Allergenicity and Linear Epitope Analysis of Scy p 8, an Allergen from Mud Crab.

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

Composition of breast milk from mothers of premature and full-term infants and its influence in Z-Scores for infant physical growth.

BACKGROUND: Breast milk contains various crucial nutrients and biologically active substances and is ideal for newborns. This study aimed to analyze the composition of breast milk from mothers of premature and full-term infants and its influences on the growth of infants. METHODS: Infant-mother dyads examined at our Hospital (March 2016 to May 2017) were included. Milk was collected at 0-1 month, 2-3 months, and 5-6 months and analyzed using a MIRIS human milk analyzer. Z-scores of weight-for-length (WLZ), weight-for-age (WAZ), and length-for-age (LAZ) were calculated. RESULTS: This study included full-term (> 37 weeks of gestation, n = 177) and premature (< 37 weeks, n = 94) infant-mother dyads. The premature infants showed higher ΔWAZ, ΔLAZ, and ΔWLZ from infancy to toddlerhood for the physical growth speed, compared with term infants (P < 0.001). All proteins and true protein components of breast milk decreased with infants' age (P < 0.001). For premature and full-term infants, differences in ΔWAZ and ΔLAZ from birth to infancy and the difference in ΔLAZ, WAZ, and LAZ in toddlerhood were positively associated with non-protein nitrogen (NPN) (all P < 0.05), while the Z-score differences in ΔWLZ from birth to infancy were negatively associated with NPN (all P < 0.05). For premature babies, from birth to infancy stage, ΔWAZ was positively correlated with NPN and carbohydrates while negatively correlated with dry matter (all P < 0.05), and ΔLAZ correlated with NPN (ß = 0.428, P = 0.005). CONCLUSION: Breastfeeding helped premature infants compensatory growth when compared to term infants. Whileduring early infancy stage ΔWLZ gain was negatively associated with increased amounts of NPN in breast milk. This might mean although NPN increase the Z-scores of weight-for-age and length-for-age, with no rise in adipose tissue mass.

Immunoglobulin E Epitope Mapping and Structure-Allergenicity Relationship Analysis of Crab Allergen Scy p 9.

Filamin C is an allergen of Scylla paramamosain (Scy p 9), and six IgE linear epitopes of the allergenic predominant region had previously been validated. However, the IgE epitope and structure-allergenicity relationship of Scy p 9 are unclear. In this study, a hydrophobic bond was found to be an important factor of conformation maintaining. The critical amino acids in the six predicted conformational epitopes were mutated, and the IgE-binding capacity and surface hydrophobicity of four mutants (E216A, T270A, Y699A, and V704A) were reduced compared to Scy p 9. Ten linear epitopes were verified with synthetic peptides, among which L-AA187-205 had the strongest IgE-binding capacity. In addition, IgE epitopes were mapped in the protruding surface of the tertiary structure, which were conducive to binding with IgE and exhibited high conservation among filamin genes. Overall, these data provided a basis for IgE epitope mapping and structure-allergenicity relationship of Scy p 9.

IgE Epitope Analysis and Hypo-Immunoreactivity Derivative of Arginine Kinase in Mantis Shrimp (Oratosquilla oratoria).

As the main allergenic food, shrimp can trigger allergic reactions in various degrees. In this study, arginine kinase (AK) was identified as an allergen in Oratosquilla oratoria by LC-MS/MS. The open reading frame of AK was obtained, which included 356 amino acids, and recombinant AK (rAK) was expressed in Escherichia coli. The results of immunological analysis and circular dichroism showed that rAK displayed similar IgG-/IgE-binding activity and structure as native AK. Besides, five IgE linear epitopes of AK were verified by serological analysis, on the basis of which an epitope-deleted derivative was obtained and named as mAK-L. It has been shown that mAK-L displayed hypo-immunoreactivity compared to rAK, and the contents of secondary structures were different. In conclusion, these discoveries enrich the overall understanding of crustacean allergens and epitopes and set the foundations for food allergy diagnosis and immunotherapy.

Effects of three processing technologies on the structure and immunoreactivity of α-tropomyosin from Haliotis discus hannai.

The subunit of tropomyosin (α-TM) from Haliotis discus hannai is an important allergen. The methods to reduce the immunoreactivity of α-TM are worth investigating. Thus, this study confirmed the reacted conditions of α-TM with transglutaminase (TG)-catalyzed cross-linking reaction, TG-catalyzed glycosylation, and glycation. Three processing technologies reduced significantly the contents of α-helix and hydrophobic force of α-TM and increased the surface hydrophobicity. A serological experiment confirmed that the glycated α-TM with xylose showed the lowest IgG/IgE-binding capacity. The inhabitation dot blot displayed that five epitope peptides could bind with the site-specific IgE prepared by the glycated α-TM. Three in nine glycated sites (M68, N202, and N203) were verified to modify-two epitopes (L-HTM-3 and L-HTM-7) of α-TM, which affected the immunoreactivity of α-TM during glycation. These results indicated that glycation would be desired for developing hypo-allergenic abalone products.

Prenatal second-hand smoke exposure and the risk of suspected developmental coordination disorder in preschoolers: A nationwide retrospective cohort study in China.

Prenatal exposure to second-hand smoke (SHS) is associated with increased neurodevelopmental problems in children, however, its impact on the risk of developmental coordination disorder (DCD) in preschoolers have not been studied thoroughly. Herein, we probed this association based on a nationwide retrospective cohort study of 149,005 preschoolers in China. We divided the objects into the prenatal SHS-exposed group or the no prenatal smoke exposed group (NS-exposed group). Preschoolers were assessed for motor proficiency by the Chinese version of Little Developmental Coordination Disorder Questionnaire (LDCDQ). Multivariable logistic regression was used to evaluate the associations. The prevalence of prenatal SHS exposure was 23.89%. Generally, the prevalence of suspected DCD was significantly higher in prenatal SHS-exposed group (16.38% VS. 14.19%, P < 0.001). With the increase of age, the mean total scores of LDCDQ of both boys and girls increased gradually; and the prevalence of suspected DCD in girls was higher than that in boys in the same age group. After adjusting for covariates, prenatal SHS exposure had the negative association with the total score of LDCDQ and increased the risk of suspected DCD. Our results suggest a need for interventions designed to reduce maternal SHS exposure during pregnancy, early screen for DCD and increase targeted movement and coordination skill training for vulnerable children.

Hypoallergenic derivatives of Scylla paramamosain heat-stable allergens alleviated food allergy symptoms in Balb/c mice.

The design of hypoallergenic derivatives is a new strategy for allergen-specific immunotherapy. Although hypoallergenic derivatives of Scylla paramamosain (mud crab) heat-stable tropomyosin (TM) and myosin light chain (MLC) have been preliminarily explored, their allergenicity in vivo needs to be further studied. In this work, recombinant allergens (wtTM, wtMLC) and hypoallergenic derivatives (mtTM, mtMLC) were purified. IgE-binding frequencies of wtTM and wtMLC in 177 crab-sensitised patients were 32.8% and 11.9%, respectively. In the Balb/c mouse model, mtTM and mtMLC caused mild intestinal inflammation, did not activate T-helper (Th) 2 immune response (interleukin-4, anaphylactic mediator, IgE, and IgG1 antibodies were not significantly increased) but could significantly promote the production of interleukin-10, which equilibrated Th1/Th2 cells, thus alleviating allergic symptoms. Moreover, mtTM and mtMLC-induced rabbit/mice anti-IgG antibodies could effectively block wtTM and wtMLC binding to patients' sera IgE in vitro. These results indicate that hypoallergenic derivatives offer the promise for an immunotherapeutic regimen for crab allergy.

IgE Epitope Analysis for Scy p 1 and Scy p 3, the Heat-Stable Myofibrillar Allergens in Mud Crab.

Tropomyosin (Scy p 1) and myosin light chain (Scy p 3) are investigated to be important heat-stable allergens in Scylla paramamosain. However, the epitopes of Scy p 1 and Scy p 3 are limited. In this study, recombinant Scy p 1 and Scy p 3 had similar IgE-binding capacity to natural proteins. Mimotopes of Scy p 1 and Scy p 3 were analyzed by bioinformatics, phage display, and one-bead-one-compound technology. Ten linear epitopes of Scy p 1 and seven linear epitopes of Scy p 3 were identified by synthetic peptides and inhibition dot blot. Meanwhile, three conformational epitopes of Scy p 1 and seven conformational epitopes of Scy p 3 were verified by site-directed mutagenesis and the serological test. Furthermore, strong IgE-binding epitopes of Scy p 1 and Scy p 3 were conserved in multiple crustaceans. Overall, these epitopes could enhance our understanding of crab allergens, which lay the foundation for a cross-reaction.

Identification of linear epitopes and their major role in the immunoglobulin E-binding capacity of tropomyosin from Alectryonella plicatula.

Tropomyosin (TM) is an important allergen in molluscans. However, there was a lack of information about TM as an allergen in oysters. TM was purified and identified from Alectryonella plicatula (ATM), and its primary sequence was cloned and encoded with 284 amino acids (AAs). Chemical denaturants were used to destroy the structure to confirm that linear epitopes played a major role in the immunoglobulin E-binding capacity of ATM. Subsequently, nine linear epitopes were identified using a serological test. The peptide with AA27-41 was regarded as the key epitope because it could be recognized strongly by most sera of oyster-sensitive individuals in comparison to other epitope peptides. Finally, the epitopes and the primary sequence of TM among shellfish were aligned to find the two conserved epitopes (AA117-132 and AA164-178) in oyster, octopus, abalone, scallop, clam, shrimp, and crab. Overall, these data provide a foundation for the allergenicity and cross-reactivity of TM.

Optimization and application of a method to determine 24 perfluorinated compounds in umbilical cord serum by using liquid chromatography tandem mass spectrometry.

Perfluorinated compounds (PFCs) are a group of widely used synthetic chemicals. Owing to their unique chemical properties, PFCs can accumulate in the environment and living organisms. In vitro and in vivo studies have demonstrated the adverse effects of exposure to PFCs, resulting in increased concern. Therefore, a fast, reliable analytical method is crucial for human biomonitoring and health risk assessment. This study used two isotope internal standards to identify and quantify 24 PFCs in umbilical cord serum samples, based on classical liquid-liquid extraction (LLE) with liquid chromatography tandem mass spectrometry (LC-MS/MS). According to our review of the literature, this study is the first to determine the TFHSA, S4hPDS, S4hPOS, S4hPHS, SPHeS, SPNoS, and SPPeS by using this developed method. The average spiked recoveries of 24 PFCs were acceptable, ranging from approximately 64.0% to 124%; RSDs ranged from 0.74% to 11.2%; LOD and LOQ ranged from 0.013 to 0.248 µg/L and from 0.030 to 0.747 µg/L, respectively. This method was applied to measure the PFCs in umbilical cord serum samples; 24 PFCs were detected in the investigated samples, which are comparable to those reported in the literature. TFHSA, S4hPDS, S4hPOS, S4hPHS, SPHeS, SPNoS, and SPPeS were also detected in the samples, which should be investigated in further research. The sensitivity, accuracy, and precision of the developed method are sufficient for its application in large-scale biomonitoring studies.

Characterization, Epitope Identification, and Cross-reactivity Analysis of Tropomyosin: An Important Allergen of Crassostrea angulata.

Oyster is a common shellfish product in China, which is associated with food allergy. However, there is still lack of research on allergens in oysters. In this study, tropomyosin (TM), an important allergen of Crassostrea angulata, was purified and identified by mass spectrometry. Subsequently, TM was cloned and expressed, with a sequence of size 852 bp, encoding 284 amino acid residues. The results of circular dichroism, digestion assay, inhibition enzyme-linked immunosorbent assay, and basophil activation test showed that recombinant TM had similar physicochemical properties and immunological properties to native TM. Furthermore, two conformational mimotopes were obtained and 10 IgE linear epitopes were verified. Meanwhile, different degrees of cross-reactivity were observed between C. angulata TM and the other 8 shellfish TMs using antibodies and serological analysis, which may relate to the 3 conserved epitope regions. These findings are expected to provide a theoretical basis for the molecular diagnosis of oyster allergy and cross-reactivity among shellfish.

Probiotics for the prevention of atopic dermatitis in infants from different geographic regions: a systematic review and Meta-analysis.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease during infancy, but the preventive effect of probiotics on AD remains unclear. AIM: To evaluate the efficacy of probiotic supplementation for the prevention of AD in infants. METHODS: PubMed, Cochrane Library, and Embase databases were reviewed for relevant randomized controlled trials (RCTs). Two authors independently extracted the data. The primary endpoint was the risk of AD in infants. Risk ratio (RR) and 95% confidence interval (CI) were calculated. RESULTS: A total of 20 related articles including 22 studies were included. A significant reduction in AD risk was revealed for the probiotic group compared to the control group (RR 0.76; 95% CI 0.67-0.87). Subgroup analyses by participant receiving intervention revealed that not only probiotics given to mother (RR 0.70; 95% CI 0.57-0.85) or given to mother and infant (RR 0.76; 95% CI 0.61-0.95) were effective in preventing AD in infants, and probiotics given to infants alone (RR 0.76; 95% CI 0.60-0.96) still effectively decreased the risk of AD. CONCLUSION: Probiotic supplementation reduced the risk of developing AD in infants. Furthermore, probiotic supplementation given to mother or to mother and infant could effectively prevent AD in infants.

Crystal Structure Analysis and IgE Epitope Mapping of Allergic Predominant Region in Scylla paramamosain Filamin C, Scy p 9.

Filamin C (FLN c) is a novel allergen in shellfish. In this study, FLN c from Scylla paramamosain was divided into three regions for recombinant expression based on the number of domains and amino acids. Using dot blot and basophil activation tests, the allergic predominant region of FLN c was determined to be 336-531 amino acid positions (named FLN c-M). It was confirmed that by X-ray diffraction, the crystal structure of FLN c-M with immunoglobulin-like folding at a resolution of 1.7 Å was obtained. The monomer was a barrel structure composed of 16 ß-strands and 2 α-helices. Three conformational epitopes were predicted, six linear epitopes were verified by serological test, and they were positioned on the crystal structure of FLN c-M. For the first time, the crystal structure of the allergic predominant region of FLN c was determined, and it provided an accurate template for the localization of IgE epitopes.

Two hypo-allergenic derivatives lacking the dominant linear epitope of Scy p 1 and Scy p 3.

Scylla paramamosain frequently elicits IgE-mediated type-I hypersensitivity reactions. Molecular candidates for crab allergen-specific immunotherapy have not been studied previously. In this study, reduced and alkylated (red/alk) derivatives with destroyed conformational epitopes and mutant derivatives (mtALLERGEN) with deleted heat/digestion-stable linear epitopes were produced of tropomyosin and myosin light chain. Structural changes and the allergenicity of derivatives was analyzed. Compared with wild-type allergens, red/alk derivatives had dramatically altered protein structures, whereas mtALLERGEN showed slightly structural effects. Enzyme linked immunosorbent assay revealed the heterogeneous epitope-recognition patterns with derivatives among 29 crab-sensitised patients, of whom 13% and 62% recognised conformational and linear epitopes, respectively, whereas 25% recognised both epitope types to the same extent. Furthermore, mtALLERGEN could not bind to IgE or induce basophil activation in some patients. These results imply that hypo-allergenic derivatives of crab myofibril allergens that specifically lacked linear epitopes may serve as viable candidates for immunotherapy.

Association of Gestational Age at Birth With Subsequent Suspected Developmental Coordination Disorder in Early Childhood in China.

Importance: It remains unknown whether children born at different degrees of prematurity, early term, and post term might have a higher risk of developmental coordination disorder (DCD) compared with completely full-term children (39-40 gestational weeks). Objective: To differentiate between suspected DCD in children with different gestational ages based on a national representative sample in China. Design, Setting, and Participants: A retrospective cohort study was conducted in China from April 1, 2018, to December 31, 2019. A total of 152 433 children aged 3 to 5 years from 2403 public kindergartens in 551 cities of China were included in the final analysis. A multilevel regression model was developed to determine the strength of association for different gestational ages associated with suspected DCD when considering kindergartens as clusters. Main Outcomes and Measures: Children's motor performance was assessed using the Little Developmental Coordination Disorder Questionnaire, completed by their parents. Gestational age was determined according to the mother's medical records and divided into 7 categories: completely full term (39 to 40 weeks' gestation), very preterm (<32 weeks), moderately preterm (32-33 weeks), late preterm (34-36 weeks), early term (37-38 weeks), late term (41 weeks), and post term (>41 weeks). Results: A total of 152 433 children aged 3 to 5 years (mean [SD] age, 4.5 [0.8] years), including 80 370 boys (52.7%) and 72 063 girls (47.3%), were included in the study. There were 45 052 children (29.6%) aged 3 years, 59 796 (39.2%) aged 4 years, and 47 585 (31.2%) aged 5 years. Children who were born very preterm (odds ratio [OR], 1.35; 95% CI, 1.23-1.48), moderately preterm (OR, 1.18; 95% CI, 1.02-1.36), late preterm (OR, 1.24; 95% CI, 1.16-1.32), early term (OR, 1.11; 95% CI, 1.06-1.16), and post term (OR, 1.17; 95% CI, 1.07-1.27) were more likely to be classified in the suspected DCD category on the Little Developmental Coordination Disorder Questionnaire than completely full-term children after adjusting for the same characteristics. Additionally, there was no association with suspected DCD in younger (aged 3 years) early-term and postterm children by stratified analyses. Conclusions and Relevance: In this cohort study, every degree of prematurity at birth, early-term birth, and postterm birth were associated with suspected DCD when compared with full-term birth. These findings have important implications for understanding motor development in children born at different gestational ages. Long-term follow-up and rehabilitation interventions should be considered for children born early and post term.

Linear Epitopes Play an Important Role in the Immunoglobulin G (IgG)/Immunoglobulin E (IgE)-Binding Capacity of Scy p 4.

Sarcoplasmic calcium-binding protein is a stable allergen in Scylla paramamosain and named Scy p 4. To explore the importance of linear epitopes in the immunoglobulin G (IgG)/immunoglobulin E (IgE)-binding capacity of Scy p 4, chemical denaturants were used to destroy the structure. Scy p 4 was reduced with dithiothreitol and subsequently alkylated with iodoacetamide (IAA). Furthermore, the structural analysis indicated that IAA-Scy p 4 was an unstructured protein. The inhibition enzyme-linked immunosorbent assay showed that IAA-Scy p 4 could inhibit the binding of Scy p 4 to sensitize serum, with inhibition rates reached 55%. Moreover, the linear mimotopes of Scy p 4 were predicted in silico. Three linear epitopes were verified by serological tests and named L-Scy p 4-1 (AA76-91), L-Scy p 4-2 (AA111-125), and L-Scy p 4-3 (AA137-146). Overall, these data provide an understanding of the relationship between the structure and allergenicity about Scy p 4, and the identified linear epitopes can be used for diagnosis and food processing of shellfish allergy.

IgE epitope analysis of sarcoplasmic-calcium-binding protein, a heat-resistant allergen in Crassostrea angulata.

Sarcoplasmic-calcium-binding protein (SCP) has been investigated as a novel allergen in Crassostrea angulata. Nevertheless, knowledge of its effector-cell-based allergic relevance and epitopes is limited. In this study, the heat-resistant allergen SCP was able to induce significant upregulation of CD63 and CD203c (p < 0.05), which showed obvious allergenicity in a basophil activation test. Furthermore, immunoinformatic tools, a one-bead-one-compound peptide library, and phage display technology were combined to analyze the allergenic epitopes of SCP. Five linear epitopes named L-SCP-1 (AA22-33), L-SCP-2 (AA64-75), L-SCP-3 (AA80-90), L-SCP-4 (AA107-116), and L-SCP-5 (AA144-159) were verified using serological tests. Additionally, two conformational epitopes (C-SCP-1 and C-SCP-2) were determined, and C-SCP-1 was located at one of the calcium-binding sites (AA106-117). Moreover, SCP showed weaker typical α-helical features and higher hydrophobicity after Ca2+ depletion, which reduced its IgE-binding capacity. Overall, these epitope data could enhance our understanding of oyster allergens, which could be used to develop hypoallergenic shellfish products.

Combination Processing Method Reduced IgE-Binding Activity of Litopenaeus vannamei by Modifying Lysine, Arginine, and Cysteine on Multiple Allergen Epitopes.

Allergic reactions occur after the whole food is ingested, rather than the purified allergen. The present study explores the low-allergenic food processing for Litopenaeus vannamei by analysis of macrostructure, digestibility, and immunoreactivity. Furthermore, the presence of modified amino acids on the reported IgE epitopes was analyzed by mass spectrometry. Results showed that the combination processing of Maillard reaction (shrimp meat with galactose) with high temperature-pressure at 115 °C obviously changed the macrostructure and increased the digestibility for the shrimp meat. Meanwhile, the processing significantly reduced the IgG/IgE-binding activity of the shrimp meat. The hypo-IgE-binding activity in processed shrimp may be due to the modification of lysine, arginine, and cysteine residues in antigen epitopes. This is a comprehensive assessment of the specific amino acid residues modified by glycation of multiple allergens in processed L. vannamei, which provides a new research method to explore the hypo-IgE-binding activity in food.

Changes of Serum Zinc-α2-Glycoprotein Level and Analysis of Its Related Factors in Gestational Diabetes Mellitus.

Previous studies have discovered that zinc-α2-glycoprotein (ZAG) is related to insulin resistance and lipid metabolism. The aim of the study is to explore the change of serum ZAG and its related factors in gestational diabetes mellitus (GDM). Eighty newly diagnosed GDM patients were enrolled in the case group, and 80 normal pregnant women were selected as the control group. The differences of baseline data between the two groups were compared, and the change of serum ZAG level and its relationship with related indexes was analyzed. Compared to the control group, the level of serum ZAG in GDM women decreased (P < 0.001). What is more, the serum ZAG level of overweight and normal subjects in two groups was also found to have statistical differences. The Pearson correlation (or Spearman correlation) analysis showed that serum ZAG level was negatively correlated with FPG, FINS, HOMA-IR, and TG (all P < 0.05) and positively correlated with HDL (P < 0.05). Multiple linear regression showed that HDL and HOMA-IR were independent factors of serum ZAG (P < 0.05). The level of serum ZAG in patients with gestational diabetes mellitus decreased, and HDL and HOMA-IR are the influencing factors in the case group.

Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.