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Comparative quantitative immunoproteomic study of raw and roasted peanut major allergen modifications
Get PDFPeanut allergy affects approximately up to 3 % of children and up to 2 % of the adult world population, causing reactions ranging from mild to severe. Major peanut allergens are well characterized but little is known about their post-translational modifications and even less is known about the influence of thermal treatment on their profile. Protein post-translational modification patterns may differ between raw and thermally treated peanuts, which could affect its functional properties, such as allergic potential. In this study we combined proteomic and immunological methods to characterize the modifications or proteoforms of four major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in raw and roasted peanut. Bottom-up high-resolution accurate mass spectrometry and a specialized proteomics software package to identify, map and compare modifications of major peanut allergens between differently treated peanut kernels. Modification-specific antibody western blot was used to confirm the presence of modifications on major allergens in both extracts. Twenty different post-translational modifications in four prominent peanut allergens (Ara h 1-3, 6) were identified, while twelve were quantitatively compared between raw and roasted peanuts by high-resolution mass spectrometry and a proprietary proteomics software. post-translational modification specific antibodies confirmed the presence of these modifications in western-blots of raw and roasted peanuts. This study initiates appreciation of modifications and thermal processing affecting food quality, and development of state-of-the-art methodology in the risk assessment of allergen contamination.Book of Abstract
Effects of lysin's and arginige's modifications on trypsin proteolytic efficacy imposed before and after the peanut roasting
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C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae
No full textScreening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein alpha-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein alpha-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor
Detection and characterization of tropomyosin from Anadara Seashells using immunoproteomic aproach
Get PDFIntroduction: Shellfish allergy is one of the most common food allergies with a prevalence of 0.5%-2.5% in the general population. The most common allergen present in shellfish is tropomyosin. Detection of tropomyosin in seashells is a challenge because there are no specific antibodies for seashells’ tropomyosin. Our aim was to verify the presence of tropomyosin in Anadara seashells using an immunoproteomic approach and to investigate the level of cross-reactivity with shrimps.Methods and Results: Proteins from lyophilized seashells Tegillarca granosa (TG) and Anadara broughtonii (AB) were extracted in: RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0,1% SDS, 150 mM NaCl, 50 mM Tris-HCl, 1mM EDTA) and Rehydration buffer (7M urea, 2M thiourea, 2% CHAPS and 10mM DTT). Protein concentration of extracts was determined by Bradford assay and SDS-PAGE. The presence of tropomyosin has been supported by commercial tropomyosin standard in 1D SDS-PAGE. With 1D immunoblot, it was possible to confirm the reactivity of seashells’ tropomyosin to rabbit anti-shrimp tropomyosin polyclonal antibodies, confirming its presence. Tropomyosin’s presence was also validated with 1D immunoblot using monoclonal antibodies. 2D electrophoresis showed that most of samples’ proteins are in acidic pI range with prevalence of spots in the range 35-50kDa, and, by comparing spots to 2D immunoblot with polyclonal antibodies, it is possible to confirm tropomyosin’s presence in Anadara seashells.Conclusions: We found that tropomyosin is present in both blood clam species. Both monoclonal and polyclonal antibodies raised against shrimp tropomyosin can detect seashells tropomyosin by immunoblot pointing to a potential antibodies cross-reactivity of allergic subjects to shrimps and seashells.Book of Abstract
Characterization of potential probiotic strain, L. reuteri B2, and its microencapsulation using alginate-based biopolymers
Get PDFIn this study, Lactobacillus reuteri B2was isolated fromthe feces of C57BL/6 mice and assessed on probiotic activity.L. reuteri B2was identified by 16S rDNA sequencing, which the cell viability in acidic conditions at pH 2.0was64% after 2 h, and in the presents of 0.30% of the bile salts, after 6 h, was 37%. Antimicrobial assay with L. reuteri B2showed maximumdiameters against Klebsiela oxytoca J7 (12.5±0.71mm).Wefurther hypothesized if L. reuteriB2 strain in the free form can survive all conditions in the gastrointestinal tract (GIT) then the utilization of theappropriate biomaterials would ameliorate its stability and viability in GIT. L. reuteri B2 was microencapsulatedinto sodium alginate-(Na-alg) and different content of Na-alg and sodium maleate (SM) beads. Characterizationmaterials enveloped their thermal characteristics (TGA/DTA analysis) and structure using: scanning electron microscopy(SEM), FTIR, and particle size distribution. The high survival rate of L. reuteri B2 at lowpH from2.0 to 4.0and in the presence of the bile salts, at concentrations up to 0.30%, was obtained. L. reuteri B2 showed strong antimicrobialactivity and the best protection microencapsulated with Na-alg + SM in simulated gastric juices(SGJ).This is the peer-reviewed version of the article: Popović, M., Stojanović, M., Veličković, Z., Kovačević, A., Miljković, R., Mirković, N.,& Marinković, A. (2021). Characterization of potential probiotic strain, L. reuteri B2, and its microencapsulation using alginate-based biopolymers. International Journal of Biological Macromolecules, Elsevier, 183, 423-434. [https://doi.org/10.1016/j.ijbiomac.2021.04.177]Published version: [http://intor.torlakinstitut.com/handle/123456789/626]Supplementary material: [https://hdl.handle.net/21.15107/rcub_intor_627
Sex differences in the effects of early‐life probiotic treatment on TNBS‐induced colitis in rats
No full textWe tested the effects of early‐life probiotic treatment on the induction of colitis in female and male adult rats. Rat pups were fed an aqueous solution of Lactobacillus rhamnosus (from
day 4 to day 30). Feces were collected for microbial analysis. Colitis was induced at day 85. Seven days later rats were graded for histological damage in colon, and samples of
mesenteric lymph node (MLN) and peritoneal exudate cells were analyzed. Female rats developed slightly less severe symptoms of colitis than males, whereas early‐life probiotic
treatment had a more pronounced effect on males in nearly every analyzed parameter. Namely, it increased fecal bacterial diversity and ameliorated colon tissue damage, as well
as increased percentage of resident peritoneal macrophages (CD163+), decreased peritoneal monocyte (HIS48+CD43+) influx, reduced production of IFNγ and IL10 by MLN cells,
attenuated NO production in stimulated peritoneal macrophages and unstimulated MLN cells of male rats. Our findings reveal that effects of probiotic treatment are sex‐specific to an
extent. While microbial diversity was impacted by probiotic treatment in both sexes at an early age, the effect was more pronounced in young males, and it lasted to their adulthood.
The change in microbial diversity correlated with improved outcome of TNBS‐induced colitis, confirming the importance of microbiota for local inflammatory processes. It remains to
be elucidated whether the sex differences in the effect of probiotic treatment on development of colitis may be a consequence of sex differences in early‐life microbial diversity and
severity of colitis symptoms in untreated rats.Supplement: 1; Meeting Abstract: P-069
Brevibacillus laterosporus supplementation diet modulates honey bee microbiome
Get PDFAmong them, bacterial and fungal pathogens Paenibacillus larvae, Melissococcus pluton, Ascosphera apis andNosema ceranae play a major impact on honey bees colonies. Thus, developing alternative prophylactic andcurative strategies are urgently needed. The use of probiotic bacteria in honey bee supplemental feeding istherefore promising to treat or prevent diseases. Brevibacillus laterosporus, Gram-positive endospore formingbacilli, is recognised as one of the promising antibacterial and antifungal agents producer.The aim of this study was to examine the short-therm effects of B. laterosporus supplemented diet on workerhoney bee microbiome.Dry spores of B. laterosporus strain BGSP11 have been administrated through a sugar syrup diet to tencolonies and a representative specimen of worker honey bees was taken before the start of the treatmentand immediately after the syrup was consumed. The microbial diversity was assessed before and after thetreatment using Illumina MiSeq sequencing platforms (ID Genomics service, Seattle, WA, USA). 16s rRNAsequencing for bacterial community profiling and fungal Internally Transcribes Spacer for mycological taxaprofiling were used. The next-generation microbiome bioinformatics platform QIIME2 v 2021.4 was used forfiltering and denoising obtained sequences, calculation of diversity metrics and taxonomy assignment. Thefeature classifier was trained using the Greengenes v 13_8 for bacterial taxa and fungal UNITE database v 8.3.The results obtained in this study indicated statisticaly significant alfa diversity between control and experimentalgroup honey bee microbiota composition. The diversity abundance was higher in control comparingto the group treated with B. laterosporus strain BGSP11 spores. There was no significant diference in Bray-Curtis distance among two groups of analysed samples. Regarding to mycological abundance, compositionwas completely different between two groups; control group had Claviceps as predominant genus, while intreated group of honey bee microbiome Metschnikowia genus was prevalent, indicating that the presence offungal pathogens in treated group is highly diminished.Book of Abstracts: Belgrade BioInformatics Conference 202
Characterization of antibiotic resistance in Escherichia coli isolates from Black-headed gulls (Larus ridibundus) present in the city of Novi Sad, Serbia
No full textDespite common resistance to antimicrobials in Escherichia coli isolates from farm animals in Serbia, no data are currently accessible on its occurrence in E. coli isolated from gulls. Therefore, 67 cloacal swabs and 70 fecal samples from black-headed gulls were investigated for the presence of antibiotic-resistant E. coli isolates. Ninety-nine isolates were obtained during the study. Resistotyping and resistance gene typing has shown that 44 isolates harbor resistance to one or more antibiotics. Multidrug resistance was detected in 24 E. coli isolates. Ten isolates were resistant to extended-spectrum cephalosporin antibiotics and were studied in detail including virulence gene typing, phylogenetic and multilocus sequence typing, and mating. These ten isolates belonged to phylogenetic groups B2 (five isolates), D (four isolates) and B1 (one isolate). Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in E. coli isolates with AmpC phenotype and genotype. One isolate carried the Inc I2/FIB replicon type plasmid with the bla(CTX-M-1) gene. Nine isolates had bla(CMY-2) genes, which were detected on conjugative plasmids in seven isolates. The virulence genes hly, iroN, iss, ompT and cvaC were detected in one transconjugant. Ten isolates were found to be resistant to ciprofloxacin, whose MIC ranged from 4 to 32 mg/L. Genotyping revealed single or double mutations in the quinolone resistance determining region (QRDR) of the gyrA or gyrA, parC and parE genes, respectively. So, Black-headed gulls from Serbia may be colonized by multidrug-resistant E. coli, some of which are resistant to critically important antibiotics in medicine
Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases
Get PDFThe incidence of multiple sclerosis (MS) and susceptibility of animals to experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, decrease with aging. Generally, autoimmune diseases develop as the ultimate outcome of an imbalance between damaging immune responses against self and regulatory immune responses (keeping the former under control). Thus, in this review the age-related changes possibly underlying this balance were discussed. Specifically, considering the central role of T cells in MS/EAE, the impact of aging on overall functional capacity (reflecting both overall count and individual functional cell properties) of self-reactive conventional T cells (Tcons) and FoxP3+ regulatory T cells (Tregs), as the most potent immunoregulatory/suppressive cells, was analyzed, as well. The analysis encompasses three distinct compartments: thymus (the primary lymphoid organ responsible for the elimination of self-reactive T cells – negative selection and the generation of Tregs, compensating for imperfections of the negative selection), peripheral blood/lymphoid tissues (“afferent” compartment), and brain/spinal cord tissues (“target” compartment). Given that the incidence of MS and susceptibility of animals to EAE are greater in women/females than in age-matched men/males, sex as independent variable was also considered. In conclusion, with aging, sex-specific alterations in the balance of self-reactive Tcons/Tregs are likely to occur not only in the thymus/”afferent” compartment, but also in the “target” compartment, reflecting multifaceted changes in both T-cell types. Their in depth understanding is important not only for envisaging effects of aging, but also for designing interventions to slow-down aging without any adverse effect on incidence of autoimmune diseases
Sex and strain-specific changes of macrophage cytokine profile in female rats during reproductive aging
Get PDFStarenje se povezuje sa razvojem sistemskog, sterilnog hroničnog zapaljenja (engl. „inflammaging”). Malo je podataka o uticaju genetskih faktora i pola na sposobnost makrofaga (Mϕ), kao ključnih ćelija urođenog imunskog odgovora, da tokom starenja “kontrolišu” rezoluciju akutnog zapaljenja i time razvoj hroničnog zapaljenja. Ciljevi ove disertacije su bili da se ispita 1) uticaj rane faze reproduktivnog starenja na fenotipske osobine (ekspresija markera povezanih sa aktivacijom i poreklom/funkcijom) i funkcijska svojstva (fagocitoza, sinteza inflamatornih medijatora) Mϕ “mirne” i inflamirane (delovanjem tioglikolata) peritonealne duplje ženki Albino Oxford (AO) pacova, 2) značaj genetskih faktora za reproduktivnim starenjem uslovljene promene peritonealnih Mϕ od značaja za uspešnu rezoluciju akutnog zapaljenja i 3) uloga polnih steroida u nastanku ovih promena uporednom analizom promena kod mužjaka i ženki AO pacova, njihovom analizom kod ženki AO pacova kojima su na kraju reproduktivnog perioda uklonjeni jajnici i ispitivanjem delovanja estradiola na Mϕ mladih i sredovečnih ženki AO pacova in vitro. Rezultati su pokazali da: 1) se sposobnost Mϕ ženki AO pacova da “kontrolišu” rezoluciju akutnog zapaljenja menja već tokom rane faze reproduktivnog starenja, kao i da su ove promene sojno specifične (Mϕ sredovečnih ženki AO pacova koje “uspešnije” stare od ženki Dark Agouti pacova pokazuju svojstva koja se mogu povezati sa boljom “kontrolom” inflamacije); 2) su ove promene polno specifične (Mϕ sredovečnih ženki imaju svojstva koja ukazuju na veći kapacitet da “kontrolišu” inflamaciju od Mϕ mužjaka istog uzrasta) i 3) u nastanku promena relevantnih za sposobnost Mϕ ženki AO pacova da “kontrolišu” akutnu inflamaciju važnu ulogu imaju promene u delovanju i estradiola i progesterona. Dodatno, ispitavanja in vitro su ukazala da su za uzrasno zavisne promene u sposobnosti Mϕ da “kontrolišu” inflamaciju pored promene u koncentraciji estradiola važne i one u samim Mϕ, koje menjaju njihov odgovor na delovanje estradiola.Aging is associated with the development of systemic, sterile chronic inflammation ("inflammaging"). Little is known about the influence of genetic factors and sex on the ability of macrophages (Mϕ), as key innate immune cells, to "control" the resolution of acute inflammation and thus the development of chronic inflammation during aging. The objectives of this dissertation were to examine 1) the influence of the early phase of reproductive aging on phenotypic characteristics (expression of markers associated with activation and origin/function) and functional characteristics (phagocytosis, synthesis of inflammatory mediators) of Mϕ isolated from “naive” and inflamed (thioglycollate-induced) peritoneal cavity of females Albino Oxford (AO) rats; 2) the significance of genetic factors for reproductive aging-related changes of peritoneal Mϕ, especially those important for successful resolution of acute inflammation and 3) the role of sex steroids in the occurrence of these changes by comparative analysis in males and females of AO rats, their analysis in female AO rats whose ovaries were removed at the end of the reproductive period and by examining in vitro effect of estradiol on Mϕ from young and middle-aged female AO rats. The results showed that: 1) the ability of Mϕ from AO females to “control” the resolution of acute inflammation changes during the early phase of reproductive aging, and these changes are strain-specific (Mϕ from middle-aged AO females that “age more successful” than Dark Agouti females, show properties which may be associated with better "control" of inflammation); 2) these changes are sex-specific (Mϕ from middle-aged females have a greater capacity to “control” inflammation than Mϕ from males of the same age group) and 3) both estradiol and progesterone play important roles in the ability of Mϕ from AO females to “control” acute inflammation. In vitro studies revealed that, in addition to changes in estradiol concentration, intrinsic changes in Mϕ that regulate their response to estradiol action are important for agedependent changes in Mϕ ability to „control“ inflammation