Sangivamycin is preferentially incorporated into viral RNA by the SARS-CoV-2 polymerase.

Sangivamycin (S) is an adenosine (A) nucleoside analog with low nanomolar antiviral activity against SARS-CoV-2 in vitro. Previously, low nanomolar antiviral efficacy was revealed when tested against multiple viral variants in several cell types. SARS-CoV-2 RNA isolated from live virus infected cells and the virions released from these cells was analyzed by mass spectrometry (MS) for S incorporation. Dose-dependent incorporation occurred up to 1.8 S per 1,000 nucleotides (49 S per genome) throughout the viral genomes isolated from both infected cells and viral particles, but this incorporation did not change the viral mutation rate. In contrast, host mRNA, affinity purified from the same infected and treated cells, contained little or no S. Sangivamycin triphosphate (STP) was synthesized to evaluate its incorporation into RNA by recombinant SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) under defined in vitro conditions. SARS-CoV-2 RdRp showed that S was not a chain terminator and S containing oligonucleotides templated as A. Though the antiviral mechanism remains to be determined, the data suggests that SARS-CoV-2 RdRp incorporates STP into SARS-CoV-2 RNA, which does not significantly impair viral RNA synthesis or the mutation rate.

Congruent aero-tactile stimuli bias perception of voicing continua.

Multimodal integration is the formation of a coherent percept from different sensory inputs such as vision, audition, and somatosensation. Most research on multimodal integration in speech perception has focused on audio-visual integration. In recent years, audio-tactile integration has also been investigated, and it has been established that puffs of air applied to the skin and timed with listening tasks shift the perception of voicing by naive listeners. The current study has replicated and extended these findings by testing the effect of air puffs on gradations of voice onset time along a continuum rather than the voiced and voiceless endpoints of the original work. Three continua were tested: bilabial ("pa/ba"), velar ("ka/ga"), and a vowel continuum ("head/hid") used as a control. The presence of air puffs was found to significantly increase the likelihood of choosing voiceless responses for the two VOT continua but had no effect on choices for the vowel continuum. Analysis of response times revealed that the presence of air puffs lengthened responses for intermediate (ambiguous) stimuli and shortened them for endpoint (non-ambiguous) stimuli. The slowest response times were observed for the intermediate steps for all three continua, but for the bilabial continuum this effect interacted with the presence of air puffs: responses were slower in the presence of air puffs, and faster in their absence. This suggests that during integration auditory and aero-tactile inputs are weighted differently by the perceptual system, with the latter exerting greater influence in those cases where the auditory cues for voicing are ambiguous.

Sangivamycin is highly effective against SARS-CoV-2 in vitro and has favorable drug properties.

Sangivamycin is a nucleoside analog that is well tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin's potential for clinical administration, pharmacokinetic; absorption, distribution, metabolism, and excretion (ADME); and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 therapeutic.

Cyclic peptides with a distinct arginine-fork motif recognize the HIV trans-activation response RNA in vitro and in cells.

RNA represents a potential target for new antiviral therapies, which are urgently needed to address public health threats such as the human immunodeficiency virus (HIV). We showed previously that the interaction between the viral Tat protein and the HIV-1 trans-activation response (TAR) RNA was blocked by TB-CP-6.9a. This cyclic peptide was derived from a TAR-binding loop that emerged during lab evolution of a TAR-binding protein (TBP) family. Here we synthesized and characterized a next-generation, cyclic-peptide library based on the TBP scaffold. We sought to identify conserved RNA-binding interactions and the influence of cyclization linkers on RNA binding and antiviral activity. A diverse group of cyclization linkers, encompassing disulfide bonds to bicyclic aromatic staples, was used to restrain the cyclic peptide geometry. Thermodynamic profiling revealed specific arginine-rich sequences with low to submicromolar affinity driven by enthalpic and entropic contributions. The best compounds exhibited no appreciable off-target binding to related molecules, such as BIV TAR and human 7SK RNAs. A specific arginine-to-lysine change in the highest affinity cyclic peptide reduced TAR binding by tenfold, suggesting that TBP-derived cyclic peptides use an arginine-fork motif to recognize the TAR major groove while differentiating the mode of binding from other TAR-targeting molecules. Finally, we showed that HIV infectivity in cell culture was reduced in the presence of cyclic peptides constrained by methylene or naphthalene-based linkers. Our findings provide insight into the molecular determinants required for HIV-1 TAR recognition and antiviral activity. These findings are broadly relevant to the development of antivirals that target RNA molecules.

A new mode of cyclobutenedione ring opening for the synthesis of 2-oxobut-3-enamides and tetrasubstituted furans.

A dichotomy between the additions of organolithiums and lithium amides to cyclobutenediones is described wherein the former give carbonyl addition products while the latter induce ring opening by enone cleavage via O- to C-lithium transfer. This distinct mode of ring scission gives access to 2-oxobut-3-enamides and tetrasubstituted furans.

Non-technical skills in paramedicine: A scoping review.

This scoping review aims to identify which non-technical skills have been empirically identified in the literature, to create the first list of empirically identified non-technical skills for paramedics and allied health personnel. A five-stage scoping literature was undertaken in March 2020. The search retrieved a total of 4756 citations. A total of 93 studies met the inclusion criteria and were analyzed for data charting. A total of 26 non-technical skills were identified in the literature. The top five non-technical skills included decision-making (33%, n = 31), communication (24%, n = 23), empathy (17%, n = 16), leadership (12%, n = 12), and ethics (10%, n = 10). Furthermore, only five studies investigated the assessment or measurement of non-technical skills. This scoping review identified 26 non-technical skills that had been investigated in the paramedic literature to create the first list of empirically based desirable non-technical skills for a paramedic. Subsequently, research can then begin to focus on identifying the link that these have to paramedic practice and patient safety.

A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

Translation of the Canadian Cardiovascular Society/Canadian Heart Rhythm Society Cardiovascular Screening and Care of Athletes Program Into Practice.

In the absence of systematic cardiovascular preparticipation screening (PPS) practices in Canada, the Canadian Cardiovascular Society and Canadian Heart Rhythm Society published a joint position statement outlining PPS recommendations for competitive athletes in an effort to standardize screening. The Queen's University Cardiovascular Screening and Care of Athletes Program aimed to translate these recommendations into practice. Screening packages were administered to athletes in 2017 and 2018. Each package required completion of a medical/history questionnaire, a physician's examination, and 12-lead electrocardiogram performed by the athlete's primary care physician. A tiered approach to screening, as recommended by the position statement, was implemented. A multidisciplinary team flagged at-risk athletes and determined the need for follow-up testing and participation eligibility. Over a 2-year period, 517 varsity athletes were screened, with 438 (84.7%) completing all recommended screening components. Analysis of completed packages resulted in 114 (26.0%) athletes flagged for initial review. After subsequent review by an emergency care physician, only 12 (2.7%) athletes required further referral to cardiology for assessment or further testing. All athletes referred for cardiology assessment were cleared for participation, except for one, who was eventually cleared after a shared decision-making process and cardiovascular intervention. The Queen's University Cardiovascular Screening and Care of Athletes Program shows the successful implementation of a cardiovascular PPS program that used shared decision-making and a multidisciplinary approach to screening, allowing for efficiency and effective resource utilization.

Regulation of Antiviral Innate Immunity Through APOBEC Ribonucleoprotein Complexes.

The DNA mutagenic enzyme known as APOBEC3G (A3G) plays a critical role in innate immunity to Human Immunodeficiency Virus-1 (HIV-1 ). A3G is a zinc-dependent enzyme that mutates select deoxycytidines (dC) to deoxyuridine (dU) through deamination within nascent single stranded DNA (ssDNA) during HIV reverse transcription. This activity requires that the enzyme be delivered to viral replication complexes by redistributing from the cytoplasm of infected cells to budding virions through what appears to be an RNA-dependent process. Once inside infected cells, A3G must bind to nascent ssDNA reverse transcripts for dC to dU base modification gene editing. In this chapter we will discuss data indicating that ssDNA deaminase activity of A3G is regulated by RNA binding to A3G and ribonucleoprotein complex formation along with evidence suggesting that RNA-selective interactions with A3G are temporally and mechanistically important in this process.

Contextual predictability influences word and morpheme duration in a morphologically complex language (Kaqchikel Mayan).

The probability is one of the many factors which influence phonetic variation. Contextual probability, which describes how predictable a linguistic unit is in some local environment, has been consistently shown to modulate the phonetic salience of words and other linguistic units in speech production (the probabilistic reduction effect). In this paper the question of whether the probabilistic reduction effect, as previously observed for majority languages like English, is also found in a language (Kaqchikel Mayan) which has relatively rich morphology is explored. Specifically, whether the contextual predictability of words and morphemes influences their phonetic duration in Kaqchikel is examined. It is found that the contextual predictability of a word has a significant effect on its duration. The effect is manifested differently for lexical words and function words. It is also found that the contextual predictability of certain prefixes in Kaqchikel affects their duration, showing that contextual predictability may drive reduction effects at multiple levels of structure. While the findings are broadly consistent with many previous studies (primarily on English), some of the details of the results are different. These differences highlight the importance of examining the probabilistic reduction effect in languages beyond the majority, Indo-European languages most commonly investigated in experimental and corpus linguistics.

The Effect of Foam Rolling for Three Consecutive Days on Muscular Efficiency and Range of Motion.

BACKGROUND: Foam rolling (FR) has been shown to alleviate some symptoms of exercise-induced muscle damage and has been suggested to increase range of motion (ROM) without negatively impacting strength. However, it is unclear what neuromuscular effects, if any, mediate these changes. METHODS: In a randomized, crossover design, 16 healthy active males completed 2 min of rest or FR of the knee extensors on three consecutive days. Mechanical properties of vastus lateralis (VL) and rectus femoris (RF) were assessed via Tensiomyography. Knee extension maximal voluntary contraction (MVC) and knee flexion ROM were also assessed, and surface electromyography amplitude (RMS) was recorded during a submaximal isometric contraction (50% of MVC). Measures were performed before and after (0, 15, and 30 min) FR or rest. RESULTS: MVC was reduced on subsequent days in the rest condition compared to FR (p = 0.002, pη2 = 0.04); ROM was not different across time or condition (p = 0.193, pη2 = 0.01). Stiffness characteristics of the VL were different on the third day of FR (p = 0.002, pη2 = 0.03). RMS was statistically reduced 0, 15, and 30 min after FR compared to rest (p = 0.006, pη2 = 0.03; p = 0.003, pη2 = 0.04; p = 0.002, pη2 = 0.04). CONCLUSIONS: Following FR, MVC was elevated compared to rest and RMS was transiently reduced during a submaximal task. Excitation efficiency of the involved muscles may have been enhanced by FR, which protected against the decline in MVC which was observed with rest.

A New Class of Antiretroviral Enabling Innate Immunity by Protecting APOBEC3 from HIV Vif-Dependent Degradation.

The infectivity of HIV depends on overcoming APOBEC3 (A3) innate immunity, predominantly through the expression of the viral protein Vif, which induces A3 degradation in the proteasome. Disruption of the functional interactions of Vif enables A3 mutagenesis of the HIV genome during viral replication, which can result in a broadly neutralizing antiviral effect. Vif function requires self-association along with interactions with A3 proteins, protein chaperones, and factors of the ubiquitination machinery and these are described here as a potential platform for novel antiviral drug discovery. This Review will examine the current state of development of Vif inhibitors that we believe to have therapeutic and functional cure potential.

HIV-1 Frameshift RNA-Targeted Triazoles Inhibit Propagation of Replication-Competent and Multi-Drug-Resistant HIV in Human Cells.

The HIV-1 frameshift-stimulating (FSS) RNA, a regulatory RNA of critical importance in the virus' life cycle, has been posited as a novel target for anti-HIV drug development. We report the synthesis and evaluation of triazole-containing compounds able to bind the FSS with high affinity and selectivity. Readily accessible synthetically, these compounds are less toxic than previously reported olefin congeners. We show for the first time that FSS-targeting compounds have antiviral activity against replication-competent HIV in human cells, including a highly cytopathic, multidrug-resistant strain. These results support the viability of the HIV-1 FSS RNA as a therapeutic target and more generally highlight opportunities for synthetic molecule-mediated interference with protein recoding in a wide range of organisms.

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315.

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

The APOBEC Protein Family: United by Structure, Divergent in Function.

The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

Structural and functional assessment of APOBEC3G macromolecular complexes.

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins.

N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.

When Do DCD Donors Die?: Outcomes and Implications of DCD at a High-volume, Single-center OPO in the United States.

OBJECTIVE: The objective of this study was to determine the fate of patients who attempted to donate organs after circulatory death (DCD) using a standardized DCD protocol. BACKGROUND: Successful donation is not always possible after attempted DCD. METHODS: Data were collected for all DCD donors between 1/2011 and 9/2014. DCDs were carried out using a uniform protocol at a single-center organ procurement organization. RESULTS: During the timeframe considered, DCD donation was attempted in 169 patients. In 46 patients (27.2%), no organs were recovered because the patients did not die within 2 hours. Successful donation was more likely if withdrawal of support occurred in the operating room versus the intensive care unit (P = 0.006). Time from extubation to death was available for 161/169 donors (95.3%). Of 161 donors, 111 (66.9%) died in under 1 hour. The mean time from withdrawal of support to patient death for unsuccessful donations was 33 hours, 37 minutes (range, 24 minutes-242 hours) versus 29 minutes (range, 5 minutes-2 hours, 4 minutes) for successful donations. Twenty-seven patients who unsuccessfully donated (67.5%) died within 24 hours. Were unsuccessful donations converted to successful donations, as many as 837 abdominal transplants could have been carried out in the United States, during the study period. CONCLUSIONS: DCD is an important form of organ donation. A large number of abdominal transplants are not possible due to unsuccessful DCD organ donation. It may be useful to explore DCD donor family satisfaction to identify other options for improving DCD donation.