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.

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.

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.

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.

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.

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.

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.

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.

Effect of tablet structure on controlled release from supersaturating solid dispersions containing glyceryl behenate.

The objective of this study was to evaluate the use of glyceryl behenate as a plasticizer and release modifier in solid dispersion systems containing itraconazole and carbamazepine. Amorphous solid dispersions of high molecular weight polyvinylpyrrolidone were prepared by hot-melt extrusion, the processing of which was improved by the inclusion of glyceryl behenate. Dispersions were milled and subsequently compressed into tablets. Solid dispersions were also prepared by KinetiSol Dispersing, which allowed for the manufacture of monolithic tablets of the same composition and shape as compressed tablets. Tablets without glyceryl behenate and all compressed tablets were observed to have an incomplete release profile likely due to drug crystallization within the tablet as this occurred at conditions in which dissolution concentrations were below saturation. Monolithic tablets formulated to be more hydrophobic, by including glyceryl behenate, allowed for sustained release below and above saturation conditions.

The incorporation of low-substituted hydroxypropyl cellulose into solid dispersion systems.

While the use of amorphous solid dispersions to improve aqueous solubility is well documented, little consideration has traditionally been given to the finished dosage form. The objective of this study was to evaluate the dissolution performance of amorphous solid dispersions containing a dispersed superdisintegrant with binding properties. KinetiSol® dispersing was used to thermally process hypromellose acetate succinate-based compositions containing the drug substance nifedipine (NIF) and a highly compressible grade of low-substituted hydroxypropyl cellulose (New Binder Disintegrants; NBD-grade). Solid-state analysis demonstrated that compositions were rendered amorphous during processing. Tablets containing intra-dispersion NBD were found to exhibit non-sink dissolution performance similar to milled intermediate, demonstrating excellent disintegration characteristics. Conversely, tablets without intra-dispersion NBD were found to release significantly less NIF during dissolution analysis due to particle agglomeration. It was determined that compressibility and particle wetting increased as the level of intra-dispersion NBD increased.

Preparation of amorphous solid dispersions by rotary evaporation and KinetiSol Dispersing: approaches to enhance solubility of a poorly water-soluble gum extract.

Acetyl-11-keto-ß-boswellic acid (AKBA), a gum resin extract, possesses poor water-solubility that limits bioavailability and a high melting point making it difficult to successfully process into solid dispersions by fusion methods. The purpose of this study was to investigate solvent and thermal processing techniques for the preparation of amorphous solid dispersions (ASDs) exhibiting enhanced solubility, dissolution rates and bioavailability. Solid dispersions were successfully produced by rotary evaporation (RE) and KinetiSol® Dispersing (KSD). Solid state and chemical characterization revealed that ASD with good potency and purity were produced by both RE and KSD. Results of the RE studies demonstrated that AQOAT®-LF, AQOAT®-MF, Eudragit® L100-55 and Soluplus with the incorporation of dioctyl sulfosuccinate sodium provided substantial solubility enhancement. Non-sink dissolution analysis showed enhanced dissolution properties for KSD-processed solid dispersions in comparison to RE-processed solid dispersions. Variances in release performance were identified when different particle size fractions of KSD samples were analyzed. Selected RE samples varying in particle surface morphologies were placed under storage and exhibited crystalline growth following solid-state stability analysis at 12 months in comparison to stored KSD samples confirming amorphous instability for RE products. In vivo analysis of KSD-processed solid dispersions revealed significantly enhanced AKBA absorption in comparison to the neat, active substance.

Functions and regulation of the APOBEC family of proteins.

APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein-protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins.

Safety and efficacy of varenicline to reduce positive subjective effects produced by methamphetamine in methamphetamine-dependent volunteers.

Methamphetamine use is increasing in the US. Although there are no Food and Drug Administration (FDA)-approved medications for methamphetamine dependence, preclinical and clinical studies suggest that methamphetamine users may benefit from treatments that enhance cholinergic neurotransmission. Consequently, we determined the safety and the efficacy of varenicline treatment, a partial agonist at α4ß2 and a full agonist at α7 nicotinic acetylcholine receptors, to reduce positive subjective effects produced by smoked methamphetamine. Additionally, the effects of treatment with varenicline on the cardiovascular and reinforcing effects of methamphetamine were determined. We conducted a double-blind, placebo-controlled, within-subjects trial of varenicline vs. placebo in methamphetamine-dependent volunteers who were not seeking treatment. Participants were randomly assigned to receive one dose of varenicline (0, 1, or 2 mg) po BID, titrated up to the target dose over days 1-7, during each of three separate inpatient phases. Safety measures included the frequency, duration, severity, and relatedness of adverse events reported. Positive subjective effects included 'Any drug effect', 'High', 'Good effects', 'Stimulated', and 'Drug liking', which were rated by participants before and for 1 h after smoking methamphetamine (0, 10, and 30 mg). There were no serious adverse events and no differences in adverse events reported during the three phases. Varenicline (2 mg) significantly reduced ratings of 'Any drug effect' and 'Stimulated', as well as attenuated ratings of 'High', 'Drug liking', and 'Good effects', produced by methamphetamine (30 mg). The ability of varenicline to attenuate the positive subjective effects of methamphetamine in the laboratory suggests that varenicline should continue to be explored as a treatment for methamphetamine dependence.

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.

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.

Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918.

The H1N1 subtype of influenza A virus has caused substantial morbidity and mortality in humans, first documented in the global pandemic of 1918 and continuing to the present day. Despite this disease burden, the evolutionary history of the A/H1N1 virus is not well understood, particularly whether there is a virological basis for several notable epidemics of unusual severity in the 1940s and 1950s. Using a data set of 71 representative complete genome sequences sampled between 1918 and 2006, we show that segmental reassortment has played an important role in the genomic evolution of A/H1N1 since 1918. Specifically, we demonstrate that an A/H1N1 isolate from the 1947 epidemic acquired novel PB2 and HA genes through intra-subtype reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the 1951 influenza epidemic may also have been associated with reassortant A/H1N1 viruses. Intra-subtype reassortment therefore appears to be a more important process in the evolution and epidemiology of H1N1 influenza A virus than previously realized.

Density-dependence of functional development in spiking cortical networks grown in vitro.

During development, the mammalian brain differentiates into specialized regions with distinct functional abilities. While many factors contribute to functional specialization, we explore the effect of neuronal density on the development of neuronal interactions in vitro. Two types of cortical networks, namely, dense and sparse with 50,000 and 12,500 total cells, respectively, are studied. Activation graphs that represent pairwise neuronal interactions are constructed using a competitive first response model. These graphs reveal that, during development in vitro, dense networks form activation connections earlier than sparse networks. Link entropy analysis of dense network activation graphs suggests that the majority of connections between electrodes are reciprocal in nature. Information theoretic measures reveal that early functional information interactions (among three electrodes) are synergetic in both dense and sparse networks. However, during later stages of development, previously synergetic relationships become primarily redundant in dense, but not in sparse networks. Large link entropy values in the activation graph are related to the domination of redundant ensembles in late stages of development in dense networks. Results demonstrate differences between dense and sparse networks in terms of informational groups, pairwise relationships, and activation graphs. These differences suggest that variations in cell density may result in different functional specializations of nervous system tissue in vivo.

Dissolution enhancement of a drug exhibiting thermal and acidic decomposition characteristics by fusion processing: a comparative study of hot melt extrusion and KinetiSol dispersing.

In this study, hot melt extrusion (HME) and KinetiSol Dispersing (KSD) were utilized to prepare dissolution-enhanced solid dispersions of Roche Research Compound A (ROA), a BCS class II drug. Preformulation characterization studies showed that ROA was chemically unstable at elevated temperatures and acidic pH values. Eudragit L100-55 and AQOAT LF (HPMCAS) were evaluated as carrier polymers. Dispersions were characterized for ROA recovery, crystallinity, homogeneity, and non-sink dissolution. Eudragit L100-55 dispersions prepared by HME required the use of micronized ROA and reduced residence times in order to become substantially amorphous. Compositions containing HPMCAS were also prepared by HME, but an amorphous dispersion could not be obtained. All HME compositions contained ROA-related impurities. KSD was investigated as a method to reduce the decomposition of ROA while rendering compositions amorphous. Substantially amorphous, plasticizer free compositions were processed successfully by KSD with significantly higher ROA recovery values and amorphous character than those achieved by HME. A near-infrared chemical imaging analysis was conducted on the solid dispersions as a measure of homogeneity. A statistical analysis showed similar levels of homogeneity in compositions containing Eudragit L100-55, while differences were observed in those containing HMPCAS. Non-sink dissolution analysis of all compositions showed rapid supersaturation after pH adjustment to approximately two to three times the equilibrium solubility of ROA, which was maintained for at least 24 h. The results of the study demonstrated that KSD is an effective method of forming dissolution-enhanced amorphous solid solutions in cases where HME is not a feasible technique.