MDR1, is a well-known multidrug efflux transporter involved in drug resistance development. Pygo2 overexpression has been identified in several cancers. Previous studies have shown that abnormal expression of Pygo2 is related to tumorigenesis, chemoresistance, and tumor progression. In this study, to evaluate the underlying relationship between Pygo2 and MDR1 in GC, we constructed GC drug-resistant cell lines, SGC7901/cis-platinum (DDP), and collected tissue from GC patients' pre-and post-chemotherapy. We found that Pygo2 was overexpressed in GC, especially in GC drug-resistant cell lines and GC patients who underwent neoadjuvant DDP-based chemotherapy. Pygo2 overexpression may precede MDR1 and correlates with MDR1 in GC patients. Furthermore, knock-down of Pygo2 induced downregulation of MDR1 and restored SGC7901/DDP's sensitivity to DDP. Further mechanistic analysis demonstrated that Pygo2 could modulate MDR1 transcription by binding to the MDR1 promoter region and promoting MDR1 activation. The overall findings reveal that Pygo2 may be a promising biomarker for monitoring drug resistance in GC by regulating MDR1.Chemotherapy is the main therapy for gastric cancer (GC) both before and after surgery, but the emergence of multidrug resistance (MDR) often leads to disease progression and recurrence. P-glycoprotein, encoded by  Gastric cancer (GC) has relatively high morbidity and mortality rate around the world. Although surgery is the first choice of treatment, chemotherapy also plays an important role in treating GC as GC has a low incidence of early diagnosis. Neoadjuvant chemotherapy can diminish tumor volume, degrade tumor stage, and increase the resection rate. Postoperative chemotherapy can prevent tumor recurrence and metastasis, thus prolonging the overall survival. Palliative chemotherapy can alleviate clinical symptoms and improve the quality of life of patients. However, multidrug resistance (MDR) can eventually result in chemotherapy failure. Therefore, early detection of MDR in GC patient is essential.MDR1, can induce multidrug resistance by increasing drug efflux and decreasing intracellular drug concentration Cancer cells can pump various chemotherapeutic drugs out of the cell by drug efflux transporters DrosophilaPygopus2 (Pygo2) was initially identified as a novel functional protein of the Wingless pathway downstream in Cis-platinum (DDP) was obtained from the first affiliated hospital of Xiamen University. MDR1  and β-actin (Cat# sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Pygo2 (Cat# 11555-1-AP) antibody was obtained from Proteintech and TIAN pure Midi Plasmid Kit (Cat# DP107) from TIANGEN. TurboFect Transfection Reagent (Cat# R0531) was from Thermo Scientific. E.coli pLL3.7-hPygo2-KD (knock-down) and pGL3.3-control were a gift from Xiamen University School of Life Sciences.The GC cell lines, SGC7901 and SGC7901/DDP were cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The GC tissues were collected from 40 GC patients using gastroscopy and surgery. All the GC patients were treated with platinum-based neoadjuvant chemotherapy, including combined with capecitabine, tegafur gimeracil oteracil potassium capsule (S-1), doxorubicin (ADM) and 5-fluorouracil (5-Fu). Importantly, all subjects were given written informed consent in accordance with the recommendations of Ethics and Scientific Committee of The First Affiliated Hospital of Xiamen University and the Declaration of Helsinki, the approved number from the Ethics committee of Xiamen University is 2019118.5/well) were seeded in 96-well plates for 24 h, then treated with different concentrations of DDP for 24 h. Thereafter, cells were labeled with 20 μL MTT labeling reagent, and absorbance at 570 nm was determined with a microplate reader.Cell viability was measured by using the MTT method. Briefly, SGC7901 and SGC7901/DDP cells . 5×10Total protein extracts were prepared, and the protein concentration was measured as described previously TM RT reagent kit with gDNA Eraser . The expression levels of MDR1 and Pygo2 were detected using SYBR GREEN MIXTURE kit  and analyzed by comparing to β-actin. The primers are listed below: Pygo2, Forward Primer: 5'-GTTTGGGCTGTCCTGAAAGTCTG-3', Reserve Primer: 5'-ATAAGGGCGCCGAAAGTTGA-3'; MDR1, Forward Primer: 5'-AGCTCGTGCCCTTGTTAGACA-3', Reserve Primer: 5'-GTCCAGGGCTTCTTGGACAA-3'; β-actin, Forward Primer: 5'-CGAGCGGGAAATCGTGCGTGACATTAAGGAGA-3', Reserve Primer: 5'-CGTCATACTCCTGCTTGATCCACATCTGC-3'.Total RNAs were homogenized in Trizol and isolated, as described in our previous study GC tissues were fixed and cut into slices, then processed and incubated with primary antibodies and probed with secondary antibody. The detailed protocol could refer to our previous work SGC7901/DDP cells were transfected with pGL3.3-Control and pLL3.7-hPygo2-KD plasmid using TurboFect Transfection Reagent following the manufacturer's instructions. Briefly, short hairpin targeted Pygo2 (Pygo2 shRNA) and the scramble control sequences (SCR shRNA) were designed based on hPygo2 sequences and further synthesized for the construction of plasmids The pGL6-MDR1 reporter plasmid, which contained the MDR1 promoter region was constructed in our lab and described in our previous study The primers for ChIP on MDR1 were designed as described previously The experiments such as MTT assay, WB and RT-PCR were performed in triplicate and data was expressed as the mean ± SD. Collective data was then analyzed using student's t-test and chi-square test. Correlation analysis was processed by using GraphPad Prism 7.0. P<0.05 was defined as the statistical significance and represented with *. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.Fig. ). SGC7901/DDP was insensitive to a low concentration of DDP when cells were exposed to different concentrations of DDP . The median IC50 value of SGC7901/DDP in 24 h was 0.89 μg/mL, which was significantly higher than SGC7901 with a median IC50 value in 24 h was 0.35 μg/mL . ADM and 5-Fu were also commonly prescribed to treat GC, we found that SGC7901/DDP was also insensitive to 5-Fu and ADM compared to SGC7901. The median IC50 values of 5-Fu and ADM in SGC7901/DDP for 24 h were 109.1 μg/mL and 1.3 μg/mL, which were higher than 20.5 μg/mL and 0.5 μg/mL in SGC7901 .We successfully constructed GC drug resistance cell SGC7901/DDP after six months utilizing the method described above. Comparing to SGC7901, SGC7901/DDP had a smaller cell volume, and its cell shape had changed from long spindle to short polygon. Furthermore, SGC7901/DDP had a slower breeding rate and showed monoclonal colony growth . Next, we analyzed Pygo2 and MDR1 expression levels in normal-appearing tissue adjacent to GC (NaT), GC tissue (GcT) and GC tissue after neoadjuvant chemotherapy (NeC). Our data revealed that MDR1 and Pygo2 was low or rarely expressed in NaT but were highly expressed in GcT and with highest expression levels in NeC .Firstly, we detected the protein and mRNA expression levels of Pygo2 and MDR1 in SGC7901/DDP by WB and RT-PCR. Our results showed that SGC7901/DDP had higher expression levels of Pygo2 and MDR1 compared with SGC7901 (Table 1). Furthermore, analyzing the two genes across online data sets showed that a strong correlation between Pygo2 and MDR1 expression levels after chemotherapy in GC tissue .We analyzed 40 GC patients in the first affiliated hospital of Xiamen University who had undergone neoadjuvant DDP-based chemotherapy. Our results showed that 15% (6/40) of GC patients were Pygo2 overexpression positive, and 5% (2/40) were MDR1 overexpression positive pre-chemotherapy. While amongst the group, 62.5% (25/40) of GC patients were Pygo2 overexpression positive, and 45% (18/40) were MDR1 overexpression positive post-chemotherapy. The results indicated that Pygo2 overexpression might precede MDR1 . The results suggested that when Pygo2 was inhibited, MDR1 expression was also downregulated . In addition, GC drug resistance cell SGC7901/DDP became sensitive to chemotherapeutic drugs when Pygo2 was knocked down .To clarify the relationship between Pygo2 and MDR1 in GC, we suppressed Pygo2 expression in SGC7901/DDP by transfecting pLL3.7-hPygo2-KD plasmid. We proceeded to perform WB to determine whether Pygo2 was successfully suppressed . Further mechanism analysis by ChIP assay verified the association of Pygo2 with the MDR1 promoter region . One other point worth emphasizing is our previous study also demonstrated Pygo2 could modulate MDR1 activation in breast cancer by directly binding to β-catenin, thus regulating the Wnt/β-catenin signaling pathway To illuminate the role of Pygo2 in the activation of MDR1, we performed luciferase reporter analysis. We found that Pygo2 could promote the activation of MDR1 promoter , but also results in the ineffectiveness of the second-and third-line solutions Studies exploring the mechanism of drug resistance are mainly divided into the following categories: alterations in drug efflux Pygo2 is a novel effector protein, downstream of the Wnt/β-catenin signaling pathway, that acts as a co-activator with β-catenin to promote β-catenin/LEF/TCF activation These recent findings verified that Pygo2 and MDR1 were overexpressed in GC, especially in GC patients after chemotherapy. Pygo2 overexpression precedes MDR1 and Pygo2 strongly correlated with MDR1 in GC patients who underwent neoadjuvant chemotherapy. The Knock-down of Pygo2 could restore the drug-resistant cell SGC/7901 sensitivity to chemotherapeutic drugs. Further mechanism analysis indicated that Pygo2 could promote MDR1 activation by binding to the MDR1 promoter region. These results demonstrated that Pygo2 might be a novel biomarker for monitoring drug resistance by upregulating MDR1. This study also provided a potential therapeutic target in the treatment of cancers where Pygo2 is overexpressed.In conclusion, Pygo2 and MDR1 were overexpressed in GC patients after chemotherapy. Pygo2 played a pivotal role in drug resistance in GC by promoting MDR1 activation. Pygo2 may be a novel biomarker for monitoring drug resistance and a potential therapeutic target for cancer treatment in the future.


Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin . Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1β in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways. Obviously, to achieve ideal therapeutic efficiency, stem cell therapy should seek a new strategy that adopts a synergistic manner to target the “seed” to improve the survival potential and the “soil” to modify the hostile microenvironment in myocardial infarction (MI)10. However, the research on dual protective strategies for stem cell therapy remains limited.The success of the cell therapy has been compromised by a dual dilemma of the low survival rate of donor cells (the “seed”) and hostile host microenvironment (the “soil”)11, has been identified as a novel endogenous bioactive factor to protect endothelial function through the pro-survival reperfusion injury salvage kinase (RISK) pathway12, which consists of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular regulated protein kinases (ERK) 1/2 pathways13. Recently, we reported that adropin may offer in vitro protection against hypoxia/reoxygenation-induced cardiomyocyte injury by suppressing apoptosis, inflammation, and oxidative stress through the RISK pathway14.Adropin, a secreted protein encoded by the energy homeostasis-associated gene2O2)-induced apoptosis of MSCs through the pro-survival kinase pathway. This may represent an optimized strategy to improve the feasibility of MSCs transplantation in MI treatment.In this study, on the basis of the potential cytoprotective effects by activating pro-survival kinases in multiple cell types, adropin was in vitro used to improve the survival potential of mesenchymal stem cells (MSCs), and meanwhile, in vivo to attenuate the inflammation in rat MI. Then, the adropin-treated MSCs (modified “seed”) were transplanted into the adropin-treated injured myocardium (modified “soil”), thus implementing dual protective effects to enhance the therapeutic potential of MSCs via the paracrine mechanism. We also in vitro tested whether adropin may attenuate hydrogen peroxide  and was approved by the Experimental Animal Care Committee of Fujian Medical University Union Hospital.The study was in compliance with the Guid4. Flow cytometry  was used to detect the superficial markers of MSCs, including the cells positive for CD44 (Biolegend), CD90 (Biolegend), and CD29 (Biolegend) and negative for CD45 (Biolegend), CD34 (Santa), and CD11b (Biolegend)4. MSCs at passage 3 were labeled with bromodeoxyuridine (BrdU)  48 h at a final concentration of 10 μmol/L, and the efficiency of labeling was evaluated under a laser scanning confocal microscope  equipped with blue argon  and red krypton (for BrdU) lasers. Before transplantation, BrdU-labeled MSCs were pretreated with adropin for 24 h at a final concentration of 25 ng/ml.In this study, male Sprague-Dawley (SD) rats weighing (100 ± 10) g  were used to isolate bone marrow MSCs as described previously2O2  (Aladdin), characterized by oxidative damage induction15, were used to induce MSCs apoptosis for the indicated periods , and a final concentration of 100 μmol/L for 4 h was used to investigate the protective effect of adropin against MSCs damage  the apoptosis model group (H2O2): treated with 100 μmol/L H2O2 for 4 h; (2)–(4) the low-, moderate-, and high-dose adropin groups : treated like the apoptosis model group, but pretreated with 10, 25, and 50 ng/ml adropin  for 24 h, respectively; (5) the LY294002 + Ad-M group (LY): treated like the Ad-M but pretreated with PI3K specific inhibitor LY294002  at 50 μmol/L for 2 h; (6) the PD98059 + Ad-M group (PD): treated like the Ad-M but pretreated with ERK1/2 specific inhibitor PD98059 at 50 μmol/L for 30 min. Cell viability  was assessed by Cell Counting Kit-8 (CCK-8), apoptosis rate by flow cytometry, and the protein expression of Akt, ERK1/2, and antiapoptotic proteins [B-cell lymphoma-2 (BCL-2) and B-cell lymphoma extra-large (BCL-XL)] by western blotting.Different final concentrations of HMSCs from various groups were seeded into 96-well plates at the same density and cultured. At the end of each culture, the medium was removed and replaced with 100 μl of fresh medium and 10 μl of CCK-8 reagent . Plates were incubated at 37 °C for 1.5 h, and then absorbance was measured at 450 nm using a SpectraMax 190 microplate reader (Molecular Devices). The reference wavelength was 600 nm. Background absorbance (from wells without cells) was subtracted from all values.An Annexin-V-FLUOS Staining Kit (Roche) was used to quantitatively determine the percentage of cells undergoing apoptosis and survival according to the manufacturer’s instructions. Briefly, MSCs were collected and resuspended in the binding buffer. Annexin-V-FITC and propidium iodide (PI) were added to the suspended cells, and the reaction was incubated in the dark for 10 min. The percentage of apoptotic cells was analyzed by flow cytometry . The experiment was repeated three times.n = 16 per group): the Sham group, with a silk suture placed under the left main coronary artery but not ligated, and the same solvents of MSCs and adropin intramyocardially and intravenously injected, respectively; the MI group, with the same solvents of MSCs and adropin intramyocardially and intravenously injected, respectively, and without any other intervention; the MI + MSC group, treated like the MI group but given an intramyocardial injection of BrdU-MSCs at 10 min after ischemia and an intravenous injection of the same solvents of adropin; the MI + MSC + Adropin group, treated like the MI group but given an intramyocardial injection of adropin-pretreated BrdU-MSCs, following an injection of 0.2 mg/kg adropin via tail veins at 10 min after ischemia.A total of 64 male SD rats (weighed 200 ± 50 g), were enrolled in the experiment. The left main coronary arteries (LCA) of rats were occluded for 3 days or 28 days to establish a MI model. All rats were randomly divided into four groups , other mice underwent ischemia (I) for 45 min followed by reperfusion (R) for 4 h, and were assigned to four groups : the I/R group: treated with ischemia followed by reperfusion; the low-, moderate-, and high-dose adropin groups : receiving the same treatment as I/R and a jugular venous injection of 0.1, 0.2, and 0.4 mg/kg adropin , respectively, 5 min before reperfusion.The choice of the adropin dose 0.2 mg/kg) was based on an additional dose-effect cardioprotective study . The groups without cell therapy received the same volume of medium. At day 3 or day 28 of MI, rats (n = 8 for each time point in each group) were sacrificed by an intravenous bolus injection of 10% KCl under deep anesthesia, following venous blood sample collection and echocardiographic assessment. The heart tissues were stored at −80 °C for histological studies and assessment of vascular endothelial and basic fibroblast growth factor (VEGF and bFGF) by real-time (RT)-PCR and western blotting.To establish the in vivo model of MI, rats were intraperitoneally sedated with 75 mg/kg ketamine and 7.5 mg/kg diazepam before surgery and ventilated by a rodent ventilator 16. After pericardiotomy, silk 8-0 sutures were placed under the left main coronary artery, and the ends of the ties were threaded through small polyethylene tubes to form snares for reversible coronary artery occlusion. At 4 h R, to assess the area at risk, the left ventricle at risk was determined by injecting 1 ml of 0.1% Evan’s blue  after the left main coronary artery was ligated again. The heart was harvested and cut transversely into three slices parallel to the atrioventricular groove at a thickness of 1–2 mm for triphenyltetrazolium chloride staining, and the area at risk and infarct size were determined17.Surgical preparations of mice were adapted from procedures previously described4.Cardiac function was assessed by transthoracic echocardiography at day 3 and day 28 after cell transplantation. Short-axis two-dimensional view of the left ventricle was taken at the level of the papillary muscles to obtain the M-mode recordings with a 10 MHz electronic-phased-array transducer . The left ventricular end-systolic diameter (ESd) and end-diastolic diameter (EDd) were measured from at least three consecutive cardiac cycles. Fractional shortening (FS) (%) was calculated as (EDd − ESd)/EDd × 100; end-diastolic volume (EDv) as 7.0 × EDd3/(2.4 + EDd); end-systolic volume (ESv) as 7.0 × ESd3/(2.4 + ESd); and ejection fraction (EF) (%) as (EDv − ESv)/EDv × 1004. After blockage of endogenous peroxidase activity by 3% H2O2, antigen retrieval with sodium citrate, and blockage of nonspecific immunoreactivity by normal goat serum, paraffin sections (4 μm) were incubated with anti-BrdU antigen (Sigma) and anti-factor VIII-related antigen  and horseradish peroxidase-conjugated second antibodies. Color development in immunoperoxidase staining was performed with diaminobenzidine and sections were counterstained using hematoxylin. To quantify capillary regeneration, three sections from each heart were selected for analysis and five fields from each section were randomly counted. Capillary density was quantified as the number of factor VIII-related antigen-positive vessels (diameter <10 μm) per high-power field (0.06 mm2). Surviving transplanted MSCs were quantified as the number of BrdU-positive cells per 1 mm2 field. Myocardial fibrosis at day 28 was observed by collagen-specific Masson’s trichrome staining. The quantification analysis was performed by two observers blinded to the treatment of the animals.Immunohistochemical staining was used to detect the surviving BrdU-positive cells at day 3 and 28, and capillary regeneration at day 28 after cell transplantationThe protein expression of phosphor (p)-Akt, total (t)-Akt, p-ERK1/2, t-ERK1/2, BCL-2, BCL-XL, VEGF, and bFGF was analyzed by western blotting. Protein extract was loaded onto a polyacrylamide gel used for electrophoresis. Then, the protein was transferred onto a polyvinylidene fluoride membrane. The membrane was incubated in TBST containing 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated with antibodies of Akt, p-Akt, ERK1/2, p- ERK1/2, BCL-2, BCL-XL, VEGF and bFGF , or β-actin antibody (EMAR BIO) at 4 °C overnight followed by secondary antibody for 1 h at room temperature. The ratios of p-Akt/t-Akt and p-ERK/t-ERK were calculated, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Target signals of BCL-2, BCL-XL, VEGF, and bFGF were normalized relative to the β-actin expression.−ΔΔCt.The gene expression of VEGF and bFGF at day 28 after cell transplantation was detected by real-time PCR . The extraction of total RNA from heart tissues and their subsequent first-strand cDNA synthesis were performed with a TRIzol reagent  and a M-MLV Reverse Transcriptase kit , respectively. The following primer sequences were used: VEGF, 5′-GCAATGATGAAGCCCTGGAG-3′ (F), 5′-CGCTCCAGGATTTAAACCGG-3′ (R); bFGF, 5′-GAACCGGTACCTGGCTATGA-3′ (F), 5′-CAGTTCGTTTCAGTGCCACA-3′ (R); GAPDH, 5′-CACTAAAGGGCATCCTGGGCTACAC-3′ (F), 5′-GGAGGCCATGTAGGCCATGAGG-3′ (R). After the activation of the AmpliTaq Gold (Applied Biosystems) for 2 min at 95 °C, 40 cycles were carried out with each cycle consisting of 10 s at 94 °C followed by 30 s at 60 °C. The dissociation curve for each amplification was analyzed to confirm that there were no nonspecific PCR products. All cDNA samples were amplified in triplicate and normalized against a triplicate of GAPDH in the same plate. The data were expressed as 2The concentration of rat plasma pro-inflammatory cytokines, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and anti-inflammatory cytokine IL-10 at 3 d MI was quantitatively determined with quantitative ELISA kits  according to the manufacturer’s instructions in a blinded manner. Briefly, antibodies specific for rat cytokines were pre-coated onto microplates. Standards, Control, and samples were pipetted into the wells and any rat cytokine present was bound by the immobilized antibody. After the removal of any unbound substances, an enzyme-linked polyclonal antibody specific for rat cytokine was added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. The intensity of the color measured was in proportion to the amount of rat cytokine bound in the initial step. The sample values were then read off the standard curve.p < 0.05 was considered statistically significant.All data were presented as mean ± SD. Statistical analysis was performed with SPSS 17.0 for Windows. For comparisons between multiple groups, data were analyzed by ANOVA. When a statistical difference appeared, the least significant difference procedure was applied. A value of The characterization of cultured bone marrow MSCs was shown in Supplementary Fig. 2O2-induced apoptosis of MSCs. Compared with that of the H2O2 group, the adropin pretreatment significantly increased the MSCs viability of the H2O2 + Ad-L group, the H2O2 + Ad-M group, and the H2O2 + Ad-H group in a dose-dependent manner , without any difference between the H2O2 + Ad-M (25 ng/ml) and H2O2 + Ad-H (50 ng/ml) groups , which indicates notable antiapoptotic effects of adropin on H2O2-induced oxidative stress damage of MSCs in vitro , which was significantly reversed by adropin treatment, while no significant difference was found in total Akt and ERK1/2 (t-Akt and t-ERK1/2)  and PD98059 (ERK1/2 specific inhibitor), respectively  Fig. . In addi/2) Fig. . The sam/2) Fig. .Fig. 1InThese results indicate that adropin exerts antiapoptotic and pro-survival effects on oxidative stress-injured MSCs by activating the RISK pathway.Since adropin may in vitro exert antiapoptotic and pro-survival effects on MSCs, we further evaluated the adropin-induced dual beneficial effects on cardiac functional recovery by modifying both transplanted MSCs (“seed”) and host MI microenvironment (“soil”).p < 0.01, respectively) and FS (p < 0.01), and the MI + MSC + Adropin group  had a higher EF and FS than the MI + MSC group   Fig. , suggestp = 0.407, by Log Rank test; p = 0.447, by Breslow test).Consistent with the echocardiographic data, H&E staining showed that cardiac injury was significantly improved by adropin-based dual treatment when compared with MSCs transplantation alone Fig. . Masson’p < 0.01). Compared with the MI group, the MI + MSC and MI + MSC + Adropin groups showed a lower protein expression of IL-1β (p < 0.05) and TNF-α (p < 0.05), but a higher protein expression of IL-10 (p < 0.05). Compared with the MI + MSC group , the MI + MSC + Adropin group  had lower IL-1β and TNF-α, and higher IL-10.As mentioned above, the attenuated myocardial fibrosis and pathologic changes suggest that the cardiac microenvironment at the late stage of cell transplantation (at day 28) was improved by adropin-induced dual effects. We further evaluated the role of systemic inflammation, which is the most important characteristic of MI-induced hostile microenvironment at the early stage of cell transplantation (at day 3). The systemic plasma concentration of pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and anti-inflammatory cytokine (IL-10) was detected by ELISA Fig. . The ShaAs important protective mechanisms for the improvement of cardiac microenvironment in stem cell therapy, paracrine factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] were further evaluated in heart tissues by RT-PCR and western blotting at day 28 after cell transplantation Fig. . CompareThese data demonstrate that the adropin-based dual treatment with transplanted MSCs can produce a better effect on the secretion of local paracrine factors at a late stage and on the systemic inflammatory reaction at an early stage after cell therapy, which may exert beneficial effects on cardiac repair by neovascularization after MI.2 vs. (63 ± 14)/mm2, p < 0.05; at day 28, (51 ± 11)/mm2 vs. (17 ± 3)/mm2, p < 0.05, respectively]. In both groups, the number of surviving transplanted cells at the late stage was less than that at the early stage (p < 0.05). The data indicate that adropin-based dual treatment promotes the survival of transplanted MSCs during the experiment.At day 3 and 28 after cell therapy, immunohistochemical staining was performed to determine whether the survival of transplanted stem cells may improve in response to adropin-based dual treatment Fig. . Survivi2O2-induced MSCs apoptosis model, the antiapoptotic effects conferred by adropin are demonstrated to be involved in the increased transplanted cell survival via the pro-survival pathways.In this study, using an in vivo rat MI model, we demonstrated that the transplantation of adropin-treated MSCs into adropin-treated infarcted myocardium, an adropin-based dual protective strategy, may enhance the therapeutic potential of MSCs to improve cardiac function by promoting the survival of transplanted cells and modifying host microenvironment through paracrine mechanism. Meanwhile, in vitro using a H18. The weak “seed” and the hostile “soil” constitute a dual dilemma. Many efforts have been made to target either the “seed”20, or the “soil”22. However, all these may remain insufficient, clinically inaccessible, or even potentially detrimental24. Therefore, it is reasonable to perform a dual protective strategy targeting the dual dilemma (“seed” and “soil”). Moreover, bioactive substances may be an attractive strategy.The therapeutic effects of stem cell transplantation on cardiac repair depend on a number of influencing factors, including donor cells (the “seed”), host microenvironment (the “soil”), and the use of stem cells as a therapeutic remains challenging11. It is expressed in the liver, brain, kidney medulla, muscles, gastrointestinal tract, coronary artery endothelial cells, and heart26. Furthermore, it is present in the circulatory system of animals and humans, and is negatively associated with the severity of coronary atherosclerosis27.Adropin was identified in 2008 as a secreted protein, which is composed of 43 amino acids, and is produced by the proteolytic cleavage of 76 amino acid precursors25. The biological effects of adropin are mainly mediated through the activation of the orphan G protein-coupled receptor 1926. Adropin has a protective effect on the endothelial function and cardiomyocyte survival via the RISK pathway14. Since antiapoptosis by the activation of pro-survival cascades is an important strategy in optimized cell therapy28, in this study, adropin was used to produce dual protective effects.It has been demonstrated that adropin contributes to glucose and lipid homeostasis, and cardiovascular system functions2O2, adropin protected MSCs against apoptosis. Moreover, with the decrease in the protein expression of t-Akt, t-ERK1/2, BCL-2, and BCL-XL, adropin-induced antiapoptotic effects were blocked by the inhibitors of the RISK pathway. These data demonstrate that the RISK pathway may be involved in the adropin-induced in vitro cytoprotection of MSCs, which is consistent with our previous study of cardiomyocyte14.This study demonstrated that in simulative in vitro cardiac hostile microenvironment induced by H17 and the therapeutic effects of MSCs29. It has been shown that adropin protects endothelium by the RISK pathway12. In this study, adropin was used to in vivo modify cardiac microenvironment, together with the transplantation of adropin-treated MSCs, constituting an adropin-based dual treatment.On the other hand, the RISK pathway has also been well demonstrated to be involved in in vivo myocardial protectionOur data showed that at day 28, compared with the MSCs therapy alone, the adropin-based dual treatment conferred more significant improvement in cardiac function and ventricular remodeling. However, the dual treatment did not improve the survival rate, which may be attributed to the insufficient cases, inadequate observation time, and limited effect of the intervention on mortality.The present study showed that systemic inflammation, which is an important characteristic after acute MI, was attenuated by adropin-based dual treatment, indicating the improvement of cardiac microenvironment.4. The present study confirmed that in comparison with the MSCs therapy alone, adropin-based dual treatment increased the number of surviving BrdU-positive MSCs by 37% at day 3 after the transplantation and further salvaged surviving transplanted cells by 51% at the late stage (day 28). From 3 days to 28 days, 73% of transplanted cells were lost in the group receiving the MSCs therapy alone, but only 49% in the group receiving the adropin-based dual treatment. This evolving pattern was consistent with our previous study4.It has been well documented that the transplanted MSCs may improve cardiac function by secreting various paracrine cytokines, which may in turn promote the survival of MSCs and host cardiomyocytes by attenuating inflammation and apoptosis and accelerating microvessel regeneration to enhance regional blood flow4, in this study, we did not evaluate the transdifferentiation of transplanted MSCs to regenerate cardiomyocytes. Our data confirmed that at day 28 after stem cell therapy, MSCs significantly promoted the gene and protein expression in VEGF and bFGF, which were enhanced by adropin-based dual treatment through promoted capillary regeneration.Since the paracrine effects have been documented as a more important mechanism than myocardial regeneration in stem cell therapy26. Because the half-lives of peptide hormones vary between 3 and 30 min, it is assumed that the half-life of adropin is as short as several minutes. We postulate that in this study, a single intravenous injection of adropin after ischemia may modulate cardiac microenvironment at the early stage of MI by suppressing inflammatory reaction, and adropin-pretreated MSCs may have stronger survival potential in the injured ischemic tissue, thus conferring the effects observed at day 28.The plasma half-life of adropin has not been identified yetIn this study, in consideration of potential indeterminate outcome targeting either “seed” or “soil” alone, we did not in vivo verify the respective effect of the transplantation of adropin-treated MSCs or an intravenous injection of adropin on cardiac outcome, which is a limitation. In spite of the potential effect of adropin administration itself on cardiac function, the promoted cardiac function by adropin-based dual treatment may not be a simple accumulation of respective effects of modified “seed” and “soil”, since the increased survival of transplanted cells by dual treatment-induced improvement of host microenvironment may at least partly contribute to the final promotion of cardiac function. However, it can not be determined whether the dual treatment actually generates synergistic effects in this study.Even so, the present data demonstrate that adropin-induced dual protective effects may improve cardiac function and ventricular remodeling. Moreover, both the transplantation of MSCs and the administration of adropin were performed at 10 min after ischemia, thus favoring the clinical application and the early inhibition of ventricular remodeling process.4. Third, importantly, we did not reproduce the main key in vitro findings in a hypoxia–reoxigenation model.There are other limitations in this study. First, we evaluated the expression of VEGF and bFGF only at one time point. Second, the expression of apoptosis-related factors in infarcted myocardium was not evaluated, since their evolving pattern was consistent with the expression of systemic inflammation-related cytokinesIn conclusion, by using in vitro and in vivo models, we developed an adropin-based dual protective strategy to enhance therapeutic potential of stem cells by promoting transplanted cell survival (modifying “seed”) and improving host microenvironment (modifying “soil”) via the paracrine mechanism and the pro-survival pathways. This may be a novel optimized strategy for stem cell transplantation to repair infarcted myocardium.supplementary informationSupplemental Figure Legendssuppl Figure 1suppl figure 2suppl figure 3Suppl Figure 4Suppl Figure 5


An association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been reported in Mexican populations. The aim of this study was to compare the response of T cells from MS patients, during relapse and remission, to in vitro stimulation with VZV, adenovirus (AV) and Epstein–Barr virus (EBV). Proliferation and cytokine secretion of T cells from 29 relapsing-remitting MS patients and 38 healthy controls (HC) were analyzed by flow cytometry after stimulating with VZV, AV or EBV. IgG and IgM levels against VZV and EBV were quantified using Enzyme-Linked Immunosorbent Assay. Relapsing MS patients showed a higher percentage of responding CD4+ and CD8+ T cells against VZV compared to AV. In HC and remitting MS patients, proliferation of CD4+ T cells was higher when stimulated with VZV as compared to EBV. Moreover, T cells isolated from remitting patients secreted predominantly Th1 cytokines when cell cultures were stimulated with VZV. Finally, high concentration of anti-VZV IgG was found in sera from patients and controls. The results support previous studies of an VZV-MS association in the particular population studied and provide additional information about the possible role of this virus in the pathogenesis of MS. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) . AlthougThe disease begins with the peripheral activation by unknown stimuli of self-reactive CD4+ T cells, which cross the blood-brain barrier into the CNS ,6,7. OncInitial activating stimuli have been related to infectious agents ,14,15, mIn our laboratory, we have previously confirmed the presence of VZV in both cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC), during the first 10 days of an acute relapse in MS patients ,22. In cTo further analyze the role of VZV in the pathogenesis of MS, we here extend the analysis to evaluate the in vitro proliferative and secretory response of T lymphocyte subpopulations to VZV stimulation in MS patients both during relapse and remission.Twenty-nine patients with definite diagnosis of relapse-remitting MS according to McDonald 2010 criteria and 38 healthy controls (HC) were included in the study . T cellsp = 0.0023). For CD8+ T lymphocytes, patients in both relapse and remission showed significantly higher proliferation following VZV stimulation, compared to HC.  and remission (REM-MS), 7 relapsing patients and 32 HC, were cultured and stimulated with VZV. There was a higher proliferative response of CD4+ T cells from REM-MS, compared to HC , a virus unrelated to VZV A–C. Prolp = 0.025) and CD8+ (p = 0.012) T cells from REL-MS patients and CD4+ T cells (p = 0.049) from REM-MS patients . Compared to AV, stimulation with VZV induced higher proliferation in CD4+ . A paired Wilcoxon test comparing T cell responses to VZV and EBV revealed higher proliferation after stimulation of CD4+ T cells from REM-MS patients and HC with VZV   D,E. No dWith the data obtained from all proliferations, we ran paired Wilcoxon tests to compare the response of relapse and remission cells from each subject  to stimulation with each virus . Higher proliferative responses tended to be observed in REM-MS patients, although statistical significance was only reached in CD8+ T cells after AV stimulation .We quantified IL-17, IFN-γ, TNF, IL-10, IL-4, IL-6, and IL-2 in cell culture supernatants of PBMC from patients and controls stimulated either with VZV or EBV. Only IFN-γ, TNF and IL-2 were detected in supernatants of PBMC from REM-MS patients stimulated with VZV A. On theIL-6 was secreted at high concentrations in both patients and controls, without statistical differences after stimulation with VZV or EBV (data not shown).Antibodies against VZV and EBV were measured in the sera of all individuals. We found no difference between HC, REL-MS, and REM-MS for anti-VZV IgM and IgG A,B. In cInterestingly, 95% of the HC were positive for anti-VZV IgG. However, only 82% of them were positive for IgG against EBV. Except for one patient, none of the patients or controls had received the VZV vaccine at the time the blood sample was taken. Therefore, the immunological memory found from the quantification of anti-VZV IgG reflects a previous natural infection.In this study we found high proliferative responses of CD4+ and CD8+ T cells in MS patients after VZV stimulation in comparison to HC, suggesting reactivation of this virus in these patients. Considering that VZV establishes latency in dorsal root ganglia, an increase of memory T cells against VZV could be present in MS .There was a predominance of CD8+ T cell response upon stimulation with each of the three viruses . These cells recognize MHC class I-peptide complexes on the surface of any infected cell, not only antigen-presenting cells, which might contribute for a greater CD8+ T cell expansion .The lower percentage of proliferating T cells after viral stimulation observed in REL-MS compared to REM-MS may reflect either the selective migration of T cells to the CNS  or a T cWith respect to proliferation assays, the difference between stimulation with VZV or EBV was not as evident as between VZV or AV for MS patients. However, serology results suggest a greater immunological memory against VZV compared to EBV in this particular Mexican population. A total of 108,602 cases of VZV were reported in Mexico in the 2019 Epidemiological Bulletin , and altRemarkably, in HC we found a lower percentage of proliferating CD4+ T cells in cultures stimulated with EBV compared to VZV. This correlates with the serological assay since there was a lower frequency of HC positive to anti-EBV IgG compared to VZV.The cytokine quantification in culture supernatants of PBMC of patients and controls stimulated with VZV confirm the results obtained from proliferation assays. A Th1 cytokine profile was predominant in cell cultures of REM-MS patients stimulated with VZV, in agreement with studies of systemic memory T-cells expressing IFN-γ, TNF and IL-2 in latent VZV infected individuals  and coinHigh production of TNF-α in REM-MS cell cultures after VZV stimulation could be related to a significant increase in TNF mRNA expression in PBMC of 29 MS patients, prior to a relapse , suggestAlthough proliferation of effector T cells from control subjects was higher when stimulated with VZV compared to EBV, cytokine secretion in these cultures was low. This might suggest the participation of an unknown factor that favors the production of pro-inflammatory cytokines in the presence of VZV in MS patients.A failure in the suppressive function has been described in CD4 + CD25 + FoxP3+ T cells from MS patients, mainly with a deficient synthesis of immunosuppressive cytokines  ,42. HoweOur results are consistent with the immunopathology of MS described so far, since peripheral CD4+ T cells have been identified as responsible for initiating the inflammatory process in MS , but theAltogether, these results support previous studies of a VZV-MS association and provide additional information about the role of this virus in the pathogenesis of MS.Twenty-nine patients with definite diagnosis of relapse-remitting MS according to McDonald 2010 criteria were studied within the first 10 days of an acute relapse (REL-MS) before receiving steroid treatment. A second blood sample was obtained from 22 of them at least one month after, while they were on remission (REM-MS). Clinical, demographic and T cell proliferation data of MS patients are detailed in The main objective of this study was to evaluate the response of T cells from MS patients to VZV stimulation, given the association of this virus with MS in the Mexican population. We initially compared the response to VZV stimulation with AV, since this virus, as with other respiratory viruses, has been associated with an increased risk of relapse ). After g to obtain serum, which was stored at −80 °C until use.Peripheral blood samples were obtained by venipuncture. For each subject, two tubes of four milliliters were extracted, with and without anticoagulant. After blood clot formation, the second tube was centrifuged for 3 min at 3000× PBMC were isolated by density gradient centrifugation on Ficoll, then resuspended in Recovery Cell Culture Freezing medium , and stored at −80 °C for further analysis.® III , AV suspension (ATCC® VR-1516™) and EBV suspension (ATCC® VR-603™) was determined by triplicate using the Qubit 3.0 fluorometer. Average concentrations were as follows: VZV vaccine 4.3 µg/µL, AV suspension 1.32 µg/µL and EBV suspension 9.4 µg/µL.Quantification of total proteins in VZV vaccine (Oka) Varivax5 PBMC in 200 µL of complete medium. Plates were incubated 24 h at 37 °C in a 5% CO2 humidified atmosphere, for total cell recovery. After incubation, four conditions were analyzed: (1) virus-free medium (negative control), (2) Concanavalin A  at a concentration of 10 µg/mL (positive control), (3) VZV vaccine , and (4) 1.25 µg of total protein from either AV or EBV. Four wells were seeded for each condition (two for CD4+ and CD8+ T cells determination and the other two for regulatory T cells). Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere for 5 days. At the third day of incubation, 10 µM of the thymidine analog BrdU  was added to each well to evaluate T cell proliferation.PBMC from the Cell Culture Freezing medium were thawed, washed with RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic (complete medium), and counted using trypan blue. For each patient (relapse and remission) and control, 16 wells of a 96-well round-bottom culture plate were seeded with 1 × 10After 5-day incubation, cells were stained with anti-CD8 PerCP-Cy5.5, CD4 PE, CD25 APC and anti-BrdU FITC ; while regulatory T cells (Treg) were identified with anti-CD4 PE, FoxP3 PerCP-Cy5.5, CD25 APC . Samples were analyzed by flow cytometry. 10,000 events were registered within the lymphocyte region identified according to their size  and complexity , considering their activation status .Data were analyzed with FlowJo software .5 PBMC in 200 μL of complete medium, stimulated as in the proliferation assays , and incubated for 48 h at 37 °C in a humid atmosphere and 5% CO2. Cell cultures were harvested and centrifuged at 10,000× g for 3 min. Supernatants were collected for quantification of IL-2, IFN-γ, TNF-α, IL-17, IL-10, IL-4, and IL-6, using a CBA Human Th1/Th2/Th17 Cytokine Kit  according to the manufacturer’s instructions.Four wells of a 96-well round-bottom culture plate were seeded with 1 × 10IgG and IgM antibodies against VZV and EBV were determined in sera from patients and controls using commercial enzyme-linked immunosorbent assay (ELISA) kits . Concentration in U/mL was obtained using the Four Parameter Logistic (4PL) regression model .Statistical analysis was performed with GraphPad Prism 6.01 software. Due to the age range in the study group, we evaluated whether this covariant could influence our results using linear regression analysis on MS patients and controls. There was no correlation between the age of patients or controls and the proliferative response or anti-VZV or -EBV antibody concentration.p < 0.05.Differences in proliferation and IgG and IgM concentration between two groups were evaluated using the non-parametric Mann-Whitney ranked test. Paired samples  were tested with the non-parametric Wilcoxon ranked test. Significance was considered with The response of T cell subpopulations to stimulation with Concanavalin A confirmed the ability of cells to proliferate (positive control) but was not included in the statistical analysis.p < 0.05 was considered significant.Cytokine concentrations were compared with two-way ANOVA tests, using Bonferroni correction for multiple comparisons. A value of Values obtained in unstimulated cells were subtracted to those obtained in the cultures stimulated with VZV, AV and EBV.


Enterobacter spp. are part of the ESKAPE pathogens that have been recognized as a threat to human health. Among this genus, E. cloacae species complex (ECL) is the most common species that causes human infections. ECL can develop resistance to ß-lactams and other antimicrobial classes due to alterations in gene regulatory pathways. We evaluated the activity of meropenem-vaborbactam, ceftazidime-avibactam, and comparator agents against 235 multidrug resistant (MDR) ECL isolates collected in Europe and the US during 2017-2019.A total of 2,459 ECL clinical isolates were collected in 40 European and 33 US hospitals. Isolates were susceptibility tested by reference broth microdilution methods and results were interpreted using CLSI, EUCAST, and US FDA breakpoints. MDR was defined as resistant to 3 or more drug classes when applying the CLSI breakpoints.MDR ECL were observed among 9.6% of the overall isolates. The MDR rate in Europe  was considerably higher than in the US . Meropenem-vaborbactam inhibited 94.5% and 97.4% of the MDR ECL isolates applying CLSI and EUCAST breakpoints, respectively (Table). Meropenem inhibited 77.9%/85.5% of the isolates (CLSI/EUCAST breakpoints). Cefepime inhibited only 26.0%/16.2% of the MDR ECL isolates while piperacillin-tazobactam inhibited only 13.2%/6.4%. Ceftazidime-avibactam inhibited 93.6% of the MDR ECL isolates. Amikacin and tigecycline were the most active non-beta-lactam comparators, inhibiting 91.9% and 80.0% of these isolates using CLSI/US FDA breakpoints. A total of 93.1% of the isolates were intermediate to colistin applying CLSI breakpoints or susceptible using the EUCAST criteria. Meropenem-vaborbactam inhibited 73.5% and 87.8% of the MDR ECL isolates nonsusceptible to meropenem and cefepime, the main therapeutic option against ECL isolates. Ceftazidime-avibactam inhibited 73.5% of these isolates.Enterobacterales species/species complex displaying MDR and carbapenem-resistance phenotypes, behind only Klebsiella pneumoniae. Meropenem-vaborbactam and ceftazidime-avibactam can be important options to treat infections caused by MDR ECL.In a global surveillance, ECL is the second most common Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)


Dear Editor,We developed a gasdermin (GSDM) signature score to evaluate immune status and predict outcomes for luminal‐B breast cancer (BRCA). BRCA has become the most commonly diagnosed cancer (11.7%) and is the leading cause of cancer‐related mortality in women.Since the luminal‐B subtype had the most significant genetical changes in GSDMs, we aimed to investigate the impact of GSDMs on outcomes of this subtype. The Cox regression model was performed to evaluate each GSDM in the NCC cohort. Univariate and adjusted‐multivariate analyses revealed GSDMB, GSDMC and GSDMD as independent prognostic factors Table . We usedTo understand the biological differences, we explored the differentially expressed genes (DEGs) between the high and low score groups using edgeR and limma packages Figure . The repGSDMs could modulate immune microenvironment through the release of cytokines and inflammatory contents.To determine the driver mutations regulating the different biological behaviours between the two groups, we analysed the somatic mutations using the whole exosome sequencing data from the TCGA cohort. The high score group had 84.72% of patients carrying somatic mutations, while the low score group had 73.61% Figure . InteresThe immune checkpoint inhibitors (ICIs) have revolutionized the treatment for various tumours, yet their applications are limited in BRCA and require further understandings.The authors declare that they have no competing interests.National Natural Science Foundation of China, Grant Number: 82072097; National Key Research and Development Project, Grant Number: 2019YFE0110000; Clinical and Translational Medicine Research Foundation of Chinese Academy of Medical Sciences, Grant Numbers: 2020‐I2M‐C&T‐B‐069, 2017‐I2M‐3‐004; Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences, Grant Numbers: 2018PT32013, 2017PT32001 and 2016ZX310178; Beijing Hope Run Special Fund of Cancer Foundation of China, Grant Numbers: LC2017B15 and LC2020A18.Supporting InformationClick here for additional data file.


P < 0.001 for all). These results indicate the importance of the localization and area of the fluid for VA in DME.This retrospective study was performed to classify diabetic macular edema (DME) based on the localization and area of the fluid and to investigate the relationship of the classification with visual acuity (VA). The fluid was visualized using en face optical coherence tomography (OCT) images constructed using swept-source OCT. A total of 128 eyes with DME were included. The retina was segmented into: Segment 1, mainly comprising the inner nuclear layer and outer plexiform layer, including Henle’s fiber layer; and Segment 2, mainly comprising the outer nuclear layer. DME was classified as: foveal cystoid space at Segment 1 and no fluid at Segment 2 (n = 24), parafoveal cystoid space at Segment 1 and no fluid at Segment 2 (n = 25), parafoveal cystoid space at Segment 1 and diffuse fluid at Segment 2 (n = 16), diffuse fluid at both segments (n = 37), and diffuse fluid at both segments with subretinal fluid (n = 26). Eyes with diffuse fluid at Segment 2 showed significantly poorer VA, higher ellipsoid zone disruption rates, and greater central subfield thickness than did those without fluid at Segment 2 ( It is characterized by fluid accumulation and retinal thickening and can occur at any stage of DR. Several treatment modalities for DME, such as retinal photocoagulation, administration of anti-vascular endothelial growth factor (anti-VEGF) agents and steroids, and vitrectomy, are used in clinical practice; therefore, assessment of pathological retinal alterations and DME progression is indispensable for the selection of treatments and their timing.Diabetic macular edema (DME) is the most common cause of visual loss in patients with diabetic retinopathy (DR)5. Subsequently, the advent of fluorescein angiography (FA) and optical coherence tomography (OCT) allowed detailed characterization of DME10. In recent years, classifications based on the pattern of retinal thickening on B-scan images have been used, and these include diffuse retinal thickening, cystoid macular edema, and subretinal fluid (SF)11. However, these classifications are not practical for classifying DME progression because the elucidated patterns are not exclusive of each other and may co-exist with one another. It has also been reported that as DME progresses, the localization of the fluid expands from the inner retinal layer to the outer retinal layer, and the area of the fluid in each retinal layer increases13. However, it is difficult to evaluate the localization and area of the fluid simultaneously on B-scan images because the information obtained from the limited number of retinal cross-sectional images is not sufficient for such evaluations.To date, various classifications have been used to assess disease progression in patients with DME. On the basis of fundoscopy, DME was exclusively classified as focal or clinically significant depending on its distance from the fovea17. For example, we recently investigated epiretinal membrane (ERM) using en face imaging and revealed significant associations of the distribution of ERM and the depth of retinal folds due to retinal traction by ERM with visual functions17.The recent advent of swept-source OCT (SS-OCT), which is more penetrating and has a higher scan speed than does conventional spectral domain OCT, has enabled the acquisition of high-resolution, three-dimensional (3D) images of the retinal structure. By using en face images constructed from the 3D images, the changes in the retinal structure can be visualized at an arbitrary retinal depth from a bird’s-eye viewIn the present study, we utilized en face imaging to simultaneously evaluate the localization and area of the fluid in patients with DME and proposed a classification based on the findings. We also examined the relationship of the DME type with visual acuity and the structural features of the retina.In total, 128 eyes of 93 Japanese patients with center-involving DME were included. The mean age of the subjects was 64.0 ± 10.6 years (35–87 years), and there were 38 women (40.8%). The average HbA1c value was 7.6% ± 1.7%.We segmented the retina as follows: Segment 1, which mainly comprised the inner nuclear layer (INL) and outer plexiform layer (OPL), including Henle’s fiber layer; and Segment 2, which mainly comprised the outer nuclear layer (ONL). We then classified the findings in the segments as follows: Segment 1, no fluid (NF), foveal cystoid space (FC), parafoveal cystoid space (PC), and diffuse fluid (DF); and Segment 2, NF, DF, SF, and DF with SF. Thus, there were 15 combinations in total Table . The fouP = 0.001, Table P = 0.062, 0.324, 0.521 and 0.185, respectively; Table P < 0.001; Fig. P < 0.001; Table P < 0.001; Table P < 0.001; BCVA: odds ratio, 72.17; 95% confidence interval, 7.73–673.71; P < 0.001). There was no significant difference in the incidence of ERM and maximum depth of retinal folds (MDRF) among the different DME types  was significantly different among the different types of DME  and the area of the fluid. To date, the process of DME progression has been primarily studied using autopsied eyes, FA, and B-scan OCT imaging18. However, in autopsied eyes, which only provide information for a particular section, postmortem changes and changes occurring during the process of specimen fixation need to be considered in order to determine the localization of the fluid13. With regard to FA, although the retina can be visualized from a bird’s eye view, and the existence of vascular leakage can also be visualized, it is difficult to determine the precise area of the fluid because the area of fluorescence leakage expands as time elapses during photography. Furthermore, we cannot locate the layer at which the fluid exists or visualize the SF. With regard to B-scan images, the information obtained from the limited number of retinal cross-sectional images is not sufficient for simultaneous evaluation of the location and area of the fluid. Furthermore, when B-scan images are constructed using SD-OCT, the signal intensity is attenuated because of the short wavelength of the light source, and the deep part of the retina cannot be fully visualized in eyes with severe DME. In this study, we utilized en face images constructed using SS-OCT, which has two advantages over conventional SD-OCT. The first advantage is its high scanning speed , which enables the capture of 3D images of the retina with high resolution in a short period of time; this allowed us to construct the en face images used in this study20. The second advantage is the long wavelength of the light source (1060 nm); this light shows better tissue penetration and allows for deeper retinal visualization, even in situations where fluid is present in the retina20. On the en face images, the fluid itself was clearly visualized as a well-demarcated, low-intensity region. Furthermore, considering the fact that the inner plexiform layer (IPL) and retinal pigment epithelium (RPE) are hardly disrupted in DME because the layers with fluid accumulation are limited to INL, Henle’s fiber layer, ONL, and the subretinal space13, we found that the division into Segment 1  and Segment 2 (mainly from the ONL to the RPE) can be automatically and objectively performed by utilizing the boundary between the IPL and INL (IPL/INL) and RPE as reference planes for flattening the 3D retinal images. As a result, it was possible to separately investigate the area of the fluid at Segments 1 and 2 . One possible explanation for this difference is the lower possibility of missed ERM on en face images than on B-scan images because the former provide a bird’s eye view of the retina.In this study, the proposed en face image-based classification of DME was not significantly associated with incomplete PVD, the presence of ERM, and the strength of retinal traction due to ERM. It is important to consider the status of retinal traction, which is mediated by the taut, thickened posterior hyaloid, vitreoretinal adhesion, and ERM, when considering treatment options for DME3. Chronic inflammation induces and strengthens the adhesion of the posterior vitreous cortex to the internal limiting membrane (ILM) and promotes the proliferation of Müller cells, myofibroblast-like cells, and macrophages, which are the main cellular components of ERM associated with DME36. Accordingly, we hypothesized that, in cases of severe DME, inflammation would increase, ERM would be likely to develop, and the retinal traction due to thickening and contraction of ERM would be likely to increase. Contrary to our expectations, however, the severity of DME was not associated with the incidence of ERM or retinal traction due to ERM, which was estimated from MDRF. Because MDRF for each DME type  to Henle’s fiber layer, which shows lower resistance relative to that shown by the other layers36. When leakage from the intermediate capillary plexus and DCP increases, the fluid is primarily trapped between the IPL and the synaptic portion of the OPL, i.e., the INL, because both the IPL and the synaptic portion of the OPL act as barriers to fluid movement37. Fluid accumulation at the outer retinal layer has been reported to occur later than fluid accumulation at the inner retinal layer during the course of DME progression38. Fluid accumulation at the ONL is caused by the influx of fluid from the INL37, and this phenomenon in the subretinal space is thought to be caused by inefficient fluid removal by the dysfunctional RPE or fluid movement from the ONL through a weakened and permeable ELM41. Although the present study was not longitudinal, our classification appears to be in good agreement with the process of DME progression  device and the accompanying software, we cannot say whether other OCT machines will be similarly successful. Particularly, when delineating the IPL/INL for the segmentation of Segment 1, the boundary was delineated according to Triton’s original algorithm, which has not been released to the public. Furthermore, the en face images of Segment 2 may have shown outward extension of the fluid in Henle's fiber layer or shadowing by the hyperreflective lesion at the inner retina. Fourth, only eyes with treatment-naïve DME were included, and eyes with end-stage DME, such as those with atrophic diabetic maculopathy, were not included. Fifth, the detection power for incomplete PVD and EZ disruption was limited because the judgment was only based on horizontal and vertical B-scan images centered at the fovea. Finally, the present study did not examine the effect of retinal ischemia on visual functions in DME39, the presence of fluid in Segment 2 should be precisely determined using en face imaging.In conclusion, we proposed a classification for DME using en face imaging, with focus on both the area and localization of the fluid, and showed that the presence of fluid at Segment 2 was significantly associated with visual acuity, EZ continuity, and CST. Because it has been shown that fluid at the outer retinal layer is less responsive to anti-VEGF drugs or steroids and intractable40, and glaucoma. All the investigative procedures conformed to the tenets of the Declaration of Helsinki. The study was approved by the Ethics Committee of Okayama University Hospital, Okayama, Japan. Informed consent was obtained from all participants.This retrospective, cross-sectional study included 128 eyes of 93 Japanese patients diagnosed with center-involving DME from January 2018 to April 2020. Only eyes that had never received anti-VEGF drugs, steroids, or vitrectomy were included. Eyes with a history of laser treatment for peripheral retinal nonperfusion were also included. The exclusion criteria were as follows: a history of macular diseases that cause edema, such as retinal vein occlusion; cataract with a density of grade 3 or higher according to the Emery–Little classification, high myopia All patients underwent comprehensive ophthalmological examinations, including BCVA testing with refraction using a 5-m Landolt C acuity chart, indirect and contact lens slit-lamp biomicroscopy, and SS-OCT.For construction of B-scan and en face images, 3D volume data of the retina were obtained over a 6 × 6-mm area consisting of A-scans with 512 lines × 256 lines. The en face images were analyzed using IMAGEnet6 , which was installed on Triton. On the basis of the retinal layer boundary information, IMAGEnet6 can align the 3D OCT volume data along a specific retinal layer boundary and generate an en face image at an arbitrary depth.43, and we considered it necessary to ensure that the scan depth was a little greater than the thickness of the INL for visualization of the OPL, including Henle’s fiber layer, in addition to the INL  centered at the fovea. We considered incomplete PVD to be present when the posterior hyaloid detached from the retina partially or completely but remained attached to the optic disc47.CST was defined as the average thickness of the retina within the foveal area on the ETDRS map. It was automatically measured with IMAGEnet617. For eyes with ERM, we measured the MDRF according to previous reports17. Briefly, we visualized the black lines corresponding to the retinal folds due to retinal traction by ERM on the en face image below the ILM level. Then, we measured the depth from the ILM just before the deepest retinal fold disappeared.To visualize the en face image of the retinal surface, we flattened the 3D OCT volume scan data at the level of ILM. Using the en face image of the retinal surface, we investigated the presence of ERM, defined as irregular and hyperreflective membrane-like structures2 test of independence was used to analyze the relationships of the en face image-based classification of DME with the number of cases, sex, incomplete PVD, EZ continuity, and incidence of ERM, while one-way analysis of variance was used to analyze the relationships of the classification with age, HbA1c, CST, and MDRF. The Kruskal–Wallis test was used to determine the relationship between the classification and BCVA. With regard to fluid at Segment 2, its relationships with BCVA and CST were analyzed using Student’s t-test, while its relationships with EZ continuity and the incidence of ERM were analyzed using the χ2 test. To clarify the factors associated with EZ disruption, we performed logistic regression analysis with classification of DME, BCVA, and CST as independent variables. A P value of < 0.05 was considered statistically significant.All data are expressed as the mean ± standard deviation. BCVAs were recorded as decimal values and converted to logarithm of the minimal angle of resolution units for statistical analysis. All statistical analyses were performed using SPSS version 22.0 . The χSupplementary Figure S1.Supplementary Figure Legend.


P < 0.05). We also observed fewer scores of the Acute Physiology and Chronic Health Evaluation (APACHE II) and the sequential organ failure assessment (SOFA) in patients undergoing restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI than those undergoing restricting volumes of resuscitation fluid strategy alone (P < 0.05). According to the above data, it is concluded that UTI as an adjuvant therapy for restricting volumes of resuscitation fluid strategy in treating septic shock may decrease the LA level, attenuate the inflammatory response, reduce vascular permeability, prevent pulmonary edema, and restore cardiac and renal functions.Septic shock is the most serious complication of sepsis, leading to unacceptably high morbidity and mortality worldwide. Fluid resuscitation using crystalloids has become the mainstay of early and aggressive treatment of severe sepsis and septic shock, while increased daily fluid balances from day 2 until day 7 have been related with increased mortality. Recently, pharmacological management has been recommended to combine with appropriate fluid resuscitation for the treatment of septic shock. In this study, we compared the clinical efficacy of restricting volumes of resuscitation fluid strategy with or without intravenous infusion of ulinastatin (UTI) in treating patients with septic shock and additionally examined the patient's changes of the extravascular lung water index (EVLWI), pulmonary vascular permeability index (PVPI), systemic vascular resistance index (SVRI), cardiac function, lactic acid (LA) level, coagulation function, and renal function. The study included 182 patients with septic shock, among which 89 patients had undergone restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI and 93 patients had undergone restricting volumes of resuscitation fluid strategy alone. It was found that patients with septic shock after restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI showed an increased SVRI concomitant with declined PVPI and EVLWI, increased mean artery pressure (MAP), cardiac output (CO), left ventricular ejection fraction (LVEF), stroke volume (SV), and heart rate (HR), declined levels of cardiac troponin I (cTnI), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and C-reactive protein (CRP), reduced LA level along with shortened prothrombin time (PT) and partially activated thrombin time (PATT), and decreased levels of blood urea nitrogen (BUN), creatinine (Cr), and uric acid (UA) when comparable to those after restricting volumes of resuscitation fluid strategy alone ( Sepsis is considered as a process of infection in systemic inflammatory response syndrome. Nowadays, sepsis has been redefined as life-threatening organ dysfunction induced by a prolonged imbalance of innate immune response to infection . Septic The pathogenesis of sepsis is very complex and remains unclear, which leads to limited treatment . Since sThis investigation is a retrospective cohort study of 182 patients with septic shock admitted to the ICU of our hospital from December 2018 to December 2020. All 182 patients fulfilled the International Guidelines for Management of Sepsis and Septic Shock (2016)  within t2   >  92%. Antishock treatments were performed using crystalloid or colloid solutions based on the ratio of 2 : 1. The patients were supplemented by initial resuscitation fluid with Ringer's solution at a total volume of 500–1000 ml within 1 h to maintain mean arterial pressure (MAP) between 50 and 60 mmHg and urine discharge 0.5–1.0 ml/kg/h. Continued fluid boluses (at a maintenance <3000 ml per day) via the infusion pump were given in case of severe hypoperfusion defined as either plasma concentration of lactate of at least 4 mmol/L or MAP below 50 mmHg in spite of the infusion of norepinephrine. UTI  was dissolved in normal saline and intravenously injected or pumped into the patients for consecutive 7 days.All included patients were given conventional antishock and anti-infection treatments, and vital signs were monitored after admission. For anti-infection treatments, third-generation cephalosporin combined with vancomycin or carbapenem antibiotics was used for the treatment of septic shock with unknown reasons, and the antibiotics were changed according to the experimental results of the blood culture later. The vital signs of patients were monitored with supplemental oxygen to maintain SpOEach patient was scored using the Acute Physiology and Chronic Health Evaluation (APACHE II)  and the t-test. Categorical variables are expressed as proportions and analyzed using the chi-square test. A possibility of significant difference is shown as P < 0.05.SPSS 22.0 software was employed to perform data management and analysis. Continuous variables are displayed by mean ± standard deviation and analyzed by the n = 89) and fluid restriction + UTI treatment group (n = 93) according to restricting volumes of resuscitation fluid strategy with or without intravenous infusion of UTI. As shown in P > 0.05).The study included 182 patients with septic shock, and they were split into the fluid restriction group (P > 0.05). After treatment, patients with septic shock exhibited an increased SVRI concomitant with declined PVPI and EVLWI (P < 0.05). More importantly, patients with septic shock after restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI showed an increased SVRI concomitant with declined PVPI and EVLWI when comparable to those after restricting volumes of resuscitation fluid strategy alone . After treatment, patients with septic shock displayed elevated MAP, CO, SV, LVEF, and HR (P < 0.05). It was found that patients with septic shock after restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI showed increased MAP, CO, SV, LVEF, and HR when comparable to those after restricting volumes of resuscitation fluid strategy alone . The patients in both fluid restriction group and fluid restriction + UTI treatment group had declined plasma levels of cTnI, NT-proBNP, and CRP after treatment (P < 0.05). Besides, the plasma levels of cTnI, NT-proBNP, and CRP were lower in patients after restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI than in those after restricting volumes of resuscitation fluid strategy alone . The patients in the fluid restriction group and fluid restriction + UTI treatment group showed a decreased LA level along with prolonged PT and PATT after treatment (P < 0.05). Besides, the LA level was lower, but PT and PATT were shorter in patients after restricting volumes of resuscitation fluid strategy with intravenous infusion of UTI than in those after restricting volumes of resuscitation fluid strategy alone . The patients in both fluid restriction group and fluid restriction + UTI treatment group had increased volume of 24 h urine and declined levels of BUN, Cr, and UA after treatment (P < 0.05). We also found that, except the volume of 24 h urine, reduced levels of BUN, Cr, and UA were determined in the fluid restriction + UTI treatment group compared with the fluid restriction group . We also observed fewer APACHE II and SOFA scores in the fluid restriction + UTI treatment group than the fluid restriction group. After treatment, the mortalities of the two groups did not differ (P > 0.05, At last, we compared the clinical efficacy and safety between restricting volumes of resuscitation fluid strategy with or without intravenous infusion of UTI. As shown in At the very beginning, the term sepsis was thought to be a process of flesh decay, which could be traced back to Hippocrates . In 2016The early fluid resuscitation is the essential treatment to patients with septic shock or increased blood LA level, and the initial fluid resuscitation should be done within 3 hours after the diagnosis of sepsis. At least 30 ml/kg of intravenous crystalloids is recommended initially . HoweverMAP, CO, SV, LVEF, and HR are usually used to evaluate cardiac function, which were approved as close relevance with mortality in cardiogenic shock . In thisThe concentration of blood LA has been considered as an indicator of tissue perfusion changes in critically ill patients . Some reTaken together, the findings obtained from our results support the notion that UTI as an adjuvant therapy for restricting volumes of resuscitation fluid strategy in treating septic shock may decrease the LA level, attenuate inflammatory edema, reduce vascular permeability, prevent pulmonary edema, and restore cardiac and renal functions. The outcomes in our study might change a little bit due to the small number of included patients and the shorter observation time. Therefore, further investigations with a large cohort of patients with septic shock are required.


Homemade diets are a worldwide trend, and many recipes are currently available on websites but may not be considered balanced. This study aimed to evaluate if the number of ingredients, supplement, or vegetarian/vegan-only ingredients included in a recipe influence the nutrient content of homemade diets for dogs and cats. Chemical analyses were performed on 75 diets for dogs and 25 for cats prepared according to websites’ recipes, and minerals were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES).p < 0.05). Non-supplemented canine and feline diets had calcium and calcium:phosphorus ratio below recommendations (p < 0.05). Non-supplemented feline diets had lower sodium and iron, and zinc levels were below recommendation in diets for both species. Diets with animal products for dogs had higher levels of protein and zinc, although zinc was deficient in both groups, and there were higher amounts of crude fiber, magnesium, and manganese in vegetarian/vegan diets (p < 0.05). Diets with animal products for cats had higher levels of protein (p = 0.003), but there was a higher amount of crude fiber (p = 0.014) in vegetarian/vegan diets.Canine diets that met fat requirements had more ingredients than those below recommendations. None of the cat diets met iron requirements, and feline diets that met requirements of manganese had fewer ingredients and those that supplied requirements of protein and sodium had more ingredients than the diets below recommendations (The number of ingredients and vegetarian/vegan preparation did not guarantee nutritional adequacy of diets, and the presence of supplement did not ensure a balanced diet. To provide an adequate diet is an important part of pet care, and is essential to maintain the animal’s health, improve quality of life, and increase the lifespan. Conventional diets, such as dry kibble or canned diets, are the most common types of diets fed to pets. However, there is a recent trend of feeding unconventional diets to dogs and cats, which includes homemade and vegetarian or vegan diets. A survey conducted in the United States and Australia  observedFeeding unconventional diets, however, may put the animals at risk. The World Small Animal Veterinary Association has published a set of guidelines regarding nutritional assessment , which rThe vegetarian and vegan population seems to be increasing worldwide. A survey in Brazil  indicateRegardless of being meat- or plant-based, homemade diets need to supply all nutrient requirements. One of the items that is considered essential in homemade diets for pets is the vitamin-mineral supplement, which guarantees that all nutrients are present in the diet since common ingredients do not provide all the nutrient requirements. If a diet is formulated without proper supplementation, it can be deficient, especially in minerals and vitamins , 11. HowTherefore, this study hypothesizes that vegetarian or vegan diets and diets without supplements will be nutritionally deficient and that the number of ingredients will not influence the nutritional adequacy of diets. This study aimed to evaluate the influence of the number of ingredients, the presence of supplement, or vegetarian/vegan recipes on the nutrient content of homemade diets according to FEDIAF  and NRC None of the diets were considered complete and balanced when compared to NRC  and FEDIp < 0.05). None of the cat diets met iron requirements.The results regarding the influence of the number of ingredients in diets for dogs are presented in Table n = 25/75) did not indicate mineral or vitamin-mineral supplementation. If we consider the diets that only included salt, this percentage increases to 44.4% (n = 33/75). Of the diets supplemented, 14.0% (n = 7/50) only included calcium sources such as calcium carbonate or eggshell powder. The supplements that contained minerals indicated in the recipes for dogs were: salt (n = 15/75), veterinarian vitamin-mineral supplements of different brands (n = 15/75), eggshell powder (n = 9/75), calcium carbonate (n = 8/75), dicalcium phosphate (n = 3/75), potassium chloride (n = 2/75), zinc (n = 2/75), calcium citrate (n = 1/75), and vitamin-mineral supplement for children (n = 1/75).Of the 75 recipes for dogs, 33.3% (n = 10/25), calcium carbonate (n = 9/25), eggshell powder (n = 5/25), and vitamin-mineral supplement for children (n = 1/25).Veterinarian vitamin-mineral supplements were not indicated in any of the recipes for cats. Only one recipe contained a children’s vitamin-mineral supplement, 8 (32.0%) did not include any type of mineral supplementation, and 2 (8.0%) only included calcium sources. The supplements that contained minerals indicated in the recipes for cats were: salt (p < 0.05) below recommendations. Non-supplemented feline diets had lower sodium and iron, and zinc levels were below recommendations in both groups. The results regarding the influence of mineral supplementation in diets for dogs are presented in Table Non-supplemented canine and feline diets had calcium and calcium:phosphorus ratio (Ca:P) (n = 1/25) were vegetarian and 8.0% (n = 2/25) were vegan.Twelve percent (n = 9/75) of recipes for dogs were vegetarian and 6.6% (n = 5/75) were vegan, as they did not contain ingredients from animal sources. As for the recipes for cats, 4.0% (p < 0.05). Protein was increased in non-vegetarian diets for cats (p = 0.003), but there was a higher amount of crude fiber (p = 0.014) in vegetarian and vegan diets. The results regarding the influence of preparations with or without animal products in diets for dogs are presented in Table Protein and zinc were increased in non-vegetarian diets for dogs, although zinc was deficient in both groups. There were higher amounts of crude fiber, magnesium, and manganese in vegetarian and vegan diets p < 0.0. ProteinThis study evaluated if three factors can influence the nutritional composition of diets: number of ingredients, presence of mineral or vitamin-mineral supplements, and the fact that the recipe is vegetarian or vegan.The number of ingredients influenced the concentration of macro and micronutrients, especially in recipes for cats. For dogs, differences were only observed in fat and magnesium, for which recipes that met NRC  and FEDIA study conducted by Dodd et al.  observedIn the present study, vegetarian and vegan diets for dogs and cats presented lower concentrations of crude protein and higher concentrations of crude fiber than diets with meats. The lower protein levels of these diets may be due to the higher inclusion of vegetable ingredients that may not necessarily be selected based on protein content , 23, 24.The presence of mineral and vitamin-mineral supplements was also evaluated in the present study. In recipes for dogs, the concentrations of ash and calcium and the calcium:phosphorus ratio were lower in recipes without mineral supplementation. In recipes for cats, the concentrations of ash, calcium, sodium, and iron, and the calcium:phosphorus ratio were lower in diets without mineral supplementation. The higher calcium concentrations in recipes with mineral supplements were expected since most diets contained at least one calcium-rich supplement such as vitamin-mineral supplements, calcium carbonate, and eggshell powder. According to the results of this study, it can be suggested that the presence of a supplement alone does not guarantee a balanced diet because none of the diets were complete regardless of the inclusion of supplements. This is an important result since it demonstrates that a diet should be formulated as a whole, considering the inclusion of all ingredients and their combination of nutrients, including the supplement. Even when a multi-mineral supplement was added according to the manufacturer’s instructions, the diets remained unbalanced.In the present study, none of the recipes evaluated supplied all nutrients analyzed, including protein, fat, and essential minerals. The number of ingredients did not expressively influence the nutritional composition of diets, as well as a vegetarian or vegan preparation. The presence of supplement on its own does not ensure a balanced diet for dogs and cats. If a food is to be properly formulated the nutritionist must consider the target species and the nutritional profile of all ingredients including those used in supplementation.Recipes for healthy adult dogs and cats published in Portuguese were searched with the terms “home-prepared diet”, “home-cooked diet”, “homemade food”, “home-prepared diet recipe”, “home-cooked diet recipe” and “homemade food recipe”, all followed by the terms “dog” or “cat”, in the Google browser. The Recipes up until the 10th page of the browser for each search term were included.The exclusion criteria were: the same recipe presented in more than one website; recipes not clearly stated for healthy adults; recipes stated not for daily use; recipes considered by its author as a snack or milk replacer; and if amounts of one or more ingredients were not specified. After applying exclusion criteria, 75 recipes for dogs and 25 recipes for cats were randomly selected using a random drawing generator.Ingredients were acquired from three different markets in the city of Sao Paulo, Brazil, and preference was given to fresh foods. Preparation of samples with 500 g for each recipe was done according to recipe’s instructions, considering ingredients, quantities of ingredients, and cooking mode .For each recipe, all ingredients were prepared, weighed in a digital cooking scale, and then blended with the use of a food processor. When recipes indicated units of an ingredient instead of weight, USDA FoodData Central  measuresThe recipe’s samples were dehydrated in a forced circulation oven at 55 °C for 72 h , and the3 and 2 mL of hydrogen peroxide (H2O2) were added to each tube, which was left to rest for 30 min. After resting, 4.5 mL of ultrapure water was added to each sample. The tubes were then placed in a microwave  and were heated in two phases. In the first phase, the samples were heated for 20 min until reaching 180 °C at 400 W. In the second phase, the samples were heated for 10 min at 180 °C and 800 W. After the second phase, the samples were left to cool for 10 min.Closed vessel microwave digestion was used to process samples for mineral analyses at the laboratory of Biorigin Brazil . After dehydration, samples of 0.5 g of each recipe were placed in polypropylene tubes, and 1.5 mL of HNOThe analyses of minerals were performed in triplicate by inductively coupled plasma optical emission spectrometry [ICP-OES ] at the Multiuser Laboratory of Animal Nutrition and Bromatology of the Department of Nutrition and Animal Production of the School of Veterinary Medicine and Animal Science of University of Sao Paulo . Operational conditions are presented in Table Preparation of external calibration curves was done by using multielement standard solutions at concentrations of 100 mg/L for calcium (Ca), copper (Cu), iron (Fe), mercury (Hg), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), selenium (Se), and zinc (Zn) . The curves were prepared in a range of concentrations from 0.1 to 5 mg/L for Cu, Zn, Na, and Mn, from 0.5 to 100 mg/L for Ca, P, Mg, and K, and from 0.001 to 2 mg/L for Fe and Se. For the determination of selenium, a hydride generator  was coupled to the ICP-OES.Based on the results of the analysis of macronutrients, the Atwater method was used to calculate the metabolizable energy of the diets , conside0.75 for dogs and 75 kcal/kg0.67 for cats, both equations referring to inactive animals.The Shapiro-Wilk test was used to test the normality of variables. For normally distributed variables, Student’s t-test was used to evaluate significance between the results and NRC  and FEDIp ≤ 0.05.Data were analyzed using SAS version 9.4  and statistical significance was accepted if