As one of the most important post-translational modifications (PTMs), phosphorylation refers to the binding of a phosphate group with amino acid residues like Ser (S), Thr (T) and Tyr (Y) thus resulting in diverse functions at the molecular level. Abnormal phosphorylation has been proved to be closely related with human diseases. To our knowledge, no research has been reported describing specific disease-associated phosphorylation sites prediction which is of great significance for comprehensive understanding of disease mechanism. In this work, focusing on three types of leukemia, we aim to develop a reliable leukemia-related phosphorylation site prediction models by combing deep convolutional neural network (CNN) with transfer-learning. CNN could automatically discover complex representations of phosphorylation patterns from the raw sequences, and hence it provides a powerful tool for improvement of leukemia-related phosphorylation site prediction. With the largest dataset of myelogenous leukemia, the optimal models for S/T/Y phosphorylation sites give the AUC values of 0.8784, 0.8328 and 0.7716 respectively. When transferred learning on the small size datasets, the models for T-cell and lymphoid leukemia also give the promising performance by common sharing the optimal parameters. Compared with other five machine-learning methods, our CNN models reveal the superior performance. Finally, the leukemia-related pathogenesis analysis and distribution analysis on phosphorylated proteins along with K-means clustering analysis and position-specific conversation profiles on the phosphorylation site all indicate the strong practical feasibility of our easy-to-use CNN models. Post-translational modifications (PTMs) of proteins are a pivotal mechanism regulating cellular functions by the covalent and generally enzymatic modification, which plays vital roles in regulating various biological processes . ProteinBased on the available phosphorylation site data, the machine/deep-learning methods have been proposed for phosphorylation site prediction. Among them, traditional machine-learning models were developed by manually extracting effective features to represent phosphorylation site information, such as Shannon entropy, relative entropy, information gain, protein disordered property, the average cumulative hydrophobicity, etc. ,11,12. NDespite the tremendous potential of the deep-learning, the challenge in developing deep-learning-based prediction methods is the requirement of large amounts of data . Due to It has been confirmed that phosphorylation sites are associated with altered protein functions  and abnoAs we know, leukemia is a common blood malignant tumor caused by the anomalous growth of leukocytes and diseased leukemia cells can enter blood to affect the metabolism of normal cells in the body . LeukemiMost existing computational methods for phosphorylation site prediction are based on manual feature extraction. Existing manual feature extraction techniques are directed at one-sided material information of the phosphorylation site, which cannot adequately describe the complex biological properties of phosphorylated modification sites, and this would probably result in incomplete or biased feature representation . Deep leIn this work, with only one convolution, pooling and dense layer respectively, an easy-to-use CNN architecture was developed for predicting leukemia-related phosphorylation sites just using the protein primary sequences. Here, the phosphorylation site data were collected for three leukemia classes, including myelogenous, lymphocytic and T-cell leukemia. Since myelogenous has the largest dataset and could provide sufficient samples to achieve a CNN model, myelogenous-related S/T/Y phosphorylation site prediction models were constructed by our CNN architecture. To evaluate the performance and prove the advantage of this method, we also used other five machine-learning methods to construct the prediction models. Through comparisons, our CNN model yields promising performance, which are superior to the five other models, when all models are based on sequence information. Finally, the pre-models derived from myelogenous-related datasets were used to construct prediction models for lymphocytic and T-cell leukemia with the small size datasets by a deep transfer-learning framework and the good performance on small datasets proves the strong feasibility of our model.p-value, respectively. Meanwhile, the detailed information of the top 20 enriched terms are listed in In our three leukemia-related datasets, all phosphorylation sites are from 8011 phosphorylated proteins. In order to understand an in-depth knowledge about genes associated with leukemia, we performed functional pathway enrichment analysis using Metascape database  and 1707In addition, the distribution of the phosphorylated proteins in different types of leukemia was also given in Finally, we performed K-means clustering analysis on all the phosphorylation sites of myelogenous leukemia. By using the K-means algorithm, all samples can be divided into K clusters according to the distances between them. The optimal categorical K value makes samples within one cluster be the closest connected and those across clusters having with the largest distances. Here, each phosphorylation peptide with length of 201 was transformed into a one-hot vector. As shown in Neighboring residues are not equally important to the functional sites. Some are essential for the proper structure and function of the proteins, whereas others can be readily replaced . In factp < 0.05 by t-test) was generated by Two Sample Logo [The work of Nakariyakul et. al. has indicated that a protein domain is basically between ten residues . Here, fple Logo . Figure By means of parallel contrast, it obviously shows that distributions of the same residues have a large enough difference among flanking positions. As described by Ding et al. , same reN-terminal sequence, it functions as motif detector and becomes activated when certain position matches its preference. By the fraction of filters activated at positions, we observed that the majority of filters were activated when they convolved a continuous region including the positions from 17 to 25 in Y dataset, the positions from 30 to 39 in S dataset and the positions from 11 to 18 in T dataset. These activation sequences were aligned to obtain the learned basis sequences represented by the position weight matrix for deep-learning. The results show that the discovered amino acid motifs revealed by deep CNN yield high-similarity shape to known motifs for three classes datasets. We used WebLogo tool [One of the distinctive advantages of the deep-learning model is the ability to automatically extract predictive features from inputs during the model training . We explogo tool  to generBecause of the spatially folded structures of proteins, the sequentially distant residues may be in close proximity to the variant site in the spatial structure, which affect the environmental information of the phosphorylation sites. Here, we used different phosphorylation peptide lengths to build prediction model and aimed to find the optimal peptide length for S/T/Y phosphorylation sites, respectively. In fact, 10 different segments in n-site units  were utilized. Each fragment peptide contains intermediate phosphorylation sites and n-site elements on both upstream and downstream sides. The prediction performance of CNN models based on 10 different peptides lengths are shown in In order to demonstrate the superiority of the CNN models, based on the optimal peptide lengths for S, T and Y, other five machine-learning algorithms were also used to construct the prediction models, including support vector machine (SVM), naive Bayes (NB), K-nearest neighbors (KNN), random forest (RF) and eXtreme gradient boosting (XGBoost) since these algorithms are commonly used in studies of phosphorylation site prediction. Here, the CNN models can be implemented depending solely on protein primary sequences, requiring no prior knowledge of the variants on sequences . But macIn order to achieve convincing comparisons, the performance of each machine-learning model was given by the average of 100 random selections of testing sets. The comparison results of our method with other five methods are visually shown in Cross-validation test can reduce the contingency caused by the single division of dataset and improve generalization ability. Based on the optimal peptide lengths, the final phosphorylation site prediction models were constructed for S, T and Y respectively. Based on the whole dataset,10-fold cross validation was used to achieve the optimal parameters of CNN models, as shown in Finally, the specificity of the final models was further tested. Since amount of remaining negative samples are not included in the final prediction model, they can be used as the independent negatives. The number of independent negative samples for S, T and Y are 17,207, 25,340 and 18,149, respectively and our models give the prediction accuracy of 0.8905, 0.8009 and 0.8484, respectively, which indicates that about 10–20% of them are predicted as the phosphorylation sites. However, it is reasonable that the potential phosphorylated sites from the current negatives would be probably discovered in the future.Currently, very limited phosphorylation sites for T-cell and lymphocytic leukemia are available, so the model and the prediction parameters cannot be fully trained and optimized based on such small size datasets. Based on the large S, T and Y datasets of myelogenous, we have achieved three reliable CNN models, as shown in Homo sapiens were collected from PhosphositePlus [Protein phosphorylation data for sitePlus . PhosphoN-terminal residues appear to provide the targeting information for protein functional expression. Some effectors, e. g. PopD in Pseudomonas aeruginosa [Protein sequence information are from The UCSC Genome Browser Database . Reportsruginosa , only deruginosa . Due to CD-HIT stands for “cluster database at high identity with tolerance”. The program takes a fasta format sequence database as input and produces a set of ’non-redundant’ representative sequences as output. Since redundant sequences would lead to overfitting, we used cd-hit  to removFor negative samples, we extracted the S, T and Y residues data that were not experimentally confirmed from the 8011 proteins that contain experimentally determined phosphorylation sites. After removing redundancy with the peptide length of 201, the number of negative samples is much larger than that of the positive ones, so the equivalent number of negative samples were randomly extracted.Like traditional neural network architecture, CNN transmits information from the input to the output layer by layer . CNN is Transfer-learning means the ability of a system to recognize and apply knowledge and skills learned in previous domains/tasks to novel domains/tasks, which is one of the major types in machine-learning. In general terms, transfer-learning is commonly used to find similarities between existing knowledge and new knowledge, and then use existing knowledge to learn the new knowledge . TransfeOur pre-model of CNN architecture is shown in After determining the pre-model, the parameter settings are shared with the small sample dataset and all layers  retrained on the small sample datasets. The new model is fine-tuned by validation method to fit the T-cell leukemia-related and lymphocytic leukemia-related phosphorylation data. In this work, we aim to use the simple model to avoid manual feature extraction, the overfitting problem caused by complex models and improve the suitability of deep- learning models for small sample datasets.Five traditional machine-learning methods were used to compare with our new CNN model, including RF, XGB, NB, KNN and SVM. SVM, proposed by Vapnik , is consTP, FP, TN and FN are true positive, false positive, true negative and false negative, respectively. In addition, receiver operating characteristic (ROC) curve was also employed to assess the performance of prediction models. A ROC curve is a plot of true-positive rate (SE) versus false-positive rate (1-SP). A classifier can get a true-positive rate and a false-positive rate point pair based on its performance on the test sample, and this point pair can be mapped to a point on the ROC plane. The ROC curve is an intuitive way to indicate the performance of a classifier, and the area under the curve (AUC) is used to achieve a value to mark the performance of a classifier. AUC ranges from 0 to 1. Typically, larger AUC indicates better performance of the model, AUC values of 0.5 represents a random classifier.Four effective performance metrics including sensitivity (SE), specificity (SP), accuracy (ACC) and Matthew’s correlation coefficient (MCC) were used for performance assessment. SE, SP, and ACC indicate the predictive success rates on positive, negative, and overall samples, respectively. MCC accounts for true and false positives and negatives and is usually considered a balanced measure that can be used even if the classes are of very different sizes. These metrics are defined as follows:Studies have indicated that many diseases are closely related with abnormal phosphorylation. Therefore, the large-scale identification of phosphorylation sites has potential applications in disease treatment and drug design. But existing wet-lab technologies for targeting phosphorylation sites are costly and time consuming. Thus, computational algorithms can efficiently accelerate the annotation of unknown phosphorylation sites. Numerous machine learning-based methods have been implemented for phosphorylation sites prediction. However, no method focuses on specific disease-associated phosphorylation sites prediction. We know that disease pathogenesis is specific, so we analyzed three leukemia subtypes to achieve the effective computational predictions of leukemia-related phosphorylation sites, so as to provide reference information for predicting potential phosphorylation sites and leukemia treatments.When using machine-learning methods to solve prediction problems from biological sequences, we usually face challenges in selecting appropriate machine-learning algorithms, extracting effective features and carrying out reasonable optimization. Especially feature extraction is a key step that mainly determines the prediction performance. As a cutting-edge machine-learning method, deep learning has the ability to automatically discover complex representations of phosphorylation patterns from the raw sequences, and hence it provides a powerful tool for improvement of leukemia-related phosphorylation site prediction.In this paper, a new transfer-learning-based deep CNN was proposed for predicting leukemia-related phosphorylation sites from protein primary sequences. Focusing on three types of leukemia, we firstly analyzed the gene pathways of the phosphorylated proteins with leukemia-related phosphorylation sites, revealing that the pathways of these genes are mainly related to cellular morphological changes, especially WP3888  may play an important role in the progression of leukemia. The distribution analysis on the phosphorylated proteins in three different types of leukemia shows that there are few sharing proteins between them, so it is of practical significance to construct the prediction model for each specific type of leukemia. K-means clustering analysis on all the phosphorylation sites of myelogenous leukemia reveals the obvious variability between samples, so in process of the random division of training and testing sets, samples in all clusters should be partly extracted to ensure the completeness of the data distribution. Meanwhile, the position-specific conservation difference analysis was performed between positive and negative sample for all, S, T and Y sites respectively and the amino acid motifs revealed by CNN have a very high-similarity shape to the known motifs which validates the feasibility of the CNN to automatically extract sequence feature information.Based on the largest S/T/Y datasets of myelogenous-related phosphorylation sites, the optimal peptide lengths for S, T and Y models were achieved and they are 141, 41 and 121, respectively. By comparing with other five machine-learning methods, CNN yields the best performance on 100 times testing. By 10-fold cross validation, the final models for S/T/Y phosphorylation sites were achieved and give the AUC values of 0.8784, 0.8328 and 0.7716 for S, T and Y, respectively. Finally, the optimal parameter of the final S/T/Y myelogenous-related models were transferred to construct the prediction models for the small datasets of lymphocytic and T-cell. The satisfactory prediction results prove that the transfer-learning based CNN is an effective strategy for leukemia-related phosphorylation site prediction.We expect that with the accumulation of newly discovered leukemia-related phosphorylation sites data, the models constructed in this work can be further verified and will be a useful supplementary tool for identifying leukemia-related phosphorylation sites.


Autosomal recessive (AR) DOCK8 deficiency is a well-known actinopathy, a combined primary immune deficiency with impaired actin polymerization that results in altered cell mobility and immune synapse. DOCK8-deficient patients present early in life with eczema, viral cutaneous infections, chronic mucocutaneous candidiasis, bacterial pneumonia, and abscesses, together with eosinophilia, thrombocytosis, lymphopenia, and variable dysgammaglobulinemia that usually includes Hyper-IgE. In fact, before its genetic etiology was known, patients were described as having a form of Hyper-IgE syndrome, a name now deprecated in favor of genetic defects. We describe a school-age male patient with a clinical picture suggestive of DOCK8 deficiency, except for high serum IgE or a family history: early onset, failure to thrive, eczema, warts, condyloma, bronchiolitis, pneumonia, recurrent otitis media, bronchiectasis, candidiasis, leukocytosis, eosinophilia, high IgA, low IgG, and low CD4+ T cells. We were able to confirm the diagnosis through protein expression and whole-exome sequencing. We review the clinical, laboratory, and genetic features of 200 DOCK8-deficient patients; at least 4 other patients have had no elevated IgE, and about 40% do not have Hyper-IgE . Despite this, the constellation of signs, symptoms, and findings allow the suspicion of DOCK8 deficiency and other actinopathies. An estimated 7,000 individually rare diseases together afflict about 1 in every 17 humans . DiagnosActinopathies are CIDs that prevent the polymerization of actin, thus impairing cell mobility and the immune synapse of hematopoietic cells . AutosomBacille Calmette-Guérin (BCG) vaccine shortly after birth, and his family history was significant only for two spontaneous abortions. He started at age 1 month old with extensive eczema attributed to cow's milk protein allergy. Later, the patient also developed one episode of bronchiolitis, three of pneumonia, and three of otitis media with effusion, as well as twice-a-month intermittent diarrhea, disseminated flat warts, chronic mucocutaneous candidiasis, and condyloma acuminatum around the scrotum and groin folds  and hyper-eosinophilia , with Hb 12.7 g/dL, neutrophils 9,570, lymphocytes 4,890, monocytes 1,520, and 252,000/mm3 platelets. Serum immunoglobulin G (IgG) was low at 408 mg/dL, with high IgA (550) and normal IgM (66 mg/dL); total serum IgE was marginally high at 95 IU/mL. CD4+ T cells were also low at 365 cells (33%), with the rest of lymphocyte subsets within the normal range . Serologies for HIV, EBV, and CMV were all negative, as was a skin prick allergy test. The mitogen-induced carboxyfluorescein (CFSE) lymphoproliferation assay was equivocal: both the patient and a healthy (travel) control showed a limited response , HIV/AIDS (CD4+ lymphopenia), and leukemia (hypereosinophilia). However, a bone marrow aspirate and a CGL myeloid mutation panel were reported as normal. Despite an almost normal serum IgE, DOCK8 deficiency was included in the differential diagnosis, considering the constellation of early onset, failure to thrive, eczema, warts, condyloma, bronchiolitis, pneumonia, recurrent otitis media, bronchiectasis, candidiasis, leukocytosis, eosinophilia, high IgA, low IgG, and low CD4+ lymphocytes.Flow cytometry expression for WASp was slightly reduced in peripheral blood mononuclear cells, whereas DOCK8 expression was null see . ThroughWe describe a school-age male patient with an early onset of eczema, respiratory, and cutaneous infections caused by bacteria, viruses, and fungi. He developed hepatosplenomegaly, failure to thrive and bronchiectasis, hypereosinophilia, CD4+ lymphopenia, and dysgammaglobulinemia. Despite him not having Hyper-IgE, a high index of suspicion for DOCK8 deficiency led to a genetic diagnosis at age 7. DOCK8 expression was null, and a large genomic deletion, spanning introns 14 through 26, was identified through WES. Although this is a single atypical case, we were able to confirm the molecular (no protein expression) and genetic (large genomic deletion) diagnoses, while the patient awaits HSCT.The differential diagnosis of suspected DOCK8 deficiency includes the Wiskott–Aldrich , Loeys–Dietz , complete DiGeorge , Comel–Netherton , IPEX , and Omenn  syndromes; ARPC1B deficiency , IRAK4/MyD88 deficiency , CARD11 negative dominance (combined immune deficiency + atopy + high IgE), STAT3 negative dominance , and its phenocopy ZNF341 deficiency; PGM3  and IL6ST  deficiencies; MST1 (STK4) deficiency ; IL2RB deficiency ; and GATA2 haploinsufficiency .gestalt, which supports the diagnostic suspicion of DOCK8 deficiency in the absence of Hyper-IgE, as in this case. Our patient had up to 56,680 eosinophils per mm3 at one point.To date, more than 250 DOCK8-deficient patients have been described, 98% of which had high serum IgE , 14. At In 2016, Kienzler et al. described a DOCK8-deficient female patient without high serum IgE, who had compound heterozygous variants that resulted in a hypomorphic expression . FurtherThe Hyper-IgE syndrome was first called “Job syndrome” and was then renamed when IgE was discovered and found to be markedly high in the serum of the originally described patients , whose pMore than once , the conTo conclude, patients with DOCK8 deficiency may not have high total serum IgE levels, yet the astute clinician will maintain the diagnosis in his/her differential based on (any combination of) early onset eczema, food allergy, unusual infection, abscess, autoimmunity, eosinophilia, dysgammaglobulinemia, and CD3+ lymphopenia. “Hyper-IgE syndrome” is an incorrect name for several quite different diseases, from a time before their true nature was known, and we should abandon the misnomer in favor of more descriptive diagnoses and genetic etiologies.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Research and Ethics Committee, National Institute of Pediatrics. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.EV-M and AS-B cared for the patient, directed the diagnostic approach, wrote the manuscript, and reviewed the final version. LS-S, DM-Z, JG-C, RC-G, and YS-G cared for the patient and reviewed the final version of the manuscript. MG-S performed and analyzed the CFSE lymphoproliferation and protein expression by flow cytometry assays and reviewed the final version. SL processed and analyzed the whole-exome sequencing and conceived and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Limosilactobacillus fermentum UCO-979C is a probiotic strain possessing anti-Helicobacter pylori and immunomodulatory activity. The aim of this work was to examine if this strain maintains its probiotic properties and its viability when added to dairy-based ice creams  or to fruit-based ice creams (pineapple and raspberry) stored at −18 °C for 90 days. The probiotic anti-H. pylori activity using the well diffusion test, its immunomodulatory activity was measured using transforming growth factor beta 1 (TGF-β1) cytokine production by human gastric adenocarcinoma (AGS) cells, and its viability was measured using the microdrop technique. Assays were performed in triplicate. The L. fermentum UCO-979C strain maintained strong anti-H. pylori activity in dairy-based ice creams and mild activity in fruit-based ice cream. The production of pro-inflammatory cytokine TGF-β1 on AGS cells was higher in the probiotic recovered from Greek yogurt ice cream, maintaining a viability exceeding 107 colony-forming units/mL. The addition of the probiotic to ice creams did not significantly influence the physicochemical properties of the product. These data show the great potential of the L. fermentum UCO-979C strain in producing probiotic dairy-based and fruit-based ice creams. Helicobacter pylori infection is considered a major factor in the development of 44.4% of gastric cancer, killing nearly 800,000 people worldwide in 2018 [Lactobacillus fermentum UCO-979C, now classified as Limosilactobacillus fermentum UCO-979C, isolated in 2007 from a gastric biopsy, has anti-H. pylori activity [H. pylori [L. fermentum UCO-979C strain decreased the production of cytokines and inflammatory chemokines in a human gastric adenocarcinoma (AGS) and in the THP-1 cell lines and that it increased the production of immunoregulatory cytokines, such as TGF-α, in those same cell lines [L. fermentum UCO-979C strain was able to colonize the stomach of this animal model and to decrease the count of yjr H. pylori SS1 pathogenic strain [L. fermentum UCO-979C are involved in the immunomodulatory effect, interacting with certain receptors capable of modulating the production of inflammatory mediators [ in 2018 . A study in 2018 ,6,7,8, a in 2018 . Probiot in 2018 . That is in 2018 , and it  in 2018 . However in 2018 . Severalactivity . This st. pylori . Furtherll lines . An in vc strain . AdditioH. pylori or immunomodulatory characteristics. Hence, we are interested in investigating the behavior of the probiotic L. fermentum UCO-979C strain when added to various ice cream bases. In particular, the aim of this study was to determine whether the viability, anti-H. pylori activity, and immunomodulatory activity of the L. fermentum UCO-979C strain would be maintained after it was added to ice cream and whether it would influence the physicochemical properties of ice cream stored at −18 °C for 90 days.Currently, to our knowledge, there are no commercial ice creams with anti-All bacterial strains used in this study were available at the Laboratory of Bacterial Pathogenicity, Department of Microbiology, University of Concepcion, Chile.L. fermentum UCO-979C of human gastric origin was used. It was cultured using Mann–Rogosa Sharpe (MRS) broth  under microaerobiosis at 37 °C for 24 h followed by culturing in MRS agar  under similar conditions for another 24–48 h. The H. pylori J99 strain , originally obtained from American Type Culture Collection (ATCC) , was cultured in Columbia blood agar base (CA)  supplemented with 5% horse blood and selective supplement DENT  in a microaerobic atmosphere  at 37 °C for 72–96 h. To confirm the purity of H. pylori J99 culture, Gram staining and urease, catalase, and oxidase tests were performed.The probiotic strain L. fermentum UCO-979C strain was produced at the premises of the Processes and Foods Laboratory, Faculty of Chemical Engineering, University of Concepcion, Concepción (Chile). The culture medium contained whey cheese, lactase, casein peptone, yeast extract, potassium dihydrogen phosphate, sodium acetate, and tween 80, and its patent number is 1940–2005 , its pH was 6.8. The culture was maintained under aerobic conditions at 37 °C in a 15 L fermenter. The biomass generated was filtered using a sterile 0.22 µm filter and a vacuum pump model FD-10 Bench-top type , resuspended in 20% skim milk, frozen at −45 °C, and freeze-dried in a 50 L model BK-FD10PT freeze dryer . The biomass was stored at 4 °C in the darkness until use. The count of the strain in the milk powder was 1010 colony-forming units CFU/g of lyophilized powder, which was kept at 4 °C in the darkness until use.The biomass of the probiotic L. fermentum UCO-979C strain at a final concentration of 108 CFU/mL, and the second portion, lacking the probiotic, was used as the control. The mixtures were deposited in stainless steel molds placed in a freezing machine. The portions with or without probiotic were cooled to 4 °C for 12 h, then deposited in stainless steel molds, and frozen at −20 °C. The final product (ice cream) was finally stored at −18 °C. For each assay, three independent replicates of the portions with or without probiotic for each one of the five varieties of ice cream were analyzed and frozen at −20 °C. The final product (ice cream) was finally stored at −18 °C. For each assay, three independent true replicates of the portions with or without probiotic for each one of the five varieties of ice cream were analyzed.The ice creams were supplied by the company Sociedad Comercial Acuña Aguayo Limitada , using five varieties of ice cream: two natural fruit varieties (raspberry and pineapple) and three dairy varieties . The manufacturer kept the preparation of the different varieties of the ice creams confidential. Each ice cream variety was divided into two portions: one of them was supplemented with the freeze-dried probiotic 2) for 24 h. The counts were expressed as log (CFU/mL).The probiotic strain was recovered by dissolving 2 g of ice cream in 20 mL of sterile water. From this mixture, 10 µL were added to 900 µL of 0.9% saline solution; then, two serial dilutions prepared. The dilutions were used to count the CFU using the microdrop technique following the protocol of Sanhueza et al. , as descThe colonies were counted for each of the dilutions. The CFU count was performed at day 15 (time 0 = T0), corresponding to the day when the ice creams were transferred by the manufacturer to our laboratory. All analyses were performed in triplicate for each variety of ice cream flavors.H. pylori activity of the probiotic L. fermentum UCO-979C strain was determined using the well diffusion test according to Vega et al. [H. pylori J99 strain was seeded at a concentration of approximately McFarland 2 scale (6 × 108 CFU/mL) on CA agar supplemented with 5% horse blood. The agar was punctured with 6 mm glass Pasteur pipettes . The wells were filled with 50 µL of non-citrate MRS (negative control), 50 µL of 0.9% saline (negative control), 50 µL of non-citrate MRS plus L. fermentum UCO-979C (0.5 McFarland (1.5 × 108 CFU/mL)) or the lyophilized strain (positive control), 50 µL of non-citrate MRS plus L. fermentum UCO-979C (0.5 McFarland (1.5 × 108 CFU/mL)) obtained from the different varieties of ice creams, 50 µL of pellets, and 50 µL of the supernatant obtained from a L. fermentum UCO-979C culture in MRS previously centrifuged at 3500× g during 1 min. Then, the dishes were incubated at 37 °C at 10% CO2 for 72 h. This assay was performed in triplicate, sampling each ice cream variety after 90 days of storage at −18 °C. The probiotic strain was obtained from the same dilution of ice cream in distilled water that was seeded on MRS agar and incubated at 37 °C with 10% CO2 in an incubator  for 24 h. Then, a colony was taken and reseeded in MRS broth and  without citrate and incubated at 37 °C with 10% CO2 for 15 h in the same equipment. To determine the size of the inhibition halo, the criteria described by Gaudana et al. [The anti-a et al. , with mia et al.  was usedL. fermentum UCO-979C strain was measured after 90 days of storage of the ice creams at −18 °C using the ELISA technique. L. fermentum UCO-979 was obtained from the lyophilizate (control sample) and from the ice cream sample . The probiotic strain was seeded on MRS agar and incubated for 18 h at 37 °C in the presence 10% CO2. The AGS cells were seeded in 24-well plates  at a concentration of 7 × 104 cells/mL in  supplemented with 10% v/v of inactivated fetal bovine serum (FBS) , 100 U/mL of penicillin, and 100 μg/mL of streptomycin . The plates were incubated for 24 h at 37 °C and at 5% CO2 until reaching an 80 to 90% confluence. The probiotic obtained was then suspended in DMEM medium without antibiotics at a concentration of 105 or 107 CFU/mL and added to the 24 well plate containing the AGS cells  at a concentration of 7 × 104 cells/mL and incubated for 24 h at 37 °C and in the presence of 5% CO2.Additionally, the immunomodulatory activity was determined as described by García et al. . The effL. fermentum were then recovered, and the cytokines were measured in duplicate using the TGF-β1 cytokine kit Invitrogen Human/Mouse TGF-β1 Uncoated  at a wavelength of 450 nm using an Infinite M200 PRO equipment .The supernatants of the AGS cells plus the probiotic Proximal chemical analysis of the ice creams was performed using the methods described by Latimer G.W .Moisture was determined by the method AOAC 931.04, ash was determined by the gravimetric method (AOAC 945.46–942.05), and total protein was determined by the Kjeldahl method with a conversion factor of 6.38 (AOAC 928.08–930.33) . The perThe melting speed and the first drop fall time was performed according to the methodology proposed by Abd El-Ramh , with ceThe color of probiotic and control ice cream stored at −18 °C for 90 days was measured with a handheld colorimeter, model Cs-280  . The res variety ,27. A vaThis procedure was performed for each variety of ice cream in triplicate.t-test for independent samples (two groups) or by a two-way analysis of variance (ANOVA); in the latter case, the Tukey multiple comparison test was applied. In all cases, a significance level of 0.05 was used.The statistical analysis was performed using the SPSS software version 24.0 . The values of the physicochemical determinations were expressed as means and standard deviation. Bacterial counts were analyzed by the Student’s L. fermentum UCO-979C strain in the different fruit-based and dairy-based ice cream bases containing the probiotic strain showed that both types of ice cream matrices allowed for the probiotic to retain its viability, with counts above 107 CFU/mL during the three months of its storage at −18 °C  in the growth of H. pylori J99, similar to that of the total culture of the probiotic strain, while the inhibition caused by the pellet was not strong for H. pylori J99. It should be noted that the greatest inhibition of H. pylori growth was observed in the Greek yogurt-based ice cream. In the fruit-based ice creams supplemented with probiotics, the probiotic inhibition activity was mild in the supernatant and the culture medium, while for the pellet, the inhibition was considered null. When analyzing the lyophilized probiotic strain that was not added to any ice cream base or subjected to low temperatures, its anti-H. pylori activity was mild  in the supernatant as in the culture medium and null in the pellet , it was possible to observe that both treatments were able to retain the immunomodulatory activity (5 CFU/mL of the probiotic strain obtained directly from the lyophilizate showed cytokine production without significant difference when compared with the Greek yogurt ice cream (p > 0.05). Therefore, this ice cream flavor caused no alterations on the immunomodulating activity of L. fermentum UCO-979C when compared with the strains obtained from the other ice cream flavors.The effect on the production of pro-inflammatory cytokines TGF-β1 in AGS cells by activity  with no p < 0.05)  .Proximal analysis was performed on the control and probiotic ice cream after 30 days of storage . It was p < 0.05). The pineapple probiotic ice creams showed lower resistance to the first-drop process, with a lower melting speed (p < 0.05). Probiotic dairy-based ice creams, such as chocolate with brownie, and cookies and cream had higher or faster melting speeds (p < 0.05)  .p < 0.05), with a color variation greater than that of the control ice cream. The raspberry ice cream luminosity (L*) increased, with less intensity and a red with yellow tendency. In the case of pineapple ice cream, the luminosity values decreased, tending to become more saturated than the control ice cream; however, its color remained yellow . As for the saturation of the cookies and cream and the Greek yogurt ice creams, it decreased significantly (p < 0.05) every month  in three dairy-based ice cream formulations after processing or during storage for 40 days at −16 °C, maintaining a concentration of more than 108 CFU/g.When comparing the viability of the probiotic strain in dairy-based ice creams with those in fruit-based ice creams, a significant difference was observed; both matrices supported the viability of  product . The res product , who mads et al.  also repL. fermentum UCO-979C, to retain their viability. Hence, ice cream is an ideal vehicle to deliver these microorganisms into the human diet [Therefore, the results reported by the present work, supported by those of the abovementioned authors, support that dairy-based ice creams constitute a favorable environment that allows probiotic bacteria, such as man diet ,31.The high survival of probiotic bacteria in dairy-based ice cream may be due to the high total solids level in ice cream, including fat and milk solids, which provide protection to probiotic bacteria . ProbiotAdditionally, sugars , which are used as key ingredients in dairy and ice cream desserts, have also been reported to increase the viability of probiotics because they act as cryoprotectants . Short-cL. fermentum UCO-979C during the 90 days of storage at −18 °C in fruit-based ice cream can be attributed to the percentage of dietary fiber in raspberry and pineapple ice creams, benefiting the growth and viability of the probiotics strains. Ayar et al. [L. acidophilus and Bifidobacterium animalis subsp. lactis in ice cream samples supplemented with sources of dietary fiber from different fruits act by protecting the probiotics during the storage period and by maintaining the viability of the probiotic strains in the range of 7.0 log CFU/g. Therefore, the presence of fiber may also be a factor benefiting the viability of L. fermentum UCO-979C in the fruit-based ice creams analyzed in our study. Other investigations, such as the one by Di Criscio et al. [Lactobacillus casei and Lactobacillus rhamnosus strains contained in fruit-based ice creams. The results reported by these authors showed that both probiotic strains analyzed maintained viabilities between 6.5 and 6.9 log CFU/g after freezing and during the storage period at −20 °C, even if only small doses of additional fiber, such as inulin (3%), were added to the formulation [On the other hand, since fibers acts as prebiotics, the viability of r et al.  determino et al. , also sumulation .L. fermentum UCO-979C in pineapple ice cream is consistent with that observed by Nguyen et al. [Lactobacillus incorporated in pineapple juice remained constant during the first month of storage and subsequently decreased. On the other hand, the loss of viability of bacteria in fruit-based ice cream may be the result of the freezing process, which would affect the cell wall of the probiotic due to the formation of ice crystals, temperature stress, and dehydration of the cells. Thus, the probiotic could be affected by freezing because, when a juice that will not freeze is added to the ice cream, the viability of the probiotic strain will be maintained, but when the probiotic and fruit extract mixture freezes, there will be a decrease in CFU counts [L. acidophilus La-5 had a survival rate of 87%, with a count of 2.1 × 106 viable CFU/g and Bifidobacterium lactis Bb-12 at a concentration of 9.0 × 106 CFU/g with a survival rate of 90% for 2 months [The viability of n et al.  because U counts . This wa2 months . Thus, i2 months .H. pylori activity of L. fermentum UCO-979C, it was possible to observe that the matrix containing the probiotic strain had an effect on its inhibitory capacity, having a better anti-H. pylori effect in dairy-based ice creams. This may be due to the individual properties of each ice cream, which might increase the anti-H. pylori activity of the L.fermentum UCO-979C strain. Regarding anti-H. pylori activity in dairy-based ice cream, the greatest inhibition of the pathogenic bacterium was observed in Greek yogurt ice cream, probably because yogurt is already considered an ideal vehicle for the viability of probiotics [L.fermentum UCO-979C, support that asseveration , even after being frozen for 90 days at −18 °C. Although the probiotic retained its immunomodulating activity in dairy-based ice creams, it was possible to observe that L. fermentum UCO-979C strains isolated from chocolate with brownie, or cookies and cream ice creams supplemented with 105 or 107 CFU/mL of the probiotic caused a significant reduction in TGF-β1 when L. fermentum 979C was compared with the lyophilized strain (p < 0.05). In the case of Greek yogurt ice cream supplemented with 105 or 107 CFU/mL of the probiotic strain, there was no significant changes in the production of TGF-β1 when compared with the control. Therefore, since it allows us to retain both the immunomodulatory activity and the viability of this probiotic strain, Greek yogurt ice cream can be considered an ideal matrix for the administration of L. fermentum UCO-979C. These results coincide with those reported by Garcia et al. [L. fermentum UCO-979C caused a higher production in TGF-β1 than in the other cytokines on AGS cells. In addition, studies by Yang et al. [1. As for probiotic fruit ice creams, there was a significant decrease (p < 0.05) in the immunomodulatory activity detected by a reduction of cytokine production.The immunomodulating activity of o et al. , and thea et al. , providig et al.  also repp < 0.05) when compared with the control ice creams (ice creams without probiotic). This may be the direct consequence of a high lactic acid production by L. fermentum UCO-979C [p < 0.05) ice creams. The presence of lactic acid may explain the decrease in the pH values of the ice cream matrices [Regarding the proximal analysis performed for the different flavors of ice creams with or without probiotic supplement in both dairy-based and fruit-based ice creams, there was a significant pH reduction  first-drop compartment and a longer melting time when compared with ice cream without probiotics. This is possibly due to the influence of pH, since the decrease in pH occurs with increased production of lactic acid due to the fermentation in dairy-based ice creams [L. fermentum UCO-979C strain synthesizes exopolysaccharides capable of forming a biofilm. Exopolysaccharides are capable of acting as stabilizers, emulsifiers, gelling agents, and cell protectors in dehydration processes. Emulsifiers have the function of increasing the resistance to melting by destabilizing the fat [On the other hand, with respect to the first-drop compartment tests in probiotic ice creams, it was observed that they had a significantly lower or faster  with respect to the control. The L*C*h* values in pineapple probiotic ice cream decreased; this is related to what Ferreira et al. [Finally, in the changes in color parameters in control ice cream (ice creams not supplemented with the probiotic strain) and ice cream supplemented with ed color . This coa et al.  reporteda et al.  showed tp < 0.05) every month. This probably occurred due to the thin ice layer formed in the surface of the ice cream when transferred from −18 °C to room temperature, changing the appearance of the color. Since the high temperatures lead to a surface melting that causes a separation of the components of the ice cream matrix, the water crystals melt and flow. As a result, there will be more or less color concentrated on certain surfaces of the ice cream depending on the type of pigment used [As for the saturation of the cookies and cream and the Greek yogurt ice creams, color decreased significantly (ent used .The color difference (ΔE) in the fruit ice cream was ΔE = 3.920 for raspberry and ΔE = 2171 for pineapple. Considering that the values for a notable difference of color magnitude should be ΔE ≥ 2, in the case of the dairy-based ice creams , the variations had no major significant changes . This suL. fermentum UCO-979C strain, when incorporated into fruit-based (raspberry or pineapple) or dairy-based  ice creams is capable of maintaining its viability above 107 CFU/mL during 90 days of storage at −18 °C. This probiotic also maintained its anti-H. pylori activity in all ice cream bases; nevertheless, H. pylori inhibition was strong in dairy-based ice cream and a mild in fruit ice cream. Both fruit and dairy-based ice creams supplemented with L. fermentum UCO-979C were able to maintain the immunomodulatory activity of the probiotic on AGS cells, which produced the cytokine TGF-β1. The probiotic strain recovered from Greek yogurt ice cream caused a production of TGF-β1 close to that of the control, even at a concentration below 105 CFU/mL after storage at −18 °C for 90 days. On the other hand, the addition of the L. fermentum UCO-979C strain in ice creams did not significantly modify the physicochemical properties of ice cream when compared with control ice creams, and it does not negatively affect the manufacture or nutritional composition of the ice creams.The results obtained in the present work demonstrate that L. fermentum UCO-979C strain has a great potential in the production of dairy-based and fruit-based probiotic ice creams. Furthermore, considering that the functional properties of the probiotic strain are better in dairy-based ice creams, they can be considered more suitable candidates for incorporation in the probiotic biomass.These data show that the


Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) for youth in community-based settings in Zimbabwe.Point-of-care testing for sexually transmitted infections (STIs) may improve diagnosis and treatment of STIs in low- and middle-income counties. We explored the facilitators and barriers to point-of-care testing for This study was nested within a cluster randomised trial of community-based delivery of integrated HIV and sexual and reproductive health services for youth aged 16 to 24 years. On-site CT/NG testing on urine samples using the Xpert® CT/NG test was piloted in four intervention clusters, with testing performed by service providers. On-site testing was defined as sample processing on the same day and site as sample collection. Outcomes included proportion of tests processed on-site, time between sample collection and collection of results, and proportion of clients receiving treatment. In-depth interviews were conducted with nine service providers and three staff members providing study co-ordination or laboratory support to explore facilitators and barriers to providing on-site CT/NG testing.p = 0.61). The median (IQR) number of days between sample collection and collection of positive results was 14 (7–35) and 14 (7–52.5) for samples processed on and off-site, respectively,Of 847 Xpert tests, 296 (35.0%) were performed on-site. Of these, 61 (20.6%) were positive for CT/NG; one (1.6%) received same day aetiological treatment; 33 (54.1%) presented later for treatment; and 5 (8.2%) were treated as a part of syndromic management. There was no difference in the proportion of clients who were treated whether their sample was processed on or off-site (64% (39/61) vs 60% (66/110); The interviews revealed four themes related to the provision of on-site testing associated with the i) diagnostic device ii) environment, iii) provider, and iv) clients. Some of the specific barriers identified included insufficient testing capacity, inadequate space, as well as reluctance of clients to wait for their results.In addition to research to optimise the implementation of point-of-care tests for STIs in resource-limited settings, the development of new platforms to reduce analytic time will be necessary to scale up STI testing and reduce the attrition between testing and treatment.trials.gov (NCT03719521).Registered in clinical The online version contains supplementary material available at 10.1186/s12913-022-07557-7. The World Health Organization (WHO) African Region was estimated in 2016 to have the highest prevalence for chlamydia (CT) in men, and gonorrhoea (NG) and trichomoniasis in both men and women . ImportaThe WHO has recommended syndromic management of STIs in resource-limited settings since 1991, predominantly due to a lack of high quality, affordable diagnostics . HoweverThe WHO Sexually Transmitted Diseases Diagnostics Initiative developed a set of criteria as a benchmark for tests to meet STI control needs, recommending them to be affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free and deliverable to end-users (The “ASSURED” criteria) . CrucialThe Xpert CT/NG has been trialled as a point-of-care platform in resource-limited settings in antenatal care –25, primtrials.gov: NCT03719521) investigating the impact of a community-based package of integrated HIV and sexual and reproductive health (SRH) services for youth on HIV and other health outcomes  you will be in trouble.”when they were busy inside you had to wait for them”.Furthermore, due to shortage of space and for safety the instrument was placed in one of the booths, which was simultaneously used by nurses for clinical consultations. This sometimes resulted in sample processing and testing being delayed, as ““simple and straightforward”. Some providers noted that they did find using the machine difficult initially, however with experience they became more confident in running tests.Training was an important facilitator ensuring that providers were able to use the GeneXpert machine. The training received was generally felt to be adequate and most providers found using the machine The introduction of on-site CT/NG testing led to an increase in workload for CHIEDZA providers. This included both a higher intensity of work at the sites as providers had to juggle CT/NG testing with other duties, as well as the need for excess samples to be processed outside of normal working hours.“What it meant was that a person had to be in the booth and at the same time run the test, this put everyone under unnecessary pressure and the number of samples kept increasing whilst people were doing exchanges in the booths. We ended up being forced to run the samples over the weekend and we developed a roster to run these samples over the weekend.”Some providers did not view on-site GeneXpert testing as providing same day results and did not think it was feasible to do so. When results were given out on the same day as sample collection, this was seen as an exception rather than the rule. Providers generally informed clients to return the following week to collect their results.“They would leave the samples and be told to come back next week, but you will find out that because of that some of the clients up to now they haven’t come back to collect their results.”“cannot wait for 90 minutes” for their results. Clients who did receive same day results were noted to either spend time in the social area or go home and return.There was a strong perception amongst providers that there was limited demand from clients for same day results, given the time clients had to wait, except in specific limited circumstances. Providers perceived clients’ choice to wait for their results to be influenced by their level of interest in the results, worry surrounding a potential positive result and the time commitment to receive results. There was a perception that clients simply “Most clients did not want to wait for their results they just said they will leave their details and they will collect next time when they come. They were few who were willing to wait, and it was mainly boys who then went to the social area to play but no girls waited for their results. They said they had to go back home and do other chores that needed to be done.”This study has demonstrated some of the challenges and limitations associated with on-site CT/NG testing for youth in community-based settings, using the GeneXpert platform. Our operational environment was challenging with an intermittent electricity supply, and testing performed in a non-laboratory environment by staff who were not laboratory technicians. Despite this, we have demonstrated the feasibility of processing samples on-site in such an environment. Furthermore, the number of error codes was low and crucially the providers generally felt that their training was adequate and became more confident with time. Of note, feasibility of GeneXpert as part of a mobile testing unit has previously been shown for tuberculosis screening . HoweverWe reported a previous study also nested within the CHIEDZA trial that investigated uptake of STI testing in youth, which showed that only two-thirds of those with positive CT/NG tests were treated, the remainder being LTFU. In that study, STI testing was performed in a central laboratory with results available in a week . The maiWhile other studies in resource-limited settings have demonstrated the feasibility of offering same day results and treatment using the Xpert® CT/NG , 25, 28,The barriers to on-site STI testing were multifactorial and highlight the importance of understanding the local context in the design of interventions. Less than a quarter of samples were processed on-site using the two-module device. As a result, samples not processed on the same day had to be processed during the following days. This in turn affected testing capacity for the remainder of the week, hindering the ability to provide same day results for clients. Increased testing capacity led to 20% more samples being processed on-site. However, a larger machine is more difficult to transfer between sites. Testing capacity could potentially be optimised through risk stratification of clients, to either only test higher risk individuals, or to prioritise sample processing. However, if high prevalence is found amongst individuals without risk factors, such as were found amongst young people in Harare, then such risk prediction tools will be insufficiently sensitive .Even with increased capacity, the 90-min analytic time was still prohibitive to providing same day results to many young people. Providers highlighted that young people often had competing priorities and were generally unwilling to wait for results. Waiting time is a frequently mentioned barrier to youth accessing health services in resource-limited settings, and young people may be less willing to wait than adults for test results . Importaio assay , which has an analytic time of thirty minutes [There are a number of point-of-care tests for CT and NG in the pipeline with shorter analytic times . Their d minutes . Even gr minutes , 41.To our knowledge, this is the first study to evaluate the implementation of the GeneXpert platform for on-site STI testing for youth in a non-clinical, community-based setting in sub-Saharan Africa. This study used mixed methods enabling exploration of issues related to STI testing in both breadth and depth. We interviewed each member of the service provider team including the team that developed the point-of-care testing and collected detailed data on the diagnostic cascade. We acknowledge several limitations. We did not interview clients, and so we can only infer their perceptions from themes elicited from CHIEDZA study personnel and providers. As a result, although waiting time for clients is undoubtedly a barrier, its perceived relative influence on the feasibility of providing on-site testing is potentially skewed. We must also consider that testing was only performed for chlamydia and gonorrhoea, and so the results may not reflect some of the additional barriers that may become apparent if offering testing for other common STIs. Data on testing date and location was missing for 89 samples and recording of sample errors required providers to document an error on a log. Given the busy service, this may not always have happened and so the 60 errors that occurred may be an underestimate.Syndromic management is insufficient for STI control as asymptomatic infections cannot be detected and treated. While mobile diagnostic platforms for CT/NG testing have been developed, analytic capacity and testing time are barriers to their use as a true point-of-care test. Some of these barriers are likely to be addressed as newer platforms are developed that meet the ASSURED criteria. More generally, approaches to improve access to results and linkage to treatment are needed, particularly in youth who have high burdens of STIs, in order to develop strategies that are scalable. As shown with other conditions such as HIV and TB, strategies for delivery of testing and treatment must be diverse, nuanced, and take account of the local context.Additional file 1. Interview topic guide.Additional file 2.


There were significant differences between the last two deglaciations, particularly in Atlantic Meridional Overturning Circulation (AMOC) and Antarctic warming in the deglaciations and the following interglacials. Here, we present transient simulations of deglaciation using a coupled atmosphere–ocean general circulation model for the last two deglaciations focusing on the impact of ice sheet discharge on climate changes associated with the AMOC in the first part, and the sensitivity studies using a Northern Hemisphere ice sheet model in the second part. We show that a set of abrupt climate changes of the last deglaciation, including Bolling–Allerod warming, the Younger Dryas, and onset of the Holocene were simulated with gradual changes of both ice sheet discharge and radiative forcing. On the other hand, penultimate deglaciation, with the abrupt climate change only at the beginning of the last interglacial was simulated when the ice sheet discharge was greater than in the last deglaciation by a factor of 1.5. The results, together with Northern Hemisphere ice sheet model experiments suggest the importance of the transient climate and AMOC responses to the different orbital forcing conditions of the last two deglaciations, through the mechanisms of mass loss of the Northern Hemisphere ice sheet and meltwater influx to the ocean. Based on geological reconstructions from multiple lines of evidence, there were significant differences in the climate events of the last two deglaciations and the climate states of the following interglacials4. In particular, during the last deglaciation , there was abrupt strengthening in the Atlantic Meridional Overturning Circulation (AMOC), which caused the Bolling–Allerod (BA) warming event during the middle stage6. After the BA warming, the climate of the Antarctic region followed a cooling trend known as Antarctic Cold Reversal , followed by the Younger Dryas , characterized by weakening in the AMOC and Antarctic warming until the onset of the present interglacial. In contrast, during the penultimate deglaciation , the AMOC was weak for about 4000 years (~ 133 to 129 ka BP) during the middle and later stage of T2, followed by the strengthening of AMOC in the last interglacial 10. There were significant differences in the climate state of this interglacial compared with the present interglacial, as the sea surface temperature (SST) of the Southern Ocean and surface temperature over the Antarctic continent during the LIG were about 2° higher than those of the present interglacial11. There were also differences in the global mean sea level, which was higher during the LIG than in the present by at least 5 m14, presumably because of mass loss of the West Antarctic Ice Sheet (WAIS)16. It has been noted that a weakened AMOC during the early LIG caused the Antarctic warmth18 as the longer duration of weakened AMOC contributed to Antarctic warming22. Moreover, it has been hypothesized that greater boreal summer insolation caused faster retreat of Northern Hemisphere ice sheets and thus caused a weak AMOC throughout T224 based on the reconstructed sea level rise of T2, indicating that it was significantly faster than that of T18. Therefore, it is of importance to investigate the role of orbital forcing on different climate events during the last two deglaciations through melting of Northern Hemisphere ice sheets and AMOC changes.The climate system of the Earth underwent glacial–interglacial cycles associated with growth and decay of continental ice sheets, driven by insolation changes as the external forcing, and by climate system feedbacks. The transition from the glacial state to the interglacial state (deglaciation) typically occurred within about 10,000 years, characterized by global warming and melting of continental ice sheets26. Clark et al.27 conducted transient simulation of T2 using reconstructed climate forcing and greater meltwater flux than T1. They compared the results with the simulation of T125 and showed that the greater meltwater flux of T2 corresponding to Heinrich event 11 led to longer weakening in the AMOC and significant Antarctic warming that was sufficient to cause the mass loss of the WAIS. However, it is still unclear which phase of the deglaciation was important for the AMOC and Antarctic warming, and what the cause of significant ice sheet retreat during deglaciation was. Recently, abrupt AMOC recovery and associated ACR have been simulated with constant meltwater flux by transient simulation of the last deglaciation using the atmosphere–ocean coupled General Circulation Model (GCM) MIROC .Transient simulations of the last deglaciation have been conducted using climate models, and climate events associated with variations in the AMOC have been represented by applying meltwater flux to the oceanIn this study, first, we conducted sensitivity experiments of deglaciation with different levels of meltwater influencing the AMOC and surface temperature mainly over the Antarctic region, using an atmosphere–ocean coupled GCM. Second, we conducted sensitivity experiments of the effect of insolation on melting of the Northern Hemisphere ice sheets, using a three-dimensional Northern Hemisphere ice sheet model. We discuss the roles of orbital forcing and climate system feedbacks in the differences of the last two glacial terminations and the following interglacials.27. The simulation of T1 (T1-like experiment) is from OA201928, and was extended to 9 ka BP to serve as a T1-like experiment covering the YD event and onset of the Holocene. In the sensitivity experiment to imitate T2 (T2-like experiment), we changed meltwater flux while maintaining identical orbital and atmospheric greenhouse gas forcings of T1  around Antarctica continued warming in the T2-like experiment until 128 ka BP, and the simulated peak in Antarctic SAT was higher than the interglacial level by about 2°  in response to gradual warming even under continuous meltwater  at 133 and 132 ka respectively; the meltwater flux was at the same level as in the T2-like experiment  before T260 was one primary source of meltwater during the early stage of T2. The dynamics of marine ice sheets and the extensive Eurasian ice sheet at the PGM were not represented in our ice sheet model, but it is important that the land-based ice sheet alone could be a source of differences after the middle stage of the deglaciation, when there were significant differences in the surface mass balances of the ice sheet caused by differences in insolation . The resolution of the atmospheric component was T42 (about 2.8° × 2.8°) with 20 vertical levels, and that of the ocean component was about 1.4° × 1° with 43 vertical levels. MIROC 4 m produced vigorous AMOC under the LGM when submitted to PMIP273. The coefficient of the horizontal isopycnal layer thickness diffusivity of the ocean model was changed to 7.0 × 106 cm2/s from 3.0 × 106 cm2/s, and the present model produces a weak AMOC under the LGM because of enhanced dense Antarctic Bottom Water (AABW) formation34. MIROC has been used to investigate the climate of the LGM with radiative forcing and climate feedback74, the effect of ice sheets on the climate and the AMOC75, ocean biogeochemical cycles78, and mass balance of the Antarctic ice shelves80.We used the MIROC4m AOGCM, the same model used in the simulation of the last deglaciation  lasting for more than 30 thousand years34, and time-evolving insolation, atmospheric greenhouse gas concentrations, and meltwater were applied based on reconstructions81. The continental ice sheets and land–sea mask were fixed to those of the LGM (ICE-5G82) as used in PMIP283; therefore, the climate system feedback associated with changes of ice sheets was excluded. Ice sheet reconstructions30, referred from PMIP431, were used as constraints on meltwater flux in the deglaciation experiments. The meltwater was uniformly applied to 50–70° N in the North Atlantic as in previous studies84. OA2019 showed an increase in the AMOC during BA under a constant meltwater flux of ~ 0.06 Sv, and the last deglaciation experiment was extended to 9 ka BP to serve as a T1-like experiment covering the YD event and onset of the Holocene. The time series of meltwater flux in the T1-like and T2-like experiments are summarized in Supplementary Table The transient simulations were mostly based on the protocols proposed by the Paleoclimate Modelling Intercomparison Project Phase 4 (PMIP4) for the last and penultimate deglaciations38. The horizontal resolution is 1 × 1 degrees, and ice sheet dynamics are represented by the Shallow Ice Approximation. The climate factors that control the surface temperature and surface mass balance of ice sheets, such as the lapse rate, albedo feedback and stationary wave feedback, were obtained from a suite of experiments using GCM snapshots to obtain a climate parameterization85. The surface temperature distribution was parameterized as the sum of the present-day climatology and anomaly due to changes in insolation, atmospheric CO2, ice sheet area, and nonlinear effects estimated from climate model experiments38. The surface melt of the ice sheets was calculated with a positive-degree-day method86. The Control (CTL) experiment used an experimental design identical to that of Abe-Ouchi et al.38; the ice sheet model was initialized with the present-day ice sheet, and was forced by the orbital parameters37 and atmospheric CO2 concentration87 for the last 400,000 years , as the climatic precession was negative in the early deglaciation  used in this study corresponds to that described by Abe-Ouchi et al.Supplementary Information.


We herein describe a cascade enzymatic reaction (CER)-based IgE detection method utilizing a personal glucose meter (PGM), which relies on alkaline phosphatase (ALP) activity that regulates the amount of adenosine triphosphate (ATP). The amount of sandwich assay complex is determined according to the presence or absence of the target IgE. Additionally, the ALP in the sandwich assay catalyzes the dephosphorylation of ATP, a substrate of CER, which results in the changes in glucose level. By employing this principle, IgE was reliably detected at a concentration as low as ca. 29.6 ng/mL with high specificity toward various proteins. Importantly, the limit of detection (LOD) of this portable PGM-based approach was comparable to currently commercialized ELISA kit without expensive and bulky analysis equipment as well as complexed washing step. Finally, the diagnostic capability of this method was also successfully verified by reliably detecting IgE present in a real human serum sample with an excellent recovery ratio within 100 ± 6%. Food allergy is a deleterious immune response against a component present in food and frequently occurs in many people worldwide ,2. Most Recently, monitoring IgE levels using conventional technologies, such as radioimmunoassay (RIA) ,14,15, eTo overcome these limitations, Xiang et al. developed a novel personal glucose meter (PGM)-based sensing system for detecting various non-glucose targets, such as nucleic acids, proteins, enzymes, and small biological molecules, and has several advantages, including portability, cost-effectiveness, and simplicity ,30,31,32As an alternative to invertase-based methods, Ahn et al. proposed a label-free and washing-free biosensing method based on a cascade enzymatic reaction (CER) combined with hexokinase and pyruvate kinase . In thisIn this study, we described a CER-based IgE detection method utilizing PGM, which relies on alkaline phosphatase (ALP) activity to regulate the amount of ATP. This method basically depends on the PGM-based ALP assay system , however2), diethanolamine (DEA), 2-(N-morpholino)ethanesulfonic acid monohydrate (MES), tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), phosphoenolpyruvic acid (PEP), adenosine 5′-triphosphate disodium salt hydrate (ATP), casein, sodium chloride (NaCl), hexokinase, pyruvate kinase, Tween-20, bovine serum albumin (BSA), human serum albumin (HSA), human serum, p-nitrophenyl phosphate (p-NPP) substrate solution, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution, rabbit anti-goat IgG antibody-alkaline phosphatase (Ab-ALP) were purchased from Sigma-Aldrich . Target human IgE was supplied by NIBSC . Mouse anti-human IgE antibody-alkaline phosphatase (Ab-ALP) was purchased from SouthernBiotech . Goat anti-human IgE antibody purchased form Thermo-Fisher Scientific . Troponin I cardiac antibody (anti-cTnI) was purchased from Hytest . Human IgE ELISA core kit was purchased from Komabiotech . Aqueous solutions were prepared by use of ultrapure DNase/RNase-free distilled water (DW) purchased from Bioneer . Carboxyl-modified magnetic nanoparticles  were provided by Amo Lifescience . The portable glucose meter (PGM) was purchased from Accu-Chek .D-Glucose, magnesium chloride  with 0.1% Tween-20, pH 7.4) and resuspended and incubated at 4 °C for 12 h for stabilization. The stabilized capture particles (CPs) were stored at 4 °C after being suspended in TBS-T  in a concentration of 2 mg/mL.First, 200 μL of MNPs (10 mg/mL) was added to a 1.5 mL microtube, the supernatant was discarded using a magnetic separator, and resuspended in 133.4 μL MES buffer . Then, 66.6 μL goat anti-human IgE antibody (primary antibody) (1.5 mg/mL) and 100 μL EDC (1 mg/mL in MES buffer) were added and incubated at room temperature for 30 min. After the reaction, the mixture was washed three times with 1 mL of PBS-T  were added to a 1.5 mL microtube and incubated at room temperature for 30 min. The CPs were washed three times with 1 mL of PBS-T and then transferred to microplate well. After the addition of 100 μL of p-NPP substrate solution to the well, the absorbance was measured at 405 nm using the Infinite M 200 PRO  after 10 min. The MNPs and CPs were analyzed to verify the modification of the antibody on the surface using Perkin Elmer Spectrum BX FT-IR System .The antibody immobilization statuses of the CPs were analyzed using anti-goat IgG antibody-alkaline phosphatase. First, 10 μL CPs (2 mg/mL), 30 μL rabbit anti-goat IgG antibody-alkaline phosphatase (2 μg/mL) or mouse anti-human IgE antibody-alkaline phosphatase (secondary antibody) (2 μg/mL) and 160 μL tris buffer  at various concentrations or human IgG, BSA, HSA, anti-cTnI (10 μg/mL) was first mixed with 50 μL of Mouse anti-human IgE Fc-alkaline phosphatase (2 μg/mL) and incubated at room temperature for 30 min; then, 50 μL of CPs (2 mg/mL) was added and incubated for another 30 min. The CPs were collected with a magnet and washed four times with 100 μL of TBS-T.2, 60 mM DEA, 1 mM ATP, pH 9.8) and incubated at 37 °C for 2 h. Next, 30 μL of the above reaction solution was transferred to a PCR tube, 20 μL of reaction mixture II  was added and then incubated at 30 °C for 30 min. Finally, the glucose level of the reactant was measured by a PGM.After discarding the supernatant of the sandwich complexes prepared in 2.4., the CPs were resuspended in 40 μL of reaction mixture I  was added, the absorbance was measured at 450 nm using the Infinite M 200 PRO.Primary antibody (100 μL) (included in kit) (2 μg/mL) was added to each well and incubated at 4 °C for overnight. After the antibody was coated, it was washed three times using a 300 μL of PBS-T and then washed in the same manner at the end of each step. To prevent non-specific binding of well surfaces, 200 μL of blocking solution (1X PBS with 0.1% casein) was added to each well and incubated at room temperature for 1h. After washing step, 100 μL of human IgE at various concentrations was added, incubated at room temperature for 1 h, and the washing step was repeated. Then, 100 μL of horseradish peroxidase (HRP)-modified secondary antibody (200 ng/mL) was added, and the mixture was incubated at room temperature for 1 h. After the washing step, 100 μL of TMB substrate solution was added to the wells and then incubated at room temperature for 20 min. Finally, to terminate the reaction, 100 μL of stop solution  and analyzed directly following the same detection procedure used in the buffer solution. The calibration curve was created with a set of standards where human IgE at a known amount is contained in the non-diluted human serum. Based on the calibration curve, the unknown amount of human IgE was examined.The conceptual design of the PGM-based human IgE assay is shown in p-NPP by ALP. In addition, the absorbance of CPs reacted with secondary antibody was similar to the absorbance of bare MNPs or CPs, indicating that a non-specific reaction does not occur between the prepared CPs and secondary antibody. Moreover, antibody conjugation to MNPs was confirmed by FTIR analysis. As shown in −1 reflected the C = O bond and antibody amide I, and the peak at 1550 cm−1 was assigned to the N–H bond of antibody amide II. These results confirmed that the antibodies were covalently bound to the MNPs surface.CPs were prepared by chemical conjugation of primary antibody with MNPs. As shown in The detection feasibility of the PGM-based human IgE assay was validated by measuring glucose levels using PGM in different samples. As shown in To optimize the detection performance of the PGM-based IgE assay, the enzyme concentrations (hexokinase and pyruvate kinase) and the CER time were investigated. First, we optimized the concentrations of hexokinase and pyruvate kinase. Enzymes at too low a concentration may inhibit the CER. Conversely, those at too high a concentration may provide the small signal difference between the presence and absence of a target IgE, which causes low sensing capability. As shown in The results demonstrated that the change in the PGM signal increased with increasing reaction time up to 20 min but decreased over 20 min . Based o2 = 0.9796) existed in the range of 10–400 ng/mL, and the limit of detection (LOD) (3σ/slope) was ca. 29.59 ng/mL, which is comparable to or higher than the limits of other human IgE assay methods . In addition, the zeta potential of CPs and interfering proteins was investigated. As shown in The selectivity of the present method for the PGM-based human IgE assay was also investigated by the addition of human IgE and four interfering proteins, including human IgE, BSA, HSA, and anti-cTnI. As shown in The human IgE contained in non-diluted human serum was analyzed to demonstrate the practical applicability of the PGM-based human IgE detection method. In the study described above, we proposed an IgE detection method-based portable PGM that serves as an analysis equipment to quantitatively detect target IgE. Our strategy employs CER combined by hexokinase and pyruvate kinase, leading to changes in glucose level that is measurable by a portable PGM. Our PGM-based method to detect IgE can be conducted even in facility-limited environment because it does not need a bulky and expensive device that is generally needed in conventional fluorescent and colorimetric methods, which enables the realization of convenient and cost-effective system to detect IgE. By employing this design principle, IgE was detected quantitatively with high comparable sensitivity to recent reports and commercialized ELISA assay kit. The practical utility of the proposed methods was successfully verified by reliably determining IgE present in human serum samples. The basic signaling strategy developed in this study would serve as a new platform to construct novel immunoassay systems to detect other various biological molecules.


In recent years, angiotensin-converting enzyme (ACE) inhibitory peptide has become a research hotspot because of its essential role in maintaining human blood pressure balance. In this study, two novel ACE inhibitory peptides of Val-Glu-Arg-Gly-Arg-Arg-lle-Thr-Ser-Val  and Phe-Val-Ile-Glu-Pro-Asn-Ile-Thr-Pro-Ala  were isolated and purified from defatted walnut meal hydrolysates through a series of preparation processes including ultrafiltration, Sephadex G-15 gel chromatography, and reverse high performance liquid chromatography (RP-HPLC). Both peptides showed high ACE inhibitory activities. The molecular docking study revealed that VERGRRITSV and FVIEPNITPA were primarily attributed to the formation of strong hydrogen bonds with the active pockets of ACE. The binding free energies of VERGRRITSV and FVIEPNITPA with ACE were −14.99 and −14.69 kcal/mol, respectively. Moreover, these ACE inhibitory peptides showed good stability against gastrointestinal enzymes digestion and common food processing conditions . Furthermore, animal experiment results indicated that the administration of VERGRRITSV or FVIEPNITPA exhibited antihypertensive effects in spontaneously hypertensive rats. Our results demonstrated that walnut could be a potential source of bioactive peptides with ACE inhibitory activity. Juglans regia L.) is known as one of the world’s famous “four dried fruits” together with almonds, cashews, and hazelnuts. It has a long history of cultivation and a large planting area in China. At present, edible oil extraction is the main processing method for walnut kernels.Walnut  and essential amino acids . HoweverHypertension is a significant risk factor for heart diseases, stroke, arteriosclerosis, and other diseases. This condition threatens the health of approximately 20% of adults worldwide ,4. StudiIn general, ACE inhibitory peptides directly bind or indirectly induce conformational changes at enzyme active sites . The molAt present, the preparation methods of food-derived antihypertensive peptides primarily include microbial fermentation, enzymatic hydrolysis, and gene synthesis ,16,17, aFor this study, we have prepared glutelin-1 from a walnut meal. After being hydrolyzed by pepsin, the glutelin-1 hydrolysate was separated and purified by membrane separation, gel chromatography, and high-performance liquid chromatography (HPLC–MS/MS). Finally, highly active ACE inhibitory peptides could be obtained. The amino acid sequences of the ACE inhibitory peptides were identified through N-terminal sequence analysis. Moreover, we evaluated the antihypertensive effects of walnut glutelin-1 ACE inhibitory peptides in vivo using a spontaneously hypertensive rat (SHR) model. The ACE inhibitory mechanism of the peptide was explored through molecular docking simulation. We have also investigated the stability of walnut glutelin-1 ACE inhibitory peptides under different food processing conditions and simulated GI digestion environments. The outcomes of this study can improve the deep processing and comprehensive utilization of walnuts and provide theoretical and technical references for the research and development of walnut antihypertensive peptides.3 U/g) was obtained from Solarbio Science & Technology, Co., Ltd. . High-performance liquid chromatography grade acetonitrile and trifluoroacetic acid were purchased from Fischer Scientific Co. . The enzyme-linked immunosorbent assay kit for the detection of ACE, angiotensinogen (AGT), aldosterone (ALD), and Endothelin-1(ET-1) was purchased from Jiangsu Jingmei Biotechnology Co., Ltd. . The enzyme-linked immunosorbent assay kit for the detection of Ang II was obtained from Shanghai Kexing Biotechnology Co., Ltd., . and the nitric oxide (NO) detection kit was a product of Nanjing Jiancheng Institute of Biological Engineering , and other chemical reagents used in the experiment were of analytical grade.The Grain, Oil and Protein Engineering Laboratory of Shaanxi Normal University  provided the defatted walnut meal. ACE (from rabbit lung) and hippuryl-histidyl-leucine (HHL) were purchased from Sigma Co., St. Louis, MO, USA. Pepsin (3 × 10v/v) ethanol, 50% (v/v) glacial acetic acid, and 0.1 M NaOH. After centrifugation, each extract went through dialysis and lyophilization, the final power obtained was stored at −20 °C until use.The glutelin-1 fractions were prepared according to our previously published method . In shorw/v). The pH of the mixture was adjusted to 1.65 with 1 M HCl and enzymatically hydrolyzed at 45 °C for 6 h. The reaction was terminated by heating the solution in a 95 °C water bath for 10 min. Subsequently, the enzymatic hydrolysate was centrifuged at 10,000 rpm/min for 20 min at 4 °C, and the supernatant was freeze-dried and stored at −20 °C for further analysis.We prepared glutelin-1 hydrolysate according to our previous method . In shorv/v) TFA) and solvent B (acetonitrile with 0.1% (v/v) trifluoroacetate) were eluted linearly at a flow rate of 1.0 mL/min for 30 min. The monitoring wavelengths at this stage were 220 nm and 280 nm, respectively. All fractions’ ACE inhibitory activity and peptide content could be determined after being collected and lyophilized.An ultrafiltration unit  was used to obtain three fractions named A1 with molecular weight (MW) > 5000 Da, A2 with MW 3000–5000 Da, and A3 with MW < 3000 Da from the glutelin-1 hydrolysate by an ultrafiltration unit. The fraction with the highest ACE inhibitory activity was sequentially isolated and purified by a Sephadex G-15 gel column  and eluted with distilled water at a flow rate of 0.6 mL/min. Its loading concentration was 100 mg/mL, and the absorbance was measured at 280 nm. Then, with specific conditions, a reversed-phase high-performance liquid chromatography (RP-HPLC) equilibrated with a C18 column . First, two eluents consisting of solvent A  and a mass spectrometer .The synthetic peptides were obtained by a solid phase procedure  according to the previously known amino acid sequence. They were purified by RP-HPLC-MS/MS (purity > 95%) and stored at −20 °C for further analysis.50 value (the concentrationof the inhibitory peptide (μM) had to inhibit 50% of the ACE activity) was calculated by linear regression of the curve.Following a previous method reported by Zheng et al. (2017) , we coulThe following formula was used to calculate the ACE inhibition rate (%):https://www.rcsb.org, accessed on 2 February 2021) was used to download the crystal structure of the ACE (Protein Data Bank identification (PDB ID): 1O86, Resolution: 2.00 Å). The peptide structure was processed on the molecular manipulation environment platform (Molecular operating environment (MOE) 2019.1), including steps such as removing water and ions, protonation, addition of missing atoms, and completion of missing groups. Moe|Dock Module was used, and Amber10 was selected as the force field. One hundred conformations were generated after docking, and we selected the most negative conformations using the London DG scoring function. Finally, molecular visualization was processed by Pymol 2.3 software .The RCSB Protein Data Bank  and five normal male rats of the same age were used for the experiment. The rats were placed at 25 °C ± 1 °C, relative humidity of 45–55% and a light/dark cycle for 12 h. SHRs were randomly divided into 6 groups after one week of adaptation, while rats in the normal group (NG) were used as controls. The administration was continued for 28 days.Rats in the normal and SHR groups were given 1.0 mL of 0.9% saline per day, while the positive control group was given captopril (10 mg/kg body weight) per day. The ACE inhibitory peptides treatment groups’ low and high doses were fed orally with 5 and 15 mg/kg (based on their body weight) by dissolving the peptides in 1.0 mL of 0.9% normal saline. The rats’ BP was measured by the tail-cuff method. The systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate of all rats were measured after a single oral administration (0–8 h) and weekly (0–4 weeks). All measurements were repeated at least three times so the average value could be calculated.g at 4 °C for 10 min after the blood was taken from the abdominal aorta of all rats. The samples were stored at −20 °C, and the serum expression was detected using the ELISA kit (double antibody sandwich enzyme-linked immunosorbent assay).The upper serum samples were obtained by centrifugation at 3000× g.We conducted a GI digestion stability analysis of the synthesized peptides according to the method proposed by Khueychai et al. (2018)  with sli2, ZnSO4, MgSO4, and FeSO4 solution at 37 °C for 2 h; with glucose solutions of 2%, 4%, 8%, and 12% at 37 °C for 30 min; with NaCl solutions of 2%, 4%, 8%, and 12% at 37 °C for 30 min, respectively. The ACE inhibition rate was measured immediately after incubation.The effect of temperature, pH, metal ion, sugar, and salt treatments on the stabilities of two synthesized peptides, were determined by incubating 1.0 mg/mL of VERGRRITSV and FVIEPNITPA at 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C for 2 h; 37 °C for 2 h at pH 2, 4, 6, 8, and 10; with 100 μg/mL of KCl, CaClp < 0.05). GraphPad Prism 6.0  and Microsoft Office programs, including Word and Excel, were used for statistical analysis and pictures drawing.Each experiment had to be repeated at least three times, and the results were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to analyze the significant difference between the groups (p < 0.01).As shown in Many studies have found a specific correlation between the molecular weight of peptides and ACE inhibitory activity. Low molecular weight (less than 3 kDa) peptides often had higher ACE inhibitory activity . Our resp < 0.01) (The A3 fraction was divided into four main fractions by Sephadex G-15 gel chromatography A. The AC < 0.01) B. In add < 0.01) A. Ten ma < 0.01) B. P5 and < 0.01) C. RP-HPL50 values of 6.82 μM and 6.36 μM, respectively.The amino acid sequences of peptides P5 and P8 that showed the highest ACE inhibitory activity peptides, were analyzed using the Edman degradation method. As shown in Previous studies have evidenced that the ACE inhibitory activity of peptides is primarily related to molecular weight, hydrophobicity, amino acid composition, and peptide sequence . Most of2) with Glu411, His387, and His383 [The interactions between the two purified bioactive peptides (VERGRRITSV and FVIEPNITPA) and ACE were characterized by molecular docking technology. As an important thermodynamic property, binding free energy is usually calculated with a biological system model . As it cd His383 . VERGRRIIt has previously been reported that some ACE inhibitory peptides have good inhibitory activity in vitro. However, the antihypertensive effect in vivo is not so evident, and some ACE inhibitory peptides actually have the opposite effect ,32,33. Wp < 0.01). In the first week, the SBP of the VERGRRITSV-L-treated group decreased from 175.3 ± 10.1 mmHg to 137.7 ± 8.2 mmHg; the SBP remained basically stable in the following three weeks.It can be seen from p < 0.01). On the contrary, the DBP of the captopril, low-dose ACE inhibitory peptide VERGRRITSV, and FVIEPNITPA groups began to decrease after one week of continuous administration, and the decline reached a maximum in the second week. The DBP of the high-dose ACE inhibitory peptide FVIEPNITPA group began to decrease in the second week after administration. Besides, the high-dose ACE inhibitory peptide VERGRRITSV could inhibit the rapid increase in DBP, but its DBP did not decrease . These findings demonstrated that the walnut glutelin-1 ACE inhibitory peptide could inhibit the ACE activity and over-activated RASS, thereby reducing the production of AngⅡ, preventing the excessive secretion of ALD, slowing the reabsorption of sodium by the kidneys, and lowering BP levels of SHRs.The renin-angiotensin system (RAS) is a vital body fluid system of the human body. Renin secreted by the parabulbar cells in the glomerulus enters the bulbar artery and can catalyze AGT conversion into angiotensin I (AngI). AngI then produces AngⅡ, which has a solid hypertension-increasing effect under the influence of ACE . AngⅡ cap < 0.01), and the NO content was significantly lower than that of the treated groups (p < 0.05). In addition, the VERGRRITSV and FVIEPNITPA-treated groups had low ET-1 levels, and the NO content was equivalent to that of the captopril group. Therefore, the walnut glutelin-1 ACE inhibitory peptide could decrease the activity of ACE, slow down the degradation of bradykinin, improve the function of vascular endothelium, and regulate the vasomotor state effectively, consequently lowering the BP level.The balance of NO and ET-1 synthesis and release is the key to maintaining vascular endothelium’s normal function. ET-1 is considered a potent vasoconstrictor secreted by endothelial cells . NO can After entering the human digestive system, the peptides are degraded by digestive enzymes in the GI tract, which have uncertain effects on the spatial structure, amino acid sequence, molecular weight and other aspects of ACE inhibitory peptides . TherefoSome environmental factors during food processing, such as temperature, pH, metal ions, salt concentration and sugar concentration, will affect the stability of ACE inhibitory peptides and determine their potential applicability in functional foods .The inflThe change of pH value had no significant effect on the activity of walnut glutelin-1 ACE inhibitory peptides C, indica+, Ca2+, and Mg2+, whereas such peptides had poor stability under Zn2+ and Fe2+ conditions. Potassium, calcium, and magnesium are important components of daily food, and walnut glutelin-1 ACE inhibitory peptides can be consumed with foods containing these nutrients without affecting their activity. However, to contain other potential changes in the peptides’ activity, food rich in Zn2+ and Fe2+, e.g., animal liver, kidney, blood, lean meat, shellfish, kelp, and fungus should be avoided [Metal ions directly affected the ACE inhibitory activity of walnut glutelin-1 D. We hav avoided ,46,47.Glucose is a common preservative that is widely used in food and health products. It also has varying degrees of influence on the color, taste, and flavor of food. Considering that the Maillard reaction occurs when there are reducing sugars and amino compounds in food, it is necessary to study the effect of glucose on the inhibitory rate of peptides . The ACEThe results of this study showed that VERGRRITSV and FVIEPNITPA had good stability under low-concentration salt conditions, but they became less stable under high-concentration salt conditions F. This i+, Ca2+, and Mg2+) under low concentration of salt and glucose. These results indicated that the activity of ACE inhibitory peptides (VERGRRITSV and FVIEPNITPA) would not be greatly affected by appropriate processing conditions, which suggested the possible therapeutic use of these peptides as antihypertensive agents in vivo.The ACE inhibitory peptides (VERGRRITSV and FVIEPNITPA) isolated from the hydrolysate of walnut glutelin-1 showed stability across a range of pH values, at temperatures < 60 °C, and in the presence of metal ions  were successfully isolated and identified from walnut glutelin-1 hydrolysate. The results of animal experiments showed that the oral administration of the two ACE inhibitory peptides could significantly decrease systolic and diastolic blood pressure in SHRs. On the one hand, the two ACE inhibitory peptides could reduce the AGT, ACE, Ang II, and ALD levels in SHRs by regulating the RAS system. On the other hand, the level of ET-1 decreased, whereas the level of NO in serum increased by regulating the kinin system, lowering the BP level as a consequence. These results suggested that the two ACE inhibitory peptides have the potential to treat hypertension. Moreover, the molecular docking study revealed that the ACE inhibition of VERGRRITSV and FVIEPNITPA were mainly attributed to forming very strong hydrogen bonds with the active pockets of ACE. In addition, the two ACE inhibitory peptides displayed good resistance to digestive enzymes, and they could maintain structural stability in ordinary food processing environments. We have extensively verified that the walnut meal glutelin-1 ACE inhibitory peptides (VERGRRITSV and FVIEPNITPA) exhibited significant ACE inhibitory activity in vitro and in vivo, and concluded that this is a valuable feature to develop antihypertensive drugs and food. Meanwhile, walnut meal may have a potential active ingredient with antihypertensive properties, and its development and utilization as an antihypertensive agent will improve the value of this underutilized protein resource. However, the clinical application of ACE inhibitory peptides is still very limited, and further studies on the clinical efficacy and bioavailability of these peptides in humans needs to be further investigated.


Treponema pallidum, and Chlamydia trachomatis infections. A microscopic analysis by Giemsa staining showed negative results for Tzank or Haemophilus ducreyi; however, it showed Donovan bodies that are characteristic of donovanosis  and microscopic examinations of swabs of ulcer material were conducted. Serological examinations gave positive results for human immunodeficiency virus (HIV-1), herpes simplex virus (HSV-1/2), ovanosis . -The global incidence of STIs exceeds millions of cases per year, mostly affecting people aged 15-49 years. In tropical and developing areas, donovanosis, also known as granuloma inguinale, is an endemic genital ulcerative disease frequently associated with sexual transmissionKlebsiella granulomatis, an intracellular gram-negative bacterium. Clinically, it manifests as painless, slowly progressive ulcerative lesions in the genitals or perineum without regional lymphadenopathy. The infection can extend to the pelvis or disseminate to the intra-abdominal organs, bones, and mouth. Additionally, secondary bacterial infections in the lesions and co-infections with other sexually transmitted pathogens can be observed-Donovanosis is caused by K. granulomatis is an extremely fastidious organism that is difficult to isolate in artificial culture media. Therefore, the laboratory diagnosis of donovanosis is based on the microscopic visualization of Donovan bodies, dark-staining encapsulated bacteria inside macrophages measuring between 0.6 and 2.5 µm in size-Persons with both granuloma inguinale and HIV infection should receive the same regimens as those without HIV infections