Comparison of Gene Editing Versus Conventional Breeding to Introgress the POLLED Allele Into the Tropically Adapted Australian Beef Cattle Population

Dehorning is the process of physically removing horns to protect animals and humans from injury, but the process is costly, unpleasant, and faces increasing public scrutiny. Genetic selection for polled (hornless), which is genetically dominant to horned, is a long-term solution to eliminate the need for dehorning. However, due to the limited number of polled Australian Brahman bulls, the northern Australian beef cattle population remains predominantly horned. The potential to use gene editing to produce high-genetic-merit polled cattle was recently demonstrated. To further explore the concept, this study simulated introgression of the POLLED allele into a tropically adapted Australian beef cattle population via conventional breeding or gene editing (top 1% or 10% of seedstock bulls/year) for 3 polled mating schemes and compared results to baseline selection on genetic merit (Japan Ox selection index, $JapOx) alone, over the course of 20 years. The baseline scenario did not significantly decrease the 20-year HORNED allele frequency (80$8.00/year). Compared to the baseline, the conventional breeding scenarios where polled bulls were preferentially used for breeding, regardless of their genetic merit, significantly decreased the 20-year HORNED allele frequency (30%), but resulted in a significantly slower rate of genetic gain ($6.70/year, P ≤ 0.05). The mating scheme that required the exclusive use of homozygous polled bulls, resulted in the lowest 20-year HORNED allele frequency (8$5.50/year). The addition of gene editing the top 1% or 10% of seedstock bull calves/year to each conventional breeding scenario resulted in significantly faster rates of genetic gain (up to $8.10/year, P ≤ 0.05). Overall, our study demonstrates that, due to the limited number of polled Australian Brahman bulls, strong selection pressure on polled will be necessary to meaningfully increase the number of polled animals in this population. Moreover, these scenarios illustrate how gene editing could be a tool for accelerating the development of high-genetic-merit homozygous polled sires to mitigate the current trade-off of slower genetic gain associated with decreasing HORNED allele frequency in the Australian Brahman population

Systemic Anticancer Therapy and Thromboembolic Outcomes in Hospitalized Patients With Cancer and COVID-19

IMPORTANCE: Systematic data on the association between anticancer therapies and thromboembolic events (TEEs) in patients with COVID-19 are lacking. OBJECTIVE: To assess the association between anticancer therapy exposure within 3 months prior to COVID-19 and TEEs following COVID-19 diagnosis in patients with cancer. DESIGN, SETTING, AND PARTICIPANTS: This registry-based retrospective cohort study included patients who were hospitalized and had active cancer and laboratory-confirmed SARS-CoV-2 infection. Data were accrued from March 2020 to December 2021 and analyzed from December 2021 to October 2022. EXPOSURE: Treatments of interest (TOIs) (endocrine therapy, vascular endothelial growth factor inhibitors/tyrosine kinase inhibitors [VEGFis/TKIs], immunomodulators [IMiDs], immune checkpoint inhibitors [ICIs], chemotherapy) vs reference (no systemic therapy) in 3 months prior to COVID-19. MAIN OUTCOMES AND MEASURES: Main outcomes were (1) venous thromboembolism (VTE) and (2) arterial thromboembolism (ATE). Secondary outcome was severity of COVID-19 (rates of intensive care unit admission, mechanical ventilation, 30-day all-cause mortality following TEEs in TOI vs reference group) at 30-day follow-up. RESULTS: Of 4988 hospitalized patients with cancer (median [IQR] age, 69 [59-78] years; 2608 [52%] male), 1869 had received 1 or more TOIs. Incidence of VTE was higher in all TOI groups: endocrine therapy, 7%; VEGFis/TKIs, 10%; IMiDs, 8%; ICIs, 12%; and chemotherapy, 10%, compared with patients not receiving systemic therapies (6%). In multivariable log-binomial regression analyses, relative risk of VTE (adjusted risk ratio [aRR], 1.33; 95% CI, 1.04-1.69) but not ATE (aRR, 0.81; 95% CI, 0.56-1.16) was significantly higher in those exposed to all TOIs pooled together vs those with no exposure. Among individual drugs, ICIs were significantly associated with VTE (aRR, 1.45; 95% CI, 1.01-2.07). Also noted were significant associations between VTE and active and progressing cancer (aRR, 1.43; 95% CI, 1.01-2.03), history of VTE (aRR, 3.10; 95% CI, 2.38-4.04), and high-risk site of cancer (aRR, 1.42; 95% CI, 1.14-1.75). Black patients had a higher risk of TEEs (aRR, 1.24; 95% CI, 1.03-1.50) than White patients. Patients with TEEs had high intensive care unit admission (46%) and mechanical ventilation (31%) rates. Relative risk of death in patients with TEEs was higher in those exposed to TOIs vs not (aRR, 1.12; 95% CI, 0.91-1.38) and was significantly associated with poor performance status (aRR, 1.77; 95% CI, 1.30-2.40) and active/progressing cancer (aRR, 1.55; 95% CI, 1.13-2.13). CONCLUSIONS AND RELEVANCE: In this cohort study, relative risk of developing VTE was high among patients receiving TOIs and varied by the type of therapy, underlying risk factors, and demographics, such as race and ethnicity. These findings highlight the need for close monitoring and perhaps personalized thromboprophylaxis to prevent morbidity and mortality associated with COVID-19-related thromboembolism in patients with cancer

Investigating the relationship between imputation accuracies and relatedness

Single Step Best Linear Unbiased Prediction (ssBLUP) is used in the Australian beef industry's genetic evaluation, BREEDPLAN, for the prediction of Estimated Breeding Values (EBVs), which uses genomic information in a Genomic Relationship Matrix (GRM). Imputation of missing Single Nucleotide Polymorphisms (SNPs) and imputation of low density to high density genotypes is essential to combine various SNP densities for building the GRM. EBV accuracies are dependent on an individual's relationship to the rest of the population. Similarly, a target population's imputation accuracy is dependent on relatedness to the reference population. This study introduces a 'relatedness score', calculated in a similar way to EBV accuracies, to indicate an animal's relatedness to the reference population. The objective of this study was to identify how well the relatedness score can predict imputation accuracies. For this purpose QMSim was used to simulate 10 generations of genotypes (20 chromosomes and 2000 SNPs – 200 cM) with 40 males and 800 females in the historical population. Generations 4 to 10 were used to evaluate the relationship between imputation accuracies and relatedness score. The results demonstrated a non-linear correlation between imputation accuracies and relatedness score when individuals exist across multiple generations and with densities greater than 1000 SNPs were used. These results indicate a relatedness score may explain low EBV accuracies and EBV instability within BREEDPLAN data, due to low imputation accuracies

Changes to poll DNA testing for Australian beef cattle

The poll microsatellite DNA marker test has been used for poll testing Australia’s beef industry since 2011, and largely relied on the submission of phenotypes (horned, polled, and scurred) as a reference dataset to estimate genotypes with an associated probability. Recently poll SNP based testing has become available, which does not require phenotype submission and provides a more conclusive genotype result. The following summarises changes to poll DNA testing, how tests differ and potential changes in genotype results

An Efficient Method to Calculate Genomic Prediction Accuracy for New Individuals

Diagonal elements of the coefficient matrix are necessary to calculate the genomic prediction accuracy. Here an improved methodology is described, to update the inverse of the coefficient matrix (C) for new individuals with a genotype, with and without phenotypes. Computational performance is significantly improved by re-using parts of the coefficient matrix inverse calculations that do not change from one animal to another, in combination with updated calculations for those that do change. This method expedites calculation of accuracy for new individuals with genotypes, without re-doing the whole population, by using the previously calculated matrices

Synthetic biology design to display an 18 kDa rotavirus large antigen on a modular virus-like particle

Virus-like particles are an established class of commercial vaccine possessing excellent function and proven stability. Exciting developments made possible by modern tools of synthetic biology has stimulated emergence of modular VLPs, whereby parts of one pathogen are by design integrated into a less harmful VLP which has preferential physical and manufacturing character. This strategy allows the immunologically protective parts of a pathogen to be displayed on the most-suitable VLP. However, the field of modular VLP design is immature, and robust design principles are yet to emerge, particularly for larger antigenic structures. Here we use a combination of molecular dynamic simulation and experiment to reveal two key design principles for VLPs. First, the linkers connecting the integrated antigenic module with the VLP-forming protein must be well designed to ensure structural separation and independence. Second, the number of antigenic domains on the VLP surface must be sufficiently below the maximum such that a "steric barrier" to VLP formation cannot exist. This second principle leads to designs whereby co-expression of modular protein with unmodified VLP-forming protein can titrate down the amount of antigen on the surface of the VLP, to the point where assembly can proceed. In this work we elucidate these principles by displaying the 18.1 kDa VP8* domain from rotavirus on the murine polyomavirus VLP, and show functional presentation of the antigenic structure. (C) 2015 The Authors. Published by Elsevier Ltd

Improved fusion tag cleavage strategies in the downstream processing of self-assembling virus-like particle vaccines

Virus-like particle (VLP) vaccines are emerging as an exciting platform for the delivery of antigenic modules to enable engineering of desirable immunological outcomes, both in therapeutic and prophylactic settings. The processing of these macromolecular assemblies raises new challenges not previously encountered for simpler protein therapeutics. Microbial expression of viral precursor protein complexes (capsomeres) and their subsequent cell-free self-assembly represent a new and technologically interesting pathway to VLP vaccines, yet significant scientific and engineering challenges remain. Among these, the use of thrombin within the existing laboratory process must be eliminated, for regulatory, cost and product quality reasons. Here, the use of alternatives to thrombin is explored. It is shown that tobacco etch virus protease (TEVp) is a viable alternative to thrombin, and leads to higher-quality VLP product; as TEVp has no known human physiological or biochemical role, its bioprocessing acceptability is expected to be higher than for thrombin. The results presented here enhance the scale-up potential of microbial VLP processing, and suggest that TEVp may be a preferable enzyme for use in other bioprocess settings, including those related to the processing of less complex biologics

Predicting recombinant protein expression experiments using molecular dynamics simulation

Soluble expression of de novo-designed proteins in Escherichia coli (E. coli) remains empirical. For given experimental conditions expression success is determined in part by protein primary sequence. This has been previously explored with varying success using a variety of statistical solubility prediction tools though without taking fold stability into account. In the present study, the three-dimensional structure of proteins in molecular dynamics (MD) simulations is used to predict expression as a new approach with a set of four-helix bundles. Stability-related parameters for ten structures were determined in a thermal unfolding MD simulation and used to build statistical models with a support vector machine (SVM) classifier. The most accurate models were identified by their performance on five independent four-helix bundle sequences. The final model provided accurate classification prediction for this test set and was successfully applied in a model challenge with two newly designed sequences. The combination of simulation-derived parameters and an SVM classifier has potential to predict recombinant expression outcome for this set of four-helix bundles. With further development, this approach of utilizing higher-dimensional protein structural information to predict expression may have potential to advance recombinant biotechnology through modern computational and statistical science

Biomolecular engineering of virus-like particles aided by computational chemistry methods

Virus-like particles (VLPs) are repetitive organizations of viral proteins assembled in an appropriate physicochemical environment. VLPs can stimulate both innate and adaptive immune responses, due to their particulate structure enabling uptake by antigen presenting cells. These characteristics have led to successful development of VLP-vaccine products, and will ensure their vast potential in years to come. Future success of VLP therapeutic products will be determined by advances in their bioengineering, and also by the development of tools to design for their stability, function and application. This review focuses on approaches for VLP assembly in controlled chemical environments in vivo and in vitro, and the application of computational tools for improved chemical sequence design, and fundamental understanding of assembly

Computational study of elements of stability of a four-helix bundle protein biosurfactant

Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants. DAMP4, a member of a four-helix bundle biosurfactant protein family, can be produced in soluble form and at high yield in Escherichia coli, and can be recovered using a facile thermal phase-separation approach. As such, it encompasses an interesting synergy of biomolecular and chemical engineering with prospects for low-cost production even for industrial sectors. DAMP4 is highly functional, and due to its extraordinary thermal stability it can be purified in a simple two-step process, in which the combination of high temperature and salt leads to denaturation of all contaminants, whereas DAMP4 stays stable in solution and can be recovered by filtration. This study aimed to characterize and understand the fundamental drivers of DAMP4 stability to guide further process and surfactant design studies. The complementary use of experiments and molecular dynamics simulation revealed a broad pH and temperature tolerance for DAMP4, with a melting point of 122.4 °C, suggesting the hydrophobic core as the major contributor to thermal stability. Simulation of systematically created in silico variants of DAMP4 showed an influence of number and location of hydrophilic mutations in the hydrophobic core on stability, demonstrating a tolerance of up to three mutations before a strong loss in stability occurred. The results suggest a consideration of a balance of stability, functionality and kinetics for new designs according to their application, aiming for maximal functionality but at adequate stability to allow for cost-efficient production using thermal phase separation approaches