Infection with mpox virus via the genital mucosae increases shedding and transmission in the multimammate rat (Mastomys natalensis).

The 2022 mpox virus (MPXV) outbreak was sustained by human-to-human transmission; however, it is currently unclear which factors lead to sustained transmission of MPXV. Here we present Mastomys natalensis as a model for MPXV transmission after intraperitoneal, rectal, vaginal, aerosol and transdermal inoculation with an early 2022 human outbreak isolate (Clade IIb). Virus shedding and tissue replication were route dependent and occurred in the presence of self-resolving localized skin, lung, reproductive tract or rectal lesions. Mucosal inoculation via the rectal, vaginal and aerosol routes led to increased shedding, replication and a pro-inflammatory T cell profile compared with skin inoculation. Contact transmission was higher from rectally inoculated animals. This suggests that transmission might be sustained by increased susceptibility of the anal and genital mucosae for infection and subsequent virus release.

Molnupiravir inhibits SARS-CoV-2 variants including Omicron in the hamster model.

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC), which contains a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here, we assessed the efficacy of MK-4482 against the earlier Alpha, Beta, and Delta VOCs and Omicron in the hamster COVID-19 model. Omicron replication and associated lung disease in vehicle-treated hamsters was reduced compared with replication and lung disease associated with earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of hamsters infected with Alpha, Beta, or Delta VOCs. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious titers compared with viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model.

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

Three-Week Old Pigs Are Not Susceptible to Productive Infection with SARS-COV-2.

As the COVID-19 pandemic moves into its third year, there remains a need for additional animal models better recapitulating severe COVID to study SARS-CoV-2 pathogenesis and develop countermeasures, especially treatment options. Pigs are known intermediate hosts for many viruses with zoonotic potential and are susceptible to infection with alpha, beta and delta genera of coronaviruses. Herein, we infected young (3 weeks of age) pigs with SARS-CoV-2 using a combination of respiratory and parenteral inoculation routes. Pigs did not develop clinical disease, nor macroscopic or microscopic pathologic lesions upon SARS-CoV-2 infection. Despite occasional low levels of SARS-CoV-2 genomic RNA in the respiratory tract, subgenomic RNA and infectious virus were never found, and SARS-CoV-2-specific adaptive immune responses were not detectable over the 13-day study period. We concluded that pigs are not susceptible to productive SARS-CoV-2 infection and do not serve as a SARS-CoV-2 reservoir for zoonotic transmission.

UK B.1.1.7 (Alpha) variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, we compared African green monkeys infected intranasally with either the UK B.1.1.7 (Alpha) variant or its contemporary D614G progenitor. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.

Coastal complexity: Ancient human diets inferred from Bayesian stable isotope mixing models and a primate analogue.

An extensive ecological literature applies stable isotope mixing models to derive quantitative dietary reconstructions from isotope ratios of consumer tissues. While this approach works well for some organisms, it is challenging for consumers with complex, varied diets, including humans; indeed, many archaeologists have avoided the use of mixing models because uncertainties in model outputs are sufficiently large that the findings are not helpful in understanding ancient lifeways. Here, we exploit an unparalleled opportunity to evaluate the feasibility of dietary quantification in a nutritionally and isotopically complex context on the Cape Peninsula, South Africa. Delta values (δ13C and δ15N) of 213 indigenous food samples enable us to characterise four food groups: terrestrial plants, terrestrial vertebrates, marine invertebrates and marine vertebrates. A recent study of baboons that consumed marine and terrestrial foods provides insight into the relationship between such foods and consumer tissue isotopes. We use this information to refine our interpretation of δ15N and especially δ13C in bone collagen from 35 archaeological hunter-gatherers, achieving better estimates of the relative importance of marine and terrestrial foods in the diet than has hitherto been possible. Based on Bayesian stable isotope mixing model (SIMM) outputs, we infer that the trophic enrichment factor (TEF) for δ13Cbone collagen in these coastal humans is closer to +3 than +5‰. In the most 13C- and 15N-rich individuals, 65-98% of bone collagen (95% credible intervals) derived from marine foods. Conversely, in 13C and 15N-poor individuals, 7-44% of bone collagen derived from marine foods. The uncertainties discussed here highlight the need for caution when implementing SIMMs in studies of consumers with complex diets. To our knowledge, this work constitutes the most detailed and most tightly constrained study of this problem to date.

Isotopic assessment of marine food consumption by natural-foraging chacma baboons on the Cape Peninsula, South Africa.

OBJECTIVES: Stable isotope analysis has been used to investigate consumption of marine resources in a variety of terrestrial mammals, including humans, but not yet in extant nonhuman primates. We sought to test the efficacy of stable isotope analysis as a tool for such studies by comparing isotope- and observation-based estimates of marine food consumption by a troop of noncommensal, free-ranging chacma baboons. MATERIALS AND METHODS: We determined δ13 C and δ15 N values of baboon hair (n = 9) and fecal samples (n = 144), and principal food items (n = 362). These values were used as input for diet models, the outputs of which were compared to observation-based estimates of marine food consumption. RESULTS: Fecal δ13 C values ranged from -29.3‰ to -25.6‰. δ15 N values ranged from 0.9‰ to 6.3‰ and were positively correlated with a measure of marine foraging during the dietary integration period. Mean (± SD) δ13 C values of adult male and female baboon hairs were -21.6‰ (± 0.1) and -21.8‰ (± 0.3) respectively, and corresponding δ15 N values were 5.0‰ (± 0.3) and 3.9‰ (± 0.2). Models indicated that marine contributions were ≤10% of baboon diet within any season, and contributed ≤17% of dietary protein through the year. DISCUSSION: Model output and observational data were in agreement, both indicating that despite their abundance in the intertidal region, marine foods comprised only a small proportion of baboon diet. This suggests that stable isotope analysis is a viable tool for investigating marine food consumption by natural-foraging primates in temperate regions.

Targeting Bacterial Nitric Oxide Synthase with Aminoquinoline-Based Inhibitors.

Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.

Beyond basins: φ,ψ preferences of a residue depend heavily on the φ,ψ values of its neighbors.

The Ramachandran plot distributions of nonglycine residues from experimentally determined structures are routinely described as grouping into one of six major basins: ß, PII , α, αL , ξ and γ'. Recent work describing the most common conformations adopted by pairs of residues in folded proteins [i.e., (φ,ψ)2 -motifs] showed that commonly described major basins are not true single thermodynamic basins, but are composed of distinct subregions that are associated with various conformations of either the preceding or following neighbor residue. Here, as documentation of the extent to which the conformational preferences of a central residue are influenced by the conformations of its two neighbors, we present a set of φ,ψ-plots that are delimited simultaneously by the φ,ψ-angles of its neighboring residues on both sides. The level of influence seen here is typically greater than the influence associated with considering the identities of neighboring residues, implying that the use of this heretofore untapped information can improve the accuracy of structure prediction algorithms and low resolution protein structure refinement.

Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

(φ,ψ)2 motifs: a purely conformation-based fine-grained enumeration of protein parts at the two-residue level.

A deep understanding of protein structure benefits from the use of a variety of classification strategies that enhance our ability to effectively describe local patterns of conformation. Here, we use a clustering algorithm to analyze 76,533 all-trans segments from protein structures solved at 1.2 Å resolution or better to create a purely φ,ψ-based comprehensive empirical categorization of common conformations adopted by two adjacent φ,ψ pairs (i.e., (φ,ψ)(2) motifs). The clustering algorithm works in an origin-shifted four-dimensional space based on the two φ,ψ pairs to yield a parameter-dependent list of (φ,ψ)(2) motifs, in order of their prominence. The results are remarkably distinct from and complementary to the standard hydrogen-bond-centered view of secondary structure. New insights include an unprecedented level of precision in describing the φ,ψ angles of both previously known and novel motifs, ordering of these motifs by their population density, a data-driven recommendation that the standard C(α(i))…C(α(i+3))<7 Å criteria for defining turns be changed to 6.5 Å, identification of ß-strand and turn capping motifs, and identification of conformational capping by residues in polypeptide II conformation. We further document that the conformational preferences of a residue are substantially influenced by the conformation of its neighbors, and we suggest that accounting for these dependencies will improve protein modeling accuracy. Although the CUEVAS-4D(r(10)є(14)) 'parts list' presented here is only an initial exploration of the complex (φ,ψ)(2) landscape of proteins, it shows that there is value to be had from this approach, and it opens the door to more in-depth characterizations at the (φ,ψ)(2) level and at higher dimensions.

Improving the quality of discharge communication with an educational intervention.

BACKGROUND: Communication between hospital providers and primary care physicians at the time of hospital discharge is necessary for optimal patient care and safety. Content of the inpatient discharge summary (DS) is not uniformly addressed by residency programs. OBJECTIVE: To improve DSs quality through a brief educational intervention. METHODS: We prospectively enrolled interns (first-year pediatric residents [PL1s]) in an educational intervention that consisted of a group session in which components of a high-quality DS were taught and a subsequent brief small-group session in which key components with distribution of a reminder card were reiterated. Six key components were identified: diagnosis; timely completion; pending laboratory work/studies; medications; length ≤3 pages; and discharge weight. DSs prepared by PL1s before and after the small-group session were objectively scored by blinded reviewers on the basis of how many DS components they contained (maximum score: 6). Scores were compared with historical controls of PL1s from the previous year. Audit scores were analyzed by using a mixed-effects linear regression model. RESULTS: Sixty-four PL1s were enrolled in the study; 477 DSs were scored. Mean score before the small-group reminder session was 3.6 in both groups. In mixed-effects models, scores in the intervention group increased by 0.56 points (P=.002) and DSs incorporating at least 5 of 6 components increased from 22% to 41% (P<.001) after the small-group session, whereas the control group's scores were unchanged. CONCLUSION: A brief, low-intensity educational intervention can improve quality of discharge communication and be incorporated into residency training. Electronic templates should incorporate prompts for key components of a DS.