Necroptosis is a type of programmed cell death, and recent researches have showed that lncRNAs could regulate the process of necroptosis in multiple cancers. We tried to screen necroptosis‐related lncRNAs and investigate the immune landscape in breast cancer (BC).The samples of breast normal and cancer tissue were acquired from TCGA and GTEx databases. A risk prognostic model was constructed based on the identified necroptosis‐related lncRNAs by Cox regression and least absolute shrinkage and selection operator (LASSO) method. Moreover, the forecast performance of this model was verified and accredited by synthetic approach. Subsequently, an accurate nomogram was constructed to predict the prognosis of BC patients. The biological differences were investigated through GO, GSEA, and immune analysis. The immunotherapy response was estimated through tumor mutation burden (TMB) and tumor immune dysfunction and exclusion (TIDE) score.A total of 251 necroptosis‐related lncRNAs were identified by differential coexpression analysis, and SH3BP5‐AS1, AC012073.1, AC120114.1, LINC00377, AL133467.1, AC036108.3, and AC020663.2 were involved in the risk model, which had an excellent concordance with the prediction. The pathway analyses showed that immune‐related pathways were relevant to the necroptosis‐related lncRNAs risk model. And the risk score was significantly correlated with immune cell infiltration, as well as the ESTIMATE score. Most notably, the patients of higher risk score were characterized with increased TMB and decreased TIDE score, indicating that these patients showed better immune checkpoint blockade response.These findings were conducive to understand the function of necroptosis‐related lncRNAs in BC and provide a potential promising therapeutic strategy for BC. Seven necroptosis‐related lncRNAs, SH3BP5‐AS1, AC012073.1, AC120114.1, LINC00377, AL133467.1, AC036108.3, and AC020663.2 were involved in the risk model. In addition, the forecast performance of this model was verified and accredited. Most notably, the patients of higher risk score were characterized with increased TMB and decreased TIDE score, indicating that these patients showed better immune checkpoint blockade response. The HTSeq‐Counts value matrix was used to search for the differentially expressed (DE) lncRNAs, while the FPKM values were transformed to TPM values for other analyses. After excluding the sample of male, 191 normal tissue samples (79 samples from GTEx dataset) and 1086 BC samples were obtained from two datasets. After ruling out the missing overall survival time, 1057 cases with survival time and 908 cases with full clinical pathology information were extracted for following analyses.2.2http://cancergenome.nih.gov/abouttcga, http://www.gtexportal.org). The DE lncRNAs were screened by DESeq2 R package with the standard of |log2 fold change (FC) | >1, false discovery rate (FDR) <0.05, and p adjusted <0.05. The Pearson correlation algorithm was used to identify necroptosis‐related DE lncRNA with correlation filter >0.4 and p < 0.001. After the completion of screening steps, 251 necroptosis‐related lncRNAs were retrieved for further analysis. The analyses were based on limma R package.According to necroptosis gene set M24779.gmt and previous literature search, 67 necroptosis‐related genes were collected for next identification.2.3p value < 0.05. Then, LASSO regression analysis was made to filter necroptosis‐related lncRNA with 10‐fold cross‐validation. Further, the necroptosis‐related lncRNAs screened by LASSO method were used for multivariate (multi‐Cox) proportional hazards regression and risk model construction. The risk score was calculated by using following formula: risk score = Σk=1n expression (lncRNAk) × coefficient(lncRNAk). The median risk score that calculated by the above formula was used to stratify the BC patients into low‐ and high‐risk groups. The chi‐square test was used to validate the correlation of the clinical features and the risk group. The independent variables were assessed by uni‐Cox and multi‐Cox regression analyses, respectively. Receiver operating characteristic (ROC) curves and concordance index(C‐index) were subsequently applied to measure the precision of the model. The analyses were based on survival, caret, glmnet, rms, survminer, and timeROC R packages.Preliminarily, the prognostic‐classified lncRNAs were selected by using univariate Cox (uni‐Cox) regression with 2.4With rms R package, the nomogram was set up based on risk group, age, and clinicopathological factors. The nomogram aimed to evaluating the predictive efficacy of risk score we got for 1‐, 3‐, 5‐, and 10‐year overall survival rates. Subsequently, the calibration curve was developed to illustrate the prediction power of the established nomogram model.2.5https://www.gsea‐msigdb.org/). The limma, org. Hs.eg.db, clusterProfiler, and enrichplot package based on R 4.1.1 were used for the analysis. The threshold of significantly enriched biological processes and pathways was set as p < 0.05 and FDR <0.25.The principal component analysis (PCA) was used to classify BC samples through necroptosis‐related lncRNAs expression patterns. Additionally, the spatial distribution of samples was displayed through three‐dimensional scatterplot. We identified the differential genes among the low‐ and high‐risk groups for subsequent Gene Ontology (GO) analysis, aiming to investigate the relevant biological process. Furtherly, differentially expressed KEGG pathways in two groups were identified by Gene Set Enrichment Analysis (GSEA). The KEGG gene set(c2.cp.kegg.v7.0.symbols.gmt) was derived from the website (2.6The correlations between risk score and tumor‐infiltrating immune cells (TIICs) were evaluated by CIBERSORT algorithm. Furthermore, with ESTIMATE R package,2.7The somatic mutation file (TCGA.BRCA.varscan.DR‐10.0.somatic) was obtained from the TCGA website. The original mutation annotation format (MAF) was divided into two groups according to the risk score. Then, we calculated the tumor mutation burden (TMB) score according to the somatic mutation data for each patient in the two groups. The foregoing analyses were based on maftools R package. Potential immune checkpoint blockade (ICB) response was assessed by tumor immune dysfunction and exclusion (TIDE) algorithm.33.1We analyzed the necroptosis‐related lncRNA expression level in 1,086 BC samples and 191 normal samples from the TCGA and GTEx datasets, and 2,848 DE lncRNAs were obtained. Then, 251 necroptosis‐related lncRNAs were identified in the DE lncRNAs by the Pearson correlation algorithm.3.2p value < 0.001)  Figure .3.3Based on risk score, age, and clinicopathological factors, a nomogram was developed for predicting the 1‐, 3‐, 5‐, and 10‐year OS incidences Figure . The cal3.4The three‐dimensional scatter diagram of the PCA respectively showed the distribution of different patterns. The samples grouped by risk score had distinct aggregation feature Figure . The res3.5Significant differences in the immune cell infiltration were observed between the two groups Figure , and the3.6Then, we analyzed the variations of the somatic mutations in two risk groups. The ten highest mutated genes were PIK3CA, TP53, TTN, CDH1, GATA3, MUC16, MAP3K1, MUC4, KMT2C, and PTEN. Patients in high‐risk group had markedly higher frequencies of TP53 mutation, and the opposite result was discovered with the alternation of PIK3CA and CDH1Figure . Compare4Necroptosis involved in immune responses and tumor microenvironment, and the benefits of activation of necroptosis pathways combined with immune checkpoint blockade have been demonstrated in recent study.In this study, we established a risk model with 7 necroptosis‐related lncRNAs, including SH3BP5‐AS1, AC012073.1, AC120114.1, LINC00377, AL133467.1, AC036108.3, and AC020663.2. Then, the patients were divided into low‐ and high‐risk groups according to the median values. The ROC and C‐index curve fatherly validated the prognostic precision of the risk score. We found that the risk score could be a yardstick for predicting prognosis. Subsequently, a nomogram was constructed for predicting prognosis of BC patients, which had an excellent concordance with the prediction.The 3D scatterplot of the PCA showed that patients categorized by necroptosis‐related lncRNAs exhibited distinct inherent biological feature. The results of Gene Ontology (GO) analysis demonstrated that activation of immune response, humoral immune response, and B‐cell receptor signaling pathway played an important role in biological pathways. Besides, the results of GSEA showed different enrichment of genes in KEGG, including cell cycle, DNA replication, pyrimidine metabolism, RNA degradation, spliceosome, and immune network. Although breast cancer is not regarded as a highly immunogenic cancer in the past, but tumor immune microenvironment impacts on a subset of breast cancers and partial patients might be suitable to immune checkpoint blockade treatment strategies after evaluation.The somatic mutation analysis showed that the samples in high‐risk group had more frequent TP53 mutation mutated and TP53 mutations may boost immunotherapy activity in BC according to previous study.Hypermutated breast cancer patients may benefit from PD‐1 inhibitors,There are still limitation and weaknesses in our study. Firstly, the analysis results were not validated in vitro and in vivo, and the biological function needs to be furtherly elucidated. Secondly, in view of complexity, we did not clarify the relationship between necroptosis‐related lncRNA and tumor‐infiltrating immune cells. Thirdly, in the retrospective study, there may be some biases in the case inclusion and data processing. The collection of clinical samples and external validation will be implemented in the future.5A well‐validated risk model was constructed based on 7 necroptosis‐related lncRNAs, including SH3BP5‐AS1, AC012073.1, AC120114.1, LINC00377, AL133467.1, AC036108.3, and AC020663.2. The BC patients in the high‐risk group had worse clinical outcomes. Besides, the high‐risk patients demonstrated higher TMB and lower TIDE score, indicating the better immune checkpoint blockade response. These findings pointed novel ways of BC prognosis estimation and optimal treatment strategy.


It was verified that DAD had activated PI3K/AKT/GSK-3β to reduce myocardial apoptosis and play a therapeutic function in MI/RI.Dried ginger-aconite decoction (DAD) is a traditional Chinese medicine (TCM) formula that has been extensively used in the treatment of myocardial ischemia reperfusion injury (MI/RI). However, its specific mechanism against MI/RI has not been reported yet. Therefore, this paper studies the potential active components and mechanism of DAD against MI/RI based on network pharmacology and experimental verification. Sixteen active components of DAD were screened according to oral bioavailability and drug similarity indices. Through Cytoscape 3.7.0, a component-target network diagram was drawn, and potential active components of DAD against MI/RI were determined. Protein-protein interaction (PPI) and compound-target-pathway (C-T-P) networks were established through the software to discover the biological processes, core targets and core pathways of DAD against MI/RI. High Performance Liquid Chromatography (HPLC) analysis identified the presence of potentially active core components for network pharmacological prediction in DAD. It was found that DAD might have played a therapeutic role in anti-MI/RI by activating the PI3K/Akt/GSK-3β signaling pathway in order to reduce mitochondrial hypoxia injury and myocardial cell apoptosis. The network pharmacological prediction was validated by Hypoxia/reoxygenation(H/R) model  Myocardial ischemia-reperfusion injury (MI/RI) denotes the further destruction of the cardiac structure, and the further aggravation of metabolic dysfunction or even irreversible damage of the myocardial cell, following the restoration of blood supply of ischemic and anoxic myocardial tissue, which mainly involves re-expansion of myocardial infarction area and life-threatening arrhythmia . A commoTreatize on Febrile and Miscellaneous Disease. Considering the efficacy of Yang for resuscitation, DAD is used to clinically treat ischemic heart diseases  plays an indispensable role in the prevention and treatment of MI/RI. It spans a long history, including Yi Qi Huoxue decoction, Gualou Xiebai Baijiu decoction and Si Ni decoction . It has diseases . Previoudiseases . Howeverin vivo and in vitro experiments are conducted to validate its mechanism on network pharmacological prediction. A graphical abstract of this study is presented in Graphical Abstract.Network pharmacology integrates biological systems and multi-directional pharmacological approaches, incorporates biological networks and drug action networks, transcends the constraints of single-target beliefs, and begins from multi-target research strategies in order to achieve a comprehensive network analysis of drug effects . It is aAconitum abietetorum W.T.Wang and L.Q.Li (No. 51078020190334YC) and Zingiber officinale Roscoe (No. 51078020191020YC) were obtained from Jiangyou City, Sichuan Province, China. The geographical location of Jiangyou is within 31°32′26′′−32°19′18′′ north and 104°31′35′′−105°17′30′′ east. Material authentication for TCM identification was carried out by Professor Gang Zhang of Shaanxi University of Chinese Medicine. The samples were deposited at the Herbal Medicine Museum of the same university.β-action, GADPH, Casp9, PI3K, AKT, Bax, Bcl-2 and phosphorylated(P)-AKT, GSK3β were also purchased from Boster Biological Technology Co., Ltd. . 6-gingerol , aconitine (No. A0608), mesaconitine (A0196) and hypaconitine (A0609) were all purchased from Chengdu Munst Biotechnology Co., Ltd. Standard purity was set as more than 98%. Methanol and triethylamine were purchased from Shaanxi Weitong Chemical Co., Ltd.Fetal bovine serum (FBS) was purchased from BI (United States). Phosphate buffer saline (PBS) and Dulbecco’s modified Eagle medium (DMEM) were procured from Gibco (United States). Penicillinstreptomycin mixture and Cell Counting Kit-8 (CCK-8) from Shanghai Biyuntian Co., Ltd. . Dimethyl sulfoxide (DMSO) and trypsin were also procured from Gibco (United States). The assay kits for malondialdehyde (MDA), superoxide dismutase (SOD)apoptosis, atpase, creatine kinase (CK), mitochondrial permeability transition pore (MPTP), lactate dehydrogenase (LDH) and glutathione peroxidase (GSH-PS) were all obtained from Boster Biological Technology Co., Ltd. . Cyt-C, Aconite and dried ginger were mixed at a 1:1 ratio. They were soaked in water for 0.5 h, then were boiled twice for 1 h each time. The filtrates were collected via gauzes, combined and concentrated to 1 g/ml to obtain the extract. For this study, 100 g of aconite and 100 g of dried ginger were prepared. Both components were completely immersed in water for 0.5 h. Then, 1.6 L water was added, letting the mixture boil for 1 h twice. The extract was then collected, filtered with gauze, and concentrated to 200 ml. The supernatant was obtained after centrifugation of the solution at 3,000 r/min, sterilized with 0.22 μm aqueous microporous membrane, and sealed.https://tcmspw.com/tcmsp.php) and the Comparative Toxicogenomic Database , with aconite and dried ginger as the keywords. Active components of DAD were screened via oral bioavailability (OB) and drug-like quality (DL)  , with DLhttps://www.uniprot.org/) to standardize their names.The primary compounds of aconite and dried ginger are alkaloids and volatile oil, both of which are irreplaceable and have good pharmacological activity. Thus, the following nine compounds were supplemented: deoxyaconitine, aconitine, hypaconitine, mesaconitin, 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, and zingerone. The targets of all active compounds were obtained and imported into the Universal Protein (UniProt) database (https://www.disgenet.org/) limited to “Homo sapiens” were obtained. The interactions of the DAD and MI/RI targets were considered as the potential therapeutic targets. The protein-protein interaction (PPI) of the common targets was accomplished in the string database (https://string-db.org/); the parameter organism was set to Homo sapiens, while the other basic settings were set as default. Using the Cytoscape 3.7.0 software, compound-target (C-T) and PPI were constructed.With “myocardial ischemia-reperfusion injury” as the keywords, MI/RI targets in the disgenet database (www.david.ncifcrf.gov/) provides a comprehensive set of functional annotation tools for researchers to understand the biological meanings behind extensive lists of genes. It was employed to undertake pathway enrichment analyses using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Pathway terms with p < 0.05 were deemed significant. Using the Cytoscape 3.7.0 software, compound-target-pathway (C-T-P) was constructed.The database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (−1. The column temperature was 30°C. The wavelength was set at 237 nm. The mobile phases were (A) methanol and (B) triethylamine aqueous solution, with gradient elution of 0–15 min (A: B = 30:70), 15–40 min (A:B = 65:35) and baseline (A:B = 30:70).DAD was filtered through a 0.22 μm nylon membrane prior to HPLC analysis. An HPLC System  was used to separate the components of DAD. All components were separated by Waters Bridge C18  and a C18 guard. Flowrate was set at 1.0 mL, min2 at 37°C. They were subjected to experimental procedures when they reached an 80% confluence level of population. They were classified into five groups: control group, Hypoxia/reoxygenation(H/R) group, DAD low-dose group (0.125 mg/ml), DAD medium-dose group (0.25 mg/ml), and DAD high-dose group (0.5 mg/ml). For all experiments, the cells were rendered quiescent by serum starvation for 24 h before treatment. Following pretreatment with DAD at varied doses for 24 h, the cells for all groups–except for the control group and the H/R group–were incubated in DMEM and glucose-free DMEM, respectively and then placed inside a hypoxia chamber . The chamber was flushed with 95% (v/v) N2 and 5% (v/v) CO2 at a flowrate of 15 L/min for 10 min, and maintained at 37°C to induce hypoxia injury. After hypoxia for 12 h, reoxygenation was conducted by replacing the medium to DMEM that contained 4.5 mM glucose (pH 7.4) and by subsequent incubation in a CO2 incubator (5% (v/v) CO2, 95% (v/v) air) for 2 h  were purchased from Wuhan Punosai Life Science and Technology Co., Ltd. . The cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin and 100 μl/ml streptomycin. They were maintained inside a humidified incubator with 95% air and 5% CO for 2 h .CCK8 assay was applied to determine the influence of DAD on the survival rate of H9C2 cells that were damaged by oxygen. The cells were briefly seeded onto 96-well plates and then cultured until they adhered. Afterward, the cells were treated with DAD at varied concentrations . Model group and control group were given DMEM (without glucose) and DMEM, respectively. Model according to the above method. Afterward, 10 μl of CCK-8 was added, and the mixture was incubated for another 2 h. Absorbance was recorded at 450 nm, and the experiments were performed in parallel in triplicate.The H/R damaged cells in each group were digested with 0.25% trypsin and centrifuged at 1,500 r/min for 5 min. The supernatant was discarded and the cells were collected. The collected cells were then resuspended with PBS (pH 7.2), washed with PBS twice, and centrifuged at 1,500 r/min for 5 min, before the supernatant was discarded. The precipitated cells were resuspended with 500 μl of binding buffer, then 5 μl of annexin V-FITC and 5 μl of PI staining solution was added. After mixing, the cells were incubated at room temperature in the dark for 5–15 min. Finally, the apoptosis for each group was detected by flow cytometry .+-K+-ATP and Ca2+-Mg2+-ATP levels were detected by their respective commercial kits.After H/R injury, the cells in each group were obtained. According to the manufacturer’s protocols, the SOD, MDA, NaSixty Sprague-Dawley male rats were purchased from Chengdu Da Shuo Experimental Co., Ltd. . They were housed in a specific-pathogen-free (SPF) environment. The rats in DAD low-dose, DAD medium-dose and DAD high-dose groups were orally administered with 1.4 g/kg, 2.8 g/kg and 5.6 g/kg DAD once daily, respectively. Those in the positive control group were orally administered with 0.09 g/kg per day of compound danshen dripping pills (CDDP), and those in the model, and sham group were orally administered with the same volume of 0.9% NaCl. A week later, all rats were operated, with the sham group only opening the chest without ligation. Left thoracotomy and pericardiectomy, followed by left anterior descending coronary artery ligation, were performed. After 40 min of ischemia, the ligature was opened for reperfusion for 2 h. The serum and heart tissue samples were prepared for future experiment. All animal experiments were performed in accordance with the Animal Care and Use Committee of the Institute of Materia Medica, China .Prior to the experiment, 2% TTC was placed in a 37°C thermostat for 0.5 h. Four rats were randomly selected from each group. Their hearts were removed, flushed with PBS, and rapidly frozen at −20°C. The specimens were uniformly cut into 1 mm slices under the line of ligature and placed in a 37°C, 2% TTC solution to dye for 20°min, and then fixed with 10% formaldehyde solution. Ultimately, the myocardial infarction area was white and the non-infarction area was red. The infarct area was calculated using ImageJ software . The applied equation was as follows,Immediately after reperfusion, the heart was removed and rinsed in precooled saline. The myocardium from the anterior wall of the left ventricle was removed. The heart was then fixed with precooled 4% paraformaldehyde and rinsed with water for 12 h. The specimens were dehydrated afterward. They were then immersed in xylene, and hematoxylin-eosin (H-E) staining was conducted after routine paraffin-embedded staining. Then, the slices were sealed with conventional resin, and the pathological changes in the myocardium were observed under an optical microscope .After MI/RI modeling, the heart tissue was removed. The tissue sections were washed in a phosphate buffer solution, and fixed in a 4% paraformaldehyde solution. They were then cut into paraffin sections with a thickness of 4 μm, and proteinase K was added. After a strict color rendering according to the kit instructions, five visual fields were randomly selected for shooting, and the color images of ten independent fields were randomly captured and digitized. The cells with clear nuclear markers were defined as TUNEL positive. Image J software was used for recording, and the apoptosis rate was calculated. The applied equation was as follows:After reperfusion, the rats were intraperitoneally anesthetized using chloral hydrate (30 mg/kg), and the blood samples were obtained from the abdominal aorta. The samples were left standing at room temperature for 30 min and then centrifuged at 3,000 r/min for 15 min. The serum was collected and stored at −80°C until used. Based on the manufacturer’s protocols, the GSH-Sp, MDA, CK and LDH levels were detected by the respective commercial kits.The fresh myocardial tissue just removed was rinsed with PBS; the excess water on the surface of the myocardial tissue was absorbed using a filter paper. The proper part of the entire heart tissue was taken; its mass was accurately measured, and the tissue homogenate was prepared using a mass-to-volume ratio of 1:9. The entire operation needed to be conducted in an ice bath. Finally, the tissue homogenate was centrifuged for 3,500 r/min for 10 min. The supernatant was collected and stored at −80°C for later use. The openness of the MPTP holes in the homogenate was determined according to the kit instructions.p-Akt , anti-p-GSK-3β , and anti-GAPDH . The membrane was rinsed thrice on the second day and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The blots were imaged under an enhanced chemiluminescence (ECL) system. The target band molecular weights and the net optical density were analyzed using the AlphaEase FC software .The myocardial tissue and the H9C2 cells were lyzed by RIPA buffer  containing cocktail protease inhibitors (1:100) and a protein phosphatase inhibitor (1:50) for 30 min on ice. The protein concentration in the supernatants was determined by BCA assay . Protein samples were loaded with 10% SDS-polyacrylamide gel , and then electrophoretically transferred onto PVDF . The membranes were blotted with 5% fat-free milk in a TBST buffer for 2 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: anti-Caspase-9 (1:600), anti-Bax (1:500), anti-Bcl-2 (1:500), anti-Cyt-c (1:500), anti-PI3K (1:500), anti-Akt , anti-p < 0.05.All data were expressed as mean ± standard deviation (SD). GraphPad Prism 7 software was employed to ascertain statistically significant differences. The differences among multiple groups were assessed using one-way analysis of variance (ANOVA). The difference between the means was considered statistically significant when From aconite and dried ginger, 16 compounds  were retTo examine the potential interactions of the 80 targets, String 11.0 database was used to build a PPI network. The minimum combined score between the targets was set as the medium confidence (0.400). The PPI network of the potential target was saved as a TSV file and then entered into Cytoscape 3.7.0 for visualization . In the p < 0.05). To realize a brief demonstration, only the top 10 significant GO entries were selected for further analysis. The top ten analyses for BP, CC and MF were selected respectively , 22 cell components (CC) and 18 molecular functions (MF) . Compared to the control group, the survival rate of the H/R group was only 58%. When the H9C2 cells were pretreated with 0.125–0.5 mg/ml DAD, cell viability was significantly restored (p < 0.01). DAD (0.25 mg/ml) had the greatest effect on cell survival rate, which increased by 25% (p < 0.01), compared to the H/R group . These results imply that the protective effect of DAD on H/R-damaged H9C2 is related to the mitochondria.The outcome of cardiomyocyte hypoxia was insufficient oxygen as required by the mitochondria, which would lead to mitochondrial damage, reduced ATP production and aggravated oxidative damage of the cardiomyocytes. SOD is an oxygen free radical scavenger in human body . The fint of ATP . After pon group -C, DAD (p < 0.01). after DAD preconditioning. The apoptosis rate of the H9C2 cells damaged by H/R significantly decreased, while the apoptosis rate of the DAD group (0.25 mg/kg) decreased by 18% (p < 0.01) compared to the H/R group . Compared to the H/R group, the expression of Bax in the H9C2 cells damaged by H/R had significantly decreased, while the expression of BCL2 had significantly increased when the H9C2 cells were pretreated by DAD (p < 0.01). In the administration group  release from the mitochondria . Therefoon group , DAD (0.p < 0.01). Compared to the MI/RI group, the infarct size of the myocardial tissue for each administration group had significantly reduced. The lowest infarct size was 9.2% in the CDDP (positive) group. Among the three DAD groups (p < 0.01), the MI area of rats in the DAD (2.8 g/kg) medium dose group was the lowest (19.3%) (p < 0.01). Meanwhile, although the high dose of DAD (5.6 g/kg) did not manifest a significant reduction in infarct size, a protective trend of infarct size reduction could be perceived.The electrocardiogram test results  of rats The degree of myocardial injury could be determined by histopathological examinations . In the p < 0.01). DAD  treatment had significantly mitigated the increased percentage of apoptotic cells compared to the model group (p < 0.01). Among the three DAD groups, the 2.8 g/kg group exhibited the lowest apoptosis rate (45%). Meanwhile, although a high dose of DAD (5.6 g/kg) did not significantly reduce the apoptosis rate, a decreasing trend in such rate was observed  of the MI/RI group had significantly increased (observed .p < 0.01), suggesting that serious heart damage might occur. After treatment, the activities of CK and LDH decreased in each dose group of DADs , while the activities of the 2.8 g/kg group had significantly decreased (p < 0.01) , the degree of mitochondrial swelling and the degree of elevated MDA level among MI/RI rats were reduced, while the activity of GSH-Px, SOD were restored (p < 0.05).Myocardial enzymes are vital indicators in the clinical detection of heart health . The act < 0.01) . Oxidati < 0.01) , and rearestored . Among tin vitro, the network pharmacologically predicted pathway and the related mitochondrial targets in vivo were further validated, namely the PI3K/AKT/GSK-3β pathway and the mitochondrial targets–Cyt-C and CASP9. Western blot analysis presented that the expression of PI3K/AKT/GSK-3β was inhibited (p < 0.05), and that the expression of Cyt-C and CASP9 was significantly increased in the MI/RI group compared to the sham group (p < 0.05). After DAD intervention, the expression of PI3K/AKT/GSK-3β was significantly activated, and the expressions of Cyt-C and CASP9 were significantly decreased in the DAD groups compared to the MI/RI group (p < 0.05).As a unique molecular target in the mitochondria, Cyt-C can activate apoptosis factors such as CASP9 and can lead to the apoptosis of damaged myocardium . ConsideRI group . Among tMI/RI is not only a primary cause of death among patients with cardiovascular and cerebrovascular diseases, but can also seriously affect the prognosis of patients with ischemic heart diseases. While the effects of DAD against MI/RI have been demonstrated, its exact mechanism is vague. In this case, pharmacological approaches are adopted to explore relevant molecular pharmacological mechanisms and validate them empirically.Sixteen active components and 171 targets of DAD were obtained using OB and DL parameters as significant evaluation indices and supplements of significant components. A higher degree of the compound in the C-T network denotes a greater significance. This study found that the degree values of aconitine, 6-ginger, hypaconitine and mesaconitine were among the top. These could be the key components of DAD against MI/RI. The HPLC method was used to determine the above components in DAD that were predicted by network pharmacology. We found that DAD contained aconitine, 6-ginger, mesaconitine, which was consistent with the results of network pharmacology.in vivo and in vitro experiments are conducted to validate its mechanism on network pharmacological prediction.Through network pharmacology, it was found that BP that was highly correlated with DAD anti-MI/RI was the negative regulation of the apoptotic process, the lipopolysaccharide-mediated signaling pathways, the regulation of mitochondrial membrane potential, the inherent apoptotic signaling pathway as a response to DNA damage, the external apoptotic signaling pathway lacking ligand, and release of cytochrome C from mitochondria. An analysis of the C-T-P network revealed that DAD anti-MI/RI acted on multiple targets and signaling pathways. The core targets of the active compounds in DAD were determined, namely AKT1, PIK3G, MAPK3, MAPK1, NFKB, TNF, NFKBA, MTOR, GSK3β and TP53. As with BP, these targets were associated with apoptosis and inflammation. Various studies have likewise confirmed that apoptosis is the key mechanism of anti-MI/RI . Apoptos2+ overload and excessive oxidative stress can induce it to open  can specifically catalyze the phosphorylation of the phosphatidylinositol-3 hydroxyl group . It phosThe innovation of this study involves the prediction of active components, BP and mechanism of action of DAD against MI/RI using network pharmacology. This study has demonstrated that DAD plays an anti-MI/RI role by activating PI3K/AKT/GSK3β to reduce cardiomyocyte apoptosis. Nevertheless, the limitations of this study should be acknowledged. Firstly, DAD at its highest concentration has either no or minimal effect against MI/RI. In the dose-setting process, the clinical equivalent dose was selected as the medium-dose group. In In this study, a comprehensive strategy that involved network pharmacological analysis, HPLC technology and experimental verification was adopted to determine the potential active components and molecular mechanisms of DAD against MI/RI. Based on the TCMSP database and on core compounds, 16 active compounds of DAD were obtained. The presence of four of these components was identified in DAD by HPLC, in which the components were potential therapeutic ingredients as predicted by network pharmacology. Through the analysis of BP, hub targets and hub signaling pathways and experimental verification, it was concluded that DAD could play an anti-MI/RI role by inhibiting myocardial apoptosis via PI3K/AKT/GSK3β. The experimental results were consistent with the network pharmacological predictions. Relatively, this study evidently clarified the anti-MI/RI mechanism of DAD, which could provide a certain basis for future studies on DAD.


Staphylococcus aureus (MRSA) osteomyelitis. All treatment groups had less MRSA recovered than saline-treated animals. The emergence of rifampin resistance was observed in 3 of 16 animals with rifampin monotherapy and none with rifampin combination therapy. After treatment, the median tibial bacterial loads were 6.04, 0.1, 4.81, and 5.24 log10 CFU/g for saline-, rifampin-, vancomycin-, and omadacycline-treated animals, respectively. Omadacycline or vancomycin administered with rifampin yielded no detectable MRSA. Omadacycline administered with rifampin deserves evaluation in humans as a potential treatment for osteomyelitis.Omadacycline, vancomycin, and rifampin, as well as rifampin combination therapies, were evaluated in an experimental rat model of methicillin-resistant  Staphylococcus aureus, the most common cause of osteomyelitis, which often requires surgical intervention and long-term antimicrobial therapy , vancomycin-resistant enterococci, extended-spectrum-β-lactamase-producing Enterobacterales strains, multidrug-resistant pneumococci, and Legionella species, as well as anaerobes and Mollicutes strains  for the study strain MRSA IDRL-6169 were 0.5 and >32 μg/ml for omadacycline, 1 and >32 μg/ml for vancomycin, and 0.004 and 1 μg/ml for rifampin, respectively . Minimumesistant .Cmax) was calculated to be 3.4 μg/ml at 0.5 h, and the area under the concentration-time curve from 0 to 24 h (AUC0–24) was 29.4 μg · h/ml. Mean plasma concentrations of omadacycline in infected rats were evaluated 2 h after administration of drug on treatment days 1, 3, and 10. Mean concentrations of omadacycline on days 1, 3, and 10 were 3.9 ± 0.84, 0.89 ± 0.78, and 0.74 ± 0.11 μg/ml, respectively, with an average of 1.8 ± 1.6 μg/ml across all days. Mean concentrations of omadacycline, when administered with rifampin, on days 1, 3, and 10 were 4.7 ± 0.62, 1.53 ± 0.89, and 2.6 ± 1.6 μg/ml, respectively, with an average of 2.9 ± 1.7 μg/ml across all days. Compared to the plasma concentration profile of healthy animals (mean omadacycline concentrations of 2.6 ± 0.92 μg/ml at 1 h and 2.0 ± 0.3 μg/ml at 3 h), these values suggest no effect of infection on omadacycline plasma concentrations in this model; while omadacycline concentrations trended slightly higher when omadacycline was administered with rifampin, conclusions cannot be drawn from the limited data set.Mean omadacycline plasma concentrations versus time after a single 20 mg/kg dose of omadacycline in healthy, uninfected rats are shown in 0–24 was previously determined to be 332 μg · h/ml, with a peak concentration of 28 μg/ml  . In rats24 μg/ml ). A vanc10 CFU/g , 0.10 log10 CFU/g , 4.81 log10 CFU/g , and 5.24 log10 CFU/g  for the saline-, rifampin-, vancomycin-, and omadacycline-treated groups, respectively (P ≤ 0.0122). The amounts of MRSA recovered with vancomycin monotherapy were lower than those with omadacycline monotherapy (P = 0.0348). Rifampin monotherapy was not significantly different than combination therapies; however, the emergence of rifampin resistance was detected in 3 animals in the rifampin monotherapy group (MICs of >16 μg/ml).Results of bone cultures for each animal are shown in ectively . No MRSAThe physical appearance of animals in the different treatment groups was not different. Yellowing of the bone (the color of the omadacycline) was noted in omadacycline-treated animals. Histopathologic examination showed that saline- and vancomycin-treated animals each had a single granuloma with central suppuration  and B. AS. aureus osteomyelitis, methicillin-resistant cases have been reported to have worse outcomes than methicillin-susceptible cases, including longer hospital stays, more surgeries, recurrent infections, and higher morbidity rates  of MRSA was still recovered after 21 days of treatment. Currently available antibiotics are often less than perfect in the treatment of orthopedic infections because of the complex nature of the bone. Addition of rifampin is recommended in some scenarios due to its activity against staphylococcal biofilms  that was isolated from a periprosthetic hip infection at Mayo Clinic  and has been shown to produce infection in previous osteomyelitis studies  by TurboIonSpray liquid chromatography-tandem mass spectrometry. Mean omadacycline plasma concentrations versus tested time points for uninfected Sprague-Dawley rats were used to estimate the pharmacokinetic parameters (Cmax and AUC0–24) of omadacycline in plasma by standard noncompartmental methods using a WinNonlin -validated SAS program.The study was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Omadacycline plasma concentrations were determined in healthy Sprague-Dawley rats  after a single 20 mg/kg intraperitoneal dose of omadacycline. Blood was collected from 4 rats via the tail vein at 0.5, 1, 3, 6, 12, and 24 h after omadacycline administration, and plasma concentrations of omadacycline were analyzed at Q8 CFU/ml MRSA IDRL-6169 were injected into the medullary cavity. The hole was sealed with dental gypsum, and the site was closed. Four weeks after the establishment of infection, rats were randomly assigned to one of six intraperitoneal treatment arms , as follows, and treated for 21 days: saline , rifampin , vancomycin , omadacycline , vancomycin plus rifampin, and omadacycline plus rifampin. Plasma was collected 2 h after treatment from 4 animals each in the omadacycline and omadacycline plus rifampin groups at 1, 3, and 10 days, to determine plasma omadacycline levels in infected animals.Experimental chronic osteomyelitis was established in 102 Sprague-Dawley rats using a previously described model of rat osteomyelitis . BrieflyTwelve hours after completion of vancomycin and rifampin therapy and 24 h after saline and omadacycline therapy, rats were euthanized, and the left tibiae were aseptically removed. One infected tibia from each group was chosen for histopathologic examination, fixed for 48 h in 10% formalin, cut in half longitudinally, paraffin embedded with the medullary cavity surface on top, sectioned longitudinally, mounted on glass slides, and stained with hematoxylin and eosin. Slides were reviewed by a board-certified pathologist with expertise in microbiology and infectious disease pathology. The remaining tibiae were cryopulverized and weighed for quantitative bacterial culture. In addition, to screen for the emergence of resistance, bone homogenates were plated on Mueller-Hinton agar (MHA) containing 4 μg/ml of rifampin, vancomycin, or omadacycline if the animals had received treatment with the cognate antibiotic. All colonies recovered on selective medium were subjected to identification and MIC testing with the relevant antibiotic.10 CFU per gram of bone. Descriptive summaries are reported as median and range. Comparisons among the six groups were first performed using the Kruskal-Wallis test. Due to statistically significant differences between the groups, further comparisons between groups were performed in a pairwise manner using the Wilcoxon rank sum test. Nonparametric tests were used because of the small sample size and non-normally distributed data. All tests were two sided, and P values of <0.05 were considered statistically significant. Analysis was performed using SAS software version 9.4 .Results were reported as log


Lactobacillus delbrueckii. The aim of the present study was to interpret the peptidomic pattern of the fermented milk by five strains of L. delbrueckii ssp. bulgaricus and ssp. lactis prior to and after the simulated gastrointestinal digestion in vitro. The results indicated variations in the peptidomics among the samples, particularly between the samples of different subspecies. The peptides originating from β-casein were abundant in the samples of ssp. bulgaricus, whereas the peptides derived from αs1-casein and αs2-casein were more likely to dominate in those of ssp. lactis. For β-casein, the strains of ssp. bulgaricus displayed extensive hydrolysis in the regions of (73–97), (100–120), and (130–209), whereas ssp. lactis mainly focused on (160–209). The digestion appears to reduce the variations of the peptidomics profile in general. Among the five strains, L. delbrueckii ssp. bulgaricus DQHXNS8L6 was the most efficient in the generation of bioactive peptides prior to and after digestion. This research provided an approach for evaluating the peptide profile of the strains during fermentation and digestion.Few studies have investigated the peptidomics of fermented milk by  Lactobacillus delbrueckii, one of the thermophilic lactic acid bacteria (LABs), plays an important role in food fermentation, including ssp. bulgaricus and ssp. lactis, two subspecies isolated from milk niches. Previous studies showed that there were differences in carbohydrate metabolism between ssp. bulgaricus and ssp. lactis [. lactis ,2. HowevL. delbrueckii ssp. bulgaricus and lactis were classified as PrtB [The proteolytic system provides peptides and free amino acids for the growth of strains during fermentation , and the as PrtB  and PrtL as PrtB . How PrtL. helveticus and could resist gastrointestinal digestion [L. delbrueckii ssp. bulgaricus and lactis. Moreover, as gastrointestinal digestion affects the absorption and function of peptides, investigating the changes of the peptide profile during digestion are critical for evaluating the function of fermented milk.Milk fermentation by LAB is known for the generation of bioactive peptides ,8 with digestion ,11,12,13L. delbrueckii ssp. bulgaricus and ssp. lactis were investigated before and after the simulated gastrointestinal digestion in vitro in order to understand the pattern of peptide formation during fermentation and digestion.Therefore, in the current study, the peptidomics of fermented milk by five strains of L. delbrueckii ssp. bulgaricus strains, DXJLHTS2M2, DQHXNS8L6, and 2038 and two L. delbrueckii ssp. lactis strains, D11M188 and ATCC12315, were used to ferment skim milk. All the strains were obtained from the Culture Collections of Food Microbiology, Jiangnan University .Three w/w) skim milk powder. The bacterial strains were incubated into skim milk with an initial culture concentration of 1–5 × 106 CFU/mL and incubated at 37 °C for 12 h. The viable cell counts of L. delbrueckii strains were determined using the plating method. Then, 0.5 mL samples were added to 4.5 mL of 0.9% (w/v) sterile saline and diluted serially as appropriate. Plates were incubated at 37 °C for 48 h under anaerobic conditions. Colonies were counted on each plate, and live-cell counts were expressed as lg(cfu∙mL−1). The pH was measured by a pH meter . The experiments were conducted in three replications.Fermented skim milks were manufactured using the previously referred to method with some modifications . Skim mig for 10 min at 4 °C and filtration with 0.45 µm nylon syringe filter. Then, ultrafiltration was applied using a membrane with a cut-off value of 10 kDa , and the whey was stored at −20 °C prior to further analysis.Whey fraction of samples was prepared according to the previous method with moderate modifications . The pH w/w) enzyme to substrate. After digestion at 37 °C for 2 h, the pH was adjusted to 8 with 0.4 M NaOH, and then trypsin  was added to digest at 37 °C for 2 h. Then, samples were heated at 95 °C for 10 min to inactivate the enzyme activity. The supernatants of the digested samples were collected by centrifuge at 14,000× g for 5 min at 4 °C and filtered by a 0.45 µm nylon syringe filter. The digested samples were finally stored at −20 °C prior to further analysis.The digestion process was conducted according to the previous study with slight modification . The pH TM Quantitative Colorimetric Peptide Assay . The absorbance was read at 480 nm and the results were expressed as mg∙mL−1.The peptide content of the samples was determined using Pierce−1. After being centrifuged for 10 min at 8000× g, supernatants were collected, and 2 µL were injected to an EASY nLC 1200  with Acclaim PepMapTM RSLC . The linear gradient of mobile phase B (acetonitrile with 0.1% FA and 10% MilliQ-treated water) with flow rate of 0.2 µL∙min−1 was performed as follows: 6–20% for 40 min, 20–32% for 12 min, 32–80% for 4 min.One milliliter of the samples was purified using C18 solid-phase extraction with Hypersep C18 100 mg microcolumns  and dried by centrifugal evaporation  for 2 h at 40 °C. Dried samples were dissolved in mobile phase A (MilliQ-treated water with 0.1% FA and 2% acetonitrile) and diluted to 0.5 mg∙mLm/z range with a scan time of 0.2 s. A maximum of 15 precursor ions with an intensity threshold of 10,000 counts was selected for ion trap collision-induced dissociation (CID) fragmentation and subjected to collision energy ramping from 8 V to 9 V for low mass and 40 V to 90 V for high mass. The MS/MS spectra were recorded on the 180 to 2000 m/z range with a scan time of 0.1 s.The chromatography device was coupled to a Q-Exactive mass spectrometer , and the liquid chromatography (LC) eluent was electrosprayed from the end of the column at an applied voltage of 2.3 kV. MS analysis was performed in sensitivity, positive ions, and data-dependent analysis (DDA) modes, and MS data were collected in the 150–2000 https://maxquant.net/maxquant/ (accessed on 5 December 2014)) were performed using the UniProt databases (Available online: https://www.uniprot.org/ (accessed on 5 January 2021)) restricted to Bos taurus organism. A mass tolerance of 35 ppm and 3 missing cleavage sites as well as an MS/MS tolerance of 0.02 Da were allowed. Variable methionine oxidation was also considered. The relevance of protein and peptide identities was judged according to their identification generated by PEAKS Studio 7.0 (p < 0.05) and a false discovery rate < 1%.Database searches via Maxquant  and partial least squares discriminant analysis (PLS-DA) were used, respectively, to visualize differences in peptidomic profiles between all samples and two subspecies groups in MetaboAnalyst ). Peptides detected in at least two of the three replicates were considered identified in samples, and the variable importance in projection (VIP) score was used to represent differences between two subspecies. The bioactivity of detected peptides was matched to the Milk Bioactive Peptide Database (available online: http://mbpdb.nws (accessed on 9 August 2021)). GraphPad Prism 8.0  was chosen to draw the figure.The mean ± standard deviation (SD) was used for the presentation of data, and multiple ANOVA was carried out with L. delbrueckii ssp. lactis, whereas the digestion appears to reduce the extent of discrimination of the fermented samples. The peptide content of the fermented milk of ssp. bulgaricus was generally higher than that of ssp. lactis, with the highest peptide content detected in the fermented milk of L. delbrueckii ssp. bulgaricus DXJLHTS2M2 and 2038 followed by DQHXNS8L6. , while only 24 differential peptides were present in that of ssp. bulgaricus DXJLHTS2M2. In total, 28 peptides were shared by six digested samples, with 22 from the five digested samples of fermented milk.After digestion, the number of peptides dramatically decreased in the samples of DXJLHTS2M2, 2038, and D11M188 b. HoweveL. delbrueckii species level, five fermented milks shared 44 peptides with a difference in abundance (bulgaricus such as β-casein f (106–119), β-casein f (103–123), and β-casein f (78–93), while 13 peptides were more abundant in the samples of ssp. lactis such as αs1-casein f (1–9), αs1-casein f (176–192), and β-casein f (193–208).At bundance a. In totbulgaricus, whereas peptides derived from αs1-casein and αs2-casein were more likely to dominate in the group of ssp. lactis as previously reported [Comparison of the fermented samples was evaluated with PLS-DA, with R2X, R2Y, and Q2Y values of 0.979, 0.998, and 0.957, respectively , confirmreported ,18.bulgaricus and 70% from αs1-, αs2-, and κ-casein for two samples of ssp. lactis (L. delbrueckii ssp. bulgaricus displayed more intensive proteolysis of milk proteins during fermentation than those of L. delbrueckii ssp. Lactis and digestion appears to significantly reduce the richness of the peptide profile (p < 0.05)  c.L. delbrueckii ssp. bulgaricus was classified as PrtB [L. delbrueckii ssp. lactis has a unique amino acid substitution at position 222 for substrate specificity and different hydrolysis patterns of chromophoric peptides compared to PrtB [bulgaricus and ssp. lactis had similar hydrolysis activity against αs1-casein, αs2-casein, and β-casein based on the pattern of SDS-PAGE [Different LABs had different CEPs, which are responsible for the specificity for the substrate ,20. The  as PrtB , while P to PrtB . A previSDS-PAGE ,18, whicbulgaricus and the latter in ssp. lactis.Comparison of the digested samples is shown in bulgaricus displayed a stronger ability in hydrolyzing β-casein while ssp. lactis showed a preference to αs2-casein.Variation on the cleavage pattern of αs2- and β-casein based on peptidomics was observed between the samples from the two subspecies in the heat map , in whicbulgaricus produced notable peptides from the region (95–115), whereas ssp. lactis generated more peptides from the region (142–207). Among these peptides from αs2-casein, 13 were unique in the samples of ssp. bulgaricus, while 12 were unique in ssp. lactis  and (142–207) were extensively hydrolyzed and lead to the generation of a large number of peptides a. Specif. lactis . Besidesbulgaricus (PrtB) might prefer the hydrophobic region while ssp. lactis (PrtL) might prefer the hydrophilic region.A previous study indicated that different CEPs may have varied preference to the cleavage regions. The cleavage sites of PrtH and PrtH2 on αs2-casein are located in the regions (1–25) and (97–162) . H-straibulgaricus had more extensive hydrolysis in the regions (73–97), (100–120), and (130–209), whereas fermented samples of ssp. lactis had narrower hydrolysis in the region (160–209) with fewer peptides from β-casein  of αs2-casein, while ssp. lactis did not, which released a notable number of peptides through the fermentation of both subspecies and had a subspecies-based profile. In addition, the region (24–35) released several peptides in all fermented samples after digestion, such as αs2-casein f (25–33) and αs2-casein f (25–35). Alternatively, peptides from regions (73–97), (124–163), and (189–202) of β-casein still existed, while regions (100–120) and (164–188) diminished after the digestion of pepsin and trypsin  of β-casein, were hydrolyzed into smaller peptides around arginine residues (position 202) at the C-terminal side b. This wL. delbrueckii generally led to the generation of more digestion-resistant and bioactive peptides in the fermented milk samples than in the skim milk [As shown in kim milk . The ferThe bioactivity of the peptides is shown in bulgaricus generated notable peptides in the region (95–115), whereas ssp. lactis formed more peptides and a more distributed pattern from the region (142–207). For β-casein, ssp. bulgaricus had more extensive cleavage distribution, including regions (73–97), (100–120), and (130–209), whereas ssp. lactis had a narrower distribution in the region (160–209) with fewer peptides. After digestion, L. delbrueckii ssp. bulgaricus DQHXNS8L6 had the most peptides, peptides resisting digestion, bioactive peptides, and short-sized peptides, together with robust growth and acidification capacity.In the present study, a difference was observed in the peptidomic feature of fermented milks before and after the simulated gastrointestinal digestion in vitro between subspecies, mainly originating from the hydrolysis of αs2- and β-casein. Regarding αs2-casein, ssp.


Developing ecological approaches for disease control is critical for future sustainable aquaculture development. White spot syndrome (WSS), caused by white spot syndrome virus (WSSV), is the most severe disease in cultured shrimp production. Culturing specific pathogen-free (SPF) broodstock is an effective and widely used strategy for controlling WSS. However, most small-scale farmers, who predominate shrimp aquaculture in developing countries, cannot cultivate SPF shrimp, as they do not have the required infrastructure and skills. Thus, these producers are more vulnerable to WSS outbreaks than industrial farms. Here we developed a shrimp polyculture system that prevents WSS outbreaks by introducing specific fish species. The system is easy to implement and requires no special biosecurity measures. The promotion of this system in China demonstrated that it allowed small-scale farmers to improve their livelihood through shrimp cultivation by controlling WSS outbreaks and increasing the production of ponds. Wang et al. develop a shrimp polyculture system that prevents white spot syndrome outbreaks by introducing specific fish species. The system is easy to implement and requires no special biosecurity measures which makes it ideal for small-scale farmers. In addition, aquaculture can improve the socioeconomic condition and livelihood of people in low-income countries by providing a highly nutritious food supply, employment, and income4. In Asian countries, shrimp are predominantly cultivated by small-scale farmers6. Furthermore, shrimp farming has been adopted as a strategy to promote economic growth and alleviate the poverty of farmers in these countries7. The increasing incidence of infectious diseases outbreaks is a major problem affecting the expansion of the shrimp aquaculture industry3. Therefore, developing convenient and ecological approaches for small-scale farmers to control the disease is critical for the future sustainable development of the shrimp aquaculture industry and poverty alleviation in developing countries.Due to the decline of wild fishery resources worldwide, aquaculture plays a critical role in meeting the increasing demand for aquatic foods, which are a major source of animal protein9. WSS pandemics primarily occur with the sequential transmission of WSSV from healthy shrimp that consume dead WSSV-infected shrimp to other healthy shrimp11. Because of the high efficiency and low negative environmental impact, culturing specific pathogen-free (SPF) shrimp is the most widely used strategy for controlling WSS outbreaks12. Disease prevention by using SPF shrimp is only likely to be successful if accompanied by stringent and sophisticated pathogen-exclusion management practices13. However, small-scale farmers, especially those from low-income countries, have limited access to or cannot afford SPF broodstock. Moreover, they do not have the infrastructure and technical skills to apply the required biosecure practices for culturing SPF shrimp15. Therefore, these limited-resource farms, which cultivate shrimp to improve livelihoods, are more vulnerable to WSS outbreaks than industrial farms. Most of these small farms have suffered from financial collapse due to production losses caused by WSS outbreaks16.White spot syndrome (WSS), which is caused by the WSS virus (WSSV), leads to catastrophic economic losses for the global shrimp aquaculture industry of over $1 billion annually, outweighing the losses due to other major crustacean diseases18. Therefore, polyculture has been considered as a promising strategy in the future sustainable shrimp aquaculture industry18. As general theory predicts that selective predation on infected individuals can reduce the prevalence of diseases in the prey population20, polyculture might prevent WSSV outbreaks by restoring the spatiotemporal interaction of predators and prey. Here we developed a cost-effective and convenient shrimp polyculture system that effectively prevents outbreaks of WSS by introducing specific fish. The system is highly robust and has been demonstrated to successfully control WSS outbreaks in the cultivation of major cultivated marine shrimp species, including Pacific white shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), kuruma shrimp (Marsupenaeus japonica), and Chinese white shrimp (Fenneropenaeus chinensis). The implementation of this polyculture system does not require taking biosecurity measures. Furthermore, the system is capable of controlling WSS outbreaks even when there are WSSV carriers in shrimp postlarvae. Thus, small-scale farms can easily adopt this system to control WSS outbreaks without additional investment.Polyculture in aquaculture, which is cultivating more than one species in the same pond, might maximize yield and reduce wastes in effluent through better utilization of the available food in the system23. The prevalence of diseases in the prey population is positively correlated with the disease transmission rate but is negatively correlated with predation pressure and predator selectivity25. Thus, to develop a shrimp culture system that controls WSSV outbreaks through selective predation, we studied WSSV transmission dynamics in an L. vannamei population by determining the relationships among the bodyweight of one initial WSSV-infected shrimp, a number of deaths, and death time distribution. One piece of dead WSSV-infected shrimp infected a large number of healthy shrimp with the same bodyweight via ingestion. The number of infected shrimp in the groups exhibiting average body weights of 1.98, 6.13, and 7.95 g was 57.3, 64.7, and 71.3, respectively. This suggests that the transmission rate of WSSV is extremely high, and the basic reproduction number (R0) of WSSV increases with the bodyweight of dead WSSV-infected shrimp , African sharptooth catfish (Clarias gariepinus) (hereafter referred to as catfish), and red drum (Sciaenops ocellatus). The daily dead shrimp ingestion rates of grass carp, catfish, and red drum were 8.26%, 4.99%, and 11.63%, respectively  for 10 days and then introduced 525–750/ha catfish with an average body weight of 0.5 kg. Shrimp were cultivated in 57 ponds (67.00 ha) without fish in zone B. In zone A, we did not observe WSS outbreaks in any of the ponds and harvested 8730 ± 1187 kg/ha of shrimp in 110 days of culture, while WSS outbreaks occurred in 53 ponds of zone B  was selected as the coculture fish in the polyculture system of P. monodon (5/ha of non-SPF shrimp in 6 ponds (1.60 ha) for 30 days and then introduced 600–750/ha of brown-marbled grouper with an average body weight of 0.1 kg. We did not observe WSS outbreaks in any of the ponds in 150 days of culture and harvested 6395 ± 427 kg/ha and 6440 ± 447 kg/ha of shrimp in 2013 and 2014, respectively. However, WSS outbreaks occurred in all 3 ponds (0.80 ha) in which shrimp were cultivated without fish in these two years.We further developed polyculture systems for three widely cultivated shrimp species,  monodon . The eff monodon . We cultChaeturichthys stigmatias) as the coculture fish in the polyculture systems of M. japonica or F. chinensis  for 30 days and then introduced 750–900/ha of branded gobies with an average body weight of 0.05 kg. We did not observe WSS outbreaks in any of the ponds in 100 days of culture and harvested 1089 ± 50 kg/ha and 1121 ± 48 kg/ha of shrimp in 2013 and 2014, respectively. Shrimp were also cultivated without fish in 5 ponds (6.70 ha) as a control. In 2013, 4 out of 5 ponds had WSS outbreaks. In 2014, WSS outbreaks were observed in all 5 ponds, resulting in an average yield of 407 ± 16 kg/ha.We selected branded gobies . Second, the implementation does not require applying any biosecurity measure, and the fish used in the system are common aquaculture species or easy to find in coastal areas. Thus, small-scale farms can easily adopt the system in earthen ponds to prevent WSS outbreaks without additional investment. During the promotion in 2015, six farmers at a farmers’ association in Nansha, China, decided to adopt the polyculture system in their earthen ponds without infrastructure renovation  for preventing WSS outbreaks. As the time window is too short, it is necessary to coculture shrimp with fish, which allows the fish to remove the moribund shrimp promptly. In addition, Model 3 demonstrated that a polyculture system can effectively control WSS outbreaks in shrimp cultivation as long as the fish can swallow the WSS-infected and dead shrimp promptly and completely. Thus, new polyculture systems can be developed based on the results of experiments and our mathematical models.39. Thus, the co-culture fish species should not carry pathogens that can infect shrimps. Otherwise, the fishes should be screened for these pathogens before introducing into the ponds.Farmers usually cultivate shrimps at low salinity in the estuary areas of China, where produces more than 50% of shrimps in China. Therefore, two freshwater fish species, grass carp and catfish, were used in our polyculture systems. We also developed polyculture systems using marine fish species (brown-marbled grouper and branded goby). Farmers can select a polyculture system that is suitable for their cultivation conditions. In addition, new polyculture systems can be developed to adapt to local cultivation conditions by changing the co-cultured fish species. Local aquaculture or native fish species are recommended to be used in the polyculture systems, as they are well-adapted to the local environment. The use of exotic fish species in the polyculture system should be highly cautious and certified by local administration agencies, as it might cause adverse environmental impacts. It is reported that shrimps can be infected by a few fish diseasesThe experimental data show the time course of death for the infected shrimp satisfies the Laplacian distribution  in cultured shrimp as influenced by the WSS epidemic with the following:Now we can develop a model for the spread and break out of WSS. For any given weight According to infection process, the decrement of healthy shrimp is caused by their infection, therefore we have In the above system of ordinary differential equations, quantity Now we explain how to set up the formulas To evaluate the performance of the model 2, we compare the simulated scenario and the biological experimental settings. Our experiments show the quantity change of dead shrimps and live shrimps with respect to time, which is consistent with the result of simulation  fish-feeding quantity of dead shrimp, and (2) fish-feeding ratio of dead shrimp over healthy shrimp. We obtained 1 kg grass carp’s feeding quantity of different body weights of shrimp and the feeding selectivity through experiments. The mathematical reasoning of Model 3 is as follows:To block the transmission of WSS, we apply fish to eat dead shrimp and infected shrimp. Let The decrement of healthy shrimp is related to the number of infected healthy shrimp and the number of shrimp eaten by fish, as expressed in s in Eq. ; for \dofrom Eq.  is that lts Fig. .3 and a salinity of 8‰. Shrimp were quarantined for seven days before the experiment started. One piece of dead WSSV-infected shrimp was then introduced to each of the experimental aquariums. In addition, one piece of frozen dead shrimp (WSSV-free) was introduced to the control aquarium. Shrimp were fed once a day with artificial feed that is 2% of their body weight. Shrimp feces were timely removed, and 50% of the water in the aquarium was exchanged every day. To prevent healthy shrimps from eating the moribund shrimp but not the initial dead WSSV-infected shrimp, shrimp were observed every 10 min to identify and remove moribund shrimp from the second day of the experiment. Moribund shrimp were identified as the ones having pleopod activity, but no response to glass rod agitation. The experiment was continued until three days after the appearance of the last moribund shrimp in each aquarium. Five pieces each of moribund and survived shrimps in each aquarium were subjected to a one-step WSSV PCR assay. All moribund shrimps showed WSSV-positive, while survived shrimps showed WSSV-negative. A mathematical model (Model 1) describing the relationship among the bodyweight of one initial WSSV-infected shrimp, number of deaths, and death time distribution was established based on the experimental results.Three groups of 430 shrimp with a bodyweight of 1.98 ± 0.03, 6.13 ± 0.16, and 7.95 ± 0.13 g, respectively, were used. In each group, 30 shrimp were randomly selected and subjected to a two-step WSSV PCR assay. All the tested shrimp showed negative in the assay. The remaining 400 shrimps were divided equally and introduced to three experimental and one control ponds. All 12 aquariums (220 cm × 60 cm × 80 cm) were set up with a water volume of 0.5 m3 and salinity of 8‰. Regarding the stocking quantity of 7.5 × 105/ha in shrimp farming production, 750 healthy shrimp with an average body weight of 7.9 g were cultured in each of the nine ponds.To determine the changes in numbers of live and dead shrimp during WSSV transmission, 9 cement ponds (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 mTo prepare the WSSV acute-infected shrimp, healthy shrimp were starved for 3 days, and then fed with parts of dead WSSV-infected shrimp that are 20% of their body weights twice a day. Five shrimp were randomly selected and subjected to a one-step WSSV PCR assay. If the tested shrimp showed WSSV positive in the assay. The rest of the shrimp in the aquarium was used as the WSSV acute-infected shrimp in the following experiments.Healthy shrimp were quarantined for seven days before the experiment started. Thirty WSSV acute-infected shrimp were then introduced in each pond. Shrimps were fed once a day with artificial feed that is 2% of their body weight. The numbers of survived shrimp were counted in three ponds on the 2nd, 4th, 8th day after WSSV infection, respectively. Five dead shrimps in each pond were subjected to a one-step WSSV PCR assay, showing WSSV-positive. Based on model 1, we established a mathematical model (Model 2) to describe the dynamic changes of healthy, infected, and dead shrimps during WSSV transmission.Ctenopharyngodon idellus). Three cement ponds (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 m3 and a salinity of 5‰. Three grass carps with an average body weight of 0.5 kg, 1 kg, and 1.5 kg were released in each of the three ponds, respectively. The fish were raised for four days and then fed with dead shrimps with an average weight of 5.3 g. In addition, to determine the dead shrimp ingestion rate of African sharptooth catfish (Clarias gariepinus). Four cement ponds (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 m3 and salinity of 3‰. One African sharptooth catfish with bodyweight of 0.262, 0.496, 0.731, and 1.502 kg was released in each of the four ponds, respectively. The fish were raised for four days and then fed with dead shrimps with an average body weight of 6.2 g. Finally, to determine the dead shrimp ingestion rate of red drum (Sciaenops ocellatus). Three cement ponds (315 cm × 315 cm × 120 cm) were set up with water volume of 5 m3 and a salinity of 5‰. One red drum with a bodyweight of 0.590, 0.654, and 0.732 kg was released in each of the three ponds, respectively. The fish were raised for four days and then fed with dead shrimps with an average body weight of 3.9 g.To determine the dead shrimp ingestion rate of grass carp . The daily ingestion rate of fish was calculated for 5 days.3 and salinity of 5‰. In total, 750 healthy shrimp with an average body weight of 5.3 g were cultured in each pond. One grass carp weighting 0.956, 1.013, and 1.050 kg was released in each of the experiment ponds, respectively. No fish was released in the control pond. Every two days, 50% of the water in each pond was changed. Live shrimp that remained in each pond were counted and weighted after 10 days of the experiment.To determine the healthy shrimp ingestion rate of grass carp, three experimental ponds and one control pond (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 m3 and salinity of 3‰. In total, 750 healthy shrimp with an average body weight of 2.2 g were cultured in each pond. One African sharptooth fish weighting 1.050 kg was released in the experiment pond. No fish was released in the control pond. Every 2 days, 50% of the water in each pond was changed. Live shrimp that remained in each pond were counted and weighted after 10 days of the experiment.To determine the healthy shrimp ingestion rate of African sharptooth catfish, one experimental pond and one control pond (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 m3 and salinity of 5‰. In total, 750 healthy shrimp with an average body weight of 2.7 g were introduced in each pond. One red drum weighting 0.519, 0.554, and 0.595 kg was released in each of the experiment ponds, respectively. No fish was released in the control pond. Every two days, 50% of the water in each pond was changed. Live shrimp that remained in each pond were counted and weighted after 10 days of the experiment.To determine the healthy shrimp ingestion rate of red drum, three experimental ponds and one control pond (315 cm × 315 cm × 120 cm) were set up with a water volume of 5 m3 and a salinity of 5‰. Grass carp weighting 1.58 kg was cultured in the aquarium for four days before the experiment started. The diseased shrimp infected with WSSV died within two days, which makes it hard to distinguish the initial dead shrimp from the ones that were died from diseased shrimp. The diseased shrimp had reduced activity, and the activity of shrimp was reduced after the endopods and exopods were removed. Thus, shrimp with endopods and exopods removed were utilized to resemble WSSV-infected shrimp. Thirty pieces each of dead, WSSV-infected (endopods and exopods removed), and healthy shrimps were introduced in the aquarium. The mean weight of shrimp used in the experiment is 3.5 g.To determine the feeding selectivity of grass carp on dead, infected, and healthy shrimp, one aquarium (220 cm × 60 cm × 80 cm) was set up with a water volume of 0.5 m3 and salinity of 3‰. African sharptooth catfish with body weight of 1.03 kg was cultured in the aquarium for four days before the experiment started. Thirty pieces each of dead, WSSV-infected (endopods and exopods removed), and healthy shrimps were introduced in the aquarium. The mean weight of shrimps used in the experiment is 8.4 g.To determine the feeding selectivity of African sharptooth catfish on dead, infected, and healthy shrimps, one aquarium (220 cm × 60 cm × 80 cm) was set up with a water volume of 0.5 mDuring the 9 days of the experiment, the dead, infected (endopods and exopods removed), and healthy shrimp that remained in the aquarium were counted and weighed every day. New shrimps were added to ensure there are 30 pieces each of dead, infected (endopods and exopods removed), and healthy shrimp in the aquarium. The daily total body weight of shrimp that were ingested by fish in each pond was calculated by subtracting the total body weight of shrimp that remained in the pond from the total weight of shrimp put in the pond. The shrimp ingestion rate of fish is quantified by the daily ingestion rate .To determine the suitable bodyweight of grass carp for controlling WSS, four experimental groups and two control groups were set up. Each group consisted of three ponds (315 cm × 315 cm × 120 cm). In total, 600 healthy and 3 WSSV-infected shrimp with an average body weight of 5 g were cultured in each pond of experimental groups. One grass carp with a bodyweight of 0.3, 0.5, 1.0, 1.5 kg was released in the ponds of each experimental group, respectively. In the positive control group, 600 healthy and 3 WSSV-infected shrimp with an average body weight of 5.0 g were cultured in each of the three ponds without introducing grass carp. In the negative control group, 600 healthy shrimp with an average body weight of 5.0 g were cocultured with one grass carp weighting 1.0 kg in each of the three ponds. The numbers of live shrimp were counted after ten days of the experiment. If there were dead shrimp in the ponds, they were subjected to a one-step WSSV PCR assay. All dead shrimps showed positive for WSSV infection.To determine the suitable bodyweight of African sharptooth catfish for controlling WSS, four experimental groups and two control groups were set up. Each group consisted of three ponds (315 cm × 315 cm × 120 cm). In total, 600 healthy and WSSV carrying shrimp and 3 WSSV-infected shrimp with an average body weight of 1.5 g were cultured in each pond of experimental groups. The WSSV carrying shrimp were determined as the ones that showed positive in a two-step WSSV assay. One African sharptooth catfish with a bodyweight of 0.25, 0.5, 0.75, 1.5 kg was released in the ponds of each experimental group, respectively. In the positive control group, 600 healthy and 3 WSSV-infected shrimp with an average body weight of 1.5 g were cultured in each of the three ponds without introducing African sharptooth catfish. In the negative control group, 600 healthy shrimps with an average body weight of 1.5 g were cocultured with one African sharptooth catfish weighting 1.0 kg in each pond. The numbers of live shrimp were counted after ten days of the experiment. If there were dead shrimps in the ponds, they were subjected to a one-step WSSV PCR assay. All dead shrimps showed positive for WSSV infection.To determine the capacity of grass carp for controlling WSS, the number of WSSV-infected shrimp that could be ingested by one grass carp weighting 1 kg was evaluated. Four groups of shrimp with different body weights  were cocultured with 1-kg grass carp in the ponds.In 1.3 ± 0.1 g group, 750 healthy shrimp were cultured in each of the nine cement ponds (315 cm × 315 cm × 120 cm). Healthy shrimps were cultured with 3, 6, 9, 12, 15, 18, and 21 pieces of WSSV-infected shrimp in each of the seven experimental ponds, respectively. One grass carp weighting 1 kg was released in each of the seven ponds. Healthy shrimp were cultured with 3 WSSV-infected shrimps in one pond as a positive control. Additionally, healthy shrimps were cultured without WSSV-infected shrimp nor grass carp in one pond as a negative control. In 2.5 ± 0.2 g group, 750 healthy shrimp were cultured with 10, 20, 30, 40, 50, 60, and 70 pieces of WSSV-infected shrimp in each of the seven experimental ponds, respectively. One grass carp weighting 1 kg was released in each of the seven ponds. Healthy shrimp were cultured with 10 WSSV-infected shrimp in one pond as a positive control. Additionally, healthy shrimps were cultured without WSSV-infected shrimp nor grass carp in one pond as a negative control. In 5.0 ± 0.3 g group, 750 healthy shrimp were cultivated with 50, 70, 90, 110, 120, 130, and 140 pieces of WSSV-infected shrimp in each of the seven experimental ponds, respectively. One grass carp weighting 1 kg was released in each of the seven ponds. Healthy shrimp were cultured with 50 WSSV-infected shrimps in one pond as a positive control. Additionally, healthy shrimps were cultured without WSSV-infected shrimps nor grass carp in one pond as a negative control. In 7.8 ± 0.5 g group, 750 healthy shrimp were cultured with 30, 40, 50, or 60 pieces of WSSV-infected shrimps in four experimental ponds, respectively. One grass carp weighting 1 kg was released in each of the four ponds. Healthy shrimp were cultured with 30 WSSV-infected shrimps in one pond as a positive control. In addition, healthy shrimps were cultured without WSSV-infected shrimps nor grass carp in one pond as a negative control.In all the ponds, shrimp were fed with artificial feed that is 2% of their body weight. And 50% of the water was changed every day. The numbers of the remaining live shrimp were counted after 15 days of the experiment. A mathematical model (Model 3) was established based on the relationship of healthy shrimp, infected shrimp, dead shrimp, and fish.The number of grass carp required for controlling WSS in shrimp production was determined in Pinggang Aquaculture Base, Yangjiang, China in 2010. Forty ponds (0.34 ± 0.04 ha/pond) were divided into eight groups; each group consisted of 5 ponds. We cultured 675,000/ha of shrimp in the ponds. Shrimp were cultured for 20 days before 45, 150, 225, 300, 450, 600, 750/ha of grass carp with an average body weight of 1.0 kg were released in the ponds of group 2 to group 8. Shrimp were cultured without fish in the ponds of group 1. These 40 ponds were managed by using the same farming method. If the WSS outbreak occurred, shrimps were harvested immediately; if not, shrimps were harvested after 110 days of cultivation.The number of African sharptooth catfish required for controlling WSS in shrimp production was determined in Pinggang Aquaculture Base, Yangjiang, China in 2010. Thirty-five ponds (0.37 ± 0.06 ha/pond) were divided into seven groups; each group consisted of 5 ponds. We cultured 675,000/ha of shrimp in the ponds. Shrimp were cultured for 10 days before 150, 300, 450, 600, 750, 900/ha of African sharptooth catfish with an average body weight of 1.0 kg were released in the ponds of group 2 to group 7. Shrimp were cultured without fish in the ponds of group 1. These 35 ponds were managed by using the same farming method. If the WSS outbreak occurred, shrimps were harvested immediately; if not, shrimps were harvested after 110 days of cultivation.L. vanmamei and grass carps was validated at a farm in Maoming, Guangdong Province, China (Farm 1). Forty-six farm ponds (17.33 ha) were divided into zone A and zone B. Zone A consisted of 18 ponds with a total area of 6.03 ha, and zone B consisted of 28 ponds with a total area of 11.30 ha. The stocking quantity of shrimp in the ponds of zone A is 900,000/ha. Shrimp were cultured in the ponds for 20 days before releasing grass carps with an average body weight of 1.0 kg. The stocking quantity of fish is 317–450/ha. Shrimp were cultured without fish in the ponds of zone B, and the stocking quantity of shrimp is 900,000/ha. In 2012, we switched zones A and B, cultivating shrimp with grass carp in zone B but without fish in zone A. The stocking quantities of shrimp and fish were the same as in 2011. If a WSS outbreak occurred, shrimps were harvested immediately; if not, shrimps were harvested after 110 days of cultivation, and yields were measured.In 2011, the polyculture system of coculturing L. vanmamei and African sharptooth catfish was validated at a farm in Qinzhou, Guangxi Province, China (Farm 2). Ninety-five farm ponds (88.2 ha) were divided into zone A and zone B. Zone A consisted of 38 ponds with a total area of 21.2 ha, and zone B consisted of 57 ponds with a total area of 67.0 ha. The stocking quantity of shrimp in the ponds of zone A is 750,000/ha. Shrimp were cultured in the ponds for 10 days before releasing African sharptooth catfish with an average body weight of 0.5 kg. The stocking quantity of fish is 525–750/ha. Shrimp were cultured without fish in the ponds of zone B, and the stocking quantity of shrimp is 750,000/ha. In 2012, we split zone B into zones B1 and B2. Shrimp were cultivated with catfish in 38 ponds of zone A and 25 ponds (27.00 ha) of zone B1, while shrimp were cultivated without fish in 32 ponds (40.00 ha) of zone B2. The stocking quantities of shrimp and fish were the same as in 2011. If WSS outbreak occurred, shrimps were harvested immediately; if not, shrimps were harvested after 110 days of cultivation, and yields were measured.In 2011, the polyculture system of coculturing We tested the effectiveness of using fish for controlling WSS in shrimp production at a farm in Maoming, Guangdong Province, China (Farm 1) from 2013 to 2019. In 2013, shrimp were co-cultured with African sharptooth catfish of body weight ranging from 0.5 to 0.6 kg in 13 ponds (3.73 ha). The stocking quantity of shrimp in these ponds ranges from 878,788/ha to 1,230,769/ha. And shrimp were co-cultured with grass carp of body weight ranges from 0.7 kg to 1.0 kg and African sharptooth catfish of body weight ranges from 0.5 kg to 0.6 kg in 10 ponds (3.7 ha). The stocking quantity of shrimp in these ponds ranges from 909,091/ha to 1,212,121/ha. Additionally, shrimp were cultured without fish in 11 ponds (3.63 ha). The stocking quantity of shrimp in these ponds ranges from 878,788/ha to 969,697/ha. If WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.In 2014, shrimp were cocultured with grass carp of body weight ranging from 0.7 to 1.0 kg in 8 ponds (2.76 ha). The stocking quantity of shrimp in these ponds ranges from 833,333/ha to 1,060,606/ha. And shrimp were co-cultured with grass carp of body weight ranges from 0.7 to 1.0 kg and African sharptooth catfish of body weight ranges from 0.5 to 0.6 kg in 12 ponds (4.03 ha). The stocking quantity of shrimp in these ponds ranges from 825,000/ha to 1,060,606/ha. Additionally, shrimp were cultured without fish in 5 ponds. The stocking quantity of shrimp in these ponds was 1,060,606/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.In 2015, shrimp were co-cultured with grass carp of body weight ranging from 0.7 to 1.0 kg in 19 ponds (7.4 ha). The stocking quantity of shrimp in these ponds ranges from 746,269 to 1,538,462/ha. In addition, shrimp were cultured without fish in 10 ponds (3.8 ha). The stocking quantity of shrimp in these ponds ranges from 750,000 to 909,091/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.In 2016, shrimp were co-cultured with grass carp of body weight ranging from 0.7 to 1.0 kg in 19 ponds (8.11 ha). The stocking quantity of shrimp in these ponds ranges from 488,372/ha to 636,364/ha. Additionally, shrimp were cultured without fish in 8 ponds (2.84 ha). The stocking quantity of shrimp in these ponds ranges from 543,478/ha to 636,364/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.In 2017, shrimp were cocultured with grass carp of body weight ranging from 0.7 to 1.0 kg in 6 ponds (1.56 ha). The stocking quantity of shrimp in these ponds was 961,538/ha. And shrimps were co-cultured with grass carp of body weight ranging from 0.7 kg to 1.0 kg and African sharptooth catfish of body weight ranges from 0.5 to 0.6 kg in 12 ponds (3.96 ha). The stocking quantity of shrimp in these ponds ranges from 848,485/ha to 909,091/ha. Additionally, shrimp were cultured without fish in 9 ponds (2.76 ha). The stocking quantity of shrimp in these ponds ranges from 848,485/ha to 961,538/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.In 2018, shrimp were cocultured with grass carp of body weight ranging from 0.7g to 1.0 kg in 22 ponds (9.24 ha). The stocking quantity of shrimp in these ponds ranges from 454,545/ha to 869,565/ha. Additionally, shrimp were cultured without fish in 9 ponds (3.36 ha). The stocking quantity of shrimp in these ponds ranges from 695,652/ha to 861,111/ha. If a WSS outbreak occurred, shrimp were harvested; if not, shrimp were harvested after 110 days of cultivation.In 2019, shrimp were cocultured with grass carp of body weight ranging from 0.7 to 1.0 kg in 30 ponds (11.31 ha). The stocking quantity of shrimp in these ponds ranges from 652,174/ha to 1,000,000/ha. Additionally, shrimp were cultured without fish in 10 ponds (3.57 ha). The stocking quantity of shrimp in these ponds ranges from 666,667/ha to 1,000,000/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 110 days of cultivation.P. monodon and brown-marbled grouper was validated at a farm in Changjiang, Hainan Province, China (Farm 3). We cultured 6 × 105/ha of non-SPF shrimp in 6 ponds (1.60 ha) for 30 days and then introduced 600~750/ha of brown-marbled grouper with an average body weight of 0.1 kg. Shrimp were also cultured without fish in 3 ponds (0.8 ha). The stocking quantity of shrimp in these ponds is 6 × 105/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 150 days of cultivation, and yields were measured.In 2013, the polyculture system of coculturing 5/ha of non-SPF shrimp in 6 ponds (1.60 ha) for 30 days and then introduced 600–750/ha of brown-marbled grouper with an average body weight of 0.1 kg. Shrimps were also cultured without fish in 3 ponds (0.8 ha). The stocking quantity of shrimp in these ponds is 6 × 105/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 150 days of cultivation, and yields were measured.In 2014, we cultured 6 × 10M. japonica and branded gobies was validated at a farm in Qingdao, Shandong Province, China (Farm 4). We cultured 1.5 × 105/ha of non-SPF shrimp in 10 ponds (13.40 ha) for 30 days and then introduced 750~900/ha of branded gobies with an average body weight of 0.05 kg. Shrimp were also cultured without fish in 5 ponds (6.70 ha). The stocking quantity of shrimp in these ponds is 1.5 × 105/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 100 days of cultivation, and yields were measured.In 2013, the polyculture system of coculturing 5/ha of non-SPF shrimp in 10 ponds (13.40 ha) for 30 days and then introduced 750~900/ha of branded gobies with an average body weight of 0.1 kg. Shrimp were also cultured without fish in 5 ponds (6.70 ha). The stocking quantity of shrimp in these ponds is 1.5 × 105/ha. If a WSS outbreak occurred, shrimp were harvested immediately; if not, shrimp were harvested after 100 days of cultivation, and yields were measured.In 2014, we cultured 1.5 × 10P.monodon postlarva to his/her earthen pond (3 ha) on March 28, May 8, and June 15, respectively. And 1350 grass carps with an average body weight of 1 kg were released in the ponds on April 30. These farmers harvested shrimp from May to November, and grass carp on December 14. The yields of shrimp and fish of these six ponds were recoded. The other farmers in the association introduced 225,000 and 360,000 of P.monodon postlarva to their ponds (3 ha) on March 28 and May 8, respectively. WSS outbreaks occur in their ponds from May 15 to May 23. Therefore, these farmers only harvested shrimp in May. Six ponds were randomly selected, and the yields of these ponds were recorded.When we promoted the polyculture system at the farmers’ association in 2015, only 6 farmers decided to adopt the system, as most of the farmers worried that fish would ingest healthy shrimp. Each of the 6 farmers introduced 225,000, 360,000, and 360,000 F. chinensis in earthen pond (5 ha) before the promotion of the polyculture system in 2015. The yields of 10 randomly selected ponds in 2014 were recorded. In 2015, farmers at the association started to culture 8,000/ha of F. chinensis in their ponds. The shrimp were cultured 20 days before 800/ha of branded gobies with an average body weight of 0.05 kg were released in the ponds. Branded gobies were cultivated for 15 days before introducing to the ponds. Shrimps were harvested after 120 days of cultivation. The yields of ten randomly selected ponds were recorded.Farmers at the farmers’ association used to culture 1500/ha of Alpha levels of 0.05 were regarded as statistically significant throughout the study. Three replicates were set up for each experiment to confirm the reproducibility of the data. All data are reported as the mean ± standard errors.Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Quantification of differential abundance between groups was done using MS1 intensity based label-free. Label-free quantitative shotgun proteomics was used for characterization. This novel dataset of proteomics data from PBMC contains 3807 proteins; 44, 42 and 65 were differently abundant (P ≤ 0.05 and FC ± 1.5), in FLX vs. CTL, FO vs. CTL and FLX vs. FO, respectively; these findings are discussed in our recent research article . The present dataset of PBMC proteome adds new information regarding the effects of n-3 FA on the immune system, while providing reference for PBMC proteome in postpartum dairy cows.This article contains raw and processed data related to research published by Kra et al.  FO cows.We examined the different abundance among all groups: FLX vs. CTL, FO vs. CTL and FLX vs. FO, and found that 44 proteins were differently abundant . The full list of peptides is presented in This data describes the proteome of PBMC at week 1 postpartum in dairy cows supplemented peripartum with different n-3 FA. Supplementary Table 1 contains the dataset of 3807 identified and quantified proteins obtained by proteomic analysis, as well as statistical analysis of differentially abundant proteins among all groups , along with the relevant guidelines and regulations. The experiment was performed at the experimental dairy farm of Volcani Center in Rishon LeZion, Israel. The data presented in this paper are part of a larger study examining the effects of different n-3 FA supplemented peripartum on production, inflammatory and immune parameters, subpopulation of white blood cells, and proteome of PBMC. Full details on animal management and handling are provided in the companion paper Forty-two multiparous dry dairy cows were assigned into 3 groups at 257 days of pregnancy and supplemented up to 60 days postpartum a basal diet supplemented either with: (i) CTL – (n = 14) encapsulated saturated fat; (ii) FLX (n = 14) – encapsulated flaxseed oil that is enriched with ALA; and (iii) FO – (n = 14) encapsulated fish oil that is enriched with EPA and DHA. Blood sample were collected using a heparin-coated tube on day 3–12 (average 7 days) PP from 24 cows (8 cows from each treatment) for PBMC. Details on cows and procedures are in Kra et al. 2.2The PBMC blood sample was taken into a heparin-coated tube on day 3–12 (average 7 days) PP from 24 cows (8 cows from each treatment). The PBMC separation was done as previously described 2.3We randomly selected PBMC from 18 cows (6 from each treatment) for the proteomic analysis. The samples were thawed and sediment by centrifuged for 15 min at 500g, 4 °C. The supernatant was discarded and 0.5-mm glass beads  were added to the bottom of the vial. The cells were then ground in a homogenizer  with 1 mL lysis buffer . Following a centrifugation for 15 min at 20,000g, 4 °C, the protein concentration of the sample was analyzed by bicinchoninic acid (BCA) standard assay , then snap-frozen and stored at −80 °C. The proteomics process was achieved by incubation at 96 °C for 5 min, followed by six 30-s cycles of sonication . Proteins were reduced using 5 mM DTT and alkylated with 10 mM iodoacetamide under cover. Each sample was loaded onto S-Trap microcolumns  according to the manufacturer's instructions. In short, the samples were washed with methanol containing 50 mM ammonium bicarbonate, 90:10 (v/v). The samples were then processed with trypsin for 1.5 h at 47 °C. The digested peptides were eluted using 50 mM ammonium bicarbonate; trypsin was added to this fraction and incubated overnight at 37 °C. Two more elusions were performed using 0.2% (v/v) formic acid and 0.2% formic acid in 50% (v/v) acetonitrile (ACN). The pull of all three elusions was vacuum-centrifuged to dry. Samples were kept at −80 °C until analysis.2.4All chromatographic steps were done using ULC/MS-grade solvents. Sample was loaded for split-less Nano-Ultra Performance Liquid Chromatography (UPLC) . The mobile phase consisted of: (A) H2O + 0.1% formic acid and (B) ACN + 0.1% formic acid. RP Symmetry C18 trapping column  was used to desalt the samples online. The peptides were then separated using a T3 HSS nano-column  at 0.35 µL/min. The following gradient was used to elute the peptide from the column into the mass spectrometer: 4% to 27% B in 155 min, 27% to 90% B in 5 min, maintained at 90% B for 5 min and then back to initial conditions.The Nano-UPLC was coupled online through a NanoESI emitter  to a Q Exactive HF mass spectrometer (Thermo Scientific). Data were acquired in data-dependent acquisition mode using the Top10 method. MS1 resolution was set to 120,000 (at 200m/z), mass range of 375–1650 m/z, automatic gain control (AGC) of 3e6 and maximum injection time was set to 60ms. MS2 was performed by isolation with the quadrupole, width of 1.7Th, 27 NCE, 15k resolution, AGC target of 60ms and dynamic exclusion of 45s.2.5Raw data were processed with MaxQuant v1.6.6.0. The Andromeda search engine was used for searching against the bovine sequences from UniprotKB, and appended with common laboratory protein contaminants. The trypsin enzyme specificity was set with up to two missed cleavages allowed. Fixed modification was set to carbamidomethylation of cysteines and variable modifications were set to oxidation of methionines, and deamidation of N or Q. Peptide precursor ions were searched with a maximum mass deviation of 4.5 ppm and fragment ions with a maximum mass deviation of 20 ppm. Peptide and protein identifications were filtered at a false discovery rate of 1% using the decoy database strategy, while minimal peptide length was 7 amino acids. The match-between-runs option was used for peptide identifications across samples. Searches were performed with the label-free quantification option selected. Perseus v1.6.2.3. Student's t-test was used for quantitative comparisons, following logarithmic transformation, in order to identify significant differences across the biological replica. Fold changes (FC) were calculated based on the ratio of geometric means of the case versus control samples. Since the PBMC lysates after proteomic analysis contained an extremely limited amount of protein, it was not possible to conduct immunoblots for protein validation in this study.2.6Proteomics data, after logarithmic transformation, were analyzed by 3-way ANOVA and by t-test  to determine the effects of treatment: FLX vs. CTL, FLX vs. FO and FO vs. CTL. Differently abundant proteins for each effect were determined at P ≤ 0.05 and absolute FC ±1.5.The experiment complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals .Gitit Kra: Methodology, Project administration, Data curation, Visualization, Formal analysis, Validation, Writing – review & editing. Nataly Nemes-Navon: Project administration, Data curation, Formal analysis. Jayasimha Rayalu Daddam: Data curation, Methodology, Visualization. Lilya Livshits: . Shamay Jacoby: Data curation, Formal analysis, Methodology. Yishai Levin: Data curation, Formal analysis, Methodology, Validation. Maya Zachut: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. Uzi Moallem: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.