Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies.Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as  Aspergillus spp., Candida spp., and the agents of mycoses such as Paracoccidioides species are among the deadliest mycoses  have been studied in cell-walled microorganisms. In fungi, they were first described in 2007 in oformans . So far,atitidis –13 and fs flavus –20.EVs function as vehicles carrying complex cargoes with diverse biological functions, including proteins, carbohydrates, pigments, nucleic acids, and lipids. EVs can contribute to fungal infection outcomes . EVs likCryptococcus model, previous studies demonstrated that the Vancouver Island outbreak strain of C. deuterogatti, namely, R265, can transfer its ability to proliferate within the host macrophages to an avirulent strain, which was attributed to an EV-regulated process  subunits  treatment  and added these EVs to P. brasiliensis yeast cells that did not receive TM treatment (HACA and IRE1 expression significantly (P. brasiliensis yeast cells (CONTROL EVs) were added to P. brasiliensis yeast cells, which did not alter HACA and IRE1 expression . The EVs obtained from UV-treated cells were named UV EVs, whereas the EVs obtained from regular cultures from A. fumigatus were named CONTROL EVs. Our data showed an overall population size in the range of 100 to 200 nm and a minor population of EVs with sizes varying from 320 to 394 nm in regular cultures or a range of 240 to 614 nm in cultures that underwent UV irradiation  from Copyright © 2022 Bitencourt et al.2022Bitencourt et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. fumigatus cultures after UV irradiation (UV EVs) or without UV irradiation (CONTROL EVs)  ortholog, known as Hog1p, plays a role in adaptive responses to different stress agents, such as osmotic stress, oxidative stress, heat shock, and cell wall damage and also acts in cellular division regulation, as demonstrated in A. fumigatus  were associated with a decrease in akuA transcript levels.EVs obtained from both ROL EVs)  caused aumigatus . Approxiumigatus . Thereafumigatus , 38. ThepkC gene . In addit levels , supportA repair . In addigulation . To provdulation . Similar10.1128/mbio.03272-21.2FIG S2A. fumigatus. (A) RT-qPCR to assess the akuA transcript levels. Relative expression was assessed using the NO EVs condition as a reference sample and 18S and βtub as the reference of normalization. (B) The colony formation was counted, and a percent value was determined after heated EV uptake compared with the NO EVs condition. Significantly different values are indicated by asterisks as determined using ANOVA followed by Tukey’s post hoc test . Download FIG S2, EPS file, 0.9 MB.Analysis of the effects triggered by heated EVs in Copyright © 2022 Bitencourt et al.2022Bitencourt et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans model would mediate communication, affecting its morphological transition. Therefore, we isolated EVs from C. albicans from hypha-inducing cultures and from yeast cultures. We analyzed the size and distribution of EVs in both cultures  had a size range from 99 to 182 nm and minor populations with sizes varying from 23 to 85 nm and 232 to 440 nm. For EVs isolated from C. albicans yeast cultures (CONTROL EVs), the majority of EVs had a size range from 107 to 154 nm and a minor population in the range of 211 to 440 nm. Previous studies have demonstrated the heterogeneity of EVs from Candida spp., showing a distribution profile with population sizes ranging from 60 to 280 nm, and in C. albicans, the sizes varied from 100 nm to 600 nm  during C. albicans growth in yeast-to-hypha transition cultures compared to yeast cultures after different incubation times, such as 0.5, 1, 2, and 4 h  and HWP1  were highly upregulated (CHT2 (endochitinase) and SEC24 (ER to Golgi transport). A prominent upregulation was verified for the HWP1 gene at all time points evaluated. The SAP5 gene was upregulated at all times analyzed. Taking advantage of this information, we compared gene expression profiles after EVs uptake in C. albicans. Thus, the C. albicans cells were incubated with TRANS EVs or CONTROL EVs , regardless of the type of EV treatment, CONTROL EVs (heated) or TRANS EVs (heated)  , it was 10.1128/mbio.03272-21.3FIG S3C. albicans for selected genes such as HWP1, SEC24, CHT2, and SAP5 after 0.5, 2, and 4 h. Control cultures were used as a reference for the modulation of transition cultures. The RPP2B and TDH3 genes were used as normalizer genes. Significantly different values are indicated by asterisks, as determined using an unpaired t test . Download FIG S3, EPS file, 1.7 MB.Transition gene expression profile in Copyright © 2022 Bitencourt et al.2022Bitencourt et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03272-21.4FIG S4C. albicans behavior after heated EV exposure. (A) The abundance of morphotypes was determined in percentage values in C. albicans underwent heated EVs in comparison with NO EVs condition. (B) RT-qPCR to assess the HWP1 transcript levels at the 1-h time point. Relative expression was assessed using the NO EVs condition as a reference sample, and the RPP2B and TDH3 genes as reference of normalization. Significantly different values are indicated by asterisks, as determined using ANOVA followed by Tukey’s post hoc test . Download FIG S4, EPS file, 0.9 MB.Analysis of Copyright © 2022 Bitencourt et al.2022Bitencourt et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the HWP1 gene at an earlier incubation time point (1 h) followed by a decay in its transcript levels later (4 h), which was observed for both uptake conditions, CONTROL EVs and TRANS EVs , with an increase in transcript levels of about 1.34-fold. No differences in gene modulation were induced by TRANS EVs (heated) compared with NO EVs  effectors. A recent study demonstrated that EVs enriched with Fks1 and Chs3 from turbance , which sHere, we evaluated whether EVs are capable of mediating intraspecies communication in fungi by applying three approaches in three different fungal pathogens. We used uptake times of 1 and 2 h, which were sufficient to allow the incorporation of radioactive EVs or at least to promote their aggregation with fungal cells, as mentioned above . It is eP. brasiliensis and A. fumigatus. In the P. brasiliensis model, the genes belonging to the UPR pathway were studied after cultures underwent EVs uptake (EVs were obtained from previous tunicamycin-exposed cultures) compared to cells directly exposed to tunicamycin. The UPR pathway is switched on to reestablish endoplasmic reticulum homeostasis by heightening the folding capability and controlling misfolded protein disposal  was cultivated as previously described  for 7 days at 37°C. Conidial suspensions were obtained from 7-day-old plates with sterilized PBS, recovered by centrifugation, and filtered through sterile Miracloth . Conidial concentration was determined using a Neubauer chamber. Approximately 1 × 103 conidia were directly inoculated into complete YAG medium or the conidial suspension was first exposed to UV light for 60 s and then inoculated into a complete YAG medium. The cultures were incubated for 2 days at 37°C. These plates were used for the following experiments: EV isolation and communication assay.The Candida albicans strain ATTC-64548 was grown at 30°C in Sabouraud medium  for 72 h. One fresh colony was inoculated into 10 ml of yeast extract-peptone-dextrose medium (YPD pH 6.3) and cultured overnight at 30°C with shaking (150 rpm). The overnight cultures of C. albicans then were diluted to an optical density at 600 nm (OD600) range of 0.100 to 0.130 with YPD medium (pH 6.3) or YPD medium (pH 7.4) and grown at 30°C or 37°C, respectively. The cultures grown on YPD at pH 6.3 represent the control cultures in which no stimuli for hypha differentiation were offered, whereas cultures grown on YPD at pH 7.4 represent the transition cultures in which a stimulus conferred by pH (7.4) and temperature (37°C) were offered to prompt hypha differentiation. After that, the cultures were used to perform the EVs isolation experiment, the gene expression profile of yeast to hyphae, and the communication assay.g for 15 min and 15,000 × g for 30 min at 4°C. The supernatants, containing EVs, were concentrated and filtered using Amicon systems  with a 100-kDa-cutoff membrane. The concentrated material was centrifuged again at 15,000 × g for 30 min at 4°C, and the supernatants were centrifuged at 100,000 × g for 1 h at 4°C to collect vesicles. The EVs pellets were resuspended in PBS for nanoparticle-tracking analysis (NTA) and experiments. EVs obtained from TM-treated Pb18 were termed TM EVs, whereas EVs obtained from nontreated cultures were termed Pb EVs.EVs from Pb18 yeast cultivated in the presence or absence of TM were isolated as previously described . Yeast cA. fumigatus EVs, the procedures were performed as previously described  at 16-cm distance for 60 s with constant shaking, and then 100 μl of this irradiated suspension was plated on YAG medium, yielding about 10% of the CFU compared to nonirradiated cultures. The cells were washed twice with 3 ml of sterile PBS, recovered from the dish plates with inoculation loops, and then transferred to centrifuge tubes. Thereafter, the cell suspension was filtered through sterile Miracloth , and then a sequential centrifugation and supernatant concentration in the Amicon system was performed. The EVs obtained from UV light exposure were named UV EVs, whereas the EVs obtained from regular cultures were called control EVs.Regarding the isolation of escribed , 41, witC. albicans EVs was performed as described previously, with slight modifications  or YPD (pH 7.4) and incubated for 4 h at 30°C or 37°C, with shaking (100 rpm). For EVs isolation, the cells and debris were removed by sequential centrifugation at 4,000 × g for 15 min and 15,000 × g for 15 min. Supernatants were concentrated using an Amicon ultraconcentration system . The resulting concentrated supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C. Pellets (EV-containing) were collected and resuspended in ultrapure water  supplemented with protease inhibitor cocktail   to prevent cargo degradation, and then the EVs were stored at −80°C. Taking into account the culture conditions used to promote yeast-to-hypha transition , an additional step was included in which the cultures were first filtered using sterile Miracloth  and then subjected to differential centrifugation. EVs obtained after C. albicans grown on YPD at pH 6.3 at 30°C were named CONTROL EVs, while EVs obtained from C. albicans grown on YPD at pH 7.4 and incubated at 37°C were named TRANS EVs.Isolation of ications , 44. TheC. albicans cultures in two different stages, a yeast culture condition (CONTROL EVs) and a yeast-to-hypha culture condition (TRANS EVs), NTAs were performed. We also performed the NTA to measure and characterize the size distribution of EVs isolated from regular and UV-exposed A. fumigatus cultures. We obtained the profiles of P. brasiliensis EVs as previously described  was pulsed with [1-14C] palmitic acid for 72 h before EVs isolation (9/ml) were added to the corresponding yeast-phase fungi (107/ml) and incubated for 0, 1, 6, 12, and 24 h at 37°C in 9.5% CO2. After incubation, culture supernatants containing EVs that were not taken up were removed, fungal cells were washed thrice with PBS and lysed in 200 μl of 25 mM deoxycholate, and the resultant material was collected for scintillation counting.To obtain radiolabeled EVs, each one of the fungal species  and incubated for 0.5, 2, and 4 h at 30°C (yeast culture condition). Thereafter, the cultures were centrifuged at 4,000 × g for 10 min at 4°C. The resulting pellet was stored at −80°C until RNA extraction. The C. albicans inoculum was also added to 5 ml of YPD (pH 7.4) and incubated for 0.5, 2, and 4 h at 37°C (hypha-yeast culture condition). The pellet obtained from these cultures was stored at −80°C until RNA extraction. We used the resulting material to create a panel that reflects the expression profiles of HWP1, SEC24, SAP5, and CHT2 genes during the C. albicans transition stage.The P. brasiliensis endoplasmic reticulum stress.(i) Effect of EVs on  The P. brasiliensis yeast cells were treated with TM as previously described (9/ml) with P. brasiliensis yeast cells (105/ml) was performed for 2 h at 37°C in brain heart infusion medium (Sigma). RNA isolation, cDNA synthesis, and quantitative PCR (qPCR) assays were performed as described previously (escribed . The EVseviously .A. fumigatus UV stress.(ii) Effects of EVs on  The assay was performed with 5 ml of A. fumigatus conidial suspension (104 conidia/ml). The conidial suspension was incubated with 5 × 108 EVs/ml obtained from A. fumigatus regular cultures or UV light-exposed cultures. Additionally, the EVs from both cultures were incubated at 90°C for 15 min, and these heated EVs were also added with fungal conidia. Uptake was performed for 1 h at 37°C in PBS with shaking (100 rpm). Therefore, the fungal cells were recovered by centrifugation at 4,000 × g for 10 min at room temperature, and the cultures were resuspended in 5 ml of PBS. Subsequently, approximately 100 μl of conidia was plated on the YAG medium. After incubation at 37°C in the dark for 2 days, the CFU were counted. CFU from conidia without EVs uptake were counted as controls. The colonies were stored at −80°C until RNA extraction.C. albicans dimorphism.(iii) Effects of EVs on  About 5 × 108 EVs obtained from C. albicans yeast cultures (CONTROL EVs) or C. albicans yeast-to-hypha transition cultures (TRANS EVs) were added into a fresh culture of C. albicans with the cell density adjusted to an OD600 of 0.10 to 0.130, as previously mentioned, in 5 ml of YPD (pH 6.3 for 1 h at 30°C), with shaking (100 rpm). Additionally, the EVs obtained from both C. albicans cultures were incubated at 90°C for 15 min, and these heated EVs were also added into the fresh yeast culture, as mentioned above. The yeast cells were then recovered by centrifugation. The pellets were resuspended in 5 ml of YPD (pH 7.4) and incubated for 1 h and 4 h at 37°C under shaking (100 rpm). After incubation, the cultures were centrifuged at 4,000 × g for 10 min at 4°C, and the pellet was stored at −80°C until RNA extraction. Similar conditions were employed to analyze cellular proliferation through the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl (phenylamino)]-2H-tetrazolium hydroxide (XTT) reduction assay. The XTT assay was performed as previously described  and incubated for 2 h at 37°C. The medium then was removed, and the pellet was resuspended in XTT solution (1 mg/ml in PBS) and menadione (1 mM in acetone) and incubated for 3 h with gentle shaking. The activity of the yeast mitochondrial dehydrogenase reduces the tetrazolium salt XTT to formazan salts, which results in a colorimetric change that might correlate with cell viability. Colorimetric changes were measured using an enzyme-linked immunosorbent assay microplate reader  at 450 nm. Additionally, we monitored the changes in yeast-to-hypha transition in C. albicans cultures after EVs uptake by microscopy analysis. Images were obtained using a Zeiss Observer Z.1 microscope with AxioVision SE64 software after 1-h, 2-h, and 4-h time points. As controls, cultures that had not undergone the EVs uptake process were analyzed.escribed , with slC. albicans cells were treated with lysis solution  for 1 h with shaking (100 rpm). Next, the supernatant was removed by centrifugation at 1,000 × g for 10 min, and total RNA was extracted using the Illustra RNAspin Mini RNA isolation kit  by following the manufacturer’s instructions. The A. fumigatus mycelia were lysed by mechanical pulverization with a pestle and mortar in liquid nitrogen, and total RNA extraction was performed using an Illustra RNAspin mini RNA isolation kit . P. brasiliensis cells were also lysed with the aid of a small mortar and pestle in liquid nitrogen before total RNA was isolated using TRIzol reagent , as described previously (www.idtdna.com/primerquest/Home/Index), and the oligonucleotide sequences are listed in −ΔΔCT method  followed by Tukey’s


Brassica napus with various Brassicaceae species can result in obtaining new forms with increased resistance to blackleg, a dangerous disease caused mainly by Leptosphaeria maculans. In this study, we aimed to correlate the field resistance of selected Brassica hybrids to blackleg with chromosomal structure revealed by Fluorescence in situ hybridization. Tested genotypes varied in the number of chromosomes and rDNA signals. The greatest variation was observed for A1-type chromosomes. Field evaluation also revealed significant differences in L. maculans resistance. Performed analyses allowed to distinguish three B. napus × Brassica fruticulosa genotypes in which variable patterns of chromosomal structure might be connected to field resistance. However, a more thorough study, including the detection of all A-genome chromosomes, is required.Hybridization of  The natural genetic variability among the Brassicaceae species results in their richness in biotic and abiotic stress resistance genes . SelecteBrassica napus L.), one of the most important sources of vegetable oil in the world [Brassica rapa L. and Brassica oleracea L. Interspecific hybridization with species of Brassicaceae may result in broadening B. napus gene pool with useful characteristics such as resistance to Leptosphaeria maculans, the causal agent of blackleg. This disease, distributed worldwide, can lead to a substantial yield loss of  – up to 60% in favourable conditions [L. maculans have been discovered: seedling resistance controlled by single major genes (R genes) and adult plant resistance conferred by multiple minor genes  [L. maculans, low genetic diversity of rapeseed, as well as reported and predicted breakdowns of qualitative resistance in B. napus, it seems crucial to introduce new sources of resistance other than rapeseed [Arabidopsis thaliana, B. elongata, B. fruticulosa, B. juncea, B. carinata, B. nigra, and Raphanus sativus [B. napus with its relatives may lead to transferring of the blackleg resistance genes to rapeseed, as previously reported [B. carinata accession, which resulted in obtaining BC2S3 DH population resistant to pathotype PG2 of L. maculans.Rapeseed  . Considerapeseed –10. Effe sativus –13. Thatreported –16. For reported  crossed in situ hybridization. Genomic in situ hybridization (GISH) is used primarily to identify parental genomes in allopolyploid species. The results of the GISH analysis allow the demonstration of intergenomic structural rearrangements  [B. napus, which might have been caused by the high homology of the aforementioned genomes. As the lack of entire chromosome arms painting limits the use of GISH technique in Brassica, another method of chromosome recognition should be applied. Fluorescence in situ hybridization (FISH) is a commonly used technique for analysing the structure of the plant genome. The use of rDNA sequences in the analysis of Brassica species enables the recognition of selected chromosomes  and consequently gives deeper insight into genome structure and composition [B. rapa genome, C4, C7, and C8 in the B. oleracea genome, as well as B4, B5, and B6/7 in the genome of B. nigra [To confirm hybridity and verify the genetic variation at the chromosomal level, genotypes obtained by interspecific crossing may be characterized by methods of genomes) –20 and pgenomes) . Howevergenomes)  observedposition . The useB. nigra .Brassica hybrids to L. maculans with chromosomal structure revealed by FISH. The comparison of field evaluation with cytogenetics study allows assessing if the presence or absence of certain chromosome regions is directly connected to plants' response to blackleg disease. The use of FISH technique will additionally allow tracking chromosome rearrangements based on various rDNA loci patterns found in hybrid parental forms.This study aimed to correlate the field resistance of chosen 21 generation of hybrids was developed at the Department of Genetics and Plant Breeding (Poznań University of Life Sciences) by performing crosses between B. napus  and various Brassicaceae genotypes  with known blackleg resistance level. The hybrids were developed using in vitro techniques. Next, chosen genotypes were self-pollinated to obtain F2 plants. The following interspecific hybrids of F2 generation were used as research material: B. napus × B. rapa ssp. pekinensis, B. napus × B. rapa ssp. trilocularis, B. napus × B. rapa ssp. chinensis, B. napus × B. fruticulosa, B. napus × Brassica carinata, B. napus × Brassica juncea. Field evaluation was performed to assess the level of hybrids resistance to L. maculans followed by FISH technique to analyse the chromosome architecture of chosen hybrids.The F2.1Seeds of studied genotypes were germinated in the dark for 3–4 days. Freshly cut seedlings’ roots were treated with 2 mM 8-hydroxyquinoline under the following conditions: 1 h at 4°C and 2 h at room temperature in the dark. Roots were fixed in ethanol–glacial acetic acid mixture (3:1) and stored at −20°C. Fixed plant material was macerated for 70–90 min depending on the genotype in an enzyme mixture consisting of 20% pectinase (Sigma), 1% cellulase , and 1% cellulase ‘Onozuka R-10’ (Serva). Next, single root tips were squashed on the glass slide in 10 µL of 45% acetic acid. After a preliminary assessment of prepared slides, coverslips were removed by liquid nitrogen freezing and stored at 4°C until needed.Triticum aestivum genome. The whole sequence (410 bp) was isolated from the plasmid vector with the use of QIAprep Spin Miniprep Kit (Qiagen), and labelled with rhodamine using PCR method under the following conditions: 94°C × 60 s, 35 cycles , 72°C × 5 min. 35S rDNA is a 2.3kbp long fragment derived from A. thaliana. Plasmid vector sequence was isolated using QIAprep Spin Miniprep Kit (Qiagen) and labelled with digoxygenin by nick-translation  under following conditions: 15°C × 95 min, 60°C × 10 min.Two probes, 5S and 35S rDNA, were used in this study. The 5S rDNA sequence was derived from The double-target FISH procedure was carried out according to Hasterok et al. . For eve2.22 with a 0.30 m row distance and a sowing density of 60 seeds/m2. The field experiment in Dłoń was carried out on typical heavy soil of III quality class, with optimal agricultural practices for local agroecological conditions, and no artificial irrigation In crop seasons 2017/2018, 2018/2019, and 2019/2020, the weather conditions were typical for this region of Poland. The seasonal rainfall in Dłoń was 372 mm in 2018, 393 mm in 2019, and 405 mm in 2020, whereas the mean annual temperatures in 2018, 2019, and 2020 were 10.8, 11.1, and 10.5°C respectively. The pre-sowing fertilization on experimental plots consisted of ammonium phosphate (200 kg/ha) and ammonium nitrate (100 kg/ha). Four fertilizers were used during vegetation: Saletrosan 26 (300 kg/ha), ammonium nitrate (200 kg/ha), magnesium sulphate and ADOB Bor, containing borum and nitrogen. No fungicides and pesticides were used on the testing fields. Disease severity was assessed once a year in autumn  on five genotypes per combination, according to a percentage scale  on the testing fields in Poznań University of Life Sciences experimental station Dłoń  located 100 km south from Poznań, Poland. The whole experiment was set up in a completely randomized block design with five replications, and each single plot size was 10 mge scale . Whole p2.3L. maculans infestation) was tested using Shapiro–Wilk’s normality test [The normality of the distribution of the studied trait . Detailed results are presented in The FISH technique was used to assess the chromosomal composition of newly generated hybrids that were created by crossing between allotetraploid B. napus × B. rapa ssp. pekinensis was invariable in the number of observed chromosomes: all five genotypes had 38 chromosomes. However, the studied hybrids differed in the number of detected rDNA signals. Two types of rDNA patterns were observed: 10 5S rDNA loci and 12 35S rDNA loci as well as 12 5S rDNA loci and 14 35S rDNA loci. In the first group, the following were observed: two A3 chromosomes, four A1 chromosomes, two A10 chromosomes, two chromosomes of the group A5/6/9, two C4 chromosomes, two C7 chromosomes and two C8 chromosomes. In the second group, a similar pattern of individual marker chromosomes was observed, except for A1 chromosomes the number of which was six.The first group of hybrids – B. napus × B. rapa ssp. trilocularis genotypes had 38 chromosomes. Among tested hybrids, we observed two genotypes containing ten 5S rDNA signals and twelve 35S rDNA signals, which allowed to identify two A3 chromosomes, four A1 chromosomes, two A10 chromosomes, two A5/6/9 chromosomes and two C4 chromosomes, two C7 chromosomes and two C8 chromosomes. For three remaining genotypes, we revealed eleven 5S rDNA signals and thirteen 35S rDNA signals, which enabled us to recognize eleven chromosomes from the A genome – one additional A1 chromosome, and six chromosomes of the C type.All studied B. napus × B. rapa spp. chinensis hybrids did not vary in the total number of chromosomes. All five genotypes with 38 chromosomes had ten 5S rDNA loci, twelve 35S rDNA loci, and the same number of chromosome types: four A1 chromosomes and two chromosomes of each of A3, A10, A5/A6/A9, C4, C7, C8 chromosome types.B. napus × B. fruticulosa group, a variable number of chromosomes was observed. Genotypes with 36 chromosomes predominated, but one genotype with 34 chromosomes was also observed. Two patterns of rDNA loci were detected in the group with 36 chromosomes: ten 5S rDNA loci and twelve 35S rDNA loci, as well as twelve 5S rDNA loci and thirteen 35S rDNA loci. In a genotype with 34 chromosomes, eight 5S rDNA loci and ten 35S rDNA loci were observed. A constant number of chromosomes was identified in all genotypes of a given combination: two A3 chromosomes, two A10 chromosomes, two A5/6/9 chromosomes, two C4 chromosomes, and two C8 chromosomes. A variable number of chromosomes was revealed for A1 type , and C7 type (one and two chromosomes).In the B. napus × B. carinata hybrids, the total number of chromosomes was consistent – 38 chromosomes were identified in all genotypes. The rDNA loci pattern was constant for all studied genotypes (ten 5S rDNA loci and twelve 35S rDNA loci). Moreover, the number of chromosomes observed was also the same for all individuals: four A1 chromosomes, two A3 chromosomes, two A10 chromosomes, two A5/A6/A9 chromosomes, two C4 chromosomes, two C7 chromosomes, and two C8 chromosomes. Genome B derived chromosomes were not detected.For B. napus × B. juncea hybrids. The same number of individual types of marker chromosomes (carrying rDNA sequences) was observed in five genotypes: four A1 chromosomes, two A10 chromosomes, two A3 chromosomes, two chromosomes from the A5/6/9 group, two C4 chromosomes, two C7 chromosomes, and two C8 chromosomes. No chromosomes derived from the B genome were detected.A constant number of chromosomes of 37 chromosomes and a constant number of 5S and 35S rDNA loci, that is, 10 and 12 rDNA loci was identified in 3.2B. napus × B. juncea combination in 2019 when the lowest level of L. maculans damage (0.00) was observed in 2019 and 2020 for B. napus × B. fruticulosa. Moreover, the latter combination belonged to the statistically best group in all three consecutive years.Field evaluation was used to determine the level of resistance to blackleg of studied genotypes in three following years. The ANOVA analysis revealed that the effect of the combination, year, combination × year was statistically significant for infestation level . Table 4B. napus × B. fruticulosa we were able to distinguish three genotypes with higher resistance to blackleg (infestation level 1.33). From all analysed plants, B. napus × B. rapa ssp. pekinensis genotype number 5 showed the highest level of infestation (13.67), and the highest resistance (1.00) was noted for B. napus × B. carinata genotype number 2.The infestation level was also compared between genotypes of certain combinations . For thi4Brassica species. This approach allowed to distinguish eight out of sixteen B genome chromosomes in B. nigra, as well as 20 out 36 chromosomes in B. juncea, including eight B genome chromosomes. In our study, B type chromosomes would be expected to appear in B. napus × B. carinata and B. napus × B. juncea combination, although such chromosomes have not been detected. This result can be explained simply by the absence of B genome derived chromosomes in analysed hybrids. However, it may be also elucidated by a great similarity in chromosome morphology between A, B, and C genomes, including rDNA-bearing chromosomes, which may consequently lead to misinterpretation of rDNA signals and incorrect recognition.Performed FISH analyses allowed to successfully distinguish parental chromosomes in hybrids genomes in most studied combinations. However, the recognition of B genome chromosomes was not accomplished. Hasterok et al.  used 5S B. napus × B. fruticulosa, as well as the variation in the number of detected 5S and 35S rDNA signals between genotypes for three combinations. This might be explained by irregular chromosome segregation, conversion of genes, or unequal crossing-over events [B. napus × B. rapa ssp. trilocularis their number varied from 4 to 5, for B. napus × B. rapa ssp. pekinensis – from 4 to 6, and for B. napus × B. fruticulosa – from 2 to 6. A study of Sosnowska et al. [B. napus also indicates the A1 chromosomes as the most variable in their number.In this study, we observed a variation in the number of chromosomes for one combination – r events . The grea et al.  on resynB. napus and B. juncea. An odd number of chromosomes can reduce plant fertility due to cytogenetic instability during meiosis (incorrect chromosome pairing). Nevertheless, it is in line with expectations, considering the number of chromosomes found in the parental components – B. napus (2n = 38) and B. juncea (2n = 36). In the B. napus (2n = 38) × B. carinata (2n = 34) combination, 38 chromosomes were observed for all genotypes. A higher number of chromosomes than expected may be due to disturbances during the meiotic division.Thirty-seven chromosomes were observed in the genotypes resulting from the crossing of B. rapa and B. oleracea genomes in the monosomic alien addition lines, which confirms the close relationship of species. Translocations may also occur in B. napus genome [It is known that the genomes of allopolyploids undergo dynamic changes, which result in intergenomic rearrangements . The pols genome ,31.Brassica species can be a good source of resistance to blackleg disease – especially the B. napus × B. fruticulosa genotypes, which showed a low and stable level of infestation in all three years of the study. Moreover, resistance genes may be successfully transferred to hybrid species, and possibly extend B. napus gene pool. As stated before, interspecific crossing can give rise to new enhanced rapeseed cultivars with improved characteristics [L. maculans resistance might be dangerous due to the possibility of rapid overcome of major gene resistance in various cruciferous hosts. This event has been described in a study by Li et al. [L. maculans isolates in Australia. The pathogen was able to overcome the resistance of most tested genotypes, which means that close attention and carefulness should be taken when introducing new sources of resistance to L. maculans into breeding programs.Our research proved that eristics . Neverthi et al. , in whicL. maculans resistance has been identified in several Brassica species including A-genome, B-genome, C-genome, and AC-genome species [B. napus × B. fruticulosa combination, the only one with the statistically important variation of L. maculans resistance might give more information regarding the correlation of this trait with chromosomal structure. The most resistant genotypes of the aforementioned hybrid combination differed in the number of detected chromosomes and rDNA loci, two of them containing 36 chromosomes, and one containing 34 chromosomes. The latter was also characterized by a lower number of rDNA signals. The variable pattern of chromosomal structure detected in B. napus × B. fruticulosa genotypes might be connected to blackleg resistance, nevertheless, it should be taken into consideration that not all resistance-bearing chromosomes were identified in this study. Major L. maculans resistance genes have been mapped on four A-genome chromosomes: A1, A10, A7, and A2 [Brassica hybrids' genomic composition. species . It woul, and A2 . The lat5Brassica hybrid combinations.The use of 5S rDNA and 35S rDNA probes allow to successfully identify parental genomes in chosen Brassica hybrid plants may be due to transposition or deletion within the chromatin containing rDNA sequences, elimination of marker chromosomes during meiosis, or deletion or amplification of rDNA loci.The observed variable number of chromosomes and the number of 5S and 35S rDNA loci in the genomes of the Brassica hybrids can be a good source of L. maculans resistance in rapeseed breeding programs.Selected B. napus × B. fruticulosa genotypes with highest resistance to L. maculans varied in chromosome number and the number of detected rDNA loci. However, a more thorough study, including the detection of all A genome chromosomes, is required.Three


ABO gene may reduce the activity of glycosyltransferase, resulting in lower levels of A or B antigen expression in red blood cells. Six known splice sites have been identified according to the database of red cell immunogenetics and the blood group terminology of the International Society of Blood Transfusion. Here, we describe six distinct splice site variants in individuals with ABO subtypes.Nucleotide mutations in the ABO gene. The ABO gene haplotypes were studied using allele-specific primer amplification or cloning technology. In silico analytic tools were used to assess the functional effect of splice site variations.The ABO phenotype was examined using a conventional serological method. A polymerase chain reaction sequence-based typing method was used to examine the whole coding sequence of the ABO gene splice sites were identified in nine individuals with ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A and c.374 + 5G > A. c.28 + 1_2delGT was detected in an Aw individual, while c.28 + 5G > A, c.28 + 5G > C, and c.204-1G > A were detected in Bel individuals. c.155 + 5G > A was detected in one B3 and two AB3 individuals, whereas c.374 + 5G > A was identified in two Ael individuals. Three novel splice site variants  in the ABO gene were discovered, all of which resulted in low antigen expression. In silico analysis revealed that all variants had the potential to alter splice transcripts.Six distinct variants in the ABO gene were identified in Chinese individuals, resulting in decreased A or B antigen expression and the formation of ABO subtypes.Three novel splice site variations in the The online version contains supplementary material available at 10.1186/s12967-021-03141-5. Karl Landsteiner discovered the ABO blood group system in 1900, and it was of great clinical significance in blood transfusion and organ transplantation –4. IncomABO gene is located on chromosome 9; its full-length sequence is approximately 24.9 kb, and the coding region is organized into seven exons with 28, 70, 57, 48, 36, 135, and 691 nucleotide base pairs [ABO blood group alleles, including ABO*A1.01, ABO*A1.02, ABO*B.01, ABO*O.01.01, and ABO*O.01.02, are common in the Chinese Han population [ABO gene encodes glycosyltransferase A (GTA) or glycosyltransferase B (GTB), which catalyse the formation of A or B antigens on red blood cells, respectively [ABO*A1.01 and ABO*B.01 alleles in the coding sequence (CDS) region, resulting in four amino acid alterations between GTA and GTB [The  and GTB .ABO gene may affect the activity and/or specificity of glycosyltransferases, resulting in the formation of ABO subtypes. According to the database of names for ABO blood group alleles (v1.1) by the red cell immunogenetics and blood group terminology of international society of Blood Transfusion (ISBT), five splice sites with Aweak or Ael and one splice site with B3 were found in populations from around the world. In this study, we described six different splice site variants of the ABO gene in individuals with ABO subtypes.Although the ABO blood group system is composed of four phenotypes, including A, B, O, and AB, the distribution of ABO phenotypes varies across populations and regions . AdditioIndividuals with ABO subtypes were either blood donors or patients. All specimens were obtained after the individuals provided informed consent. This study was approved by the ethics committee of the Blood Center of Zhejiang Province, China. The difference in these specimens was discovered during routine ABO blood group typing. The specimens placed in tubes with or without EDTA anticoagulant were sent to the immunohaematology reference laboratory in the Blood Center of Zhejiang Province for further analysis.1, anti-B, anti-AB, and anti-H antibody reagents were used . Red blood cells (RBCs) in groups A, B, and O were prepared in the laboratory using fresh blood from three donors of the same type at random.A, B, and H antigens, as well as anti-A and anti-B antibodies, were detected using a conventional serological method , 14. AntABO gene [ABO gene. The amplicons were purified, followed by sequencing and analysis using an ABI 3730 sequencer . SeqScape v2.5 software (Applied Biosystems) was used to evaluate the sequencing data. The ABO gene reference sequence was obtained from GenBank (ID number NG_006669.2), and the ABO genotype was assigned based on nucleotide polymorphism. The ABO allele was nominated in accordance with the ISBT guidelines for red cell immunogenetics and blood group terminology [According to our previous reports, we used the polymerase chain reaction sequence-based typing (PCR-SBT) technique to analyse the entire CDS of the ABO gene , 13, 14.minology .ABO gene were analysed using next-generation sequencing (NGS). First, the ABO gene sequence was amplified using two pairs of primers. In the first pair, the forward and reverse primer sequences were 5’GCGCCGTCCCTTCCTAGCAG 3’ and 5’AGCCACCAACTTCCCCTAGT3’. The primer sequences in the second pair were 5’TACTCACCTATTATTGGCCTTTGGTT3’ and 5’TAGGCTTCAGTTACTCACAACAGGAC3’. The expected lengths of the amplicons were approximately 12,763 and 7250 bp, respectively. The total volume for each PCR amplification reaction was 25 μL, which included 5 × GLX PCR buffer 5 μL , 200 μmol/L dNTP concentration, 0.2 μmol/L primer concentration, 0.625 U GLX Taq enzyme (Takara Bio Company) and 2.5 μL DNA sample. Amplification was performed on an ABI PCR 9700 instrument (Applied Biosystems). The following conditions were used for PCR amplification: predenaturation at 94 ℃ for 1 min, denaturation at 98℃ for 10 s, annealing at 68℃ for 10 min, 30 cycles, and extension at 68℃ for 10 min. The amplicons were digested with Tn5 transposase, and the index was added to construct the library using the Trans NGS Tn5 DNA library prep kit for Illumina . All procedures were carried out strictly according to the manufacturer’s instructions. Following the qualification of the library's quality, the sequences were detected on an Illumina MiSeq Sequencer using the MiSeq sequencing reagent kit . The sequencing data were analysed using the ABO reference sequence  and CLC main workbench 12.0 software , and all polymorphism nucleotides were recorded and analysed.Sequences from the start codon to the stop codon of the ABO gene [A, B, and O alleles were used to amplify the corresponding alleles, and the amplicons were then sequenced and analysed as previously reported [@TOPO plasmid vector according to the manufacturer’s instructions and transfected into competent cells to grow. As previously described, plasmid DNA was extracted as a template for sequencing analysis [Allele-specific primer amplification sequencing or cloning technology was used to haplotype the ABO gene , 13. Forreported , 13. Foranalysis , 13.www.interactive-biosoftware.com/doc/alamut-visual/2.6/splicing.html) [Alamut® software v2.10 was used in conjunction with four splice site prediction tools, SpliceSite Finder-like, MaxEntScan, NNSPLICE, and GeneSplicer, to predict the effects of these splice site variations (ng.html) –18. The ng.html) , 20. A sng.html) .1, anti-B, anti-AB, anti-H, and serum containing known A, B, O group RBCs. All individual RBCs exhibited 3 + or 4 + strength agglutination with anti-H. In the absorption and elution test, B antigen was expressed in ID numbers 2, 3, and 7 specimens, whereas A antigen was positive in ID numbers 8 and 9 specimens. Individuals with ID numbers 4, 5, and 6 had mixed-field agglutination. These individuals were classified as ABO subtypes based on serological characteristics  (sequence between start codon and stop codon in the ABO gene). The ID numbers 1, 2, and 9 specimens were not analysed using the NGS method. Except for the splicing acceptor/donor sites, which were consistent with the PCR-SBT, no variation was observed in the NGS method.Additional file The haplotyping analysis revealed six distinct splice site variants in the ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A, and c.374 + 5G > A , c.204-1G > A (NC_000009.11:g.136133523C > A) and c.374 + 5G > A (NC_000009.11:g.136132791C > T) existed in these databases with frequencies of 0.0009%, 0.0008%, and 0.0004%, respectively. However, c.28 + 1_2delGT (NC_000009.11: g.136150576_77delCA), c.28 + 5G > A (NC_000009.11: g.136150573C > T), and c.28 + 5G > C (NC_000009.11: g.136150573C > G) were identified for the first time in the ABO subtypes (Table GTgggctc) and c.28 + 267 (ggtcctgGTgagagc), with scores of 0.40 and 0.93, respectively. NetGene2-2.42 software predicted c.28 + 267 (ggtcctgGTgagagc) as a donor site but not c.28 + 168.Table GTaggagg) with a score of 0.95 and c.374 + 840 (ctccttaGTaagagg) with a score of 0.51. One of the new acceptor sites was predicted to be located at position c.204–224 (ctcttgccAGtttgtaag) with a score of 0.84. However, some additional spliceosomes resulting from variations in the splice sites might generate partial functional transferases as a result of the RBCs of the probands.In the in silico analysis using the Berkeley Drosophila Genome Project Searches Splice Site Prediction software, several new splice sites were predicted in the c.155 + 5G > A, c.204-1G > A, and c.374 + 5G > A variants. Some new donor sites were predicted at c.155 + 507 , cisAB, etc. Seltsam A et al. reported that the BW phenotype is caused by variations in the CCAAT-binding factor/NF-Y enhancer region of the ABO gene [ABO gene's erythroid cell-specific regulatory element could downregulate transcription in the B(m) allele [ABO variations have been identified in individuals with ABO subtypes to date [ABO gene are located in the CDS region, intron 1 erythroid-specific regulatory element region, splice site, promoter, cis- or trans-regulatory element, etc. Kronstein-Wiedemann R et al. found that miR-331-3p and miR-1908-5p directly target the mRNA of GTA and GTB and that overexpression of these miRNAs in haematopoietic stem cells may result in a significant reduction in the expression of A antigens [ABO gene are associated with some ABO subtypes. Chen DP et al. reported c.155 + 5G > A (IVS3 + 5G > A) in a B3 individual and c.374 + 5G > A (IVS6 + 5G > A) in an Ael individualABO gene splice site result in the formation of new RNA splice sites and therefore novel versions of ABO mRNA.The common ABO subtypes are AABO gene . Sano R ) allele . Numerou to date –29. Thesantigens . Some vaABO gene's full CDS and the sequence of the erythroid cell-specific regulatory element region for ABO subtypes using the PCR-SBT technique. We discovered over 50 novel alleles from ABO subtypes [ABO gene were identified in nine individuals with ABO subtypes. Between 2015 and 2019, our laboratory screened and obtained specimens from 369 individuals with suspected ABO subtypes using a combination of serological and molecular methods.The Chinese population has a high prevalence of ABO subtypes , 14. In subtypes , 13, 33.ABO gene was detected [ABO gene's RNA splicing [ABO*A1.01 allele in the Ael individual, currently referred to as the ABO*AEL.04 allele, is predicted to generate transcripts without exon 6 or exons 5 to 6ABO*AEL.04 individual [ABO*A1.02 allele.Multiple ABO mRNA forms were detected in the normal ABO phenotype by RT–PCR, the majority of which lacked exon 6 , 35. Howdetected , 36, 37.splicing . At leassplicing . While isplicing . The c.3dividual . HoweverCYP17A1 gene could result in cryptic splicing, with the splicing transcripts being included in exon 1 [ABO gene were found to be located in the exon/intron 1 boundary. We hypothesized that these variations would impair adjacent intron splicing and induce alternate activation of some cryptic splice donor sites within exon 1, resulting in aberrant mRNA splicing. One of the predicted splice sites closest to exon 1 can produce a protein with an additional 89 amino acids. Additionally, Kominato Y et al. found alternative exon 1a in the upstream genomic sequence of the ABO gene [Hwang DY et al. found that the IVS1 + 2 T > C in intron 1 of the n exon 1 . In thisABO gene . As a reAlternative 3′ or 5′ splice sites have been shown to be capable of skipping exons in the transcripts . The c.1ABO mRNA forms may be detected in peripheral blood leukocytes [ABO mRNAs in individuals with these variations were not analysed, and their function in vitro was not examined in our study. Further research is needed in the future to determine the actual status of ABO mRNA transcription in the presence of splice site variants.In this study, we predicted splicing transcripts for splice site variations in silico; however, various prediction results for splicing transcripts were discovered using different methods. The scores for the c.155 + 5G > A variant were 0.97 to 0.19 in the NNSPLICE tool and 9.02 to 2.59 in the GeneSplicer tool, and the change ratios were different. Therefore, multiple tools should be used in tandem to predict functional variations. In the normal phenotype, multiple ukocytes . HoweverABO gene splice site variants in individuals with ABO subtypes, including three novel variants, c.28 + 1_2delGT, c.28 + 5G > A and c.28 + 5G > C. Additionally, in silico analysis was used to estimate the potential splicing transcripts for the variants in splice sites. We found that splice site variations in the ABO gene affect splice transcripts, resulting in decreased A or B antigen expression and the formation of the ABO subtype.In this study, we identified six distinct Additional file 1: Figure S1. The heterozygous sequence of the splicing sites in the individuals with ABO subtypes. Arrows indicated the heterozygous variations in ABO gene. Figure S2. The variation sequences of the splicing sites after haploid analysis in the ABO gene. Arrows indicated the variation position.Additional file 1: Table S1. Nucleotide polymorphism of the ABO gene in specimens ID number3 to 8 by the NGS method.


Dear Editor,We previously designed and synthesized a novel benzimidazole compound, DHW‐221.The PI3K/AKT/mTOR pathway is a key mediator of receptor tyrosine kinase signal transduction. The PI3K family can be divided into three categories.50: PI3Kα,0.5 nM; PI3Kβ,1.9 nM; PI3Kγ,1.8 nM; PI3Kδ,0.74 nM), as well as that of mTOR (IC50: 3.9 nM)  than NVP‐BEZ235 Figure . DockingM) Table . CellulaM) Table . AdditioM) Table  and compM) Table , suggestM) Table . Combine50 is shown in Table To investigate the effect of DHW‐221 in vitro, SRB assay was performed. DHW‐221 exhibited excellent inhibitory activity concentration‐ and time‐dependently Figure  without The PI3K/AKT/mTOR pathway is strongly linked to the regulation of cell cycle progression and apoptosis during cell proliferation.Activated PI3K/AKT/mTOR signaling participates in tumor metastasis, invasion,In conclusion, DHW‐221 was identified as being a dual PI3K/mTOR inhibitor, and displayed excellent anti‐NSCLC activity, including the inhibition of cell proliferation, migration, invasion, and angiogenesis, by blocking the PI3K/AKT/mTOR signaling pathway both in vitro and in vivo. Additionally, DHW‐221 was found to promote cell cycle arrest and mediate apoptosis through the mitochondrial pathway. Combined, these findings indicate that DHW‐221 has potential as a novel anticancer drug targeting PI3K/mTOR in NSCLC to inhibit tumor initiation and progression.The authors declare no conflict of interest.All animal experiments were conducted in accordance with the guidelines of the internationally recognized guidelines for animal studies: reporting in vivo experiments. This study was approved by the Ethics Committee for Animal Experiments of Shenyang Pharmaceutical University.Prof. Q.C. Z. and Dr. H.W. D. for supervision and funding acquisition; X.C. Q. for conceptualization, methodology, validation, formal analysis, writing‐original draft, writing‐review and editing, and project administration; M.Y. L. and Y.T. W. for investigation and resources; others for editing and proofread. All authors read and approved the final manuscript.All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Used research material is available by request from the corresponding author or from commercial sources when applicable.Supporting informationClick here for additional data file.


Fourth, combined mass defect filter with background subtraction model in soft of compound discovery, all peaks were constructed to filter potential metabolites after retention time alignment and ion filtration, which could remove large amounts of interference ions. Besides, it can predict potential biotransformation, promoting to understand how to metabolize the drug. This provides multiple possibilities and prevents us conjecturing the potential metabolites blindly. Finally, the verification procedure was implemented through exporting the structure and MS2 spectrum to the analytical tool of Mass Frontier. The proposed strategy significantly improved the targeted detection and identification for metabolites in vivo. A total of 47 metabolites were tentatively characterized in the plasma, urine, and feces samples after oral administration of prucalopride. This study could provide a valuable reference for systematic metabolite profile of drug in vivo.Prucalopride was widely used for chronic constipation, which is difficult to be adequately relieved by laxatives in adult patients in clinic. Due to the difficulty in metabolite identification, metabolic process of prucalopride had not been investigated in vivo. In this study, an efficient strategy was proposed for comprehensive metabolite profiling of prucalopride after oral administration in rat plasma, urine, and feces samples. This strategy was composed of five steps. First, the samples at multiple time points after oral administration were collected to increase the representativeness of the samples. Second, different sample preparation methods were investigated to obtain superior extraction efficiency. Third, the raw data of test sample and blank sample were acquired using ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry under the positive and negative full-scan/dd MS The drug-related metabolites could provide abundant information related to the side effects and formation of active or other toxic metabolites . HoweverAs one of the leading edge techniques in the drug metabolite research, ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) has developed rapidly in recent decades. It could shorten the analysis time and improve the analysis efficiency by optimizing the parameters of chromatography–mass spectrometry. Meanwhile, there are several scan modes, such as information-dependent acquisition (IDA), selected reaction monitoring (SRM), and precursor and neutral loss scan, which have their own superiorities and characteristics. But due to its drawbacks, including relatively insensitive scan functions, long duty cycle, and multiple injections to monitor multiple precursors and neutral, it was used in the drug-related metabolite studies previously . While d4 receptor to act primarily on different parts of the lower gastrointestinal tract, promoting cholinergic, nonadrenergic and noncholinergic neurotransmission by enteric neurons . This strategy could be divided into five steps. The flow chart is shown in Prucalopride (purity >99%) was obtained from Sigma Chemical Co. . Acetonitrile and methanol of HPLC grade were purchased from Fisher Scientific . The chemical of formic acid was of HPLC grade and purchased from Aladdin Industrial Corporation . All other reagents were of analytical grade. Deionized water was purified via a Milli-Q system .Male Sprague–Dawley (SD) rats, weighing 200 ± 20 g, were acquired from the Experimental Animal Center of Zhengzhou University . In order to adapt to the environment, all animals were fed under the controlled temperature (20 ± 2°C), humidity (60 ± 5%), and 12-h-light/12-h-dark cycle for one week before the experiment. All protocols of animal experiments were in accordance with the Regulations of Experimental Animal Administration issued by the Animal Ethics Committee of First Affiliated Hospital of Zhengzhou University.18 column  was employed and the column temperature was maintained at 40°C. The mobile phase was composed of A (0.1% formic acid in water) and B (acetonitrile) with the following gradient elution: 0.0–3.0 min, 5%B; 3.0–10.0 min, 5–30%B; 10.0–27.0 min, 30–80% B; 27.0–28.0 min, 80–100% B; 28.0–30.0 min, 100% B; and 30.0–32.0 min, 100–5% B at a flow of 0.2 ml/min. The autosampler was conditioned at 10°C, and the injection volume was 5 µL for analysis.Chromatographic separation was performed on an UHPLC Dionex Ultimate 3,000  equipped with a binary pump solvent management system with online degasser, autosampler, and column oven. A Waters ACQUITY UPLC® BEH C2, 1) full MS: resolution: 70,000; automatic gain control (AGC) target: 3.0 e6; maximum injection time (IT): 200 ms; scan range: 80–1,200 m/z; 2) dd-MS/MS2: resolution: 17,500; AGC target: 1.0 e5; maximum IT: 50 ms; loop count: five; isolation window: 2.0 m/z; NCE/stepped: 20, 30, and 40; and dynamic exclusion: 10.0 s. Nitrogen was used for spray stabilization and as the collision gas in the C-trap. All data collected in profile mode were acquired and processed using Thermo Xcalibur 3.0 software. Regarding the sequence of analysis, all samples were randomized. Blank sample containing barely the solvent was added after every 20 samples, aiming to elute residuals or other impurities which were retained by the chromatography column in the previous spectra records.A Q-Exactive high-resolution mass spectrometry using a heated electrospray ionization (HESI) ion source was tandem to the UHPLC system. The optimized parameters of mass spectrometry were illustrated as below: spray voltage: + 3.5 kV or −2.8 kV; sheath gas pressure: 40 arb; aux gas pressure: 10 arb; sweep gas pressure: 0 arb; capillary temperature: 320°C; auxiliary gas heater temperature: 300°C; S-lens RF level: 50 V; scan mode: full MS/dd-MSBlood samples of 300 μL were collected via the orbital vein at 0 (predose); 10 min; 30 min; 1, 2, and 4 h after oral administration of 1.0 mg/kg prucalopride. The heparin sodium was added to exert the anticoagulant action, and the samples were centrifuged at 3,500 rpm for 10 min at 4°C, then all plasma samples were pooled together. Blank urine and feces samples were collected by putting the SD rats into the metabolic cages, and the rats were fasted but drunk freely. The samples after administration were collected the same as the above.Protein in rat plasma sample (100 µL) was precipitated with the acetonitrile (300 µL). After vortexing for 3 min, the sample was centrifuged at 13,000 rpm for 10 min. Then, the supernatant was transferred into a 1.5-ml centrifuge tube and evaporated to dryness in a vacuum centrifugal concentrator. The residue was dissolved in 50 µL of 50% methanol with vortex mixing for 1 min, and the centrifugation process was the same as the above for 5 min. Finally, 5 µL supernatant was injected into the UHPLC-MS/MS system for analysis.The urine sample (200 µL) was diluted with 200 µL pure water, vortexing for 1 min. After centrifuging at 13,000 rpm for 10 min, the supernatant was filtered by 0.22 µm MCE microporous filter membrane into sample bottle for analysis.The feces sample (0.2 g) was weighed precisely into the 4 ml EP tube, adding 2 ml methanol and ultrasonic dissolving, then the supernatant was transferred and centrifuged at 13,000 rpm for 10 min. After that, the supernatant was filtered by 0.22 µm nylon microporous filter membrane into the sample bottle for analysis.5 in order to differentiate the metabolites from the base peaks. Next, the structure was speculated based on the fragmentation pattern and relative purified ion lists, which possessed the message of molecular formula, molecular weight, retention time, potential biotransfromation, etc. The structure and mass spectrometry were further exported to the Mass Frontier 7.0  to observe whether the structure and fragments match.The HRMS data of all samples were processed in the Compound Discoverer 2.1  to extract the metabolite-related dataset according to the structural correlation between the drug and its metabolites. Then, the information of blank samples as control from the above dataset was removed by setting “has no value” in blank file after the retention time alignment and database search. And the peak area of ions was equal or greater than 10For different samples, the process of sample treatment was investigated. In order to extract and detect more metabolites of prucalopride from plasma samples, we systematically investigated the sample preparation method, including the commonly used protein precipitation (PP) with methanol and acetonitrile and liquid–liquid extraction (LLE) with ethyl acetate and n-butanol. In order to extract and detect more metabolites of prucalopride from urine samples, direct dilution (DD) with pure water, liquid–liquid extraction (LLE) with ethyl acetate, and solid phase extraction (SPE) with Oasis® HLB cartridges were done. In order to extract and detect more metabolites of prucalopride from feces samples, ultrasonic extraction with methanol, liquid–liquid extraction (LLE) with ethyl acetate, and solid phase extraction (SPE) with Oasis® HLB cartridges were done. By comparing the extraction efficiency, the treatment conditions of plasma samples (100 µL) were finally protein precipitation (PP) with acetonitrile (300 µL). Because there were a large number of metabolites in urine and the polarity of metabolites is quite different, the urine sample (200 µL) was diluted with 200 µL pure water in order to ensure that more metabolites could be detected. While for feces samples, the sample preparation was adopted by ultrasonic extraction with methanol.In order to obtain higher responses, suitable retention behaviors, and symmetrical peak shapes for the analyte, various mobile phase conditions  were tested as potential mobile phases. Finally, acetonitrile–water containing 0.1% formic acid was chosen for its good peak shapes. It was also noted that gradient elution could dramatically narrow the peak shape and improve the response intensity and resolution.2 mode and all-ion fragmentation (AIF). All ions entered into HCD for collision under the AIF mode. Due to no selectivity for parent ions, this data acquisition mode was rarely used. While under the full MS/dd MS2 mode, the matching fragment information of primary and secondary mass spectra simultaneously. So the full MS/dd MS2 mode was adopted for metabolite profiling of prucalopride in rats. Meanwhile, the other MS parameters, such as the gas pressure, the heated temperature, and the spray voltage, were all well optimized for maximum response. The optimal parameters settings are shown in Mass Spectrometric Conditions.For MS parameters optimizing, scans were simultaneously implemented in both positive and negative ion detection mode. In Q-Exactive instrument, there were two modes that could be used to perform an accurate qualitation. They were full MS/dd MSMetabolite identification, still, is a hard nut to crack at present. There are not only complex endogenous components but also abundant metabolites, which display a high degree of structural variability. The established strategy could solve the matters in the metabolite annotation to a great extent, which was proposed as follows. After the pretreatment, the data extraction was operated with the IDA-based data acquisition, which was based on the retention time alignment, database searching, scoring, etc. Then, the extracted ion lists were filtered by setting parameter in manual. Furthermore, the tentative identification was conducted.m/z × RT dimensions) in the input files. Then, the following parameters such as parent compound or compounds, number of dealkylation and dearylation steps, number and type of transformation steps, and list of possible adduct ions would be set based on the experimenter’s demands and metabolic type to search the metabolites of detected compounds. When the application finds a matching compound, it will mark the pathway for fast search. According to the above, an expected ion list was generated.After importing the raw files of control and samples into the soft of compound discovery, the alignment algorithm looks for matching features  can offer a number of enhancements designed to increase productivity. The generated ion list after the data handling procedure from CD contains the messages of molecular formula, molecular weight, retention time, and potential transformation. And in the same window interface, the mass spectrometry is presented along with the corresponding compound information and the parent drug is used as the reference in the bottom of the mass spectrometry with the corresponding compound in the upper, which is convenient for the structure fragment alignment. Therefore, we can deduce the metabolites’ structure by fragmentation regulation of mass spectra and transformation suggested. Then, the mass spectra called out from the Xcalibur 3.0 is exported into the MF, which automatically generated fragments and detailed fragmentation and rearrangement mechanisms from the chemical structure. If the theoretical fragmentation through the MF could match the MSIn this study, the plasma, urine, and feces were collected for the metabolite identification. The raw data files containing experiment group and control group were uploaded into the CD, a total of 2,717, 5,869, and 5,310 ion were extracted in the plasma, urine, and feces samples with the software running, respectively. Then, the remaining ions were handled further, just 340, 777, and 300 ions ultimately left. According to suggested transformation pathways, molecular formula, and mass spectrometry fragment ions to predict the underlying metabolite structures which can be confirmed by MF in turn, we identified 47 tentative metabolites in total. All the information of 47 metabolites of prucalopride detected by UHPLC-Q-Orbitrap HRMS is listed in 18H26ClN3O3) was extracted, and the excimer ion peak of m/z 368.17354 [M + H]+ was generated , as shown in In the positive ion mode, the ion flow chromatograph of prucalopride reference  was the ion at m/z 237.08664, which is the product of acetylation with the reaction site in amino group or hydroxyl group, and M2 was detected at 1.363 min with m/z 286.21222, which was the other part of parent drug and bound with ornithine in vivo. The fitted chemical formula of M3 was C14H18ClN3O2, a difference of C4H8O with prucalopride which indicated the loss of alkyl chain moiety, and the characteristic ions were in accordance with the speculative structure. The M4 was found at 8.500 with m/z 354.15756 , so this metabolite showed the characteristic fragments with m/z 279.09, 196.02, and 98.10. M5 and M6 (m/z 368.13687 and m/z 370.15259) were the oxidation products in piperidine ring or furan ring with the methyl group leaving. M7 presented at 9.432 min with m/z 382.15256, and only oxidation in furan ring was happened; thus, this metabolite had common fragments with the parent drug, m/z 173.16, 156.14, 98.10, and 70.07. M8 was yielded via oxidation in piperidine ring and furan ring and demethylation of the alkyl chain. Metabolite M9 was the oxidation product in the furan ring and presented precursor ion m/z 384.16846 with RT 8.336 min. Due to the tiny structural changes, the characteristic fragment ions  were detected.There were nine metabolites M1-M9;  identifi10–M37; 10–M12  were detected as the leaving of the alkyl chain moiety with m/z 294.09991, 296.11600, and 310.09479, respectively. And, compared with the metabolite M11, M10 took off two hydrogen atoms to generate a double bond in furan ring, and M12 was oxidized in the furan ring. Metabolite M13  was also the result of the ornithine conjugation, but different from the metabolite M2, the ornithine was combined with the acylamino group of the other side of the drug structure. The parent drug prucalopride was a chlorine-containing compound; thus, oxidative dechlorination and reductive dechlorination happened when it was metabolized and yielded the metabolite M14 and M15 at 9.711 and 8.162 min with m/z 334.21198 and 350.20676.A total of 28 metabolites M10–M37;  were fou16 was detected at 8.720 min with the mass charge ratio 352.14188. And the fitted molecular formula was C17H22ClN3O3, just a difference of 2H with the M4 (C17H24ClN3O3). The typical fragment ions  that indicated the part of furan ring had a deviation of 2 Da with the metabolite M4 suggesting that M16 lost 2H from the furan ring in the basis of M4A.Metabolite M17, M19, and M20 were the products of the demethylation and were found at 8.825, 8.560, and 6.900 min with m/z 366.12003, 368.13693, and 370.15237, while oxidation or dehydrogenation also took place in different positions of the piperidine ring or furan ring. Metabolite M18 just took off two hydrogen atoms in the basis of parent drug; therefore, m/z 277.07 , 194.00 , 173.06, 156.14, 98.10, and 70.07 were detected which were the typical ion fragments of the prucalopride.Metabolites M21 (RT 9.366 and m/z 382.15237), with oxidation, dehydrogenation, or demethylation in other regions. M22 was found at 6.859 min with m/z 384.13162, and this compound might be the same substance with the M8 according to the comparison of MS2 spectrum. And this assumption was confirmed by the MF. M23 was the oxidation product in the furan ring and presented precursor ion m/z 384.16785 with RT 8.201 min. The mass charge ratio and retention time were approaching between M9 and M23 which suggested that they might possess the same structure. The MS2 spectra of both were analogous and the result from MF confirmed the above assumption. M24 was the isomer of M23 with the oxidation position in the piperidine ring and exhibited precursor ion m/z 384.16800 with RT 10.101 min. And it showed the characteristic fragment ions .The oxidation in the “N” of piperidine ring also happened to generate metabolite M25, M26, M27, M30, and M31 all possessed two oxidation sites in piperidine ring or furan ring which was a kind of main metabolic pathway of the parent molecule, their mass charge ratios were 396.13153, 398.14740, 398.14603, 400.16302, and 400.16278, and the retention time was 8.825, 9.578, 7.990, 9.447, and 9.280, respectively. In the basis of M30, the demethylation and oxidation in the alkyl side chain happen to emerge M29, which was found at 7.532 min with m/z 400.12634. The [M + K] ion of the metabolite M28 was m/z 398.14911 and was detected at 10.303 min, the acetylation occurred with the hydroxyl group after the demethylation with the hydrogen atom dropping out in furan ring and chlorine atom leaving away the benzene ring through reductive dechlorination.Metabolites M32 was an acetylated product of the parent drug, combined with the amino group of benzene ring and detected at the 8.600 min with the m/z 410.18387. Typical fragments  were found in the MS2 of this metabolite. M33  was a ring-opening compound with the furan ring opening and oxidation conducted. M34 was an interesting metabolite which was a palmitic acid derivative exhibited at 11.117 min with m/z 453.34296. Such a way to metabolize was rare in the current literature reports and united closely with the endogenous substances. Therefore, the discovery of a metabolic mode like this was meaningful for the metabolite exploration.M35, M36, and M37 were the phase II metabolites identified as glucuronic acid conjugates preliminarily. The detailed information was summarized in 35 was 176 Da more than the parent drug, it was a glucuronic acid conjugate obviously. The fitted molecular formulas for M36 and M37 were C24H32ClN3O10 and C24H34ClN3O10, indicating that an oxidation happened in the basis of M35 (C24H34ClN3O9). And the structure was exported to the MF to confirm the matching between the structure and the MS2 spectrum, and the outcome corroborated our speculation.The metabolites of M38–M47, m/z 284.16068. M39 was detected at 8.052 with m/z 296.11591. The retention time and mass charge ratio were close to the M3, and the MS2 spectrum was analogous; thus, we assumed that the M39 and M3 were the same substance. Metabolite M40  was the reactive product of dehydrogenation and generated typical fragment ions m/z 194.00, 173.16, 156.14, and 98.10. The fitted molecular formula for metabolite M41 was C17H20ClN3O4, which was found in the urine (M17) as well. Through the comparison of both, M41 was considered as the same substance with M17. M42 and M43 were yielded via demethylation and oxidation in different sites, with RT 8.568 and 6.956 min and m/z 368.13687 and 370.15262, separately. The metabolite M44 was C18H24ClN3O4, and this metabolite was detected in the plasma and urine as well. The retention time and MS2 spectrum were matching; thus, these metabolites were the same substance. M45 was detected at 6.868 min with m/z 384.13156. This compound was generated through the demethylation and oxidation in the piperidine ring or furan ring which was found in the plasma and urine as well. The precursor ion of M46 was m/z 384.16791 along with RT 8.323, the hydroxylation reaction happened in the furan ring, and the fragment ions  were detected as the diagnostic ions. M47 was the result of the oxidative ring–opening reaction, with RT 7.880 min and m/z 384.16800. The detailed information is showed in In the feces samples, 10 metabolites M38–M47,  were fou18H26ClN3O3, with MS spectrum including the characteristic product ions of the m/z 279.09, m/z 196.02, m/z 173.16, m/z 156.12, m/z 98.10, and m/z 70.07. And based on the drug and metabolites’ structural characteristics, the changes mainly contained: oxidation or desaturation of piperidine ring or furan ring in diffident positions, demethylation of alkyl chain moiety in piperidine ring, separation of alkyl side chain, amide bond breaking, opening of piperidine ring or furan ring and conjugation reaction combined with the above.The entire process optimization strategy was applied for comprehensive in vivo metabolite profiling of prucalopride in rats, and 47 tentative metabolites were identified in total. The MS2 spectrum and corresponding fragment ions of prucalopride in In this study, an entire process optimization strategy was proposed for comprehensive in vivo metabolite profiling of prucalopride in rat plasma, urine, and feces samples after oral administration based on ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution mass spectrometry. The strategy was composed of sample collection, optimization sample preparation, and chromatographic and mass spectrometry conditions, establishment of MDF and BS model in CD, and structural annotation and metabolites identification. The proposed strategy was applied into the prucalopride and 47 metabolites were identified in total, among which 41 metabolites were not previously reported. Compared with other current methods, this strategy is time saving and easily processable. And this method not only can solve the crucial problem obstructing in the data processing and identification of metabolites more efficiently, but also provide convenience for confirmation of the structural reliability. Especially for identification of complex components’ metabolites, for example, natural medicine, this method could extract metabolite-related dataset based on the components one by one and obtain respective related data, and this is a great breakthrough in the field of the metabolite identification.


The highest TB content was observed in T2 (209.49 ± 14.91), followed by T3 (18.65 ± 1.01) and later T4 (12.96 ± 0.55). The highest AA was observed on T2 after 14 days (ABTS˙ 0.819 mM TE/100 g and DPPH˙ 0.343 mM TE/100 g), and the lowest was found on T1 at day 14 (ABTS˙ 0.526 mM TE/100 g and DPPH˙ 0.094 mM TE/100 g). A high content of TP was observed (7.13 to 9.79 mg GAE/g). The TPC varied between 11.38 to 12.56 µg/mL. The addition of beet extract significantly increased AA in yogurt, betalains being the main compounds responsible for that bioactivity.Beet has been used as an ingredient for functional foods due to its high antioxidant activity, thanks to the betalains it contains. The effects of the addition of beet extract (liquid and lyophilized) on the physicochemical characteristics, color, antioxidant activity (AA), total betalains (TB), total polyphenols (TP), and total protein concentration (TPC) were evaluated on stirred yogurt. The treatments  exhibited results with significant differences ( Dairy derivatives are the main segment among functional foods, representing around 74% of all functional products .Yogurt iBeta vulgaris sp.) represent the main commercial source of betalains [Beta vulgaris sp.) encapsulated with different carrier agents on the physicochemical characteristics, color, betalain and polyphenol content, and antioxidant capacity of yogurt during shelf life. The treatments were as follows: T1-natural yogurt as control, T2-yogurt added with beet juice, T3-added extract of beet encapsulated with maltodextrin, and T4-yogurt added with extract of beet encapsulated with inulin.Beet , compared to T1 (3.87 ± 0.31) and with T2 (3.88 ± 0.36) (p < 0.05). Regarding the percentage of fat, treatments T3 (5.49 ± 0.81) and T4 (5.36 ± 0.32) had a higher content in contrast to T1 (5.09 ± 0.45) and T2 (5.02 ± 0.86) (p > 0.05) between the treatments. Other studies indicate that yogurts added with rubas (Ullucus tuberosus) concentrate, curuba (Passiflora mollissima Bailey) extract, chocolate, and oats also showed a significant increase in protein and fat content [p < 0.05) in the four treatments, this behavior could be attributed to the fact that the beet extracts were in different forms (juice or powders), since in treatments 3 and 4 added with powders, the moisture content was lower (78.10 ± 0.17 and 78.60 ± 0.33) than in T1 (78.94 ± 0.15), likewise, the moisture percentage of T2 was higher (79.92 ± 0.12) than T1. Regarding the percentage of ash, there was no significant difference (p > 0.05) between the treatments.The treatments added with encapsulated beet extract (T3 and T4) presented higher protein content , showing a significant difference ( ± 0.86) , althoug content ,51,52,53p < 0.05). These values were 4.13 ± 0.052 on day 1, 4.04 ± 0.030 on day 7, and 3.95 ± 0.010 on day 14 (p < 0.05) between treatments and days of storage . In relation to the parameter a* (p < 0.05). In general, the b* values also showed an increase over time (p < 0.05). The degradation of BC leads to the formation of compounds with a yellow color and, this is reflected in the increase in the b* parameter [p < 0.05) between treatments and over time; however, treatments added with beet powder (T3 and T4) did not show differences (p > 0.05). The color change (∆E) between treatments and days was also different (p < 0.05).The values of the color parameters L*, a* and b* can be observed in meter a* , this waarameter . Color sp < 0.05) were found for the AA variable between the treatments (p < 0.05) in ABTS˙  powders values of 14.67 mg of GAE/g have been reported [A high content of TP was observed from 7.13 to 9.79 mg of GAE/g,  referrinreported . The conreported , where rreported . Storagereported ; it is wp < 0.05) in TB content were observed after 7 (191.65 mg/100 g) and 14 days of storage compared to its content on day 1 (209.40 mg/100 g). The TB content increased with the fermentation time, and that event could be due to acid hydrolysis and the bioconversion of condensed phenols [Several investigations have been carried out on the use of encapsulating agents to prevent the degradation of betalains ,58,63,64 phenols ,65. In a phenols . Of the Encapsulation with maltodextrin was effective in stabilizing betalains after 14 days of storage . Unlike Beets have been reported to have higher levels of betacianins (BC) than betaxanthins (BX) ,70,71,72p < 0.05) between treatments and over time. This behavior is probably due to the extracellular proteins secreted by bacteria during the fermentation time [The TPC  ranged fion time . Furtherion time .p < 0.0001) was found between L* and TB and a positive correlation  between the value of a* and TB. This indicates that the higher the TB content, the lower the luminosity and the higher the concentration of red color. These correlations have already been reported in prickly pear pulp micro particles [p < 0.0001) between TP and TB. It is important to highlight that betalains are the main polyphenols present in beets [p < 0.0001),  between TB and BC and between TB and BX, respectively. On the other hand, a high positive correlation  was found between TB and AA, which could be due to the presence of betalamic acid, since it acts as an antioxidant agent. However, if the acid molecule does not have hydroxyl groups, betalamic acid does not have antioxidant capacity [p < 0.0001) between AA and TP. The increase in AA during storage can occur because in these periods, new phenolic compounds capable of extinguishing radicals are released or formed [p < 0.0001) and TP  and TB . These correlations could be to compounds with high AA that were formed or released during storage, thanks to the adsorption of water [articles ,75,76 anarticles . Like Piarticles , observein beets ; therefocapacity . Likewisr formed . A positof water . Storageof water ,73.®, Gomez Palacio, Dgo., Mexico), whole cream , powdered milk , table sugar, and natural yogurt  were purchased in a local market in Chihuahua, Mexico. Maltodextrin (DE 10) and agave inulin were purchased from Sigma-Aldrich .Between March and July 2018, beet, whole milk  (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, ammonium salt, potassium persulfate, Folin–Ciocalteu, sodium carbonate, gallic acid, trichloroacetic acid, citrate, and phosphate standard reagent were analytical grade and obtained from Sigma-Aldrich . J. T. Baker provided high-performance liquid chromatography (HPLC)-grade methanol and HPLC-grade water . A deionizer was used to obtain deionized water .To elaborate yogurts, milk was heated to 95 °C for five minutes, then cooled to 45 °C, and then, powdered milk (8%), cream (3%), sugar (3%), and the inoculum (10%) were added. Next, yogurts were incubated  at 42 °C for approximately 4:30 h, until reaching a pH of 4.2. Once this acidity was reached, the yogurt was shaken and stored at 4 °C for 12 h. Subsequently, the beet extracts (liquid or lyophilized) were added to the yogurt; a shake was made, and finally, these were stored at 4 °C for 14 days.A household extractor  was used to extract the juice from the beets. The encapsulating agents  were added to the red beet juice at room temperature at an amount of 30 g of dry matter per 100 mL, as described by Antigo et al. . Next, tFour treatments with three repetitions were elaborated under a completely random statistical model. The description of the treatments was based on the addition of beet extracts (liquid or lyophilized and encapsulated). Control treatment was yogurt without the addition of beet extract (T1), yogurt added with 100 mL of liquid beet extract per liter (T2), yogurt added with 600 mg of beet extract encapsulated with maltodextrin per liter (T3), and yogurt added with 600 mg of beet extract encapsulated with inulin per liter (T4). Treatments were kept in refrigeration (4 °C) for 14 days, taking samples on days 1, 7, and 14 to evaluate color, pH, syneresis, betalains, and polyphenols content and antioxidant activity. On day 1, physicochemical analysis  of yogurts were determined.The chemical composition of the yogurt samples corresponding to day 1. Moisture, ash, fat, and protein content were determined in triplicate according to the methods established by the AOAC .® centrifuge, USA) at 2800× g for 10 min, at 4 ± 1 °C. The supernatant was weighed and expressed as a percentage in relation to the initial weight of the yogurt.The pH was measured in 20 g of each sample with a digital potentiometer (Orion Versa Star) previously calibrated and syneresis was evaluated using the method described by Özturk and Óner ; 10 g of®, Japan) evaluating the L*, a* and b* parameters. Where L* is an indicator of luminosity (from black to white), a* is an indicator that goes from green  to red , and b* indicates shades from blue  to yellow . The samples were placed in plastic containers with a capacity of 200 mL. The determinations were made in triplicate, and the L*, a*, and b* values were used to calculate Chroma* (C*), Hue angle (h°), and ∆E.Color was measured with a Model CR-410 colorimeter ChromHue angle refers to hues and can range from 0° (pure red), 90° (pure yellow), 180° (pure green), and 270° (pure blue). This parameter was calculated using the following equation :(2)Hue∘=E*) in each of the treatments, the average of the readings of the parameters L*, a* and b* was used using the following equation, and the control treatment was the reference of the readings [cL, c,ah and cbh = control parameters and sL, s,ah and sbh = parameters for the different treatments.For the yogurt color difference (represented as Δreadings :(3)ΔE=Ls® J-26 XPI. Beckman Coluter®, USA) at 10,000× g for 30 min at 4 °C. The supernatant (aqueous extract) was filtered with Whatman ™ filter paper  with 125mm pore diameter. Said extract was centrifuged again with the conditions mentioned above. The supernatants were filtered through a 0.22 µm pore polyethylene filters for the determination of antioxidant capacity and polyphenol content . To determine betalain content, the extract was filtered on 0.45 µm pore paper . All extracts were stored at −20 °C until analysis.To obtain the aqueous extract, the methodology reported by Torres-Llanez et al.  was appl2 = 0.9967) derived from the Trolox calibration curve.Antioxidant activity by the ABTS ) methodology was conducted according to Thaipong et al. . First, 2 = 0.9994) derived from the Trolox calibration curve.Antioxidant activity by the DPPH  methodology was realized according to Thaipong et al.  with sliThe ABTS and DPPH standard curves were executed according to Thaipong et al.  methodol2 = 0.9991) and reported in mg gallic acid equivalent (mg GAE/g).The polyphenols content was determined according to Singleton and Rossi  with somThe standard curve for polyphenols content was obtained following Xu and Chang, 2007  methodolg  for 10 min at 25 °C. Finally, using a 0.45 µm pore polyethylene filter  supernatants were filtered through. Samples were stored at −20 °C for further analysis.The extraction of betalains was done according to Güneşer . For thiA = value at maximum absorption (534 for BC and 480 for BX) at 600 nm, DF = dilution factor, MW = molecular weight (550 g/mol for BC and 308 g/mol for BX), V = volume of the solution (1000 mL), Є = molar extinction coefficient , and L = length of the reading cell (1 cm).Total betalains content was determined according to Ruíz-Gutiérrez et al. . McIlvaiBC and BX quantifications were done separately, and the findings were combined to get the BT content. These determinations were done in triplicate, and the results were expressed in milligrams per 100 g of powder.2 = 0.9973) and reported in µ/mL of BSA.The total protein concentration was evaluated following the methodology of Bradford . For thiFor the calibration curve, bovine serum albumin (BSA) at different concentrations  were prepared and the absorbance of these were linearized to obtain the regression equation of the calibration curve.Yij = response variable measured in the j-th repetition of the i-th treatment, µ = general mean common to all observations, Ti = effect of the i-th treatment, and Ɛij = random error measured in the j-th repetition of the i-th treatment, which was assumed to be identically and independently distributed in a normal way with mean µ and variance σ2. When there were differences across treatments, the Tukey’s and Duncan’s tests were used to perform a multiple comparison of means.All analyses were conducted using SAS 9.0 program . To determine the type, strength, and significance of their linear association, an analysis of correlations between pairs of response variables was performed, using the CORR procedure. Yogurts proximal analysis were analyzed using the ANOVA procedure, through the following model:Yij = response variable measured in the k-th repetition of the i-th treatment evaluated in the j-th storage time, µ = general mean common to all observations, Ti = effect of the i-th treatment, Pj = effect of j-th storage time, and Ɛijk = random error measured in the j-th repetition of the i-th treatment evaluated in the j-th storage time, which was assumed to be identically and independently distributed in a normal way with mean µ and variance σ2.For the analysis of the physical characteristics  and the bioactivities  measured over time, an analysis was carried out with the MIXED procedure, based on the following model:The results of this research confirm that beet extracts (liquid or lyophilized) can be added to yogurt to increase its functional properties. The addition of beet extracts significantly increased the antioxidant activity of the yogurts; where betalains and other polyphenols are the main compounds responsible for said bioactivity. Meanwhile, encapsulation of beet extract by lyophilization turned out to be an effective method to stabilize betalains. However, it was not possible to determine whether maltodextrin or inulin was better as an encapsulating agent, as the AA of the yogurt added with these varies during storage time. The mechanisms that affect antioxidant activity during fermentation are considerably varied; therefore, it is recommended to study the AA of yogurts added with liquid and/or lyophilized beet extract, with the same betalain content to obtain comparable results. The shelf life of the product could be increased to observe the behavior of betalains and polyphenols in yogurt after 14 days. In addition, it would be interesting to carry out the microbiological and economic study of this yogurt.