SARS-CoV-2 viral shedding in vaccinated and unvaccinated persons: A case series.

The preclinical time course of SARS-CoV-2 shedding is not well-described. Understanding this time course will help to inform risk of SARS-CoV-2 transmission. During an outbreak in a congregate setting, we collected paired mid-turbinate nasal swabs for antigen testing and reverse-transcription polymerase chain reaction (RT-PCR) every other day from all consenting infected and exposed persons. Among 12 persons tested prospectively before and during SARS-CoV-2 infection, ten of 12 participants (83%) had completed a primary COVID-19 vaccination series prior to the outbreak. We recovered SARS-CoV-2 in viral culture from 9/12 (75%) of participants. All three persons from whom we did not recover SARS-CoV-2 in viral culture had completed their primary vaccination series. We recovered SARS-CoV-2 from viral culture in 6/9 vaccinated persons and before symptom onset in 3/6 symptomatic persons. These findings underscore the need for both non-pharmaceutical interventions and vaccination to mitigate transmission.

Transmission potential of vaccinated and unvaccinated persons infected with the SARS-CoV-2 Delta variant in a federal prison, July-August 2021.

BACKGROUND: The extent to which vaccinated persons who become infected with SARS-CoV-2 contribute to transmission is unclear. During a SARS-CoV-2 Delta variant outbreak among incarcerated persons with high vaccination rates in a federal prison, we assessed markers of viral shedding in vaccinated and unvaccinated persons. METHODS: Consenting incarcerated persons with confirmed SARS-CoV-2 infection provided mid-turbinate nasal specimens daily for 10 consecutive days and reported symptom data via questionnaire. Real-time reverse transcription-polymerase chain reaction (RT-PCR), viral whole genome sequencing, and viral culture was performed on these nasal specimens. Duration of RT-PCR positivity and viral culture positivity was assessed using survival analysis. RESULTS: A total of 957 specimens were provided by 93 participants, of whom 78 (84 %) were vaccinated and 17 (16 %) were unvaccinated. No significant differences were detected in duration of RT-PCR positivity among vaccinated participants (median: 13 days) versus those unvaccinated (median: 13 days; p = 0.50), or in duration of culture positivity (medians: 5 days and 5 days; p = 0.29). Among vaccinated participants, overall duration of culture positivity was shorter among Moderna vaccine recipients versus Pfizer (p = 0.048) or Janssen (p = 0.003) vaccine recipients. In post-hoc analyses, Moderna vaccine recipients demonstrated significantly shorter duration of culture positivity compared to unvaccinated participants (p = 0.02). When restricted to participants without reported prior infection, the difference between Moderna vaccine recipients and unvaccinated participants was more pronounced (medians: 3 days and 6 days, p = 0.002). CONCLUSIONS: Infectious periods for vaccinated and unvaccinated persons who become infected with SARS-CoV-2 are similar and can be highly variable, though some vaccinated persons are likely infectious for shorter durations. These findings are critically important, especially in congregate settings where viral transmission can lead to large outbreaks. In such settings, clinicians and public health practitioners should consider vaccinated, infected persons to be no less infectious than unvaccinated, infected persons.

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Effect of Biofumigation on Population Densities of Pratylenchus spp. and Verticillium spp. and Potato Yield in Eastern Canada.

Biofumigation has been proposed as an alternative to soil fumigation to manage soil-borne diseases including potato early dying disease complex (PED). This study examined the potential of using brown mustard (Mustard juncea) biofumigation to manage PED under rain-fed potato production in New Brunswick, Canada in two trials between 2017 and 2020 in comparison with chloropicrin fumigation and a conventional barley rotation. Biofumigation increased yield in one trial, but not in a second trial where the potato crop experienced severe drought, whereas chloropicrin fumigation increased yield in both trials. Biofumigation was effective in suppressing root-lesion nematode (RLN, Pratylenchus spp.) counts in both trials, but was ineffective in suppressing V. dahliae population density. Chloropicrin fumigation was effective in suppressing RLN counts and V. dahliae population density only in the hill where injected, but the effect was short-lived as the population density of V. dahliae in the hill increased to the level of the control in one potato growing season. Biofumigation may be an alternative to chloropicrin fumigation in managing PED, particularly in fields with high RLN population but relatively low Verticillium population density. However, neither biofumigation nor fumigation used alone may be sustainable in the short-term potato rotations commonly used in New Brunswick, and additional beneficial practices are required to sustain productivity in the long-term.

La biofumigación se ha propuesto como una alternativa a la fumigación del suelo para manejar las enfermedades transmitidas por el suelo, incluido el complejo de enfermedades de muerte prematura de la papa (PED). Este estudio examinó el potencial del uso de la biofumigación de mostaza marrón (Mustard juncea) para manejar la PED bajo la producción de papa de secano en New Brunswick, Canadá, en dos ensayos entre 2017 y 2020 en comparación con la fumigación con cloropicrina y una rotación de cebada convencional. La biofumigación aumentó el rendimiento en un ensayo, pero no en un segundo ensayo en el que el cultivo de papa experimentó una sequía severa, mientras que la fumigación con cloropicrina aumentó el rendimiento en ambos ensayos. La biofumigación fue efectiva para suprimir los conteos del nematodo lesionador de la raíz (RLN, Pratylenchus spp.) en ambos ensayos, pero fue ineficaz para suprimir la densidad de población de V. dahliae. La fumigación con cloropicrina fue efectiva para suprimir los conteos de RLN y la densidad de población de V. dahliae solo en el lomo del surco donde se inyectó, pero el efecto fue de corta duración ya que la densidad de población de V. dahliae en el surco aumentó al nivel del testigo en un ciclo de cultivo de papa. La biofumigación puede ser una alternativa a la fumigación con cloropicrina en el manejo de la PED, particularmente en campos con alta población de RLN pero densidad de población de Verticillium relativamente baja. Sin embargo, ni la biofumigación ni la fumigación utilizadas por sí solas pueden ser sustentables en las rotaciones de papa a corto plazo comúnmente utilizadas en New Brunswick, y se requieren prácticas benéficas adicionales para mantener la productividad a largo plazo.

Creating Maps of the Ligand Binding Landscape for Kinetics-Based Drug Discovery.

Simulations of ligand-protein interactions can be very useful for drug design and to gain biological insight. Full pathways of ligand-protein binding can be used to get information about ligand binding transition states, which form the rate-limiting step of the binding and release processes. However, these simulations are typically limited by the presence of large energy barriers that separate stable poses of interest. Here we describe a simulation protocol for exploring and analyzing landscapes of ligand-protein interactions that makes use of molecular docking, enhanced molecular simulation with the weighted ensemble algorithm, and network analysis. It can be accomplished using a modest cluster of graphics processing units and freely accessible software. This protocol focuses on the construction and analysis of a network model of ligand binding poses and provides links to resources that describe the other steps in more detail. The end result of this protocol is a map of the ligand-protein binding landscape that identifies transition states of the ligand binding pathway, as well as alternative bound poses that could be stabilized with modifications to the ligand.

Membrane-Mediated Ligand Unbinding of the PK-11195 Ligand from TSPO.

The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is of longstanding medical interest as both a biomarker for neuroinjury and a potential drug target for neuroinflammation and other disorders. Recently, it was shown that ligand residence time is a key factor determining steroidogenic efficacy of TSPO-binding compounds. This spurs interest in simulations of (un)binding pathways of TSPO ligands, which could reveal the molecular interactions governing ligand residence time. In this study, we use a weighted ensemble algorithm to determine the unbinding pathway for different poses of PK-11195, a TSPO ligand used in neuroimaging. In contrast with previous studies, our results show that PK-11195 does not dissociate directly into the solvent but instead dissociates via the lipid membrane by going between the transmembrane helices. We analyze this path ensemble in detail, constructing descriptors that can facilitate a general understanding of membrane-mediated ligand binding. We construct a set of Markov state models augmented with additional straightforward simulations to determine pose-specific ligand residence times. Together, we combine over 40 µs of trajectory data to form a coherent picture of the ligand binding landscape. We find that multiple starting poses yield residence times that roughly agree with the experimental quantity. The ligand binding transition states predicted by these Markov state models occur when PK-11195 is already in the membrane and involves only minimal ligand-protein interactions. This has implications for the design of new long-residence-time TSPO ligands.

On Calculating Free Energy Differences Using Ensembles of Transition Paths.

The free energy of a process is the fundamental quantity that determines its spontaneity or propensity at a given temperature. In particular, the binding free energy of a drug candidate to its biomolecular target is used as an objective quantity in drug design. Recently, binding kinetics-rates of association (k on) and dissociation (k off)-have also demonstrated utility for their ability to predict efficacy and in some cases have been shown to be more predictive than the binding free energy alone. Some methods exist to calculate binding kinetics from molecular simulations, although these are typically more difficult to calculate than the binding affinity as they depend on details of the transition path ensemble. Assessing these rate constants can be difficult, due to uncertainty in the definition of the bound and unbound states, large error bars and the lack of experimental data. As an additional consistency check, rate constants from simulation can be used to calculate free energies (using the log of their ratio) which can then be compared to free energies obtained experimentally or using alchemical free energy perturbation. However, in this calculation it is not straightforward to account for common, practical details such as the finite simulation volume or the particular definition of the "bound" and "unbound" states. Here we derive a set of correction terms that can be applied to calculations of binding free energies using full reactive trajectories. We apply these correction terms to revisit the calculation of binding free energies from rate constants for a host-guest system that was part of a blind prediction challenge, where significant deviations were observed between free energies calculated with rate ratios and those calculated from alchemical perturbation. The correction terms combine to significantly decrease the error with respect to computational benchmarks, from 3.4 to 0.76 kcal/mol. Although these terms were derived with weighted ensemble simulations in mind, some of the correction terms are generally applicable to free energies calculated using physical pathways via methods such as Markov state modeling, metadynamics, milestoning, or umbrella sampling.

Predicting ligand binding affinity using on- and off-rates for the SAMPL6 SAMPLing challenge.

Interest in ligand binding kinetics has been growing rapidly, as it is being discovered in more and more systems that ligand residence time is the crucial factor governing drug efficacy. Many enhanced sampling methods have been developed with the goal of predicting ligand binding rates ([Formula: see text]) and/or ligand unbinding rates ([Formula: see text]) through explicit simulation of ligand binding pathways, and these methods work by very different mechanisms. Although there is not yet a blind challenge for ligand binding kinetics, here we take advantage of experimental measurements and rigorously computed benchmarks to compare estimates of [Formula: see text] calculated as the ratio of two rates: [Formula: see text]. These rates were determined using a new enhanced sampling method based on the weighted ensemble framework that we call "REVO": Reweighting of Ensembles by Variance Optimization. This is a further development of the WExplore enhanced sampling method, in which trajectory cloning and merging steps are guided not by the definition of sampling regions, but by maximizing trajectory variance. Here we obtain estimates of [Formula: see text] and [Formula: see text] that are consistent across multiple simulations, with an average log10-scale standard deviation of 0.28 for on-rates and 0.56 for off-rates, which is well within an order of magnitude and far better than previously observed for previous applications of the WExplore algorithm. Our rank ordering of the three host-guest pairs agrees with the reference calculations, however our predicted [Formula: see text] values were systematically lower than the reference by an average of 4.2 kcal/mol. Using tree network visualizations of the trajectories in the REVO algorithm, and conformation space networks for each system, we analyze the results of our sampling, and hypothesize sources of discrepancy between our [Formula: see text] values and the reference. We also motivate the direct inclusion of [Formula: see text] and [Formula: see text] challenges in future iterations of SAMPL, to further develop the field of ligand binding kinetics prediction and modeling.

Genetic analysis of Black Tiger shrimp (Penaeus monodon) across its natural distribution range reveals more recent colonization of Fiji and other South Pacific islands.

The Black Tiger shrimp (Penaeus monodon) has a natural distribution range from East Africa to the South Pacific Islands. Although previous studies of Indo-Pacific P. monodon have found populations from the Indian Ocean and Australasia to differ genetically, their relatedness to South Pacific shrimp remains unknown. To address this, polymorphisms at eight shared microsatellite loci and haplotypes in a 418-bp mtDNA-CR (control region) sequence were examined across 682 P. monodon from locations spread widely across its natural range, including the South Pacific islands of Fiji, Palau, and Papua New Guinea (PNG). Observed microsatellite heterozygosities of 0.82-0.91, allele richness of 6.85-9.69, and significant mtDNA-CR haplotype variation indicated high levels of genetic diversity among the South Pacific shrimp. Analysis of microsatellite genotypes using a Bayesian STRUCTURE method segregated Indo-Pacific P. monodon into eight distinct clades, with Palau and PNG shrimp clustering among others from Southeast Asia and eastern Australia, respectively, and Fiji shrimp clustering as a distinct group. Phylogenetic analyses of mtDNA-CR haplotypes delineated shrimp into three groupings, with shrimp from Fiji again being distinct by sharing no haplotypes with other populations. Depending on regional location, the genetic structures and substructures identified from the genotyping and mtDNA-CR haplotype phylogeny could be explained by Metapopulation and/or Member-Vagrant type evolutionary processes. Neutrality tests of mutation-drift equilibrium and estimation of the time since population expansion supported a hypothesis that South Pacific P. monodon were colonized from Southeast Asia and eastern Australia during the Pleistocene period over 60,000 years ago when land bridges were more expansive and linked these regions more closely.

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep.

BACKGROUND: Fleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance. RESULTS: The majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheep's response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores. CONCLUSIONS: We identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheep's resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved.