the EndoGlide and Jones tube groups was not significant (P = 0.73 and P = 0.53 after adjustment). Microscopy revealed endothelial cell loss in the area of viscoelastic use for the EndoGlide with viscoelastic group. CONCLUSIONS: Both the Jones tube and DMEK EndoGlide resulted in similar low rates of endothelial cell loss after tissue preparation, transport, and deployment. However, use of viscoelastic material to protect the endothelium using the DMEK technique actually resulted in increased cell loss in the area of its application resulting in overall higher rates of cell loss across the DMEK tissue.

Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for tRNA capture and applied it to the isolation of roughly half of the Escherichia coli tRNA isoacceptors. We then performed codon competition experiments between the five captured wild-type leucyl-tRNAs and their synthetic counterparts, revealing a strong preference for wild-type tRNA in an in vitro translation system. Finally, we compared the ability of wild-type and synthetic leucyl-tRNA to break the degeneracy of the leucine codon box, showing that only captured wild-type tRNAs are discriminated with enough fidelity to accurately split the leucine codon box for the encoding of three separate amino acids. Wild-type tRNAs are therefore enabling reagents for maximizing the reassignment potential of the genetic code.

Spontaneous, co-translational peptide macrocyclization using p-cyanoacetylene-phenylalanine.

Peptide macrocycles (PMCs) are increasingly popular for the development of inhibitors of protein-protein interactions (PPIs). Large libraries of PMCs are accessible using display technologies like mRNA display and phage display. These technologies require macrocyclization chemistries to be compatible with biological milieu, severely limiting the types of technologies available for cyclization. Here, we introduce the novel non-canonical amino acid (ncAA) p-cyanoacetylene-L-Phe (pCAF), which facilitates spontaneous, co-translational cyclization through Michael addition with cysteine under physiological conditions. This new, robust chemistry creates stable macrocycles of a wide variety of ring sizes including bicyclic structures.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming.

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.

Associations Between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants and Risk of Coronavirus Disease 2019 (COVID-19) Hospitalization Among Confirmed Cases in Washington State: A Retrospective Cohort Study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with 7 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. METHODS: Our study includes individuals with positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) in the Washington Disease Reporting System with available viral genome data, from 1 December 2020 to 14 January 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination. RESULTS: In total, 58 848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95% confidence interval [CI] 2.40-4.26), Beta (HR 2.85, 95% CI 1.56-5.23), Delta (HR 2.28 95% CI 1.56-3.34), or Alpha (HR 1.64, 95% CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95% CI .56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination. CONCLUSIONS: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.

AMSER Rad Path Case of the Month: Effect of Case-based Integration of Radiology and Pathology on Medical Student Education and Interdisciplinary Collaboration.

RATIONALE AND OBJECTIVES: Radiology and pathology are often underrepresented in undergraduate medical education. The Alliance of Medical Student Educators in Radiology (AMSER) hosts the AMSER Rad Path Case of the Month, an online archive of radiological case reports with pathology correlations submitted by medical students. The purpose of this study is to assess the value of preparing and submitting a case on medical students' educational experience. MATERIALS AND METHODS: Students who had cases accepted for publication in AMSER Rad Path Case of the Month from July 2018 to December 2019 were contacted by email to request their participation in a voluntary, anonymous 22-question survey. Surveys were sent to 35 students from seven institutions. RESULTS: Twenty three of the 35 students (65.7%) responded. Only five (21.7%) of respondents reported having previously followed a patient case through radiology and pathology during medical school, defined as interaction with a clinician in each specialty to discuss the case. When asked about their experience with AMSER Rad Path Case of the Month, most agreed or strongly agreed it was a valuable case-based learning experience (100%). Respondents also reported high satisfaction with improved understanding of disease process, increased understanding and ability to collaborate, and increased likelihood of participating in future academic work. CONCLUSION: AMSER Rad Path Case of the Month is a valuable case-based educational experience that deepens students' understanding of disease processes while affording them an opportunity for interdisciplinary and scholarly collaboration.

Non-canonical Amino Acid Substrates of E. coli Aminoacyl-tRNA Synthetases.

In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.

Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases in Washington State: a retrospective cohort study.

BACKGROUND: The COVID-19 pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with seven SARS-CoV-2 variants. METHODS: Our study includes individuals with positive SARS-CoV-2 RT-PCR in the Washington Disease Reporting System with available viral genome data, from December 1, 2020 to January 14, 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination. FINDINGS: 58,848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95%CI 2.40-4.26), Beta (HR 2.85, 95%CI 1.56-5.23), Delta (HR 2.28 95%CI 1.56-3.34) or Alpha (HR 1.64, 95%CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95%CI 0.56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination. CONCLUSION: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance. SUMMARY: Hospitalization risk following infection with SARS-CoV-2 variant remains unclear. We find a higher hospitalization risk in cases infected with Alpha, Beta, Gamma, and Delta, but not Omicron, with vaccination lowering risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.

Silicon phthalocyanines: synthesis and resurgent applications.

The intense far-red absorption and emission features have made silicon phthalocyanines (SiPcs) distinct from the structurally related porphyrin analogues. Unlike most other phthalocyanines, SiPcs possess two additional axial bonds which reduce aggregation in solution and can be synthetically tailored, thereby creating further scope for modulation of optical, chemical and electronic properties. Multiple synthetic strategies have been employed for facile construction of symmetrical or unsymmetrical SiPc variants bearing desired substitutents at the axial and the aromatic ring positions. The overarching motive of this concise review article is to highlight and summarize the key synthetic routes and the fast-emerging applications of SiPcs in photouncaging techniques, photothermal and photoimmunotherapy, photovoltaics, optoelectronics and photocatalysis.

Search and Rescue: A Case Report and Discussion of Iatrogenic Intravascular Foreign Bodies.

The following case report demonstrates an unusual case of a patient with multiple intravascular iatrogenic foreign bodies. An inferior vena cava filter was identified in the abdominal aorta, and multiple hydrophilic wire fragments were found in the pulmonary circulation. After describing the management of this patient, we discuss how radiologists can best distinguish between various intravascular iatrogenic foreign bodies. Determining the type of catheter or wire within the patient can not only establish the chronicity of the finding, but can determine whether or not endovascular retrieval is recommended, and by what method. It is of the utmost importance that providers be able to distinguish between various intravascular devices in order to prevent ongoing or further harm to the patient.

SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts.

Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an species to study mild/subclinical COVID-19 disease and with possible insights into live virus shedding. Future vaccine evaluation can be performed in AGM with correlates of efficacy being lung lesions by PET/CT, virus shedding, and tissue viral load.

Direct, Competitive Comparison of Linear, Monocyclic, and Bicyclic Libraries Using mRNA Display.

Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.

COVID-19 Respiratory Failure: Targeting Inflammation on VV-ECMO Support.

The outbreak of novel coronavirus (SARS-CoV-2) that causes the respiratory illness COVID-19 has led to unprecedented efforts at containment due to its rapid community spread, associated mortality, and lack of immunization and treatment. We herein detail a case of a young patient who suffered life-threatening disease and multiorgan failure. His clinical course involved rapid and profound respiratory decompensation such that he required support with venovenous extracorporeal membrane oxygenation (VV-ECMO). He also demonstrated hyperinflammation (C-reactive protein peak 444.6 mg/L) with severe cytokine elevation (Interleukin-6 peak > 3000 pg/ml). Through treatment targeting hyperinflammation, he recovered from critical COVID-19 respiratory failure and required only 160 hours of VV-ECMO support. He was extubated 4 days after decannulation, had progressive renal recovery, and was discharged to home on hospital day 24. Of note, repeat SARS-CoV-2 test was negative 21 days after his first positive test. We present one of the first successful cases of VV-ECMO support to recovery of COVID-19 respiratory failure in North America.