MRGPRX2 Is the Codeine Receptor of Human Skin Mast Cells: Desensitization through β-Arrestin and Lack of Correlation with the FcεRI Pathway

Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch.MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeinealone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a b-arrestin-1‒dependent manner. Codeine-triggered b-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions

The SCF/KIT axis in human mast cells: capicua acts as potent KIT repressor and ERK predominates PI3K

BACKGROUND: The SCF/KIT axis regulates nearly all aspects of mast cell (MC) biology. A comprehensive view of SCF-triggered phosphorylation dynamics is lacking. The relationship between signaling modules and SCF-supported functions likewise remains ill-defined. METHODS: MCs were isolated from human skin; upon stimulation by SCF, global phosphoproteomic changes were analyzed by LC-MS/MS and selectively validated by immunoblotting. MC survival was inspected by YoPro; BrdU incorporation served to monitor proliferation. Gene expression was quantified by RT-qPCR and cytokines by ELISA. Pharmacological inhibitors were supplemented by ERK1 and/or ERK2 knock-down. CIC translocation and degradation were studied in nuclear and cytoplasmic fractions. CIC's impact on KIT signaling and function was assessed following RNA interference. RESULTS: ≈5,400 out of ≈10,500 phosphosites experienced regulation by SCF. The MEK/ERK cascade was strongly induced surpassing STAT5>PI3K/Akt>p38>JNK. Comparison between MEK/ERK's and PI3K's support of basic programs (apoptosis, proliferation) revealed equipotency between modules. In functional outputs (gene expression, cytokines), ERK was the most influential kinase. OSM and LIF production was identified in skin MCs. Strikingly, SCF triggered massive phosphorylation of a protein not associated with KIT previously: CIC. Phosphorylation was followed by CIC's cytoplasmic appearance and degradation, the latter sensitive to protease- but not preoteasome-inhibition. Both shuttling and degradation were ERK-dependent. Conversely, CIC-siRNA facilitated KIT-signaling, functional outputs and survival. CONCLUSION: The SCF/KIT axis shows notable strength in MCs, and MEK/ERK as most prominent module. An inhibitory circuit exists between KIT and CIC. CIC stabilization in MCs may turn out as a therapeutic option to interfere with allergic and MC-driven diseases

MICROSATELLITES-BASED EVALUATION OF THE PEAR CULTIVARS SELECTED FROM NIKITSKY BOTANICAL GARDENS GERMPLASM BY THEIR ECONOMICALLY VALUABLE CHARACTERISTICS

Mobilization and preservation of genetic sources of diversity of the cultivated pear varieties and their wild relatives is one of the main aspects of new cultivars breeding for modern intensive horticulture. The crucial matter during a new cultivar creation goes to the selection of parental pairs, which obtain a complex of positive features. The aim of this work is to study the Gene Fund collection of pear plantings from Nikitsky Botanical Gardens in accordance with the main economically important traits and to select the most valuable genotypes for using them in breeding programs as a starting material, as well as to conduct a DNA-fingerprinting and the analyses of genetic polymorphism of promising cultivars from the collection of pear with implementing of microsatellite analyses. As a result of long-term studies the following cultivars were selected in accordance with the complex of features promising for the breeding program: Gvardeiskaya Zimnyaya, Izuminka Kryma, Izumrudnaya, Kelmenchanka, Krymskaya Aromatnaya, Krymskaya Medovaya, Lazurnaya, Maria, Mriya, Nadezhda Stepi, Nezabudka, Novosadovskaya, Oreanda Kryma, Otechestvennaya, Tauschaya, Yakimovskaya. The samples of these cultivars were forwarded for genotyping. For the genetic polymorphism analyses of the studied cultivars, seven microsatellite DNA-markers – EMPc108, EMPc117, EMPc115, CH04e03 and CH01d09, CH01f07a, CH01d08 – grouped into 2 multiplex sets were used. The SSR-markers were significantly different according to their level of polymorphism – from 3 (CH04e03 marker) to 11 (EMPc115) alleles per a gene locus were revealed, the effective number of alleles varying from 1.37 to 4.65. Based on SSR-markers polymorphism analysis data, the rate of genetic similarity of the studied pear cultivars was estimated. This evaluation research helped estimate genetic relations inside the studied sample collection of genotypes. The SSR-fingerprints of the cultivars obtained will be used as a starting material for the creation of DNA-passports database of the “NBG – NSC” Gene Fund collection of pear cultivars

Change of tRNA identity leads to a divergent orthogonal histidyl-tRNA synthetase/tRNAHis pair

Mature tRNAHis has at its 5′-terminus an extra guanylate, designated as G−1. This is the major recognition element for histidyl-tRNA synthetase (HisRS) to permit acylation of tRNAHis with histidine. However, it was reported that tRNAHis of a subgroup of α-proteobacteria, including Caulobacter crescentus, lacks the critical G−1 residue. Here we show that recombinant C. crescentus HisRS allowed complete histidylation of a C. crescentus tRNAHis transcript (lacking G−1). The addition of G−1 did not improve aminoacylation by C. crescentus HisRS. However, mutations in the tRNAHis anticodon caused a drastic loss of in vitro histidylation, and mutations of bases A73 and U72 also reduced charging. Thus, the major recognition elements in C. crescentus tRNAHis are the anticodon, the discriminator base and U72, which are recognized by the divergent (based on sequence similarity) C. crescentus HisRS. Transplantation of these recognition elements into an Escherichia coli tRNAHis template, together with addition of base U20a, created a competent substrate for C. crescentus HisRS. These results illustrate how a conserved tRNA recognition pattern changed during evolution. The data also uncovered a divergent orthogonal HisRS/tRNAHis pair

Research of antimicrobial activity of waterethanol extracts of yarrow (Achillea millefolium l.) and Calendula officinalis (Calendula officinalis l.) against staphylococcus

This paper presents a study of the bactericidal of water-ethanol extracts of the yarrow (Achillea millefolii L.) and сalendula officinalis (Calendulae officinalis L.) properties of the museum strain St. aureus 209 p. Standardization of plant raw materials for flavonoids was carried out in accordance with the State Pharmacopoeia of the Russian Federation of the XIV edition. As a result of the research, it turned out that St. aureus is sensitive to extracts of these plants, and waterethanol extraction of yarrow grass has a stronger bactericidal effect than calendula flowers.В настоящей работе представлено исследование бактерицидных свойств в отношении музейного штамма St. aureus 209 p водноэтанольных экстрактов тысячелистника обыкновенного (Achillea millefolium L.) и календулы лекарственной (Calendula officinalis L.). Стандартизацию растительного сырья по флавоноидам проводили в соответствии с Государственной фармакопеей РФ XIV издания. В результате исследования было выявлено, что St. aureus чувствителен к экстрактам данных растений, причём водно-этанольное извлечение из травы тысячелистника обладает более сильным бактерицидным действием, чем из цветков календулы

Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3

GATA-3 is essential for T cell development from the earliest stages. However, abundant GATA-3 can drive T lineage precursors to a non–T cell fate, depending on Notch signaling and developmental stage. Here, overexpression of GATA-3 blocked the survival of pro–T cells when Notch-Delta signals were present but enhanced viability in their absence. In fetal thymocytes at the double-negative 1 (DN1) stage and DN2 stage but not those at the DN3 stage, overexpression of GATA-3 rapidly induced respecification to the mast cell lineage with high frequency by direct transcriptional 'reprogramming'. Normal DN2 thymocytes also showed mast cell potential when interleukin 3 and stem cell factor were added in the absence of Notch signaling. Our results suggest a close relationship between the pro–T cell and mast cell programs and a previously unknown function for Notch in T lineage fidelity

Tolerance induction through early feeding to prevent food allergy in infants with eczema (TEFFA): rationale, study design, and methods of a randomized controlled trial

BACKGROUND: Up to 8% of all children in industrialized countries suffer from food allergies, whereas children with atopic eczema are affected considerably more frequently. In addition, the type and starting time of weaning foods seem to influence the development of food allergies. However, data from interventional studies on weaning are controversial. The aim of this randomized-controlled clinical trial is to investigate, whether an early introduction of hen's egg (HE), cow's milk (CM), peanut (PN), and hazelnut (HN) in children with atopic eczema can reduce the risk for developing food allergies in the first year of life. METHODS: This is a protocol for a randomized, placebo controlled, double blind, single-center clinical trial. One hundred fifty infants with atopic eczema at 4-8 months of age will be randomized in a 2:1 manner into an active group that will receive rusk-like biscuit powder with HE, CM, PN, and HN (initially approximately 2 mg of each food protein) for 6-8 months or a placebo group, whose participants will receive the same rusk-like biscuit powder without HE, CM, PN, and HN on a daily basis. During the interventional period, the amount of allergens in the study product will be increased three times, each after 6 weeks. All study participants who are sensitized to HE, CM, PN, or HN at the end of the interventional period will undergo an oral food challenge to the respective food in a further visit. Primary endpoint is IgE-mediated food allergy to at least one of the four foods (HE, CM, PN or HN) after 6-8 months of intervention (i.e., at around 1 year of age). Secondary endpoints include multiple food allergies, severity of eczema, wheezing, and sensitization levels against food allergens. DISCUSSION: This clinical trial will assess whether an early introduction of allergenic foods into the diet of children with atopic eczema can prevent the development of food allergies. This trial will contribute to update food allergy prevention guidelines. TRIAL REGISTRATION: German Clinical Trials Register DRKS00016770 . Registered on 09 January 2020

Tolerance induction through non-avoidance to prevent persistent food allergy (TINA) in children and adults with peanut or tree nut allergy: rationale, study design and methods of a randomized controlled trial and observational cohort study

BACKGROUND: Peanuts (PN) and tree nuts (TN) are among the most frequent elicitors of food allergy and can lead to life-threatening reactions. The current advice for allergic patients is to strictly avoid the offending food independently of their individual threshold level, whereas sensitized patients without allergic symptoms should frequently consume the food to avoid (re-)development of food allergy. The aim of this trial is to investigate (I) whether the consumption of low allergen amounts below the individual threshold may support natural tolerance development and (II) to what extent regular allergen consumption in sensitized but tolerant subjects prevents the (re-)development of PN or TN allergy. METHODS: The TINA trial consisting of (part I) a randomized, controlled, open, parallel group, single-center, superiority trial (RCT), and (part II) a prospective observational exploratory cohort study. Children and adults (age 1-67 years) with suspected or known primary PN and/or TN allergy will undergo an oral food challenge (OFC) to determine their clinical reactivity and individual threshold. In the RCT, 120 PN or TN allergic patients who tolerate ≥100 mg of food protein will be randomized (1:1 ratio) to consumption of products with low amounts of PN or TN on a regular basis or strict avoidance for 1 year. The consumption group will start with 1/100 of their individual threshold, increasing the protein amount to 1/50 and 1/10 after 4 and 8 months, respectively. The primary endpoint is the clinical tolerance to PN or TN after 1 year assessed by OFC. In the cohort study, 120 subjects sensitized to PN and/or TN but tolerant are advised to regularly consume the food and observed for 1 year. The primary endpoint is the maintenance of clinical tolerance to PN and/or TN after 1 year assessed by challenging with the former tolerated cumulative dose. DISCUSSION: This clinical trial will help to determine the impact of allergen consumption versus avoidance on natural tolerance development and whether the current dietary advice for PN or TN allergic patients with higher threshold levels is still valid. Trial registration: German Clinical Trials Register; ID: DRKS00016764 (RCT), DRKS00020467 (cohort study). Registered on 15 January 2020, http://www.drks.de