[Using the Matter-Element Extension Model to Assess Heavy Metal Pollution in Topsoil in Parks in the Main District Park of Lanzhou City].

The UNMIX model was used to analyze the source of heavy metals found to be present in the topsoil of parks in the main district of Lanzhou City. The Hakanson toxicity response coefficient was used concurrently to modify the traditional weights in the model, and the matter-element extension model was used to evaluate heavy metal pollution. The results of the evaluation were compared with the comprehensive pollution index (PN) and potential ecological risk index (RI). The results were as follows. ①The average heavy metal content in the topsoil at each sampling point was higher than that of the background value of soil in Lanzhou, with the proportion of Ni, Cu, and Co being 100% while the proportion of Cr, V, Pb, and As contents were 58.82%, 14.71%, 20.59%, and 2.94%, respectively. ② The results of source analysis showed that there were three major sources of heavy metal pollution in the topsoil of the parks in the study area. Source 1 is construction pollution, which contributes 56% of the Co present. Source 2 is traffic pollution, which contributes 44% and 52% of Cu and Pb, respectively. Source 3 is natural, and contributes 62%, 60%, 56%, and 56% of V, Cr, Ni, and As, respectively. Thus, this research showed that natural sources are predominant. ③ The weight correction effect for each heavy metal was significant; there was an approximately 44% reduction in both Cr and V, while the corrected weights of Ni, Cu, Pb, As, and Co increased in the order Co < Pb < Cu < Ni < As compared with the conventional weights. The most obvious change in weight was that of As, which increased by approximately 188%. ④ The results of the evaluation using the matter-element model showed that the state of 46% of the topsoil in the parks in the study area was grade Ⅴ (severely polluted), while 41% was grade Ⅳ (moderately polluted) and 3% was grade Ⅲ (lightly polluted); Co was the main pollutant. The results of the model evaluation were roughly the same as of from the PN and RI, indicating that the matter-element extension model can be used to evaluate heavy metal pollution in soil and the evaluation results are accurate and objective.

Site-Directed Mutations of Calcium-Binding Sites Contribute to Reducing the Immunoreactivity of the EF-Hand Sarcoplasmic Calcium-Binding Protein in Scylla paramamosain.

In order to reduce the immunoreactivity of sarcoplasmic calcium-binding protein (SCP), site-directed mutations were used to replace key amino acids in the conformational epitopes and calcium-binding sites. The mutant SCPs (mSCPs) were expressed in Escherichia coli, and their immunoreactivities were analyzed using iELISA and basophil activation assays. Furthermore, the structural changes of mSCPs were determined from the circular dichroism spectra. The iELISA results showed that mSCPs could effectively inhibit the binding of wild-type SCP (wtSCP) to sensitive serum, with inhibition rates that reached 90%. Moreover, mSCPs could downregulate the expression levels of CD63 and CD203c on the basophil surface. Compared with wtSCP, the peak values were significantly changed, and the calcium binding ability was impaired, which explained the decline in immunoreactivities of the mSCPs. All of the data confirmed that this approach was effective in reducing the immunoreactivity of SCP and could be applied to other shellfish allergens.

[Assessment of Heavy Metal Pollution in Surface Dust of Lanzhou Schools Based on Random Forests].

In this study, seven types of heavy metal elements and 11 types of characteristic parameters affecting heavy metal pollution and accumulation in surface dust were selected. Based on the comprehensive pollution index (PN) and potential ecological risk index (RI) calculated from the heavy metal element content of the school dust in the main urban area of Lanzhou City in 2018 as the training set, the PN and RI of the information sampling points were estimated using random forests. Then, the temporal and spatial characteristics of heavy metals in school dust in the main urban area of Lanzhou were analyzed. Finally, the correlation coefficient was used to evaluate the advantages and disadvantages of the traditional interpolation results and the random forest interpolation results. The results showed that the concentrations of heavy metals in the dust were higher than the local background values. The over standard rate of a single sample is 100%, Zn is 5 times higher than the background value, and Pb is 4 times higher than background value. PN in the study area was in the order Chengguan > Xigu > Anning > Qilihe, and RI was in the order Chengguan > Xigu > Qilihe > Anning. PN and RI exhibited very similar spatial distribution characteristics, both located in transportation hubs or downtown. In winter and summer, PN exhibited a high value, whereas RI had a high value. The reason for the high value of PN and RI in winter was the increase of coal sources in winter. The comparison of spatial interpolation results shows that the correlation coefficient between the results of random forest interpolation and traffic flow and normalized building index is greater than that of the traditional algorithm.

Inhibitory Effect of Volatiles Emitted From Alcaligenes faecalis N1-4 on Aspergillus flavus and Aflatoxins in Storage.

Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 µL/L, respectively. The effective concentration for MI against A. flavus is 200 µL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.

Experimental realization of sequential weak measurements of non-commuting Pauli observables.

Sequential weak measurements of non-commuting observables are not only fundamentally interesting in terms of quantum measurement but also show potential in various applications. Previously reported methods, however, can only make limited sequential weak measurements experimentally. In this article, we propose the realization of sequential measurements of non-commuting Pauli observables and experimentally demonstrate for the first time the measurement of sequential weak values of three non-commuting Pauli observables using genuine single photons.

Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.

Functionality of an absolutely conserved glycine residue in the chimeric relaxin family peptide R3/I5.

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.

A comprehensive analysis of the allergenicity and IgE epitopes of myosinogen allergens in Scylla paramamosain.

BACKGROUND: Scylla paramamosain is one of the most common and serious food allergens in Asia. Therefore, research on its prevalence, accurate diagnosis, and IgE-binding pattern of the allergens is crucial. OBJECTIVE: To identify the IgE epitopes of the myosinogen allergens in S. paramamosain using phage peptide library. METHODS: The prevalence of allergy to crabs (AC) and of sensitization was analysed using a questionnaire and a serological assay. BAT was performed by flow cytometry, and its diagnostic performance was evaluated in relation to allergens purified from crab myosinogen. IgE-binding epitopes were identified by phage display using the IgE from patients with AC. Sequence- and structure-based bioinformatics analyses were performed to identify allergenic epitopes. RESULTS: Crab was the most common cause of food allergies in this study. Subjects with AC (n = 30) with clear clinical symptoms were identified by immunoblotting and BAT. All of the myosinogen allergens triggered basophil activation; surface expression of CD63 and CD203c was higher in patients allergic to AK and FLN c than in patients allergic to SCP and TIM. In addition to six conformational epitopes of SCP, six linear epitopes and eight conformational epitopes of AK were identified. Five linear epitopes and three conformational epitopes of TIM, nine linear and ten conformational epitopes of FLN c were also identified, and the sequence VH(I/T) L was appeared in epitopes of both TIM and FLN c. The number of epitopes showed consistency with the value of BAT. CONCLUSIONS AND CLINICAL RELEVANCE: BAT can be used for accurate diagnosis of AC. Identification of particular allergenic motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergies.

Crystal structure determination of Scylla paramamosain arginine kinase, an allergen that may cause cross-reactivity among invertebrates.

Shellfish are one of the most common causes of food allergy. Arginine kinase (AK) is known as an important allergen in shellfish. In the present study, AK from crab (Scylla paramamosain) was purified and its crystal structure was determined. A comparison of AK from S. paramamosain to AKs of other species showed high amino acid sequence and secondary structure identity, while the superposition of crystal structures of AKs from different species revealed only slight differences. Similarity of the linear epitope regions among species was observed in the epitope alignment of AKs; conformational epitopes were located in the regions where secondary structure was conserved. The structure of S. paramamosain AK is an accurate template for the analysis of the IgE binding pattern, and the structure conservation and epitope similarity of AKs among species could help to inform our understanding of the cross-reactivity and contribute to the prediction of cross-reactivity related epitopes.

atrB-Associated Fludioxonil Resistance in Botrytis fragariae Not Linked to Mutations in Transcription Factor mrr1.

Resistance to fludioxonil in Botrytis cinerea and B. fragariae was previously found to be linked to either overexpression of the drug efflux pump atrB activated by mutations in transcription factor mrr1 or to mutations in the osmoregulation gene os1. In the present study, isolates of B. cinerea, Botrytis group S, or B. fragariae collected from strawberry fields in the United States were resistant to fludioxonil with half-maximal effective concentration values ranging from 0.04 to 0.43 µg/ml for B. cinerea, 0.03 to 1.03 µg/ml for Botrytis group S, and 0.28 to 3.48 µg/ml for B. fragariae. Analyses of mrr1 sequences revealed various mutations linked to fludioxonil resistance in B. cinerea and Botrytis group S isolates. However, no mutations in mrr1 correlated with atrB overexpression-mediated resistance in B. fragariae isolates. Neither nucleotide variations in the 1,370-bp upstream region of atrB nor increased atrB copy numbers could explain the atrB overexpression in these B. fragariae isolates. Mutations in os1 conferred resistance to iprodione in B. cinerea and Botrytis group S isolates; none correlated with resistance to fludioxonil in B. fragariae. In contrast to European isolates, U.S. B. fragariae isolates contained a 3-bp insertion in the coding region of os1. These isolates were more sensitive to osmotic stress but it is unclear whether the insertion is responsible for this phenotype. Our findings suggest that atrB overexpression-associated fludioxonil resistance is an across-species mechanism of resistance to fludioxonil that can be induced by mutations in mrr1 and other, still-unknown mechanisms.

Analysis of the Allergenic Epitopes of Tropomyosin from Mud Crab Using Phage Display and Site-Directed Mutagenesis.

Mud crab ( Scylla serrata), which is widely consumed, can cause severe allergic symptoms. Eight linear epitopes and seven conformational epitopes of tropomyosin (TM) from S. serrata were identified using phage display. The conformational epitopes were formed based on the coiled-coil structure of TM. Most of the epitopes were located in the regions where primary structures were conserved among crustacean TM. Twelve synthetic peptides were designed according to the epitopes and trypsin-cutting sites of TM, among them, three synthetic peptides (including one linear epitope and two conformational epitopes) were recognized by all of the patient sera using inhibitory dot blotting. A triple-variant (R90A-E164A-Y267A) was constructed based on the critical amino acids of the TM epitope. The IgE-binding activity of the triple-variant was significantly reduced compared with that of native TM. The results of phage display and site-directed mutagenesis offered new information regarding conformational epitopes of TM.

Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Our recent study demonstrated that selectivity of the chimeric relaxin family peptide R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of the highly conserved nonchiral B23Gly or B24Gly with some natural l-amino acids. To investigate the mechanism of this modulating effect, in the present study we incorporated unnatural amino acids into the B23 or B24 position of a semi-synthetic R3/I5 that was prepared by a novel sortase-catalysed ligation approach using synthetic relaxin-3 B-chain and recombinant INSL5 A-chain. R3/I5 was a weak agonist for RXFP3 after B23Gly was replaced by D-Ala or D-Ser, but a strong antagonist for this receptor after B23Gly was replaced by corresponding l-amino acids. However, these replacements always resulted in a weak agonist for RXFP4. Thus, configuration of the B23 residue of R3/I5 affected activation of RXFP3 but not RXFP4. For the B24 residue, both size and configuration affected receptor selectivity of R3/I5. l-amino acids with an appropriate size, such as L-Ser and L-Abu, had the greatest effect on increasing the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. Our present results provided new insights into receptor selectivity of R3/I5, and would facilitate design of novel agonists or antagonists for RXFP3 and RXFP4 in future studies.

Experimental three-party quantum random number generator based on dimension witness violation and weak measurement.

Random number generation is an important task in modern science. A variety of quantum random number generation protocols have been proposed and realized. These protocols, however, are all based on two parties. Based on the weak measurement technique, we propose and realize a quantum random number generator among three observers. The violation of a double classical dimension witness based on the determinant value is first observed in experiment. With the heralding single-photon source, our experimental setup attains the independent assumption and the dimension assumption, which means our setup is semi-device-independent (DI). This Letter sheds new light on generating DI-type random number among multi-user and it has potential application prospect on the quantum cryptography and quantum random number in network environment.

Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

The Maillard Reaction Reduced the Sensitization of Tropomyosin and Arginine Kinase from Scylla paramamosain, Simultaneously.

The Maillard reaction was established to reduce the sensitization of tropomyosin (TM) and arginine kinase (AK) from Scylla paramamosain, and the mechanism of the attenuated sensitization was investigated. In the present study, the Maillard reaction conditions were optimized for heating at 100 °C for 60 min (pH 8.5) with arabinose. A low level of allergenicity in mice was shown by the levels of allergen-specific antibodies, and more Th1 and less Th2 cells cytokines produced and associated transcription factors with the Maillard reacted allergen (mAllergen). The tolerance potency in mice was demonstrated by the increased ratio of Th1/Th2 cytokines. Moreover, mass spectrometry analysis showed that some key amino acids of IgE-binding epitopes (K112, R125, R133 of TM; K33, K118, R202 of AK) were modified by the Maillard reaction. The Maillard reaction with arabinose reduced the sensitization of TM and AK, which may be due to the masked epitopes.

Cholesterol modulates the binding properties of human relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. As an A-class G protein-coupled receptor, RXFP3 is an integral plasma membrane protein with seven transmembrane domains, yet influence of the membrane lipids on its function remains unknown. In the present study, we disclosed that cholesterol, an essential membrane lipid for mammalian cells, modulated the binding properties of human RXFP3 with its ligands. We first demonstrated that depletion of cholesterol from host human embryonic kidney (HEK) 293T cells by methyl-ß-cyclodextrin altered ligand-binding properties of the overexpressed human RXFP3, such as increasing its binding potency with some antagonists and decreasing its binding affinity with a NanoLuc-conjugated R3/I5 tracer. Thereafter, we demonstrated that two B-chain residues, B5Tyr and B12Arg, were primarily responsible for the increased binding potency of these antagonists with human RXFP3 under the cholesterol depletion condition. Our results suggest that cell membrane cholesterol interacts with human RXFP3 and modulates its ligand-binding properties, providing new insights into the influence of membrane lipids on RXFP3 function.

Fungicide Resistance in Botrytis fragariae and Species Prevalence in the Mid-Atlantic United States.

Botrytis fragariae was recently described causing gray mold of strawberry in Germany and the United States. The goal of the present study was to determine its prevalence, distribution, and sensitivity to fungicides in strawberry fields of five states. In total, 188 Botrytis isolates were obtained from flowers and fruit collected from the states of Maryland (n = 35), Virginia (n = 38), North Carolina (n = 46), South Carolina (n = 41), and Georgia (n = 28). Only 13 of these were fruit samples and came from South Carolina (n = 5) and Georgia (n = 8). B. fragariae made up 35.1% of the entire collection, and composed close to half of the Botrytis population in North Carolina (43.4%), South Carolina (61.0%), and Georgia (42.9%). One isolate of B. mali was also found, and the rest of the isolates were B. cinerea (sensu lato). B. fragariae and B. cinerea were found coexisting in 11 fields, while other field samples consisted of only B. fragariae (n = 3) or only B. cinerea (n = 10) isolates. B. fragariae isolates with resistance to one or more fungicides were found, and resistance profiles differed from those of B. cinerea, in that no resistance to cyprodinil (FRAC 8) or boscalid and other FRAC 7 botryticides was detected. We detected B. fragariae resistance to the active ingredients thiophanate-methyl, iprodione, fludioxonil, and fenhexamid. We also detected B. fragariae isolates with resistance to up to four chemical classes of fungicides, though most isolates were resistant to one or two chemical classes. In conclusion, isolates of the newly detected species B. fragariae were commonly found on strawberry flowers in the Mid-Atlantic United States, and have developed resistance to many of the most commonly used botryticides. Though the relevance of this species to pre- and postharvest fruit infections is unknown, fludioxonil applications may give this species a competitive advantage over B. cinerea. Controlling this fungus with FRAC 7 fungicides may be an effective way of limiting its spread in strawberry fields.

Genotypic and Phenotypic Variations in Botrytis spp. Isolates from Single Strawberry Flowers.

Gray mold, caused by Botrytis spp., is among the most devastating diseases affecting strawberry worldwide. The great diversity present in the pathogen enhances its ability to survive and adapt in the field. In this study, we explored the genotypic and phenotypic diversity present in single strawberry flowers. In total, 192 isolates were collected from 19 flowers and four farms, and 9 to 12 isolates were collected from each flower. Forty-two haplotypes were found using microsatellite fragment analysis. Multiple haplotypes of two different Botrytis spp. (Botrytis cinerea and B. fragariae) were found in 12 flowers. In the remaining seven flowers, the single-spore isolates examined were of identical haplotypes. In three flowers, the two Botrytis spp. were found to coexist. Isolates were either sensitive (zero chemical class resistance) or resistant to one, two, three, four, or five chemical classes of fungicides. Resistance to multiple fungicides was commonly observed in both species but resistance to boscalid and penthiopyrad was only found in B. cinerea isolates. Resistance to cyprodinil was found in B. fragariae for the first time in the United States. Each haplotype was generally linked to a single resistance profile; however, a single resistance profile often was represented by multiple haplotypes. Isolates from the same flower of multiple haplotypes were largely identical in resistance profiles. This study is a first detailed investigation of genotypic diversity combined with phenotypic analysis of Botrytis spp. at the single-tissue level. It demonstrates that high genotypic and phenotypic diversity is present not only within fields but also in individual blossoms as well. This information is important for understanding the epidemiology of Botrytis and also has implications for fungicide resistance management, particularly related to resistance monitoring practices.

Cloning, Expression, and the Effects of Processing on Sarcoplasmic-Calcium-Binding Protein: An Important Allergen in Mud Crab.

Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.

Development of a selective agonist for relaxin family peptide receptor 3.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.