CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/ß-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

Quantitative assessment of wheat quality using near-infrared spectroscopy: A comprehensive review.

Wheat is one of the most widely cultivated crops throughout the world. A great need exists for wheat quality assessment for breeding, processing, and products production purposes. Near-infrared spectroscopy (NIRS) is a rapid, low-cost, simple, and nondestructive assessment method. Many advanced studies associated with NIRS for wheat quality assessment have been published recently, either introducing new chemometrics or attempting new assessment parameters to improve model robustness and accuracy. This review provides a comprehensive overview of NIRS methodology including its principle, spectra pretreatments, spectral wavelength selection, outlier disposal, dataset division, regression methods, and model evaluation. More importantly, the applications of NIRS in the determination of analytical parameters, rheological parameters, and end product quality of wheat are summarized. Although NIRS showed great potential in the quantitative determination of analytical parameters, there are still challenges in model robustness and accuracy in determining rheological parameters and end product quality for wheat products. Future model development needs to incorporate larger databases, integrate different spectroscopic techniques, and introduce cutting-edge chemometrics methods. In addition, calibration based on external factors should be considered to improve the predicted results of the model. The NIRS application in micronutrients needs to be extended. Last, the idea of combining standard product sensory attributes and spectra for model development deserves further study.

A comprehensive review of wheat phytochemicals: From farm to fork and beyond.

The health benefits of whole wheat consumption can be partially attributed to wheat's phytochemicals, including phenolic acids, flavonoids, alkylresorcinols, carotenoids, phytosterols, tocopherols, and tocotrienols. It is of increasing interest to produce whole wheat products that are rich in bioactive phytochemicals. This review provides the fundamentals of the chemistry, extraction, and occurrence of wheat phytochemicals and includes critical discussion of several long-lasting issues: (1) the commonly used nomenclature on distribution of wheat phenolic acids, namely, soluble-free, soluble-conjugated, and insoluble-bound phenolic acids; (2) different extraction protocols for wheat phytochemicals; and (3) the chemistry and application of in vitro antioxidant assays. This review further discusses recent advances on the effects of genotypes, environments, field management, and processing techniques including ultrafine grinding, germination, fermentation, enzymatic treatments, thermal treatments, and food processing. These results need to be interpreted with care due to varied sample preparation protocols and limitations of in vitro assays. The bioaccessibility, bioavailability, metabolism, and potential health benefits of wheat phytochemicals are also reviewed. This comprehensive and critical review will benefit scientific researchers in the field of bioactive compounds of cereal grains and also those in the cereal food industry to produce high-quality functional foods.

Analysis of corn and sorghum flour mixtures using laser-induced breakdown spectroscopy.

BACKGROUND: In a world constantly challenged by climate change, corn and sorghum are two important grains because of their high productivity and adaptability, and their multifunctional use for different purposes such as human food, animal feed, and feedstock for many industrial products and biofuels. Corn and sorghum can be utilized interchangeably in certain applications; one grain may be preferred over the other for several reasons. The determination of the composition corn and sorghum flour mixtures may be necessary for economic, regulatory, environmental, functional, or nutritional reasons. RESULTS: Laser-induced breakdown spectroscopy (LIBS) in combination with chemometrics, was used for the classification of flour samples based on the LIBS spectra of flour types and mixtures using partial least squares discriminant analysis (PLS-DA) and the determination of the sorghum ratio in sorghum / corn flour mixture based on their elemental composition using partial least squares (PLS) regression. Laser-induced breakdown spectroscopy with PLS-DA successfully identified the samples as either pure corn, pure sorghum, or corn-sorghum mixtures. Moreover, the addition of various levels of sorghum flour to mixtures of corn-sorghum flour were used for PLS analysis. The coefficient of determination values of calibration and validation PLS models are 0.979 and 0.965, respectively. The limit of detection of the PLS models is 4.36%. CONCLUSION: This study offers a rapid method for the determination of the sorghum level in corn-sorghum flour mixtures and the classification of flour samples with high accuracy, a short analysis time, and no requirement for time-consuming sample preparation procedures. © 2020 Society of Chemical Industry.

Effects of stress on functional connectivity during problem solving.

AIM: Our purpose was to examine how stress affects functional connectivity (FC) in language processing regions of the brain during a verbal problem solving task associated with creativity. We additionally explored how gender and the presence of the stress-susceptible short allele of the serotonin transporter gene polymorphism influenced this effect. METHODS: Forty-five healthy participants (Mean age: 19.6 â€‹± â€‹1.6 years; 28 females) were recruited to be a part of this study and genotyped to determine the presence or absence of at least one copy of the short (S) allele of the serotonin transporter gene, which is associated with greater susceptibility to stress. The participants underwent functional magnetic resonance imaging in two separate sessions (stress and no stress control). One session utilized a modified version of the Montreal Imaging Stress Test (MIST) to induce stress while the other session consisted of a no stress control task. The MIST and control tasks were interleaved with task blocks during which the participants performed the compound remote associates task, a convergent task that engages divergent thinking, which is a critical component of creativity. We examined the relationship between stress effects on performance and effects on connectivity of language processing regions activated during this task. RESULTS: There was no main effect of stress on functional connectivity for individual ROI pairs. However, in the examination of whether stress effects on performance related to effects on connectivity, changes in middle temporal gyrus connectivity with stress correlated positively with changes in solution latency for individuals with the S allele, but anti-correlated for those with only the L allele. A trend towards a gene â€‹× â€‹stress interaction on solution latency was also observed. DISCUSSION: Results from the study suggest that genetic susceptibility to stress, such as the presence of the S allele, affects neural correlates of performance on tasks related to verbal problem solving, as indicated by connectivity of the middle temporal gyrus. Future work will need to determine whether connectivity of the middle temporal gyrus serves as a marker for the effect of stress susceptibility on cognition, extending into stress susceptible patient populations.

Ipsilateral rotational double-barrel fibula autograft for limb salvage in a pediatric patient with lower extremity intramedullary osteosarcoma: A case report.

Osteosarcoma is a relatively uncommon diagnosis that often requires limb salvage techniques in young patients. Due to the potential long-life expectancy of these patients, techniques which avoid prostheses are preferred to limit the need for future revision surgeries. This case report presents a 9-year-old male diagnosed with intramedullary well-differentiated osteosarcoma of the tibial diaphysis. This limb salvage procedure utilized a rotational ipsilateral vascularized double barrel fibula autograft measuring a total of 24.5 cm when harvested but osteotomized to lengths of 11 and 12.5 cm limbs to reconstruct an 11 cm tibial shaft defect. Anterior chamfer cuts were performed on one limb allowing it to be inserted intramedullary while the second limb was then placed with an inlay technique posterolaterally and then compressed with a carbon fiber plate. The patient recovered well initially postoperatively but was taken back for revision of the carbon fiber plate 10 weeks postoperatively secondary to asymptomatic loosening of the proximal screws. The patient subsequently healed and experienced full graft incorporation with expected hypertrophy of the graft. At most recent follow-up at 19 months, the patient reported no pain and was utilizing the extremity in a normal fashion with no activity limitations. In this report, we present a case of the use of an ipsilateral rotational double-barrel fibula autograft for limb salvage in a pediatric patient with lower extremity intramedullary osteosarcoma.

Influence of Serotonin Transporter SLC6A4 Genotype on the Effect of Psychosocial Stress on Cognitive Performance: An Exploratory Pilot Study.

BACKGROUND AND OBJECTIVE: Previous research has shown an effect of various psychosocial stressors on unconstrained cognitive flexibility, such as searching through a large set of potential solutions in the lexical-semantic network during verbal problem-solving. Functional magnetic resonance imaging has shown that the presence of the short (S) allele (lacking a 43-base pair repeat) of the promoter region of the gene (SLC6A4) encoding the serotonin transporter (5-HTT) protein is associated with a greater amygdalar response to emotional stimuli and a greater response to stressors. Therefore, we hypothesized that the presence of the S-allele is associated with greater stress-associated impairment in performance on an unconstrained cognitive flexibility task, anagrams. METHODS: In this exploratory pilot study, 28 healthy young adults were genotyped for long (L)-allele versus S-allele promoter region polymorphism of the 5-HTT gene, SLC6A4. Participants solved anagrams during the Trier Social Stress Test, which included public speaking and mental arithmetic stressors. We compared the participants' cognitive response to stress across genotypes. RESULTS: A Gene×Stress interaction effect was observed in this small sample. Comparisons revealed that participants with at least one S-allele performed worse during the Stress condition. CONCLUSIONS: Genetic susceptibility to stress conferred by SLC6A4 appeared to modulate unconstrained cognitive flexibility during psychosocial stress in this exploratory sample. If confirmed, this finding may have implications for conditions associated with increased stress response, including performance anxiety and cocaine withdrawal. Future work is needed both to confirm our findings with a larger sample and to explore the mechanisms of this proposed effect.

Using Anilines as Masked Cross-Coupling Partners: Design of a Telescoped Three-Step Flow Diazotization, Iododediazotization, Cross-Coupling Process.

The conversion of commercially available anilines into biaryl and biarylacetylene products was realized by using a telescoped, three-reactor flow diazotization/iododediazotization/cross-coupling process. The segmented flow stream created by off-gassing during the Sandmeyer sequence was restored to a continuous column of reaction solution by using a specially designed continuous-flow unit controlled by custom software created in-house. The resultant aryl iodide was then combined with a stream of cross-coupling solution that fed into the final reactor. The system proved versatile as modifications to the diazotization/iododediazotization sequence could be made rapidly to account for any substrate specificity (e.g., solubility problems), leading to a wide substrate scope of Suzuki-Miyaura and Sonogashira cross-coupled products.

Multicomponent, flow diazotization/Mizoroki-Heck coupling protocol: dispelling myths about working with diazonium salts.

A single pass flow diazotization/Mizoroki-Heck protocol has been developed for the production of cinnimoyl and styryl products. The factors that govern aryl diazonium salt stability have been examined in detail leading to the development of a MeOH/DMF co-solvent system in which the diazonium salts can be generated in the presence of all other reaction components and then coupled selectively to give the desired products. Finally the key role of the reaction quench for flow reactions has been demonstrated.

Effect of high molecular weight glutenin subunit composition in common wheat on dough properties and steamed bread quality.

BACKGROUND: Steamed bread is a popular staple food in Asia with different flour quality requirements from pan bread. Little is known about how glutenin characteristics affect steamed bread quality. This work investigated how deletions of high-molecular-weight glutenin subunits (HMW-GS) influence gluten properties and Chinese steamed bread quality using 16 wheat lines grown in Texas. RESULTS: Although similar in protein content (134-140 mg g⁻¹), gluten composition and dough properties differed widely among the lines. Compared with non-deletion lines, deletion lines had lower (P < 0.05) unextractable polymeric protein (294 vs 470 mg g⁻¹), HMW-GS/low-molecular-weight glutenin subunit ratio (0.25 vs 0.41), dough force to extend (0.16 vs 0.44 N) and mixing peak time (2.03 vs 4.52 min). Deletion lines with HMW-GS composition of 2*/17+_/5+_ and 2*/17+_/2+12 showed moderate gluten strength (mixing peak time, 1.96-2.94 min; force to extend, 0.18-0.23 N) and high dough extensibility (106-129 mm). These lines also produced good steamed bread quality (score, 60.8-65.0) with good elasticity and crumb structure. CONCLUSION: Deletion at Glu-B1y and/or Glu-D1y loci in high-strength hard wheat produced good dough properties for steamed bread. This suggests that wheat functionality for steamed bread can be improved by manipulating HMW-GS composition.

Enhancing Gluten Network Formation and Bread-Making Performance of Wheat Flour Using Wheat Bran Aqueous Extract.

Wheat bran possesses diverse nutritional and functional properties. In this study, wheat bran aqueous extract (WBE) was produced and thoroughly characterized as a functional ingredient and improver for bakery application. The WBE contained 50.3% total carbohydrate, 24.5% protein, 13.0% ash, 6.7% soluble fiber, 2.9% insoluble fiber, and 0.5% ß-glucan. Notably, adding 7.5% WBE significantly increased the bread-specific volume to 4.84 cm3/g, compared with the control of 4.18 cm3/g. Adding WBE also resulted in a remarkable improvement in dough properties. The WBE-enriched dough showed increased peak, setback, breakdown, and final viscosities, along with higher storage and loss modulus. Scanning electron microscopy analysis further revealed that the WBE promoted the aggregation of protein and starch within the dough. The extractable gliadin to glutenin ratio increased with 5 and 7.5% WBE additions, compared with the control and 2.5% WBE addition. WBE did not significantly alter the starch gelatinization temperature or dough extension properties. These findings demonstrate that the inclusion of WBE in wheat flour is a promising approach for producing high-quality bread that is enriched with dietary fiber and protein.

Fatal Food: Silver-Coated Grain Particles Display Larvicidal Activity in Culex quinquefasciatus.

Mosquitoes pose a significant risk to millions of people worldwide since they can transmit pathogens. Current methods to control mosquito populations include the use of synthetic pesticides. Nanotechnology may be a solution to develop new mosquito control. However, one barrier to expanding the impact of nanomaterials is the ability to mass-produce the particles. Here, we report a novel hybrid particle synthesis combining micro- and nanoparticles using the coprecipitation technique with the potential for mass production. These particles may have applications as a mosquito larvacide. The particles reported here were designed using a microparticle zein polymer as the core and a nanoparticle silver as the active ingredient. The hybrid NPs reported here targeted a late-stage mosquito larvae and that resulted in a high larval mortality concentration (1.0 ppm, LC90) and suppression of pupal emergence at 0.1 ppm. This research demonstrates the efficacy of a plant-based core with a metal-based AI coating (AgNPs) against larval mosquitoes.

Bread-making properties of different pulse flours in composites with refined wheat flour.

There has been a growing demand for pulses due to the nutrition, health benefits, and agronomical advantages, along with the recommendation of international organizations to diversify the use of pulses. Lentil, yellow pea, and chickpea were studied for their effect on the quality of the bread when incorporated into refined wheat flour. The pulse grains were roller milled into different particle sizes (small: 44-59 µm, medium: 70-85 µm, and large: 95-104 µm) with similar composition, and each refined pulse flour was incorporated in refined wheat flour at levels of 5, 12.5, and 20% (wt/wt). The flours were characterized for particle size distribution, starch damage, proximate composition, water retention, and dough mixing properties, followed by baking test and texture analysis. The particle size of the pulse flours did not significantly affect the bread volume or the texture, while increasing the pulse flour incorporation decreased the bread specific volume from 5.0 to 3.4 cm3 /g and the hardness of the bread increased from 3.9 to 9.9 N at the maximum incorporation level. The crust color darkened as pulse flour incorporation was increased, while the effect on crumb color was marginable. Among the three pulses, chickpea performed better compared to lentil and yellow pea, and yellow pea resulted in bread with lower volume at all levels of incorporation. Enriched bread can be produced by incorporating pulse flours at up to 12.5% (wt/wt) without significant effect on the quality of bread, with chickpeas possessing better baking properties than yellow pea or lentil.

The Relationship between Maternal Antibodies to Fetal Brain and Prenatal Stress Exposure in Autism Spectrum Disorder.

Environmental and genetic factors contribute to the etiology of autism spectrum disorder (ASD), but their interaction is less well understood. Mothers that are genetically more stress-susceptible have been found to be at increased risk of having a child with ASD after exposure to stress during pregnancy. Additionally, the presence of maternal antibodies for the fetal brain is associated with a diagnosis of ASD in children. However, the relationship between prenatal stress exposure and maternal antibodies in the mothers of children diagnosed with ASD has not yet been addressed. This exploratory study examined the association of maternal antibody response with prenatal stress and a diagnosis of ASD in children. Blood samples from 53 mothers with at least one child diagnosed with ASD were examined by ELISA. Maternal antibody presence, perceived stress levels during pregnancy (high or low), and maternal 5-HTTLPR polymorphisms were examined for their interrelationship in ASD. While high incidences of prenatal stress and maternal antibodies were found in the sample, they were not associated with each other (p = 0.709, Cramér's V = 0.051). Furthermore, the results revealed no significant association between maternal antibody presence and the interaction between 5-HTTLPR genotype and stress (p = 0.729, Cramér's V = 0.157). Prenatal stress was not found to be associated with the presence of maternal antibodies in the context of ASD, at least in this initial exploratory sample. Despite the known relationship between stress and changes in immune function, these results suggest that prenatal stress and immune dysregulation are independently associated with a diagnosis of ASD in this study population, rather than acting through a convergent mechanism. However, this would need to be confirmed in a larger sample.

High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour.

BACKGROUND: Sorghum possesses phenolic compounds that are health-promoting constituents of the grain. There are approximately 40 000 sorghum accessions, many of which have not been evaluated for the grain's health-promoting potential. Conventional methods for measuring total phenolic content, flavonoid content and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging capacity are time-consuming and labour-intensive, resulting in low overall throughput. The objective of this study was to develop a high-throughput screening assay for large sorghum sample sets to determine flavonoid and phenolic content and to modify existing DPPH and total phenolic assays. RESULTS: The 96-well assays exhibited a correlation of > 0.9 with the conventional assays. The 96-well assays allowed for up to 64 samples to be run per day compared with 20-24 samples (depending on the test) for the conventional methods. The 96-well assays had excellent accuracy (97.65-106.16% recovery), precision (1.06-8.28% coefficient of variation (CV)) and reproducibility (1.32-2.13% CV inter-day and 1.36-2.09% CV intra-day). CONCLUSION: The high-throughput 96-well plate method proved to be as robust and reproducible as the conventional method for determining total phenolic content, flavonoid content and DPPH-scavenging capacity in either sorghum bran or flour.

Chemical Composition, Fatty Acid and Mineral Content of Food-Grade White, Red and Black Sorghum Varieties Grown in the Mediterranean Environment.

Grain sorghum (Sorghum bicolor) is a gluten-free cereal grown around the world and is a food staple in semi-arid and subtropical regions. Sorghum is a diverse crop with a range of pericarp colour including white, various shades of red, and black, all of which show health-promoting properties as they are rich sources of antioxidants such as polyphenols, carotenoids, as well as micro- and macro-nutrients. This work examined the grain composition of three sorghum varieties possessing a range of pericarp colours (white, red, and black) grown in the Mediterranean region. To determine the nutritional quality independent of the contributions of phenolics, mineral and fatty acid content and composition were measured. Minor differences in both protein and carbohydrate were observed among varieties, and a higher fibre content was found in both the red and black varieties. A higher amount of total saturated fats was found in the white variety, while the black variety had a lower amount of total unsaturated and polyunsaturated fats than either the white or red varieties. Oleic, linoleic, and palmitic were the primary fatty acids in all three analysed sorghum varieties. Significant differences in mineral content were found among the samples with a greater amount of Mg, K, Al, Mn, Fe, Ni, Zn, Pb and U in both red and black than the white sorghum variety. The results show that sorghum whole grain flour made from grain with varying pericarp colours contains unique nutritional properties.

Changes in phenolic profiles and antioxidant activities during the whole wheat bread-making process.

Health benefits of whole wheat products are partially attributed to their unique phenolic profiles. This study investigated the effect of bread-making processes on the phenolic profiles and antioxidant activities of four different varieties of hard red winter wheat. The fermentation process generally increased soluble phenolic content, flavonoid content, antioxidant activities, and soluble ferulic acid of whole wheat products. The baking process increased the soluble phenolic content and antioxidant activities. Some phenolic acids were incorporated into Maillard reaction products during baking. For the insoluble fraction, fermentation and baking slightly increased phenolic content, flavonoid content, and antioxidant activities of certain wheat varieties. Ferulic acid and isomers of di-ferulic acid (DFA) were not significantly affected by the baking process. Overall, the bread-making process demonstrated positive effects on the potential health benefits of whole wheat products.

Rapid determination of total phenolic content of whole wheat flour using near-infrared spectroscopy and chemometrics.

Phenolics in whole wheat products provide many health benefits. Wheat breeders, producers, and end-users are becoming increasingly interested in wheats with higher total phenolic content (TPC). Whole wheat flour with higher phenolics may have greater marketing value in the future. However, conventional methods determining TPC are costly and labor-intensive, which are not practical for wheat breeders to analyze several thousands of lines within a limited timeframe. We presented a novel application of near-infrared spectroscopy for TPC prediction in whole wheat flour. The optimal regression model demonstrated R2 values of 0.92 and 0.90 for the calibration and validation sets, and a residual prediction deviation value of 3.4. The NIR method avoids the tedious extraction and TPC assay procedures, making it more convenient and cost-effective. Our result also demonstrated that NIR can accurately quantify phenolics even at low concentration (less than 0.2%) in the food matrix such as whole wheat flour.

Identification of gluten-like proteins in selected pod bearing leguminous tree seeds.

The protein composition, molecular weight distribution, and rheological properties of honey locust, mesquite, Kentucky coffee tree, and carob seed germs were compared against wheat gluten. Polymeric and Osborne fractionation protocols were used to assess biochemical properties. Dynamic oscillatory shear tests were performed to evaluate protein functionality. All samples had similar ratios of protein fractions as well as high molecular weight disulfide linked proteins except for the Kentucky coffee tree germ proteins, which were found to have lower molecular weight proteins with little disulfide polymerization. Samples were rich in acidic and polar amino acids (glutamic acid and arginine,). Rheological analyses showed that vital wheat gluten had the most stable network, while Kentucky coffee seed proteins had the weakest. High molecular weight disulfide linked glutenous proteins are a common, but not universal feature of pod bearing leguminous trees.

microRNAs and Gene-Environment Interactions in Autism: Effects of Prenatal Maternal Stress and the SERT Gene on Maternal microRNA Expression.

Background: Genetics and environment both are critical in autism spectrum disorder (ASD), but their interaction (G × E) is less understood. Numerous studies have shown higher incidence of stress exposures during pregnancies with children later diagnosed with ASD. However, many stress-exposed mothers have unaffected children. The serotonin transporter (SERT) gene affects stress reactivity. Two independent samples have shown that the association between maternal stress exposure and ASD is greatest with maternal presence of the SERT short (S)-allele (deletion in the promoter region). MicroRNAs play a regulatory role in the serotonergic pathway and in prenatal stress and are therefore potential mechanistic targets in this setting. Design/methods: We profiled microRNA expression in blood from mothers of children with ASD, with known stress exposure during pregnancy. Samples were divided into groups based on SERT genotypes (LL/LS/SS) and prenatal stress level (high/low). Results: Two thousand five hundred mature microRNAs were examined. The ANOVA analysis showed differential expression (DE) of 119 microRNAs; 90 were DE in high- vs. low-stress groups (stress-dependent). Two (miR-1224-5p, miR-331-3p) were recently reported by our group to exhibit stress-dependent expression in rodent brain samples from embryos exposed to prenatal stress. Another, miR-145-5p, is associated with maternal stress. Across SERT genotypes, with high stress exposure, 20 significantly DE microRNAs were detected, five were stress-dependent. These microRNAs may be candidates for stress × SERT genotype interactions. This is remarkable as these changes were from mothers several years after stress-exposed pregnancies. Conclusions: Our study provides evidence for epigenetic alterations in relation to a G × E model (prenatal maternal stress × SERT gene) in ASD.