Assessing the Link Between Survey Interview Method and Survey Outcomes: Evidence from the CPS and the COVID-19 Pandemic.

The COVID-19 pandemic significantly affected data collection for the nation's primary source of household-level labor force data, the Current Population Survey (CPS). In the first four months of the pandemic period (March-June 2020) the average month-over-month nonresponse rate increased by 58 percent, while the size of newly entering cohorts declined by 37 percent relative to the prior 15 months. Together, these factors reduced the overall sample size of the CPS by around 16 percent. We hypothesize that these changes, and significant associated shifts in the demographic composition of the sample, were caused by the cessation of in-person interviewing. Geographic variation in nonresponse over this period does not appear related to variation in COVID case rates across metro areas or states. Using this change in interview method as a natural experiment, we compare labor market outcomes of those who entered the survey pre- and post-COVID pandemic and find that the change in how individuals were recruited into the survey affected estimates of unemployment and labor force participation. In an exercise generating a counterfactual group of "missing" respondents, we estimate that, between April and August of 2020, the average unemployment rate was 0.5 to 0.7 percentage points higher, and the labor force participation rate was 0.4 to 0.8 percentage points lower than estimates using the actual sample of respondents. One implication of these results is that web-based surveys, which are increasingly relied on in empirical labor market studies, may fail to reach important subpopulations of the labor market and that reweighting is unlikely to address the selection on outcomes we document.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Measurement of endogenous allergens in genetically modified soybeans--short communication.

The measurement of endogenous allergens is required by the European Commission (EC) as part of the compositional analysis for GM products from host plants that are common causes of food allergy, such as soybean (EC Implementing Regulation No. 503/2013). In each case, the EC Implementing Regulation indicates that analysis be conducted on identified allergens as specified in the Organization of Economic Cooperation and Development (OECD) consensus documents on compositional considerations for new plant varieties. This communication discusses the methods available to measure endogenous allergens as well as the endogenous soybean allergens that should be analyzed. It is suggested herein that in conjunction with the 2012 OECD consensus document on soybean, any list of soybean allergens should be based on clinically relevant data among publicly available allergen databases and peer-reviewed scientific publications, and the ability to measure the identified allergen. Based on a detailed analysis of the scientific literature, the following key points are recommended: (1) the acceptance of serum-free, quantitative analytical method data as an alternative to traditional IgE reactivity qualitative or semi-quantitative data for evaluation of endogenous soybean allergen content; (2) eight of the 15 potential allergens listed in the OECD soybean consensus document (Gly m 3, Gly m 4, Gly m Bd28K, Gly m Bd30K, Gly m 5, Gly m 6, Gly m 8, and Kunitz trypsin inhibitor) have both appropriate supporting clinical data and sufficient sequence information to be evaluated in comparative endogenous soybean allergen studies; and (3) the remaining seven proteins (Gly m 1, Gly m 2, unknown 50kDa protein, unknown 39kDa protein, P-22-25, lipoxygenase and lectin) lack sufficient data for clear classification as confirmed allergens and/or available sequence information and should not be currently included in the measurement of endogenous soybean allergens in the compositional analysis for the EU.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.

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.

Development of a Sandwich ELISA for Quantification of Gly m 4, a Soybean Allergen.

Gly m 4 is a key soybean allergen that causes allergic symptoms in the skin, gastrointestinal tract, or respiratory tract of sensitive individuals. To understand naturally variable levels of Gly m 4 among conventional soybean varieties, a sandwich ELISA was developed and validated using a mouse anti-Gly m 4 monoclonal antibody and a goat anti-Gly m 4 polyclonal antibody as capture and detection antibodies, respectively. The ELISA shows high specificity to Gly m 4 without any cross-reactivity to other soybean proteins and has a quantification range of 7.8-250 ng/mL using an Escherichia coli-produced recombinant Gly m 4, with 2.1 ng/mL being the limit of detection. Within the quantification range, the coefficients of variation of the intra-assay and interassay precision are less than 5 and 12%, respectively. Moreover, extraction efficiency and dilutional parallelism experiments were completed to demonstrate the assay is accurate. The validated assay was used to quantify Gly m 4 levels in 128 soybean samples from 24 conventional soybean varieties grown at 8 distinct geographical locations. There was a 13-fold difference between the least and greatest amounts of Gly m 4 concentrations among the samples, and the results demonstrate that the most significant sources of variability in Gly m 4 levels in the conventional varieties were related to location and variety.

The food and environmental safety of Bt crops.

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

Dual function of novel pollen coat (surface) proteins: IgE-binding capacity and proteolytic activity disrupting the airway epithelial barrier.

BACKGROUND: The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., "de-fatted"), and, as a result, their involvement in allergy has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. CONCLUSIONS/SIGNIFICANCE: Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is warranted and could potentially lead to the development of improved diagnostic and therapeutic tools.

Quantification of transgene-derived double-stranded RNA in plants using the QuantiGene nucleic acid detection platform.

The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over ∼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples.

A new role for the Arabidopsis AP2 transcription factor, LEAFY PETIOLE, in gibberellin-induced germination is revealed by the misexpression of a homologous gene, SOB2/DRN-LIKE.

Gibberellic acid (GA) promotes germination, stem/hypocotyl elongation, and leaf expansion during seedling development. Using activation-tagging mutagenesis, we identified a mutation, sob2-D (for suppressor of phytochromeB-4 [phyB-4]#2 dominant), which suppresses the long-hypocotyl phenotype of a phyB missense allele, phyB-4. This mutant phenotype is caused by the overexpression of an APETALA2 transcription factor, SOB2, also called DRN-like. SOB2/DRN-like transcript is not detectable in wild-type seedling or adult tissues via RT-PCR analysis, suggesting that SOB2/DRN-like may not be involved in seedling development under normal conditions. Adult sob2-D phyB-4 plants have curled leaves and club-like siliques, resembling plants that overexpress a closely related gene, LEAFY PETIOLE (LEP). Hypocotyls of a LEP-null allele, lep-1, are shorter in the light and dark, suggesting LEP involvement in seedling development. This aberrant hypocotyl phenotype is due at least in part to a delay in germination. In addition, lep-1 is less responsive to GA and more sensitive to the GA biosynthesis inhibitor paclobutrazol, indicating that LEP is a positive regulator of GA-induced germination. RT-PCR shows that LEP transcript accumulates in wild-type seeds during imbibition and germination, and the transcript levels of REPRESSOR OF ga1-3-LIKE2 (RGL2), a negative regulator of GA signaling during germination, is unaffected in lep-1. These results suggest LEP is a positive regulator of GA-induced germination acting independently of RGL2. An alternative model places LEP downstream of RGL2 in the GA-signaling cascade.

The Dof transcription factor OBP3 modulates phytochrome and cryptochrome signaling in Arabidopsis.

Plants perceive subtle changes in light quality and quantity through a set of photoreceptors, including phytochromes and cryptochromes. Upon perception, these photoreceptors initiate signal transduction pathways leading to photomorphogenic changes in development. Using activation-tagging mutagenesis to identify novel light-signaling components, we have isolated a gain-of-function mutant, sob1-D (suppressor of phytochrome B-4 [phyB-4] dominant), which suppresses the long-hypocotyl phenotype of the phyB missense allele, phyB-4. The sob1-D mutant phenotype is caused by the overexpression of a Dof (DNA binding with one finger) transcription factor, OBF4 Binding Protein 3 (OBP3). A translational fusion between OBP3 and green fluorescent protein is nuclear localized in onion (Allium cepa) cells. Tissue-specific accumulation of an OBP3:OBP3-beta-glucuronidase translational fusion is regulated by light in Arabidopsis thaliana. Hypocotyls of transgenic lines with reduced OBP3 expression are less responsive to red light. This aberrant phenotype in red light requires functional phyB, suggesting that OBP3 is a positive regulator of phyB-mediated inhibition of hypocotyl elongation. Furthermore, these partial-loss-of-function lines have larger cotyledons. This light-dependent cotyledon phenotype is most dramatic in blue light and requires functional cryptochrome 1 (cry1), indicating that OBP3 is a negative regulator of cry1-mediated cotyledon expansion. These results suggest a model where OBP3 is a component in both phyB and cry1 signaling pathways, acting as a positive and negative regulator, respectively. An alternate, though not mutually exclusive, model places OBP3 as a general inhibitor of tissue expansion with phyB and cry1, differentially modulating OBP3's role in this response.

BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms.

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.