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.

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.

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.

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.

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.

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.

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic ß-amino acids.

A Platinum(II) Complex of Heptamethine Cyanine for Photoenhanced Cytotoxicity and Cellular Imaging in Near-IR Light.

Controlled generation of cytotoxic agents with near-IR light is a current focus of photoactivated cancer therapy, including that involving cytotoxic platinum species. A heptamethine cyanine scaffolded PtII complex, IR797-Platin exhibits unprecedented Pt-O bond scission and enhancement in DNA platination in near-IR light. This complex also displayed significant singlet oxygen quantum yield thereby qualifying as a near-IR photodynamic therapeutic agent. The complex showed 30-60 fold enhancement of cytotoxicity in near-IR light in various cancer cell lines. The cellular imaging properties were also leveraged to observe its significant co-localization in cytoplasmic organelles. This is the first demonstration of a near-IR light-initiated therapy involving the cytotoxic effects of both active cisplatin and singlet oxygen.

Light induced drug release from a folic acid-drug conjugate.

A major area of cancer research focuses on improving the specificity of therapeutic agents by engineering drug-delivery vehicles that target overexpressed receptors on tumor cells. One of the most commonly used approaches involves targeting of folate receptors using folic acid conjugated to a drug-containing macromolecular cargo. Once internalized via endocytosis, the drugs must be released from these constructs in order to avoid being trapped in the endosomes. Here, we describe the synthesis of a small-molecule conjugate that couples folic acid to doxorubicin via a photocleavable linker. Using HPLC we show that the doxorubicin can be released with light rapidly and with high efficiency. This approach has advantages over macromolecular systems due to its simplicity and efficiency.

Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification.

Due to the lowered pKa of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.

Role of Imaging in the Evaluation of Male Infertility.

Infertility is defined herein as the inability to achieve pregnancy after frequently engaging in unprotected sexual intercourse for 1 year. Among infertile couples, the cause of infertility involves the male partner in approximately 50% of cases. Male infertility is usually caused by conditions affecting sperm production, sperm function, or both, or blockages that prevent the delivery of sperm. Chronic health problems, injuries, lifestyle choices, anatomic problems, hormonal imbalances, and genetic defects can have a role in male infertility. The diagnostic workup of male infertility should include a thorough medical and reproductive history, physical examination, and semen analysis, followed by imaging. The main role of imaging is identification of the causes of infertility, such as congenital anomalies and disorders that obstruct sperm transport and may be correctable. Scrotal ultrasonography is the most common initially performed noninvasive examination used to image the male reproductive system, including the testes and extratesticular structures such as the epididymis. Magnetic resonance (MR) imaging is another noninvasive imaging modality used in the pelvis to evaluate possible obstructive lesions involving the ductal system. MR imaging of the brain is extremely useful for evaluating relevant neurologic abnormalities, such as pituitary gland disorders, that are suspected on the basis of hormone analysis results. Invasive techniques are usually reserved for therapeutic interventions in patients with known abnormalities. In this article, the causes and imaging findings of obstructive and nonobstructive azoospermia are discussed. In addition to detecting treatable conditions that are related to male infertility, identifying the life-threatening entities associated with infertility and the genetic conditions that could be transmitted to offspring-especially in patients who undergo assisted reproduction-is critical. ©RSNA, 2017.

Fabrication and Characterization of Poly(ε-caprolactone)/α-Cyclodextrin Pseudorotaxane Nanofibers.

Multifunctional scaffolds comprising neat poly(ε-caprolactone) (PCL) and α-cyclodextrin pseudorotaxanated in α-cyclodextrin form have been fabricated using a conventional electrospinning process. Thorough in-depth characterizations were performed on the pseudorotaxane nanofibers prepared from chloroform (CFM) and CFM/dimethylformamide (DMF) utilizing scanning electron microscopy (SEM), transmission electron microscopy (TEM), rheology, differential scanning calorimetry (DSC), thermogravimetric analyses (TGA), wide-angle X-ray diffraction (WAXD), and Instron tensile testing. The results indicate the nanofibers obtained from chloroform retain the rotaxanated structure; while those obtained from CFM/DMF had significantly dethreaded during electrospinning. As a consequence, the nanowebs obtained from CFM showed higher moduli and lower elongations at break compared to neat PCL nanowebs and PCL/α-CD nanowebs electrospun from CFM/DMF.

Photoswitchable anticancer activity via trans-cis isomerization of a combretastatin A-4 analog.

Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymerization. The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. We prepared an azobenzene analog of CA4 (Azo-CA4) that shows 13-35 fold enhancement in potency upon illumination. EC50 values in the light were in the mid nM range. Due to its ability to thermally revert to less toxic trans form, Azo-CA4 also has the ability to automatically turn its activity off with time. Azo-CA4 is less potent than CA-4 because it degrades in the presence of glutathione as evidenced by UV-Vis spectroscopy and ESI-MS. Nevertheless, Azo-CA4 represents a promising strategy for switchable potency for treatment of cancer.

Hematospermia Evaluation at MR Imaging.

Hematospermia is a challenging and anxiety-provoking condition that can manifest as a single episode or recur over the course of weeks to months. It is usually a benign self-limiting condition in younger sexually active males without a history of risk factors such as cancer, urogenital malformations, bleeding disorders, and their associated symptoms. However, patients with recurrent, refractory and painful hematospermia with associated symptoms, such as fever, pain, or weight loss, require evaluation through clinical assessment and noninvasive investigations to rule out underlying pathologic conditions such as ejaculatory obstruction, infectious and inflammatory causes, malignancy, vascular malformations, and systemic disorders that increase the risk of bleeding, especially when presenting in older men. If these investigations are negative, the patient should be reassured and treated accordingly. In the recent past, magnetic resonance (MR) imaging has assumed a major role in the investigation of hematospermia due to its excellent soft-tissue contrast and multiplanar capabilities. In this review, we will discuss the potential causes of hematospermia and its diagnostic workup, including pathophysiology, anatomic considerations, the imaging appearance of associated pathologic conditions, and management. (©)RSNA, 2016.

Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.

Microbiology for Radiologists: How to Minimize Infection Transmission in the Radiology Department.

The implementation of standardized infection control and prevention practices is increasingly relevant as modern radiology practice evolves into its more clinical role. Current Centers for Disease Control and Prevention, National Institutes of Health, and World Health Organization guidelines for the proper use of personal protective equipment, decontamination of reusable medical equipment, and appropriate management of bloodborne pathogen exposures will be reviewed. Standard precautions apply to all patients at all times and are the mainstay of infection control. Proper hand hygiene includes washing hands with soap and water when exposed to certain infectious particles, such as Clostridium difficile spores, which are not inactivated by alcohol-based hand rubs. The appropriate use of personal protective equipment in accordance with recommendations from the Centers for Disease Control and Prevention includes wearing a surgical mask during lumbar puncture. Because radiologists may perform lumbar punctures for patients with prion disease, it is important to appreciate that incineration is the most effective method of inactivating prion proteins. However, there is currently no consensus recommendation on the decontamination of prion-contaminated reusable items associated with lumbar puncture, and institutional policies should be consulted for directed management. In the event of a needlestick injury, radiology staff must be able to quickly provide appropriate initial management and seek medical attention, including laboratory testing for bloodborne pathogens.

Testicular tumors: what radiologists need to know--differential diagnosis, staging, and management.

Cryptorchidism, family history, and infertility are risk factors for testicular cancer. Most testicular cancers occur in young men aged 18-35 years, and seminoma is the most common cell type. Testicular tumors are usually diagnosed at ultrasonography (US) and are staged at computed tomography (CT) or magnetic resonance (MR) imaging. At US, testicular tumors usually appear as a solid intratesticular mass. Because the differential diagnosis includes infarct and infection, correlation with patient history and symptoms is important. At staging CT or MR imaging, retroperitoneal lymph nodes are considered regional lymph nodes, and the greatest nodal diameter is used to distinguish among N1-N3 disease. The right testicular vein drains into the inferior vena cava, and the left testicular vein drains into the left renal vein. Because of venous and lymphatic drainage pathways, retroperitoneal lymph nodes are the initial landing station for testicular cancers. Enlarged lymph nodes in the supraclavicular region, chest, and pelvis are considered distant metastases. Testicular cancer is initially treated with orchiectomy. The patient may then undergo active surveillance, chemotherapy, radiation therapy, or retroperitoneal lymph node resection, depending primarily on the clinical stage. Radiologists play an important role in initial diagnosis, staging, and imaging surveillance of testicular malignancies.

Replacing amino acids in translation: expanding chemical diversity with non-natural variants.

Here, we describe a strategy for synthesis of peptides with multiple unnatural amino acids (UAAs) using in vitro translation. Our method involves removing a natural amino acid and replacing it with an UAA variant in a reconstituted translation system. Whereas other systems require engineered components or chemical synthesis to charge UAAs onto tRNAs prior to translation, our strategy utilizes the wild-type machinery and charging occurs concomitant with translation. The design of the system allows for easy quantification of the UAA's incorporation efficiency and fidelity.

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.