Evaluation of the potential food allergy risks of human lactoferrin expressed in Komagataella phaffii.

Introduction: Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. Methods: The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera™), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. Results: The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Conclusion: Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply.

Daily energy expenditure and water turnover in female netball players from the Netball Super League: A doubly labeled water observation study.

To establish the criterion-assessed energy and fluid requirements of female netball players, 13 adult players from a senior Netball Super League squad were assessed over 14 days in a cross-sectional design, representing a two- and one-match microcycle, respectively. Total energy expenditure (TEE) and water turnover (WT) were measured by doubly labeled water. Resting and activity energy expenditure were measured by indirect calorimetry and Actiheart, respectively. Mean 14-day TEE was 13.46 ± 1.20 MJ day-1 (95% CI, 12.63-14.39 MJ day-1). Resting energy expenditure was 6.53 ± 0.60 MJ day-1 (95% CI, 6.17-6.89 MJ day-1). Physical activity level was 2.07 ± 0.19 arbitrary units (AU) (95% CI, 1.95-2.18 AU). Mean WT was 4.1 ± 0.9 L day-1 (95% CI, 3.6-4.7 L day-1). Match days led to significantly greater TEE than training (+2.85 ± 0.70 MJ day-1; 95% CI, +1.00- +4.70 MJ day-1; p = 0.002) and rest (+4.85 ± 0.70 MJ day-1; 95% CI, +3.13-+6.56 MJ day-1; p < 0.001) days. Matches led to significantly greater energy expenditure (+1.85 ± 1.27 MJ; 95% CI, +0.95-+2.76 MJ day-1; p = 0.001) than court-based training sessions. There was no significant difference in TEE (+0.03 ± 0.35 MJ day-1; 95% CI, -0.74-+0.80 MJ day-1; p = 0.936) across weeks. Calibrated Actiheart 5 monitors underestimated TEE (-1.92 ± 1.21 MJ day-1). Energy and fluid turnover were greatest on match days, followed by training and rest days, with no difference across weeks. This study provides criterion-assessed energy and fluid requirements to inform dietary guidance for female netball players.

Workshop report: A study roadmap to evaluate the safety of recombinant human lactoferrin expressed in Komagataella phaffii intended as an ingredient in conventional foods - Recommendations of a scientific expert panel.

Human milk lactoferrin (hmLF) is a glycoprotein with well-known effects on immune function. Helaina Inc. has used a glycoengineered yeast, Komatagaella phaffii, to produce recombinant human lactoferrin (Helaina rhLF, Effera™) that is structurally similar to hmLF with intended uses as a food ingredient. However, earlier FDA reviews of rhLF were withdrawn due to insufficient safety data and unanswered safety questions the experts and FDA raised about the immunogenicity/immunotoxicity risks of orally ingested rhLF. Helaina organized a panel of leading scientists to build and vet a safety study roadmap containing the studies and safety endpoints needed to address these questions. Panelists participated in a one-day virtual workshop in June 2023 and ensuing discussions through July 2023. Relevant workshop topics included physicochemical properties of LF, regulatory history of bovine LF and rhLF as food ingredients in the FDA's generally recognized as safe (GRAS) program, and synopses of publicly available studies on the immunogenicity/alloimmunization, immunotoxicology, iron homeostasis, and absorption, distribution, metabolism, and excretion of rhLF. Panelists concluded that the safety study roadmap addresses the unanswered safety questions and the intended safe use of rhLF as a food ingredient for adults and agreed on broad applications of the roadmap to assess the safety and support GRAS of other recombinant milk proteins with immunomodulatory functions.

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

A review of the safety evidence on recombinant human lactoferrin for use as a food ingredient.

Published studies on the glycosylation, absorption, distribution, metabolism, excretion, and safety outcomes of orally ingested recombinant human lactoferrin (rhLF) were reviewed in the context of unanswered safety questions, including alloimmunization, allergenicity, and immunotoxicity potential of rhLF during repeated exposure. The primary objective was to summarize current safety data of rhLF produced in transgenic host expression systems. Overall, results from animal and human studies showed that rhLF was well tolerated and safe. Animal data showed no significant toxicity-related outcomes among any safety or tolerability endpoints. The no observed adverse effect levels (NOAEL) were at the highest level tested in both iron-desaturated and -saturated forms of rhLF. Although one study reported outcomes of rhLF on immune parameters, no animal studies directly assessed immunogenicity or immunotoxicity from a safety perspective. Data from human studies were primarily reported as adverse events (AE). They showed no or fewer rhLF-related AE compared to control and no evidence of toxicity, dose-limiting toxicities, or changes in iron status in various subpopulations. However, no human studies evaluated the immunomodulatory potential of rhLF as a measure of safety. Following this review, a roadmap outlining preclinical and clinical studies with relevant safety endpoints was developed to address the unanswered safety questions.

Dose Range-Finding Toxicity Study in Rats With Recombinant Human Lactoferrin Produced in Komagataella phaffii.

The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.

Endovascular Management of A Symptomatic Giant Hepatic Artery Aneurysm.

Hepatic artery aneurysms (HAAs) are an uncommon clinical condition. Ruptured hepatic artery aneurysm carries a high incidence of mortality. Traditionally, they are treated with open surgical resection; however, endovascular aneurysm exclusion is an alternative option to open repair in select patients who have suitable anatomy. Here, we present a case of a giant hepatic artery aneurysm treated with a covered stent placement.

Reliable and Accurate Prediction of Single-Residue pKa Values through Free Energy Perturbation Calculations.

Accurate prediction of the pKa's of protein residues is crucial to many applications in biological simulation and drug discovery. Here, we present the use of free energy perturbation (FEP) calculations for the prediction of single-protein residue pKa values. We begin with an initial set of 191 residues with experimentally determined pKa values. To isolate sampling limitations from force field inaccuracies, we develop an algorithm to classify residues whose environments are significantly affected by crystal packing effects. We then report an approach to identify buried histidines that require significant sampling beyond what is achieved in typical FEP calculations. We therefore define a clean data set not requiring algorithms capable of predicting major conformational changes on which other pKa prediction methods can be tested. On this data set, we report an RMSE of 0.76 pKa units for 35 ASP residues, 0.51 pKa units for 44 GLU residues, and 0.67 pKa units for 76 HIS residues.

Insights into the molecular evolution of fertilization mechanism in land plants.

KEY MESSAGE: Comparative genetics and genomics among green plants, including algae, provide deep insights into the evolution of land plant sexual reproduction. Land plants have evolved successive changes during their conquest of the land and innovations in sexual reproduction have played a major role in their terrestrialization. Recent years have seen many revealing dissections of the molecular mechanisms of sexual reproduction and much new genomics data from the land plant lineage, including early diverging land plants, as well as algae. This new knowledge is being integrated to further understand how sexual reproduction in land plants evolved, identifying highly conserved factors and pathways, but also molecular changes that underpinned the emergence of new modes of sexual reproduction. Here, we review recent advances in the knowledge of land plant sexual reproduction from an evolutionary perspective and also revisit the evolution of angiosperm double fertilization.

Optical Sorter-Based Selection Effectively Identifies Soft Red Winter Wheat Breeding Lines With Fhb1 and Enhances FHB Resistance in Lines With and Without Fhb1.

Previous results from our lab have shown that using an optical sorter to identify Fusarium head blight (FHB) resistant breeding lines was effective at reducing the toxin deoxynivalenol (DON) and FHB-associated kernel damage. In this paper we quantified the proportion of desirable genotypes at FHB resistance QTL in lines from three selection cycles of optical sorting. Breeding lines were genotyped at loci on chromosomes 3BS, 2DL, and 5A using the following DNA markers: TaHRC, CFD233, and GWM304. TaHRC is a KASP marker for Fhb1, a major FHB resistance QTL on chromosome 3BS. CFD233 is an SSR marker for Qfhs.nau-2DL on chromosome 2DL. GWM304 is an SSR marker for Qfhs.ifa-5A on chromosome 5A. Sorter selection was effective at identifying lines that had the resistant genotype at TaHRC; in other words, the sorter was able to identify lines with resistance alleles at Fhb1. The sorter was less effective at selecting for the resistant genotype at CFD233 and GWM304. However, the proportion of lines with resistant genotypes at GWM304 did increase with additional sorter selection, just not to the degree that was observed for the Fhb1-associated marker. The proportion of lines with resistant alleles at CFD233 did not show a consistent trend. In addition to increasing the proportion of lines with Fhb1 and Qfhs.ifa-5A each selection cycle, optical sorter-based mass selection enhanced FHB resistance in different marker genotype combinations evaluated in this study. For example, there were net reductions in DON and kernel damage after two cycles of sorter selection in 15X110601S07002, a line with Fhb1, with Qfhs.nau-2DL, and with Qfhs.ifa-5A; final C3 DON levels were 63% of the resistant check (KY02C-3005-25). Kernel damage was also reduced in 15X110601A08221 a line without Fhb1, without Qfhs.nau-2DL, and without Qfhs.ifa-5A. Our findings suggest the increased resistance observed in different marker genotype combinations was conferred by QTL other than Fhb1, QFhs.nau-2DL, and Qfhs.ifa-5, and validate our previous results that the optical sorter is effective at selecting FHB-resistant breeding material.

Preoperative risk factors for massive transfusion, prolonged ventilation requirements, and mortality in patients undergoing liver transplantation.

BACKGROUND: Despite improvements in techniques and management of liver transplant patients, numerous perioperative complications that contribute to perioperative mortality remain. Models to predict intraoperative massive blood transfusion, prolonged mechanical ventilation, or in-hospital mortality in liver transplant recipients have not been identified. In this study we aim to identify preoperative factors associated with the above mentioned complications. METHODS: A retrospective observational analysis was conducted on data collected from 124 orthotopic liver transplants performed at a single institution between 2014 and 2017. A multivariable logistic regression using backwards elimination was performed for three defined outcomes (massive transfusion ≥ 10 units packed red blood cells (PRBC), prolonged mechanical ventilation > 24 h, and in-hospital mortality) to identify associations with preoperative characteristics. RESULTS: Statistically significant (P < 0.05) associations with massive transfusion ≥ 10 units PRBC were hepatocellular carcinoma and preoperative transfusion of PRBC. Significant associations with prolonged mechanical ventilation > 24 h were hepatitis C, alcoholic hepatitis, elevated preoperative ALT, and hepatorenal syndrome. Male gender was protective for requiring prolonged mechanical ventilation. End-stage renal disease and hepatitis B were significantly associated with increased in-hospital mortality. CONCLUSIONS: This study identified risk factors associated with common perioperative complications of liver transplantation. These factors may assist practitioners in risk stratification and may form the basis for further investigations of potential interventions to mitigate these risks.

Gamete Nuclear Migration in Animals and Plants.

The migration of male and female gamete nuclei to each other in the fertilized egg is a prerequisite for the blending of genetic materials and the initiation of the next generation. Interestingly, many differences have been found in the mechanism of gamete nuclear movement among animals and plants. Female to male gamete nuclear movement in animals and brown algae relies on microtubules. By contrast, in flowering plants, the male gamete nucleus is carried to the female gamete nucleus by the filamentous actin cytoskeleton. As techniques have developed from light, electron, fluorescence, immunofluorescence, and confocal microscopy to live-cell time-lapse imaging using fluorescently labeled proteins, details of these differences in gamete nuclear migration have emerged in a wide range of eukaryotes. Especially, gamete nuclear migration in flowering plants such as Arabidopsis thaliana, rice, maize, and tobacco has been further investigated, and showed high conservation of the mechanism, yet, with differences among these species. Here, with an emphasis on recent developments in flowering plants, we survey gamete nuclear migration in different eukaryotic groups and highlight the differences and similarities among species.

Relative Binding Affinity Prediction of Charge-Changing Sequence Mutations with FEP in Protein-Protein Interfaces.

Building on the substantial progress that has been made in using free energy perturbation (FEP) methods to predict the relative binding affinities of small molecule ligands to proteins, we have previously shown that results of similar quality can be obtained in predicting the effect of mutations on the binding affinity of protein-protein complexes. However, these results were restricted to mutations which did not change the net charge of the side chains due to known difficulties with modeling perturbations involving a change in charge in FEP. Various methods have been proposed to address this problem. Here we apply the co-alchemical water approach to study the efficacy of FEP calculations of charge changing mutations at the protein-protein interface for the antibody-gp120 system investigated previously and three additional complexes. We achieve an overall root mean square error of 1.2 kcal/mol on a set of 106 cases involving a change in net charge selected by a simple suitability filter using side-chain predictions and solvent accessible surface area to be relevant to a biologic optimization project. Reasonable, although less precise, results are also obtained for the 44 more challenging mutations that involve buried residues, which may in some cases require substantial reorganization of the local protein structure, which can extend beyond the scope of a typical FEP simulation. We believe that the proposed prediction protocol will be of sufficient efficiency and accuracy to guide protein engineering projects for which optimization and/or maintenance of a high degree of binding affinity is a key objective.

High resolution mapping of soil organic carbon stocks using remote sensing variables in the semi-arid rangelands of eastern Australia.

Efficient and effective modelling methods to assess soil organic carbon (SOC) stock are central in understanding the global carbon cycle and informing related land management decisions. However, mapping SOC stocks in semi-arid rangelands is challenging due to the lack of data and poor spatial coverage. The use of remote sensing data to provide an indirect measurement of SOC to inform digital soil mapping has the potential to provide more reliable and cost-effective estimates of SOC compared with field-based, direct measurement. Despite this potential, the role of remote sensing data in improving the knowledge of soil information in semi-arid rangelands has not been fully explored. This study firstly investigated the use of high spatial resolution satellite data (seasonal fractional cover data; SFC) together with elevation, lithology, climatic data and observed soil data to map the spatial distribution of SOC at two soil depths (0-5cm and 0-30cm) in semi-arid rangelands of eastern Australia. Overall, model performance statistics showed that random forest (RF) and boosted regression trees (BRT) models performed better than support vector machine (SVM). The models obtained moderate results with R2 of 0.32 for SOC stock at 0-5cm and 0.44 at 0-30cm, RMSE of 3.51MgCha-1 at 0-5cm and 9.16MgCha-1 at 0-30cm without considering SFC covariates. In contrast, by including SFC, the model accuracy for predicting SOC stock improved by 7.4-12.7% at 0-5cm, and by 2.8-5.9% at 0-30cm, highlighting the importance of including SFC to enhance the performance of the three modelling techniques. Furthermore, our models produced a more accurate and higher resolution digital SOC stock map compared with other available mapping products for the region. The data and high-resolution maps from this study can be used for future soil carbon assessment and monitoring.

The Association of Preoperative Hematocrit and Transfusion with Mortality in Patients Undergoing Elective Non-cardiac Surgery.

BACKGROUND: Patients with anemia frequently undergo surgery, as it is unclear at what threshold clinicians should consider delaying surgery for preoperative anemia optimization. The primary objective of this study was to determine whether there is an association of varying degrees of anemia and transfusion with 30-day mortality. METHODS: This is a retrospective cohort study using the American College of Surgeons National Surgical Quality Improvement Program database from 2011 to 2013. Cohorts were analyzed based on preoperative hematocrit range-patients with: (1) no anemia, (2) hematocrit ≥33% and <36% in females or <39% in males, (3) hematocrit ≥30% and <33%, (4) hematocrit ≥27% and <30%, (5) hematocrit ≥24% and <27%, and (6) hematocrit ≥21% and less than 24%. Multivariable logistic regression was used to analyze the association of anemia and transfusion with 30-day in-hospital mortality. RESULTS: The odds for 30-day mortality increased incrementally as the hematocrit ranges decreased, in which preoperative hematocrit between 21 and 24% had the highest odds for this outcome (odds ratio [OR] 6.50, p < 0.0001) compared to the reference group (no anemia). The use of transfusion increased the odds of mortality even further (OR 5.57, p < 0.0001). Among patients that received an intra-/postoperative transfusion, preoperative anemia was not predictive of mortality. CONCLUSIONS: Healthcare providers making preoperative clinical decisions for patients undergoing elective surgery should consider the degree of preoperative anemia and likelihood of perioperative transfusion.

Australian wheat production expected to decrease by the late 21st century.

Climate change threatens global wheat production and food security, including the wheat industry in Australia. Many studies have examined the impacts of changes in local climate on wheat yield per hectare, but there has been no assessment of changes in land area available for production due to changing climate. It is also unclear how total wheat production would change under future climate when autonomous adaptation options are adopted. We applied species distribution models to investigate future changes in areas climatically suitable for growing wheat in Australia. A crop model was used to assess wheat yield per hectare in these areas. Our results show that there is an overall tendency for a decrease in the areas suitable for growing wheat and a decline in the yield of the northeast Australian wheat belt. This results in reduced national wheat production although future climate change may benefit South Australia and Victoria. These projected outcomes infer that similar wheat-growing regions of the globe might also experience decreases in wheat production. Some cropping adaptation measures increase wheat yield per hectare and provide significant mitigation of the negative effects of climate change on national wheat production by 2041-2060. However, any positive effects will be insufficient to prevent a likely decline in production under a high CO2 emission scenario by 2081-2100 due to increasing losses in suitable wheat-growing areas. Therefore, additional adaptation strategies along with investment in wheat production are needed to maintain Australian agricultural production and enhance global food security. This scenario analysis provides a foundation towards understanding changes in Australia's wheat cropping systems, which will assist in developing adaptation strategies to mitigate climate change impacts on global wheat production.

Free Energy Perturbation Calculations of the Thermodynamics of Protein Side-Chain Mutations.

Protein side-chain mutation is fundamental both to natural evolutionary processes and to the engineering of protein therapeutics, which constitute an increasing fraction of important medications. Molecular simulation enables the prediction of the effects of mutation on properties such as binding affinity, secondary and tertiary structure, conformational dynamics, and thermal stability. A number of widely differing approaches have been applied to these predictions, including sequence-based algorithms, knowledge-based potential functions, and all-atom molecular mechanics calculations. Free energy perturbation theory, employing all-atom and explicit-solvent molecular dynamics simulations, is a rigorous physics-based approach for calculating thermodynamic effects of, for example, protein side-chain mutations. Over the past several years, we have initiated an investigation of the ability of our most recent free energy perturbation methodology to model the thermodynamics of protein mutation for two specific problems: protein-protein binding affinities and protein thermal stability. We highlight recent advances in the field and outline current and future challenges.

Free Energy Perturbation Calculation of Relative Binding Free Energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1.

Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been described. Here, we describe a systematic free energy perturbation (FEP) protocol and calculate the binding affinities between the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class. The protocol has been adapted from successful studies of small molecules to address the challenges associated with modeling protein-protein interactions. Specifically, we built homology models of the three antibody-gp120 complexes, extended the sampling times for large bulky residues, incorporated the modeling of glycans on the surface of gp120, and utilized continuum solvent-based loop prediction protocols to improve sampling. We present three experimental surface plasmon resonance data sets, in which antibody residues in the antibody/gp120 interface were systematically mutated to alanine. The RMS error in the large set (55 total cases) of FEP tests as compared to these experiments, 0.68kcal/mol, is near experimental accuracy, and it compares favorably with the results obtained from a simpler, empirical methodology. The correlation coefficient for the combined data set including residues with glycan contacts, R2=0.49, should be sufficient to guide the choice of residues for antibody optimization projects, assuming that this level of accuracy can be realized in prospective prediction. More generally, these results are encouraging with regard to the possibility of using an FEP approach to calculate the magnitude of protein-protein binding affinities.

Prediction of Protein-Ligand Binding Poses via a Combination of Induced Fit Docking and Metadynamics Simulations.

Ligand docking is a widely used tool for lead discovery and binding mode prediction based drug discovery. The greatest challenges in docking occur when the receptor significantly reorganizes upon small molecule binding, thereby requiring an induced fit docking (IFD) approach in which the receptor is allowed to move in order to bind to the ligand optimally. IFD methods have had some success but suffer from a lack of reliability. Complementing IFD with all-atom molecular dynamics (MD) is a straightforward solution in principle but not in practice due to the severe time scale limitations of MD. Here we introduce a metadynamics plus IFD strategy for accurate and reliable prediction of the structures of protein-ligand complexes at a practically useful computational cost. Our strategy allows treating this problem in full atomistic detail and in a computationally efficient manner and enhances the predictive power of IFD methods. We significantly increase the accuracy of the underlying IFD protocol across a large data set comprising 42 different ligand-receptor systems. We expect this approach to be of significant value in computationally driven drug design.