Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine

Background We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker–free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. Results We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. Conclusions All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors.

Characteristic of Allergen Protein of Nila Baby Fish (Oreochromis niloticus) and Its Processed Product

Allergen protein is a protein that could triggers allergy reaction. This study purposes to examine the characteristic of allergen protein of unripe, boiled, and fried nila baby fish. Characteristic of allergen protein were observed by electrophoresis and immunoblotting method, while the determination of protein concentration was observed by Bradford method. Identification of the used nila baby fish was accomplished in Faculty of Fishery and Marine Science of IPB. The result of identification showed that the used nila baby fish was Oreochromis niloticus. The proximate analysis of unripe, boiled, and fried nila baby fish resulted water content ranged from 19.16%-23.68%, fat content ranged from 1.03%-20.44% and carbohydrate content ranged from 0.16%-20.27%. Protein concentration of unripe, boiled, and fried nila baby fish extract respectively were 1963.45 mg/L; 699.82 mg/L; 607.79 mg/L. The band amount of allergen protein of unripe, boiled, and fried nila baby fish which was detected by electrophoresis, respectively were 16 of protein band, 26 of protein band and 16 of protein band. The immunoblotting showed that the sum of respondent who contained specific IgE that can bind with allergen protein of boiled and fried nila baby fish were more than allergen protein of unripe nila baby fish. It indicated that the processing process by boiling and frying would increase allergenicity toward nila baby fish.

The Toxicological Intersection between Allergen and Toxin: A Structural Comparison of the Cat Dander Allergenic Protein Fel d1 and the Slow Loris Brachial Gland Secretion Protein

Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals.

Integrative transcriptome analysis discloses the molecular basis of a heterogeneous fungal phytopathogen complex, Rhizoctonia solani AG-1 subgroups

Rhizoctonia solani is a fungal species complex that causes necrotrophic crop diseases. It comprises several anastomosis groups, some of which include intra-subgroups, such as AG-1 IA and AG-1 IB, exhibiting varying pathogenicity. Owing to its heterozygous and multinucleate features, genomic analyses of R. solani are still challenging, and understanding of its genetic diversity and genic components is limited. In this study, in order to elucidate the molecular basis of this phytopathogen complex, an integrated transcriptome analysis was undertaken for three subgroups of AG-1, i.e. AG-1 IA, AG-1 IB, and AG-1 IC. Sequence variations suggested substantial evolutionary distances within AG-1. Transcript simple sequence repeats showed comparable characteristics among AG-1, but contained polymorphic sites. Intra-subgroup polymorphisms suggested varying genic heterozygosity within AG-1, suggesting their independent evolutionary trajectory. Sequences of pathogenic factors, phytotoxin biosynthesis pathway enzymes, secreted lignocellulosic enzymes, secreted reactive oxygen species detoxification enzymes, apoplastic/cytoplasmic effector candidates, were conserved among those subgroups. dN/dS ratios of a secretome subset suggested core secreted proteins in AG-1 and distinct evolution of Cys-rich small secreted proteins after differentiation of AG-1 subgroups. Identification of likely pathogenic factors including allergen protein homologues, oxidative phosphorylation and ethylene biosynthesis pathways, and diversification of polysaccharide monooxygenases provides molecular insight into key genomic components that play a role in R. solani pathogenesis.

Transcriptome-based analysis of putative allergens of Chorioptes texanus

Background Mites of the genus Chorioptes are non-burrowing and cause mange in a wide range of domestic and wild animals including cattle, horses, sheep, goats, panda, moose, camelids, mydaus and alpacas. Molecular biology and host-parasite interactions of Chorioptes texanus are poorly understood, and only a few C. texanus genes and transcript sequences are available in public databases including the allergen genes. Methods Chorioptes texanus RNA was isolated from mites, and the transcriptome of C. texanus was analyzed using bioinformatics tools. Chorioptes texanus unigenes were compared with the allergen protein sequences from the mite allergen database website to predict the potential allergens. Chorioptes texanus putative allergen unigenes were compared with hydrolase genes by building a C. texanus hydrolase gene library with the best match of the homologous sequences. Three allergen genes were cloned and expressed, their recombinant proteins were purified and their allergenic activities were preliminarily investigated. Results Transcriptome sequencing (RNA-Seq) of C. texanus was analyzed and results demonstrated that 33,138 unigenes were assembled with an average length of 751 bp. A total of 15,130 unigenes were annotated and 5598 unigenes were enriched in 262 KEGG signaling pathways. We obtained 209 putative allergen genes and 34 putative allergen-hydrolase genes. Three recombinant allergen proteins were observed to induce different degrees of allergic reactions on rabbit skin. Conclusions The present transcriptome data provide a useful basis for understanding the host-parasite interaction and molecular biology of the C. texanus mite. The allergenic activities of recombinant Euroglyphus maynei 1-like (Eur m 1-like) protein, Dermatophagoides ptreronyssinus 1-like (Der p 1-like) protein and Dermatophagoides ptreronyssinus 7-like (Der p 7-like) protein were preliminarily investigated by intradermal skin test. Meanwhile, differences in eosinophil counts were observed in different injected sites of the skin. The identification of putative allergen genes and hydrolase genes offers opportunities for the development of new diagnostic, prevention and treatment methods.

Profil Dan Sensitivitas Protein Alergen Ikan Tongkol (Thunnus albacares) Sebagai Reagen Skin Prick Test (SPT)

Ikan tongkol merupakan salah satu produk laut penyebab alergi makanan. Gejala klinis reaksi alergi makanan adalah gatal, bentol, bengkak, sesak nafas, batuk, dan yang terparah adalah syok anafilaksis. Pengobatan dan pencegahan alergi makanan yang terbaik adalah menghindari konsumsi penyebab alergi. Menghindari konsumsi suatu makanan sebaiknya berdasarkan uji alergi seperti Skin Prick Test (SPT). SPT dilakukan menggunakan reagen SPT yang dicukitkan pada lapisan epidermis kulit lengan bagian volar. Reagen alergen pada penelitian ini merupakan protein ikan tongkol yang berasal dari laut Indonesia dan diekstrak dengan buffer fosfat, Profil ekstrak protein menggunakan elektroforesis dan immunoblotting untuk menentukan protein allergen. Sebanyak 15 pita protein terdeteksi pada ekstrak ikan tongkol yaitu protein dengan berat molekul antara 17 sampai 152 kDa. Potensi alergenik terdapat pada pita protein dengan berat molekul antara 12 sampai 50 kDa. Reagen SPT ikan tongkol mempunyai sensitivitas sebesar 81.8% dan spesifitas 100% sehingga disimpulkan bahwa reagen ekstrak ikan tongkol dapat digunakan sebagai reagen skin prick test Kata kunci: Alergi, protein, tongkol, gejala klinis, skin prick test Tuna fish is one of the marine products that can cause allergic. Clinical symptoms of allergic are a bump, swelling, shortness of breath, coughing and anaphylactic shock is the worst symptom. The best medication and treatment is avoiding the consumption of food that could cause allergy. Avoiding consumption of food should be based on an allergy test such as a Skin Prick Test (SPT). SPT using reagent which is applied on the skin of the forearm between the wrist and elbow. The reagent for SPT is a protein solution of tuna which was extracted by phosphate buffer then protein profile was detected using electrophoresis. Immunoblotting was done to determine the molecular weight of the allergen protein. Fifteen protein bands were detected on tuna fish extract; the molecular weight of the protein was obtained between 17 to 152 kDa. Protein allergenic are molecules that have protein bands with a molecular weight between 12 and 50 kDa. Tuna fish extract solution for SPT had a sensitivity is 81.8%, and specificity is 100%, it was concluded that tuna fish reagent could be used as SPT reagent. Keywords: Allergen, protein, tuna fish, skin prick test.