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Food allergies (FAs) are a crucial public health problem and a severe food safety issue, resulting in an urgent need for an accurate method to detect all of the hidden allergens that exist in food systems. Current methods for detecting allergens typically utilize ELISA, PCR, or LC-MS, which are suitable for the confirmatory analysis of allergens from ingredients rather than unintended contaminants. In this study, we demonstrate a hybridization probe cluster-targeted next-generation sequencing (HPC-NGS) platform for high-throughput screening of potential allergens in food systems. The HPC-NGS successfully captured target DNA fragments and identified 19 allergenic ingredients in a complex food system. Additionally, the HPC-NGS provided expected allergenic species matching rates of 94.24-100% in single food materials and 99.87-99.98% in processed food products. Thus, HPC-NGS enables the accurate characterization of allergenic ingredients and unintended allergenic contaminants in foods. Our results provide new perspectives on the use of HPC-NGS in the accuracy of high-throughput detection technologies for allergens imposed by the complex matrix effect.

Food allergies are global health issue with an increasing prevalence that affect food safety; hence, food allergen detection, labeling, and management are considered to be important priorities in the food industry. In this critical review, we provide a comprehensive overview of several fluorescence-based platforms based on different biorecognition ligands, such as antibodies, DNA, aptamers, and cells, for food allergen quantification. Traditional analytical methods are generally unsuitable for food manufacturers to accomplish the real-time identification of food allergens in food products. Therefore, it is important to develop simple, rapid, inexpensive, accurate, and sensitive methods to improve user accessibility. A fluorescence-based quantitative platform provides an excellent detection platform for food allergens because of its high sensitivity. This review summarizes the traditional antibody-based fluorescent techniques for food allergen detection, such as the time-resolved fluoroimmunoassay , immunofluorescence imaging, fluorescence enzyme-linked immune sorbent assay, flow injection fluoroimmunoassay, and fluorescence immunosensors. However, these methods suffer from disadvantages such as the significant rate of false-positive and false-negative results due to antibody cross-reactivity with nontarget food components in the complex food matrix and epitope degradation during food processing. Hence, different types of fluorescence-based immunoassays are suitable for standardization and quantification of allergens in fresh foods. In addition, we summarize new fluorescence-based quantitative platforms, including fluorescence genosensors, fluorescence cell sensors, and fluorescence aptamer sensors. With the advantages of high sensitivity and simple operation, fluorescence biosensors will have great potential in the future and could provide portable methods for multiallergen real-time detection in complex food systems.

Confusion persists over pathogenesis of spondylolysis. To confirm pathogenicity of the previously identified causative mutation of spondylolysis and investigate the genetic etiology, we generate a new mouse line harboring D673V mutation in the Slc26a2 gene. D673V mutation induces delayed endochondral ossification characterized by transiently reduced chondrocyte proliferation in mice at the early postnatal stage. Adult D673V homozygotes exhibit dysplastic isthmus and reduced bone volume of the dorsal vertebra resembling the detached vertebral bony structure when spondylolysis occurs, including the postzygopophysis, vertebral arch, and spinous process, which causes biomechanical alterations around the isthmic region of L4-5 vertebrae indicated by finite element analysis. Consistently, partial ablation of Slc26a2 in vertebral skeletal cells using Col1a1-Cre; Slc26a2 fl/fl mouse line recapitulates a similar but worsened vertebral phenotype featured by lamellar isthmus. In addition, when reaching late adulthood, D673V homozygotes develop an evident bone-loss phenotype and show impaired osteogenesis. These findings support a multifactorial etiology, involving congenitally predisposed isthmic conditions, altered biomechanics, and age-dependent bone loss, which leads to SLC26A2-related spondylolysis.

Backgrounds As one of the most important cereals, wheat (Triticum aestivum) can cause severe allergic reactions, such as baker's asthma, allergic rhinitis, and atopic dermatitis. A growing number of people are developing allergies to Chinese wheat; however, only a few wheat cultivars have been screened on allergenicity in China. Objective The aim of the present study was to assess the allergenicity of different Chinese wheat cultivars and characterize wheat allergen profiles of patients with allergic rhinitis. Methods We determined protein (soluble protein, gliadin, and glutenin) composition in Chinese wheat by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the immunoglobulin E (IgE) binding capacity by enzyme-linked immunosorbent assay (ELISA) and Western blot using 10 positive sera from wheat allergy patients. We identified 5 gel bands with significant IgE binding capacity using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Soluble protein, albumin, and globulin, showed the highest allergenicity, followed by gliadin, while glutenin only had slight allergenicity. In soluble protein, 5 protein bands with molecular weights of 27, 28, 53, 58, and 62 kDa showed very significant allergenicity. Meanwhile, the relative abundances of 28 kDa-protein and 58 kDa-protein were significantly positively correlated with the IgE-binding capacity of Chinese wheat cultivars, which were identified as rRNA N-glycosidase and β-amylase, respectively, among other proteins in those highly complex gel bands. Conclusion and clinical relevance 28 kDa-protein (rRNA N-glycosidase) and 58 kDa-protein (β-amylase) were speculated to be the main allergens of Chinese wheat causing baker's asthma and allergic rhinitis. These results provide new insights into the prevention and treatment of wheat allergy and the development of hypoallergenic wheat products, whose clinical significance is worth further evaluation.

Background Mutations in the SLC26A2 gene cause a spectrum of currently incurable human chondrodysplasias. However, genotype-phenotype relationships of SLC26A2-deficient chondrodysplasias are still perplexing and thus stunt therapeutic development. Methods To investigate the causative role of SLC26A2 deficiency in chondrodysplasias and confirm its skeleton-specific pathology, we generated and analyzed slc26a2−/− and Col2a1-Cre; slc26a2fl/fl mice. The therapeutic effect of NVP-BGJ398, an FGFR inhibitor, was tested with both explant cultures and timed pregnant females. Findings Two lethal forms of human SLC26A2-related chondrodysplasias, achondrogenesis type IB (ACG1B) and atelosteogenesis type II (AO2), are phenocopied by slc26a2−/− mice. Unexpectedly, slc26a2−/− chondrocytes are defective for collagen secretion, exhibiting intracellular retention and compromised extracellular deposition of ColII and ColIX. As a consequence, the ATF6 arm of the unfolded protein response (UPR) is preferentially triggered to overactivate FGFR3 signaling by inducing excessive FGFR3 in slc26a2−/− chondrocytes. Consistently, suppressing FGFR3 signaling by blocking either FGFR3 or phosphorylation of the downstream effector favors the recovery of slc26a2−/− cartilage cultures from impaired growth and unbalanced cell proliferation and apoptosis. Moreover, administration of an FGFR inhibitor to pregnant females shows therapeutic effects on pathological features in slc26a2−/− newborns. Finally, we confirm the skeleton-specific lethality and pathology of global SLC26A2 deletion through analyzing the Col2a1-Cre; slc26a2fl/fl mouse line. Interpretation Our study unveils a previously unrecognized pathogenic mechanism underlying ACG1B and AO2, and supports suppression of FGFR3 signaling as a promising therapeutic approach for SLC26A2-related chondrodysplasias. Fund This work was supported by National Natural Science Foundation of China (81871743, 81730065 and 81772377).

Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (β-turn, strang, and random coils) to α-helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products. PRACTICAL APPLICATION: In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products.

Unveiling specific interactions between nanoparticles (NPs) and proteins could benefit a better control of NPs' performance in recognition-based detection, imaging and drug delivery. Herein, we investigated the specific recognition between an aptamer modified gold nanoparticle (Apt-AuNP) and its target protein arginine kinase (AK) through a coffee-ring effect (CRE)-based approach. The evaporated droplets of the Apt-AuNP with AK featured a ring-disk-ring transition with elevated AK concentration and a disk pattern was found when the Apt was saturated by AK. Moreover, the AK concentration versus ring thickness curve below the saturation point was proved to fit in an exponential function, indicating the strong association between the Apt-AuNP and AK. In contrast, the ring thickness above the saturation point fitted in a Gompertz growth model that was similar with the Apt-AuNPs incubated with the nonspecific protein (bovine serum albumin, BSA), suggesting that AK was nonspecifically adsorbed onto the AuNPs. The impact of the specific NP-protein interaction on the translation of CRE into macroscopic patterns was further utilized to identify target food allergen AK by the Apt-AuNPs over nontarget allergens (tropomyosin, ovalbumin and β-lactoglobulin). This work provided new insight into the general NP-protein association process and demonstrated the feasibility of employing CRE as an effective tool to profile the specific interactions between NPs and proteins.

Wheat (Triticum aestivum) contains various allergenic proteins and induces diverse allergic reactions. Although many allergens are found to be glycoproteins, research on glycosylated allergens in wheat is rare. In this study, the main glycoprotein in wheat soluble protein was purified by sequential multi-column chromatography including affinity chromatography, gel filtration chromatography, and ion-exchange chromatography. The purified proteins were identified as globulin-1 S allele by liquid chromatography-tandem mass spectrometry and characterized by circular dichroism spectra. The strong IgE-binding capacity of the purified fractions was further demonstrated, suggesting globulin-1 S allele to be a novel allergen in wheat. Further de-N-glycosylated treatment by N-Glycosidase F showed that N-glycosylation enhanced the IgE-binding of globulin-1 S allele. These results identified globulin-1 S allele as a novel allergen in wheat, demonstrated the role of glycosylation in IgE-binding, and provided new insights into the prevention and treatment of wheat allergy.

Arginine kinase (AK) is one of the most important allergens in shrimp products. Herein, a novel immunoassay for quantitation of AK was developed using the antibody modified gold nanoparticle (AuNP) and quantum dot (QD). When the first antibody modified AuNP (AuNP-Ab1) was bridged by AK with the secondary antibody modified QD (QD-Ab2), fluorescence resonance energy transfer (FRET) would occur between the AuNP and QD, which led to a decrease in fluorescent signals. The decrease in fluorescence intensity was found to correlate linearly with the log of AK concentration in the range of 1.0 × 10-6-1.0 × 10-3 mg/mL (R2 = 0.9909) and the detection limit was 0.11 ng/mL. The immunoassay was further proved to have encouraging specificity, precision and accuracy. Compared with existing methods, this study provided a promising approach to develop a highly sensitive and selective detection method for AK in shrimp related food samples.

Food allergy has risen as a worldwide food safety concern due to the increasing prevalence over the last decades. It’s in urgent need to develop straight yet sensitive detection methods for food allergens to help people avoid the potential risks imposed by food allergy. Here, we developed a dual-mode aptasensor that gave both colorimetric and fluorescent readouts in response to parvalbumin (PV), a major allergen in fish. The aptasensor was constructed by hybridizing the aptamer modified gold nanoparticles (AuNP-APT), the complementary short chain modified gold nanoparticles (AuNP-CS1) and the fluorescent dye-labeled complementary short chain (FAM-CS2). The presence of PV led to disassembly of the aptasensor through competitive interaction between APT and PV, which resulted in a change in the solution color of the AuNPs and a recovery of the once quenched fluorescence signals of FAM-CS2. The aptasensor exhibited a colorimetric response in the PV concentration range of 2.5–20 μg/mL and a linear fluorescent response in the PV concentration range of 2.38–40 μg/mL. Moreover, the aptasensor showed encouraging specificity, precision, accuracy as well as efficiency in real fish sample detection, which held great potential in recognition and analysis of food allergens.

Shellfish allergy is a crucial public health problem and a severe food safety issue, resulting in an urgent need for an accurate, simple and sensitive method to detect the shellfish allergens in complex food systems. Herein, we have desighed an “on-off-on” fluorescence aptasensor based on fluorescence resonance energy transfer (FRET) using prepared carboxyl groups functionalized CQDs (cCQDs) and graphene oxide (GO) for the detection and quantification of the major shellfish allergen arginine kinase (AK). The FRET aptasensor allowed for the sensitive detection of AK in the range of 0.001–10 μg/mL with the limit of detection (LOD) of 0.14 ng/mL (S/N = 3), and the limit of quantitation (LOQ) of 0.27 ng/mL (S/N = 10). This reliable, accurate and specific aptasensor with user-friendly operation may provide new perspectives in the application of fluorescence technologies for food allergens detection in the field of food safety.

In the present study, chitosan nanoparticles were prepared, characterized and used to evaluate the embryonic toxicology on zebrafish (Danio rerio). The average particle size of chitosan nanoparticles was 84.86nm. The increased mortality and decreased hatching rate was found in the zebrafish embryo exposure to normal chitosan particles and chitosan nanoparticles with the increased addition concentration. At 120h post-fertilization (hpf), the rate of mortality were 25.0 and 44.4% in the groups treated with chitosan nanoparticles and normal chitosan particles at 250mg/L, respectively. At 72hpf, the hatching rate in the groups treated with normal chitosan particles were lower (P

Fish has been recognized as one of the “Big-Eight” allergenic food sources with parvalbumin (PV) being one of the major allergens. Here, we report a novel immunomagnetic beads-derived enzyme-linked immunosorbent assay (ELISA) method which employs antibody functionalized graphene oxide (GO) and gold nanoparticles (AuNPs) to amplify the detection signal of PV. The GO and AuNPs are modified with monoclonal antibody (mAb) and HRP-labeled secondary antibody respectively to detect the PV captured by amino-functionalized magnetic beads. With the amplification effect of the antibody modified GO and AuNPs, the proposed ELISA could selectively detect PV with a detection limit as low as 4.29 ng/mL when using pure PV. Moreover, the recovery rates are found to range from 89.82% to 115.37% in the spiked samples. The feasibility in real-world fish sample analysis further shows that the proposed immunomagnetic beads-derived ELISA is a highly promising approach for PV detection and pave a new way for food safety analysis.

Arginine Kinases (AK), known as the major allergen in shellfish, has gained increasing concerns in industrialized countries. Here, we proposed a simple but accurate analytical immunoassay that employed gold nanoparticles (AuNPs) and magnetic beads (MBs) for tracing and quantification of AK in shellfish. The limit of detection (LOD) was 3.91 ng ml−1 (S/N = 3), while the limit of quantitation (LOQ) was 13.04 ng ml−1 (S/N = 10), respectively. Moreover, promising recoveries (94.6%–106.8%) were obtained for the determination of AK in foodstuffs, indicating the good accuracy of this method. Furthermore, the results demonstrated that this method is capable of quantification of AK from nine different shellfish species. Therefore, the double-enhanced immunoassay with simple sample preparation procedures and user-friendly operation showed high applicability for allergen detection in shellfish related foodstuffs and production lines. Furthermore, the developed method can be employed to identify the adulteration of shellfish products using AK as biomarker due to the less conserved sequences of AK in phylogenetically different species.

Tropomyosin (TM), known as the major allergen in seafood, has been increasing concerns in industrialized countries. We proposed a simple, rapid and accurate analytical method using surface plasmon resonance (SPR) sensor with gold patterned biochips for detection and quantification of TM in shellfish. The limit of detection (LOD) was 1.0 μg mL−1 (S/N = 3), while the limit of quantitation (LOQ) was 2.5 μg mL−1 (S/N = 10). Moreover, the satisfactory recoveries (92.0–108.0%) was obtained for TM detection in food samples, showing the good accuracy of this method. Furthermore, the results also demonstrated that this developed method is suitable for the determination of TM from different shellfish species within 3 min. Therefore, the developed SPR sensor with gold patterned biochips will have positive significance on the rapid and accurate detection allergens in shellfish foodstuffs, related products and their production lines in the future.

A biosensor based on a chiral assembly of polymer of gold nanoparticle (AuNP) trimers was developed for the detection and quantification of the major shellfish allergen tropomyosin (TROP). TROP and anti-TROP monoclonal antibodies (mAb) were immobilized on 20 nm and 30 nm 16-mercaptohexadecanoic acid (16-MHDA) functionalized AuNPs to assemble a trimer, which has a Circular dichroism (CD) signal. The free TROP from samples was quantified as an inhibitor for the formation of the AuNP trimer. The AuNP trimer-based biosensor allowed for the selective determination of TROP in the range of 0.1–15 ng mL−1 with the limit of detection (LOD) of 21 pg mL−1 (S/N = 3) and the limit of quantitation (LOQ) of 70 pg mL−1 (S/N = 10). The use of a AuNP trimer-based biosensor with simple sample preparation functions with specificity and accuracy; this highlights its applicability for the detection of allergens in shellfish products, related products and their production lines. Furthermore, based on the less conserved sequences of TROP in phylogenetically different species, this biosensor is currently being used to identify the adulteration of shellfish products using TROP as biomarker.

The effect of chitosan coatings on quality changes of shrimp, Litopenaeus vannamei, was studied during a 10-day storage at 4 °C. Groups of treatment 1 (T-1), treatment 2 (T-2), and control were treated with carboxymethyl chitosan, chitosan nanoparticle, and deionized water, respectively. The lowest pH values (P

The freshness of Pacific white shrimp (Litopenaeus vannamei) stored for 0, 2, 4, 6, 8, and 10 days under vacuumpacking at 4°C was assessed by physicochemical, sensory, and microbiological methods. Thiobarbituric acid, total volatile basic nitrogen, pH, total viable counts, and K values increased significantly (P < 0.05) with the storage time, whereas the texture profile indexes decreased significantly (P < 0.05). Furthermore, a proteomic approach of 2-dimensional gel electrophoresis coupled with MALDI-TOF/TOF identification was used to analyze the differentially expressed muscle proteins at the different storage times. All of the 3 identified proteins (arginine kinase, phosphopyruvate hydratase, and actin T2, which were involved in muscle energy metabolism, glycolytic pathway in adenosine triphosphate generation, and organization of myofibril, respectively) were downregulated with the increase of storage time. Quantitative reverse transcription—polymerase chain reaction showed that messenger RNA ...

The aim of this study was to investigate the adhesive property of palladium-silver alloy (Pd-Ag) and the simulated clinical performance of Pd-Ag porcelain-fused-to-metal (PFM), resin-bonded, fixed partial dentures (RBFPDs). A total of 40 Pd-Ag discs (diameter=5 mm) were prepared and divided into the following four groups (n=10): a) No sandblasting, used as a control; and b, 50 µm; c, 110 µm; and d, 250 µm aluminum oxide (Al2O3) particles, respectively. Another 50 discs were pre-sandblasted and divided into five groups (n=10) subjected to different treatments: e) Sandblasting, used as a control; f) silane; g) alloy primer; h) silica coating + silane and i) silica coating + alloy primer. All 90 discs were bonded to enamel with Panavia F 2.0 and then subjected to shear bond strength (SBS) testing. The fracture surfaces were examined by scanning electron microscopy. Next, 40 missing maxillary second premolar models were restored with one of the four following RBFPD designs (n=10): I) A premolar occlusal bar combined with molar double rests (MDR); II) both occlusal bars with a wing (OBB); III) a premolar occlusal bar combined with a molar dental band (MDB); and IV) two single rests adjacent to the edentulous space with a wing (SRB) used as a control. All specimens were aged with thermal cycling and mechanical loading. Subsequently, they were loaded until broken. The data were analyzed by one-way analysis of variance. Al2O3 (250 µm) abrasion provided the highest SBS (P

Food allergies are recognized as a growing public health concern, with an estimated 3% of adults and 6-8% of children affected by food allergy disorders. Hence, food allergen detection, labeling, and management have become significant priorities within the food industry, and there is an urgent requirement for reliable, sensitive, and user-friendly technologies to trace food allergens in food products. In this critical review, we provide a comprehensive overview of the principles and applications of surface plasmon resonance (SPR) biosensors in the identification and quantification of food allergens (milk, egg, peanut, and seafood), including fiber-optic surface plasmon resonance (FOSPR), surface plasmon resonance imaging (SPRI), localized surface plasmon resonance (LSPR), and transmission surface plasmon resonance (TSPR). Moreover, the characteristics and fitness-for-purpose of each reviewed SPR biosensor is discussed, and the potential of newly developed SPR biosensors for multi-allergen real-time detection in a complex food system is highlighted. Such SPR biosensors are also required to facilitate the reliable, high-throughput, and real-time detection of food allergens by the food control industry and food safety control officials to easily monitor cross-contamination during food processing.