Biochemical and clinical studies of putative allergens to assess what distinguishes them from other non-allergenic proteins in the same family.

Many protein families have numerous members listed in databases as allergens; however, some allergen database entries, herein called "orphan allergens", are members of large families of which all other members are not allergens. These orphan allergens provide an opportunity to assess whether specific structural features render a protein allergenic. Three orphan allergens [Cladosporium herbarum aldehyde dehydrogenase (ChALDH), Alternaria alternata ALDH (AaALDH), and C. herbarum mannitol dehydrogenase (ChMDH)] were recombinantly produced and purified for structure characterization and for clinical skin prick testing (SPT) in mold allergic participants. Examination of the X-ray crystal structures of ChALDH and ChMDH and a homology structure model of AaALDH did not identify any discernable epitopes that distinguish these putative orphan allergens from their non-allergenic protein relatives. SPT results were aligned with ChMDH being an allergen, 53% of the participants were SPT (+). AaALDH did not elicit SPT reactivity above control proteins not in allergen databases (i.e., Psedomonas syringae indole-3-acetaldehyde dehydrogenase and Zea mays ALDH). Although published results showed consequential human IgE reactivity with ChALDH, no SPT reactivity was observed in this study. With only one of these three orphan allergens, ChMDH, eliciting SPT(+) reactions consistent with the protein being included in allergen databases, this underscores the complicated nature of how bioinformatics is used to assess the potential allergenicity of food proteins that could be newly added to human diets and, when needed, the subsequent clinical testing of that bioinformatic assessment.Trial registration number and date of registration AAC-2017-0467, approved as WIRB protocol #20172536 on 07DEC2017 by WIRB-Copernicus (OHRP/FDA Registration #: IRB00000533, organization #: IORG0000432).

Outcomes of a home telemonitoring program for SARS-CoV-2 viral infection at a large academic medical center.

INTRODUCTION: Telemedicine serves as a viable option during the COVID-19 pandemic to provide in-home care, maintain home isolation precautions, reduce unnecessary healthcare exposures, and de-burden hospitals. METHODS: We created a novel telemedicine program to closely monitor patients infected with SARS-CoV-2 (COVID-19) at home. Adult patients with COVID-19 were enrolled in the program at the time of documented infection. Patients were followed by a team of providers via telephone or video visits at frequent intervals until resolution of their acute illness. Additionally, patients were stratified into high-risk and low-risk categories based on demographics and underlying comorbidities. The primary outcome was hospitalization after enrollment in the home monitoring program, including 30 days after discharge from the program. RESULTS: Over a 3.5-month period, 1128 patients met criteria for enrollment in the home monitoring program. 30.7% were risk stratified as high risk for poor outcomes based on their comorbidities and age. Of the 1128 patients, 6.2% required hospitalization and 1.2% required ICU admission during the outcome period. Hospitalization was more frequent in patients identified as high risk (14.2% vs 2.7%, P < 0.001). DISCUSSION: Enrollment in a home monitoring program appears to be an effective and sustainable modality for the ambulatory management of COVID-19.

An Initiative to Improve 30-Day Readmission Rates Using a Transitions-of-Care Clinic Among a Mixed Urban and Rural Veteran Population.

BACKGROUND/OBJECTIVE: Hospital readmissions in the United States, especially in patients at high-risk, cost more than $17 billion annually. Although care transitions is an important area of research, data are limited regarding its efficacy, especially among rural patients. In this study, we describe a novel transitions-of-care clinic (TOCC) to reduce 30-day readmissions in a Veterans Health Administration setting that serves a high proportion of rural veterans. METHODS: In this quality improvement initiative we conducted a pre-post study evaluating clinical outcomes in adult patients at high risk for 30-day readmission (Care Assessment Needs score > 85) discharged from the Iowa City Veterans Affairs (ICVA) Health Care System from 2017 to 2020. The ICVA serves 184,000 veterans across 50 counties in eastern Iowa, western Illinois, and northern Missouri, with more than 60% of these patients residing in rural areas. We implemented a multidisciplinary TOCC to provide in-person or virtual follow-up to high-risk veterans after hospital discharge. The main purpose of this study was to assess how TOCC follow-up impacted the monthly 30-day patient readmission rate. RESULTS: The TOCC resulted in a 19.2% relative reduction in 30-day readmission rates in the 12-month postimplementation period compared to the preimplementation period (9.2% vs 11.4%, P = .04). Virtual visits were more popular than in-person visits among both urban and rural veterans. There was no difference in outcomes between these two follow-up options, and both groups had reduced readmission rates compared to non-TOCC follow-up. CONCLUSIONS: A multidisciplinary TOCC within the ICVA featuring both virtual and in-person visits reduced the 30-day readmission rate. This reduction was particularly notable among patients with congestive heart failure.

Evaluation of the use of untargeted metabolomics in the safety assessment of genetically modified crops.

BACKGROUND: The safety assessment of foods and feeds from genetically modified (GM) crops includes the comparison of key characteristics, such as crop composition, agronomic phenotype and observations from animal feeding studies compared to conventional counterpart varieties that have a history of safe consumption, often including a near isogenic variety. The comparative compositional analysis of GM crops has been based on targeted, validated, quantitative analytical methods for the key food and feed nutrients and antinutrients for each crop, as identified by Organization of Economic Co-operation and Development (OCED). As technologies for untargeted metabolomic methods have evolved, proposals have emerged for their use to complement or replace targeted compositional analytical methods in regulatory risk assessments of GM crops to increase the number of analyzed metabolites. AIM OF REVIEW: The technical opportunities, challenges and strategies of including untargeted metabolomics analysis in the comparative safety assessment of GM crops are reviewed. The results from metabolomics studies of GM and conventional crops published over the last eight years provide context to enable the discussion of whether metabolomics can materially improve the risk assessment of food and feed from GM crops beyond that possible by the Codex-defined practices used worldwide for more than 25 years. KEY SCIENTIFIC CONCEPTS OF REVIEW: Published studies to date show that environmental and genetic factors affect plant metabolomics profiles. In contrast, the plant biotechnology process used to make GM crops has little, if any consequence, unless the inserted GM trait is intended to alter food or feed composition. The nutritional value and safety of food and feed from GM crops is well informed by the quantitative, validated compositional methods for list of key analytes defined by crop-specific OECD consensus documents. Untargeted metabolic profiling has yet to provide data that better informs the safety assessment of GM crops than the already rigorous Codex-defined quantitative comparative assessment. Furthermore, technical challenges limit the implementation of untargeted metabolomics for regulatory purposes: no single extraction method or analytical technique captures the complete plant metabolome; a large percentage of metabolites features are unknown, requiring additional research to understand if differences for such unknowns affect food/feed safety; and standardized methods are needed to provide reproducible data over time and laboratories.

Plant Genome Editing and the Relevance of Off-Target Changes.

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.

Evaluating maize phenotypic variance, heritability, and yield relationships at multiple biological scales across agronomically relevant environments.

A challenge to improve an integrative phenotype, like yield, is the interaction between the broad range of possible molecular and physiological traits that contribute to yield and the multitude of potential environmental conditions in which they are expressed. This study collected data on 31 phenotypic traits, 83 annotated metabolites, and nearly 22,000 transcripts from a set of 57 diverse, commercially relevant maize hybrids across three years in central U.S. Corn Belt environments. Although variability in characteristics created a complex picture of how traits interact produce yield, phenotypic traits and gene expression were more consistent across environments, while metabolite levels showed low repeatability. Phenology traits, such as green leaf number and grain moisture and whole plant nitrogen content showed the most consistent correlation with yield. A machine learning predictive analysis of phenotypic traits revealed that ear traits, phenology, and root traits were most important to predicting yield. Analysis suggested little correlation between biomass traits and yield, suggesting there is more of a sink limitation to yield under the conditions studied here. This work suggests that continued improvement of maize yields requires a strong understanding of baseline variation of plant characteristics across commercially-relevant germplasm to drive strategies for consistently improving yield.

Effect of common processing of soybeans on the enzymatic activity and detectability of the protein, Dicamba Mono-Oxygenase (DMO), introduced into dicamba-tolerant MON 87708.

Assessing the safety of genetically engineered crops includes evaluating the risk (hazard and exposure) of consuming their newly expressed proteins. The dicamba monooxygenase (DMO) protein, introduced into soybeans to confer tolerance (DT) to dicamba herbicide, was previously characterized and identified to pose no food or feed safety hazards. Most agricultural commodities (e.g., soybeans, maize) enter the food supply after processing methods that can include exposure to high temperatures, harsh solvents or pH extremes that can adversely impact the structure and function of proteins. To understand the likelihood of exposure to DMO in foods from DT soy, enzymatically active and/or immunodetectable forms of DMO were measured in pilot-scale productions of two soy foods (soymilk and tofu), and eight processed fractions (full fat flour, inactivated full fat flour, defatted flour, toasted meal, protein isolate, protein concentrate, crude lecithin, and refined, bleached and deodorized oil). Western blot analysis detected DMO in tofu and in five of the eight processed fractions. DMO activity was not detected in either soymilk or tofu, nor in six of the eight processed fractions. Therefore, many commercial soy processing methods can denature and/or degrade introduced proteins, like DMO. Although the DMO protein has shown no evidence of hazard, this study demonstrates that processing further reduces any food or feed risk by limiting dietary exposure to intact DMO protein.

Safety of the Bacillus thuringiensis-derived Cry1A.105 protein: Evidence that domain exchange preserves mode of action and safety.

The lepidopteran-active Cry1A.105 protein is a chimeric three-domain insecticidal toxin with distinct structural domains derived from the naturally occurring Cry1Ab, Cry1Ac and Cry1F proteins from the soil bacterium Bacillus thuringiensis (Bt). The X-ray crystal structure of the Cry1A.105 tryptic core at 3.0 Šresolution demonstrates its high structural similarity to the tryptic core of Cry1Ac. Bioinformatics analyses demonstrate that Cry1A.105 has no significant amino acid sequence similarity to known allergens or mammalian toxins, which is the same conclusion reached for its component domains. Like its intact donor proteins, Cry1A.105 was heat labile at temperatures ≥75 °C and degraded upon exposure to gastrointestinal proteases. No adverse effects were observed in mice when Cry1A.105 was dosed orally at 2451 mg/kg body weight. Therefore, the weight of evidence supports that Cry1A.105 is safe for human and animal consumption. These results support the conclusion that the safety of a chimeric protein for human or animal consumption can be evaluated in the context of the safety of its donor proteins.

The Virtual Hospitalist: A Single-Site Implementation Bringing Hospitalist Coverage to Critical Access Hospitals.

BACKGROUND: On-site hospitalist care can improve patient care, but it is economically infeasible for small critical access hospitals (CAHs). A telemedicine "virtual hospitalist" may expand CAH capabilities at a fractional cost of an on"site provider. OBJECTIVE: To evaluate the impact of a virtual hospitalist on transfers from a CAH to outside hospitals. DESIGN, SETTING, PATIENTS: A 6-month pilot program providing "virtual hospitalist" coverage to patients at a CAH in rural Iowa. MEASUREMENTS: The primary outcome was the rate of outside transfers from the CAH Emergency Department (ED). The secondary outcomes included transfer from either the ED or the inpatient wards, daily census, length of stay, transfers after admission, virtual hospitalist time commitment, and patient and staff satisfaction. The preceding 24-week baseline was compared with 24 weeks after implementation, excluding a 2-week transition period. RESULTS: At baseline, there were 947 ED visits and 176 combined inpatient and observation encounters, compared to 930 and 176 after implementation, respectively. Outside transfers from the ED decreased from 16.6% to 10.5% (157/947 to 98/930, P < .001), and transfers at any time decreased from 17.3% to 11.9% (164/947 to 111/930, P < .001). Daily census, length of stay, and transfers after admission were unchanged. Time commitment for a virtual hospitalist was 35 minutes per patient per day. The intervention was well received by the CAH staff and patients. CONCLUSIONS: The virtual hospitalist model increased the percentage of ED patients who could safely receive their care locally. A single virtual hospitalist may be able to cover multiple CAHs simultaneously. FUNDING: Development of this project was funded through the University of Iowa Hospitalist group and the Signal Center for Health Innovations at UI Health Ventures. Virtual hospitalist clinical time was paid for by the CAH on a fractional basis of a traditional hospitalist based on projected patient volumes through analysis of baseline data. Patients were not directly billed for virtual hospitalist service but were charged for the services provided by CAH providers.

Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill).

The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure.

BACKGROUND: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. AIM: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. METHODS: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. RESULTS: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. CONCLUSIONS: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

Effect of acute nitrate ingestion on V̇O2 response at different exercise intensity domains.

While nitrate supplementation influences oxygen uptake (V̇O2) response to exercise, this effect may be intensity dependent. The purpose of this study was to investigate the effect of acute nitrate supplementation on V̇O2 response during different exercise intensity domains in humans. Eleven men ingested 10 mg·kg-1 body mass (8.76 ± 1.35 mmol) of sodium nitrate or sodium chloride (placebo) 2.5 h before cycling at moderate (90% of gas exchange threshold; GET), heavy (GET + 40% of the difference between GET and peak oxygen uptake (V̇O2peak), Δ 40) or severe (GET + 80% of the difference between GET and V̇O2peak, Δ 80) exercise intensities. Volunteers performed exercise for 10 min (moderate), 15 min (heavy) or until exhaustion (severe). Acute nitrate supplementation had no effect on any V̇O2 response parameters during moderate and severe exercise intensities. However, the V̇O2 slow amplitude (nitrate: 0.93 ± 0.36 L·min-1 vs. placebo: 1.13 ± 0.59 L·min-1, p = 0.04) and V̇O2 slow gain (nitrate: 5.81 ± 2.37 mL·min-1·W-1 vs. placebo: 7.09 ± 3.67 mL·min-1·W-1, p = 0.04) were significantly lower in nitrate than in placebo during the heavy exercise intensity. There was no effect of nitrate on plasma lactate during any exercise intensity (p > 0.05). Time to exhaustion during the severe exercise intensity was also not affected by nitrate (p > 0.05). In conclusion, acute nitrate supplementation reduced the slow component of V̇O2 only when performing heavy-intensity exercise, which might indicate an intensity-dependent effect of nitrate on V̇O2 response.

Assessment of Natural Variability of Maize Lipid Transfer Protein Using a Validated Sandwich ELISA.

Lipid transfer protein (LTP) is the main causative agent for rare food allergic reactions to maize. This paper describes a new, validated ELISA that accurately measures maize LTP concentrations from 0.2 to 6.4 ng/mL. The levels of LTP ranged from 171 to 865 µg/g of grain, a 5.1-fold difference, across a set of 49 samples of maize B73 hybrids derived from the Nested Association Mapping (NAM) founder lines and a diverse collection of landrace accessions from North and South America. A second set of 107 unique samples from 18 commercial hybrids grown over two years across 10 U.S. states showed a comparable range of LTP level (212-751 µg/g of grain). Statistical analysis showed that genetic and environmental factors contributed 63 and 6%, respectively, to the variance in LTP levels. Therefore, the natural variation of maize LTP is up to 5-fold different across a diverse collection of varieties that have a history of safe cultivation and consumption.

Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.

The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins.

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

Natural Variability of Allergen Levels in Conventional Soybeans: Assessing Variation across North and South America from Five Production Years.

Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis.

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.