Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT  lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.  Thus, clot lysis time methods are usually selected as reference methods in literatures and pharmacopoeias. Several types of lysis time methods have been developed e.g. clot lysis time  is a common disorder that is often accompanied by significant morbidity and mortality. Thrombolytic therapy, especially by plasminogen activators, is a well-established standard emergency treatment for acute myocardial infarction (AMI) and some other thromboembolic disorders. Among available thrombolytic agents, recombinant plasminogen activator , has received attention due to its lower side effects, rapid coronary patency, and prolonged half-time compared to other drugs of this group . Among vsis time , 3, euglsis time , ecarin sis time , dilute sis time  and stresis time . Clot lysis time , 3 and t2 were used as starters of coagulation reactions in the intrinsic pathway of coagulation cascade  as standard and Retelies®  as sample were purchased as lyophilized powder. Content of each vial was mixed with sterile water to concentration 1.8 mg/mL as manufacturer›s instructions, aliquoted and stored at −20 °C. Commercial reteplase RapilysinAPTT reagent2 were obtained as Hemosil® kit from Instrumentation Laboratory Company .Activated partial thromboplastin time reagent (APTT) and CaClBlood collection and plasma preparationBlood samples were collected by venipuncture without venous occlusion in tube containing 3.8% (w/v) sodium citrate resulting in 1:9 ratio of anticoagulant solution to sample. Plasma was harvested following centrifugation to sediment platelets. Hemolyzed plasma was rejected. The contents of plasma from 6 healthy, normal male/female people were mixed, then aliquoted and stored at -70 °C as pooled plasma. Other reagents were purchased from Sigma  or Merck . In the day of analysis, the aliquots of reteplase and plasma were thawed. The remaining amounts discarded following completion of each experiment.Clot lysis time assayvs. log reteplase standard concentration  and reteplase standard solution (0.001-0.1 mg/mL) were mixed on ice. In another set of tubes, plasminogen 1 mg/mL  and fibrinogen 2 mg/mL  were mixed. Definite volumes from the first series of tubes, transferred to the second series. The time interval between this time and complete lysis time of the clot in the mixture was determined for each concentration of reteplase standard. Calibration curve was plotted by log lysis time APTT lysis assay2 0.025 M. The tubes were placed in 37 °C circulating water bath. Lysis time for each vial was determined. Logarithm of lysis times as seconds were plotted against logarithm of different concentrations of reteplase standard solutions . Buffer containing 1.38 g/L of sodium dihydrogen phosphate monohydrate, 7.10 g/L of anhydrous disodium hydrogen phosphate, 0.20 g/L of sodium azide and 0.10 g/L of polysorbate 80 was used for dilution of standard and sample solutions (In a set of glass test tubes definite amount of plasma and reteplase standard solution (0.001-0.1 mg/mL) were mixed and incubated for 90 sec in circulating water bath 37 °C. APTT reagent was added then incubated for 3 min. Clotting reactions were initiated by addition of CaClMicrocon centrifugal filter tubes with cut-off 10000 Da were used to filter reteplase of excipients in the sample and standard solutions. Filtered solutions were expected to consist of excipients of these preparations. These filtered solutions were used for evaluation of excipients interactions in APTT lysis method. Statistical analysis®, version 17.0) programs. The data were expressed as mean ± SD (standard deviation). A 0.05 level of probability was taken as the level of significance and 95% level was considered for confidence intervals.The graph preparation was performed using Microsoft Excel 2007 and Statistical analysis was carried out by the one-way ANOVA, using the Statistical Package for Social Sciences . A regression of results obtained with APTT lysis method versus results obtained with reference method led to a linear graph comparable to the theoretical line with slope 1 and intercept 0 at 95% confidence level. These results indicated that the two methods were comparable to each other and accuracy of APTT lysis method was within the acceptable limit.In APTT lysis test, it was presumable that plasma as a reservoir of coagulation and lysis factors may influence the results significantly. To eliminate plasma variations, it was reasonable to design the method by pooled normal plasma. To evaluate more comprehensively about the influence of plasma variations and the preference of using pooled normal plasma instead of individual normal plasma, as shown in et al. and also Lindahl et al. have reported that the APTT method could be used for the new generation of antithrombotic agents such as dabigatran, a direct thrombin inhibitor, but the linearity could be lost in higher concentrations of the drug (In theory, APTT method is applicable for determination of all anticoagulant and thrombolytic drugs influencing on factors involve in intrinsic, common coagulation pathways or fibrinolytic processes. But studies in this regard showed limitations especially about linearity of APTT results for some of these drugs. For instance, unlike heparin and lepirudin, there is weak linear correlation for warfarin. Favaloro the drug , 13. Linthe drug , 14 or cthe drug , 16, theThe validity of APTT lysis method was evaluated against clot lysis time as a reference method. Biological activity test using APTT lysis assay was a short-time method and could be suitable for large numbers of samples. Contrary to some other antithrombotic agents, determination of biological activity of reteplase using APTT lysis assay showed to have a good dose-response correlation. APTT lysis assay proved to be a reliable, short-time, and budget-saving method.


DANCR was significantly overregulated in TNBC tissues and cell lines compared with normal breast tissues or other type of breast cancer. Knockdown of DANCR suppressed TNBC proliferation both in vitro and in vivo. Further study of underlying mechanisms demonstrated that DANCR bound with RXRA and increased its serine 49/78 phosphorylation via GSK3β, resulting in activating PIK3CA transcription, and subsequently enhanced PI3K/AKT signaling and TNBC tumorigenesis. Taken together, Our findings identified DANCR as an pro-oncogene and uncoverd a new working pattern of lncRNA to mediate TNBC tumorigenesis, which may be a potential therapeutic target for improving treatment of TNBC.Conventional therapies and novel molecular targeted therapies against breast cancer have gained great advances over the past two decades. However, poor prognosis and low survival rate are far from expectation for improvement, particularly in patients with triple negative breast cancer (TNBC). Here, we found that lncRNA  Generally, breast cancer can be classified into four subtypes, including luminal type A, luminal type B, human epidermal growth factor receptor-2 (HER2) positive type, and triple-negative breast cancer (TNBC) type3. TNBC demonstrates a lack of ER, PR, and HER2 expression by immunohistochemical results, and acts as a highly invasive subtype comprising about 20% of all breast cancer patients5. Furthermore, TNBC can exhibit high invasiveness, metastasis, high recurrence risk, and mortality rates, resulting in poor prognosis6. The lack of molecular targeted therapies and the poor survival of TNBC patients have fostered great endeavors to discover precise molecular targets for clinical treatment strategies. A growing body of evidence suggests that long non-coding RNAs (LncRNAs) are involved in TNBC progression through regulating tumor-related gene expression9. However, the precise molecular mechanisms by which lncRNA mediates TNBC progression remain unclear.Breast cancer is the most prevalent malignant tumor in women, and the efficacy of currently available therapies seems far from satisfactory, which severely threats the health of females from all over the worldDANCR), an lncRNA encoded on human chromosome 4q12, has been identified as an oncogene in multiple malignant tumors, including colon cancer10, esophageal cancer11, hepatocellular carcinoma12, osteosarcoma13, TNBC14. DANCR was previously demonstrated to contribute to suppression of cell differentiation due to acting as a negative regulator16. Recent studies demonstrated that DANCR directly interacts with miR-758-3p17 and miR-57718 in non-small cell lung cancer and colorectal cancer, respectively. DANCR represses the expression of TIMP2/3 through physical binding with EZH2 in prostate cancer19. DANCR is also shown as a direct target of MYC in cancer20. Furthermore, DANCR expression is correlated with survival and/or prognosis of patients with hepatocellular carcinoma12, gastric cancer21, and colorectal cancer10. However, due to the molecular and phenotypic heterogeneity within and between different tumor types, mechanism explorations are required to elucidate the precise biological behaviour of DANCR in tumors, especially TNBC.Differentiation antagonizing non-protein coding RNA  type, 15 HER2 type, 15 Luminal A type, and 15 Luminal B type, and 10 normal breast tissues were obtained from department of Breast Surgery in Meizhou People’ s Hospital. All these patients had not received chemotherapy and radiotherapy before the operation and all clinical samples were confirmed by pathology. This study protocol approval from the Research Ethics Committee of the Meizhou People’ s Hospital, and written informed consent from each participant were obtained.BT549, MCF7, T47D, MDA-MB-231, MDA-MB-453, and MDA-MB-468 cells were purchased from Cell Bank of the Chinese Scientific Academy , and were cultured in Dulbecco’ s modified Eagle’ s medium (DMEM)  with 10% fetal bovine serum . MCF10A was also from Cell Bank of the Chinese Scientific Academy , and was cultured in DMEM/F12  supplemented with 5% horse serum , 20 ng/ml hEGF (Sigma-Aldrich), 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 100 ng/ml cholera toxin (Sigma-Aldrich),10 μg/ml insulin (Sigma-Aldrich), and 100 U/ml penicillin-streptomycin (Sigma-Aldrich). BT549, MCF7, T47D, MDA-MB-231, MDA-MB-453, MDA-MB-468, and MCF10A were recently authenticated through using STR DNA fingerprinting from Shanghai Biowing Applied Biotechnology Co., Ltd. Moreover, by using LookOut Mycoplasma PCR Detection kit (Sigma-Aldrich), we detected the mycoplasma infection.Total RNA was isolated using TRIzol reagent  and extracted according to the manufacturers’ protocol (Sigma-Aldrich). Reverse transcription (RT) was performed using the M-MLV Reverse Transcription Kit(Thermo Fisher Scientific). Quantitative RT–PCR was performed by means of Power qPCR Premix (SYBR Green) . GAPDH was used as a control. Primers are contained in Supplenentary Table 22. The specific antibodies for WB as follows: anti-RXRA , Phospho-RXRA , GSK3β , Akt , Phospho-Akt , PIK3CA , GAPDH .Western blot was performed as we previously describedChIP assay was performed using EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore-17-408) referring to the manufacturer’s protocol.Input genomic DNA and the purified immunoprecipitated DNA were used for qRT-PCR. Primers are contained in Supplementary Table pGL3-PIK3CA promoter wild or mutant type of the RXRA binding was performed co-transfection with or without RXRA using Lipofectamine RNAiMAX Reagent (Thero Fisher) referring to the manufacturer’s protocol. pRL Renilla luciferase control reporter vector (Promega) was presented as a control. A dual-luciferase assay was analyzed 48 h after co-transfection using the Promega E1960 Dual-Luciferase® Reporter System in accordance with the manufacturer recommendation.The EZ-Magna RIP Kit  was used for RIP assay with 10 μg anti-RXRA  referring to the manufacturer recommendation. The purified immunoprecipitated RNA and input genomic RNA were detected by quantitative RT-PCR. Biotin-labeled RNA was transcribed with Biotin RNA Labeling Mix (Roche 11685597910) and T7 RNA polymerase (Roche 10881775001), mixed with DNase I recombinant (Roche 04716728001), and purified with RNeasy Mini Kit (Qiagen 74904). Cell nuclear proteins were extracted using the pierce 78833 NE-PER (R) Nuclear and Cytoplasmic Extraction Reagents. Cell nuclear extract was mixed with Biotin-labeled RNA. Washed streptavidin agarose beads (Sigma-Aldrich) were added to each reaction. The binding protein was analyzed by Western blot assay.A WST-1 Assay Kit (Roche) was used for cell proliferation assay. Cells were seeded in a 48-well plate and then incubated at 37 °C. Cell numbers were assessed with the WST-1 Assay Kit. For soft agar colony formation, cells were were split into the suspension of a single-cell, and then seeded in a media containing 0.4% top layer agar and 0.8% bottom layer agar in a 6-well plate. Cell culture media was changed every 4 days after seeding. Colonies were fixed with 4% paraformaldehyde and stained with 2% crystal violet solution after 2–3 weeks, respectively. The visible colony numbers scored and data were analyzed.DANCR, PIK3CA,GSK3β, RXRA were amplified from MCF10A cells and sequenced, and then subcloned into the pcDNA3 vector (Invitrogen), subsequently named pCDNA3-DANCR, pCDNA3-PIK3CA, pCDNA3-GSK3β, pCDNA3-RXRA. pLVX-DANCR, pLVX-PIK3CA, pLVX-GSK3β, and pLVX-RXRA was generated from pCDNA3-DANCR, pCDNA3-PIK3CA, pCDNA3-GSK3β, pCDNA3- RXRA, respectively. PIK3CA promoter was PCR-amplified from MCF10A cells and sequenced, and then subcloned into pGL3 vector (Promega). A Quik Change Site-Directed Mutagenesis Kit (Stratagene) was used for point mutations. shRNAs for DANCR were designed , and RXRA  were implanted subcutaneously into mammary fat pads of each mice. All animal experiments procedures were approved by Zhejiang Provincial People’s Hospital the Guidance of Institutional Animal Care and Use Committee (IACUC). The IVIS Lumina imaging station  was used for bioluminescence imaging. Different investigators independently performed mice allocation, surgery and the outcome assessing.BALB/c-nude mice female at an age of 4–5 weeks  were randomly divided into 4 per group, and then MDA-MB-231 cells  suffered poorer overall survival (OS) as compared to low expression group (n = 30)  Fig. . Similar30) Fig. . Gene am30) Fig. . Taken tDANCR in TNBC, we tested DANCR expression in six breast cancer cell lines  and a normal mammary epithelial cell line (MCF10A). DANCR expression was markedly upregulated in breast cancer cell lines as compared to that in MCF10A cell lines .To further demonstrate the involvement of nes Fig. . ImportaDANCR inhibition on TNBC cell growth. Here, we knocked down the DANCR expression through treating MDA-MB-231 and MDA-MB-468 cells with the short hairpin RNA-mediated DANCR silencing (shRNA) or non-silencing control (shC), and found that depletion of endogenous DANCR significantly suppressed cell proliferation in both TNBC cell lines compared with the controls  were separately injected into the mice mammary gland fat pads. Remarkably, knockdown of DANCR was significantly decreased TNBC tumor growth compared with controls , site2 (Mut2), or both sites (Mut1/2). As we predicted, re-expression of shRNA-resistant DANCR cDNA encoding the wild type restored the binding of DANCR with RXRA, whereas re-expression of a DANCR shRNA-resistant plasmid containing the DANCR-RXRA binding site1, site 2, or two sites mutants did not rescue it, suggesting that these two sites are critical for DANCR-RXRA binding  in MDA-MB-231 and MDA-MB-468 cells. As shown in Fig. DANCR significantly inhibited PIK3CA protein and mRNA expression levels in both TNBC cells. However, knockdown of RXRA significantly rescued DANCR depletion-inhibited PIK3CA expression  had been reported to promote RXRA phosphorylation in colorectal cancer cellslls Fig. . Knockdolls Fig. . Furtherlls Fig.  inhibitells Fig. . Overexpown Fig. . These dDANCR in TNBC is not well understood, we focused on the role of RXRA phosphorylation in DANCR-regulated TNBC cell proliferation. To detect whether RXRA phosphorylation is necessary to activate PI3K/AKT signaling pathway, we generated the GSK3β kinase deficient mutant plasmid, GSK3β-K58A. As shown Fig. DANCR knockdown-inhibited RXRA phosphorylation, Akt phosphorylation and PIK3CA expression, whereas transfection of GSK3β kinase deficient mutant type did not rescue it. Next, we predicted potential serine phosphorylation sites of the RXRA protein using the Phosphor Motif Finder Program. Two serine phosphorylation candidate sites at Ser49 and Ser78 were identified. Transfection of RXRA wild type in MDA-MB-231 cells restored its binding with GSK3β inhibited by DANCR knockdown, and further decreased DANCR depletion-inhibited Akt phosphorylation, PIK3CA expression, cell proliferation, and soft agar colony formation (Fig. S49A/S78A mutant did not rescued it compared with the wild type group (Fig. Since the roles of RXRA phosphorylation mediated by ion Fig. . Howeveroup Fig. . Taken tDANCR-upregulated PI3K/AKT signaling pathway through activating serine phosphorylation of RXRA protein is important for TNBC cell proliferation and tumor growth (Fig. DANCR promotes the phosphorylation of RXRA, leading to suppress RXRA induced inhibition of PIK3CA transcription, thereby activating PI3K/AKT downstream signaling and ultimately promoting TNBC tumorigenesis.In this study, we demonstrated a new mechanism by which wth Fig. . By meanDANCR promotes TNBC tumorigenesis. DANCR was previously identified to play an important role in maintaining the undifferentiated cell state16. Previous work demonstrated that DANCR interacts with EZH2 to form a complex to promote stem cell characteristics28. It has been well documented that DANCR was overexpressed and amplified in breast cancer, and knockdown of DANCR significantly inhibited cell proliferation and invasion in breast cancer through facilitating binding of EZH2 to the promoters of ABCG2 and CD44 genes14. Here, we report that DANCR is upregulated in clinical TNBC samples, and higher DANCR level is positively correlated with poorer prognosis of TNBC patients. Knockdown of DANCR significantly inhibited TNBC cell proliferation, colony formation in vitro, and tumor growth in vivo. These results strongly support that DANCR is critical for TNBC tumorigenesis.Our data demonstrate that DANCR mediates TNBC through RXRA. RXRA had been reported to play critical roles in breast cancer cell progression30. Phosphorylation of the RXRA at serine 260 decreases its coactivator recruitment ability, leading to inhibition of RXRA transcriptional activity and enhanced cancer cell proliferation32. DANCR has also been identified as a molecular sponge mediating miR-758-3p17 and miR-57718 in non-small cell lung cancer and colorectal cancer, respectively. Furthermore, some studies showed that DANCR regulate breast cancer through binding and phosphorylating EZH225. In this study, our data showed that DANCR directly bound with RXRA and mediated its phosphorylation. Knockdown of DANCR enhanced RXRA phosphorylation. Re-expression of shRNA resistant DANCR wild type impaired DANCR knockdown-promoted RXRA phosphorylation, whereas re-expression of shRNA resistant mutant type of the DANCR-RXRA binding did not affect it. Knockdown of RXRA rescued DANCR depletion-inhibited cell proliferation and soft agar colony formation. Taken together, our data demonstrate that DANCR regulate TNBC cell proliferation through RXRA phosphorylation.Our results also demonstrate that DANCR mediates TNBC through RXRA- downregulating PI3K/AKT signaling pathway. Activating PI3K/AKT signaling was reported to be associated with poor OS in patients with breast cancer33. PIK3CA activation was also demonstrated to be critical for enhancing PI3K/AKT signaling22. RXRA was reported to mediate PI3K/AKT signaling in response to stem cell differentiation and provoke tumor suppression34. Inhibition of the N-terminally truncated RXRA association with the PI3K p85α subunit also resulted in suppression of PI3K/AKT signaling activation35. Here, we found that RXRA binds with the promoter of PIK3CA and downregulate PIK3CA transcription. We also observed that knockdown of DANCR inhibited RXRA protein phosphorylation and PIK3CA expression level. Interestingly, knockdown of RXRA rescued DANCR depletion-inhibited PIK3CA expression, cell proliferation, soft agar colony formation, and the promoter transcriptional activity of PIK3CA. Furthermore, We found DANCR markedly promotes RXRA association with GSK3β. In consistent with the previous study32, our data demonstrate that DANCR-induced RXRA phosphorylation suppresses RXRA-inhibited PIK3CA transcription in TNBC cells and ultimately activates the downstream PI3K/AKT signaling, whereas overexpression of GSK3β mutant type with deficient kinase activity or RXRA with mutated binding site with GSK3β dramatically blocked it. These data suggest that DANCR promoted TNBC tumorigenesis depends on its binding and phosphorylating RXRA, which leads to PI3K/AKT signaling pathway activation.Our results further suggest that DANCR as an oncogene promotes TNBC tumorigenesis through a distinctive mechanism by which DANCR facilitated RXRA phosphorylation depending on GSK3β, thereby inhibiting the function of RXRA as a transcription repressors, and ultimately enhanced downstream PI3K/AKT signaling. Our findings have shed light into a novel roles of DANCR in TNBC tumorigenesis which have significant implications on better understanding the function of DANCR in human cancers. We hope that the newly established roles of DANCR and RXRA in tumorigenesis may provide a strong rationale for targeting them to improve the treatment of TNBC patients.In summary, our results identified Supplementry Figure 1Supplementry Figure 2Supplementry Figure 3Supplementary Table 1Rebuttal letter


Paraesophageal hernias are usually asymptomatic; however, they can cause serious complications such as necrosis or incarceration-induced perforation. Necrosis usually occurs in the incarcerated portion of the hernia. Here, we report the case of a patient with gastric necrosis secondary to an incarcerated paraesophageal hernia in which the necrotic lesion was outside the hernia sac.A 91-year-old woman presented with severe abdominal pain and vomiting. A physical examination showed hypotension and a diffusely tender and rigid abdomen. Computed tomography showed a paraesophageal hernia, massive ascites, and free air around the stomach. A laparotomy was performed to treat the upper gastrointestinal perforation. The stomach was incarcerated within the paraesophageal hernia sac. After reducing the stomach, we identified a large perforation on the posterior wall of the gastric fundus. Full-thickness necrosis involving part of the stomach necessitated total gastrectomy. She remained physiologically unstable and her condition deteriorated; she died 2 days postoperatively.A hiatal hernia can be associated with an ischemic gastric perforation outside the hernia sac. Paraesophageal hernias are usually asymptomatic; however, incarcerated hernias may cause serious complications . The estA 91-year-old woman presented with severe abdominal pain and vomiting. She revealed a 12-h history of abdominal pain that had suddenly worsened and was associated with vomiting 1 h prior to presentation. She was otherwise healthy and denied the regular use of any medications.A physical examination showed a blood pressure of 96/58 mmHg, heart rate of 95 beats/min, respiratory rate of 24 breaths/min, and hypothermia. An abdominal examination showed a diffusely rigid abdomen with rebound tenderness over the entire abdomen and reduced bowel sounds. Contrast-enhanced abdominal computed tomography showed a paraesophageal hernia in which part of the stomach was incarcerated Fig. . Free aiAn emergency laparotomy was performed through the upper abdominal midline incision, and we observed that part of the stomach was incarcerated within the paraesophageal hernia sac, which also contained ascitic fluid that appeared like gastric fluid. After reducing the stomach, we detected a perforation measuring 7 cm on the posterior wall of the gastric fundus Fig. . The affAfter the operation, the patient remained hemodynamically unstable despite maximal resuscitative efforts. We considered that the distributive shock was affected by the surgery and sepsis on postoperative day 1. However, her condition deteriorated on postoperative day 2. Although we suspected a major leakage, considering her age and hemodynamics, we did not conclude that reoperation would improve her condition effectively. Thus, we informed her family about her condition and decided on nonoperative management. She died on postoperative day 2.Four types of esophageal hiatal hernia were identified, and types II, III, and IV are all varieties of paraesophageal hernia . The preThe most notable feature in this case was that, although the perforation site was not within the incarcerated hernia sac, full-thickness necrosis of the gastric wall was evident. The stomach receives its arterial blood supply from multiple sources. Usually, a single artery can maintain adequate organ perfusion, and ischemia is rare. Gastric ischemia is usually associated with complete cessation of arterial blood flow. In this patient, most of the gastric body and 50% of the fundus were incarcerated. The antrum and the remaining portion of the fundus were outside the hernia sac at the time of surgery. Thus, all arteries including the short gastric artery were incarcerated within the sac with complete cessation of blood supply to the fundus outside the hernia sac, which could have precipitated the necrosis. This rare condition could be attributed to the fact that the perforated area was initially incarcerated but underwent spontaneous reduction before surgery. However, this appears to be less likely because there was no evidence on the outer aspect of the stomach to suggest prolonged incarceration of the area. In our opinion, the incarcerated site might not have completely blocked the fundus outside the sac, which was locally ischemic and necrotic owing to complete cessation of blood supply, so the gastric contents in the incarcerated site drained into the abdominal cavity when the fundus outside the sac was perforated and reduced. As the space between the hernia orifice and hernia content expanded owing to the decreasing gastric contents, the fluids and air likely moved into the hernia sac. If our theory is correct, preoperatively judging whether the perforation corresponds to the incarcerated site is difficult.The optimal operation in this situation remains debatable. We performed a PubMed literature search (2001 to present), and our operation was based on strategies reported in previous studies (Table We identified 2 patients (including our patient) with perforation away from the incarceration site. In these 2 patients, the incarcerated site was not always strangulated and necrotic, and fundal strangulation occurred outside the hernia sac. It would be reasonable to conclude that because the necrotic area was at the site of perforation but outside the hernia sac, only the perforated area required resection. A previous report described favorable outcomes associated with a partial gastrectomy in this context . In our Ischemia-induced gastric perforation can occur in association with a hiatal hernia, even in tissue outside the hernia sac.


KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/ Microtubules (MT) form higher-order structures such as asters, but the molecular pathway underlying aster formation remains unclear. Here authors demonstrate that the kinesin-14, HSET, clusters with soluble (nonMT) tubulin via its N-terminal tail domain and thereby promotes MT aster formation. MT organization depends on both the intrinsic dynamics of MTs and on cellular factors that govern MT positioning and assembly. MTs are polymers of αβ-tubulin that grow and shrink with a fast-growing (plus) and slow-growing (minus) end4. During all phases of the cell cycle, it is estimated that between one-half and one-third of total tubulin (termed soluble tubulin) is not incorporated into MT polymers5, and instead exists as either αβ-heterodimers or small structural intermediates that remain poorly understood6. In the absence of MT-associated proteins (MAPs), MTs are unable to organize into higher-order structures. Collectively, the molecular properties of MAPs on a ~nanometer scale determine the architecture of MT arrays on a ~micron scale through diverse mechanisms including MT cross-linking, translocation, and regulation of MT dynamics.An organized microtubule (MT) cytoskeleton is essential for a broad spectrum of biological processes, ranging from cell polarization to division. A striking example is the mitotic spindle, which drives chromosome segregation7: (i) MT bundles, which can exist in either antiparallel or parallel configurations, and (ii) MT asters, which are radial arrays of MTs focused at a pole. Both architectures are represented in the mitotic spindle. Interpolar MT bundles facilitate sliding that separates two spindle poles, which are defined by two MT asters. Although bundles minimally require MAP-induced cross-linking for their formation, aster formation requires MT end focusing or a specialized structure to impart radial symmetry9. In cells, aster formation is driven predominantly by centrosomes10. When centrosomes are either present in excess11 or absent13, cells use other factors to organize MT minus ends within the spindle to ensure its bipolar geometry.With the exception of templated structures , MT structures are built from two basic architectures17. Aster formation depends on the ability of dynein to cross-link two MTs18 and move processively toward MT minus ends, leading to a model where cargo MTs are transported along track filaments14. The minus-end-directed kinesin-14 XCTK2 is also capable of driving aster formation from growing MTs, but unlike dynein, XCTK2 requires no additional cofactors19. On a molecular level, the mechanisms underlying aster formation by both dynein and kinesin-14 are unclear. This is especially true for kinesin-14s, as they are nonprocessive motors, i.e., they do not move unidirectionally as single motors on a MT track27. Furthermore, in studies using nongrowing MTs, kinesin-14s form, slide, and sort MT bundles, rather than asters29. It thus remains to be seen how kinesin-14s can simultaneously act as promoters of both bundles and asters, and whether this behavior depends on the state of MTs .Our knowledge of aster formation mechanisms derives primarily from in vitro work. In mitotic cell extracts, the formation of MT asters requires cytoplasmic dynein, along with its cofactor NuMAKIFC1 (human kinesin-14). We determine that, consistent with previous studies, HSET is a nonprocessive motor on the single-molecule level that forms MT bundles. We show that HSET's processivity is caused by the formation of multimotor clusters upon binding to soluble (non-MT) tubulin, and that this processivity is essential for promoting aster formation. Finally, we present data suggesting that HSET’s aster-forming behavior can be activated by excess soluble (non-MT) tubulin in mitotic HeLa cells. Our work establishes a general principle of aster formation by MT-cross-linking motor proteins, and demonstrates how motor regulation on a molecular level dictates the formation of higher-order MT structures.Here, we establish the mechanism of aster formation by HSET/Xenopus homolog XCTK2, could form MT asters, we expressed and purified four HSET truncations tagged with an N-terminal enhanced green fluorescent protein (EGFP) , or the conserved C-terminal motor domain (EGFP-HSETΔMotor) eliminated aster formation , we observed bundle formation, but no MT asters  Fig.  for use ion Fig. , middle.ers Fig. , bottom.32. When we combined 100 nM full-length EGFP-HSET with preformed MTs (1 µM polymeric tubulin), we observed no aster formation or pole focusing. Rather, consistent with the established role of kinesin-14 as a MT bundling factor33, we observed MT bundling and sliding, which reached a steady state within ~20 min  increase the short association times of EGFP-HSETΔMotor to an observable level, we performed these experiments in low-salt (P12) buffer (see Methods). Consistent with previous studies on Ncd24 and a recent study on HSET29, single molecules of EGFP-HSET and EGFP-HSETΔMotor showed diffusive behavior, whereas deletion of the tail domain (EGFP-HSETΔTail) led to transient, static interactions with the MT  microscopy Fig. . In orde MT Fig. . Mean-sq MT Fig. . We concWe considered the possibility that HSET’s inability to form MT asters from preformed MTs depends on the presence of soluble (non-MT) tubulin. To investigate the effect of tubulin on the motor, we examined EGFP-HSET’s motility on stabilized MTs in the presence of 2 µM tubulin Fig. . At low In order to visualize individual motility events in these conditions of high motor concentration, we performed a spike-in TIRF experiment where 5% of HSET particles contained the N-terminal EGFP-tag at a total HSET concentration of 10 nM , and run length of 4.2 ± 0.6 µm    Fig. . These plin Fig. .To test whether the tail domain directly interacts with soluble (non-MT) tubulin, we performed a co-immunoprecipitation of purified tubulin and HSET truncations via the common EGFP-tag Fig. . We dete27; we thus wondered if the processive tubulin-HSET particles contain multiple HSET motors. We used a fluorescence intensity-based approach to quantify the number of motors in a moving particle  tubulin promotes HSET's processivity by inducing cluster formation, we tested whether soluble tubulin could promote the ability of HSET to drive the organization of asters using preformed MTs. Upon the addition of 20 µM tubulin to preformed MTs and HSET, we observed rapid formation of radially symmetric asters (~8 min) Fig. . In cont19. Here, we again observed rapid formation (~5 min) of MT asters in the presence of 20 µM tubulin  introduced saturating (100 µM) levels of colchicine, a tubulin-sequestering drug that prevents polymerization, (ii) replaced 1.5 mM GTP with 1.5 mM GDP, a guanine nucleotide that is incompatible with polymerization, and (iii) omitted taxol, a field-standard component for self-organization bufferlin Fig. . We obtalin Fig. . These rn = 185) and 4.0 ± 0.6  µm, respectively   tubulin. To construct HSET clusters without tubulin-induced activation, we incubated 6× His-tagged EGFP-HSET or EGFP-HSETΔTail with streptavidin-Quantum Dots  at a 3:1 ratio in the presence of biotin-anti-His antibody Fig. . After uely Fig. . Both coly) Fig. .Fig. 5Mu9. Alternatively, HSET’s tail may be required to bind polymer MTs as a cargo during translocation to minus ends (see Discussion). Regardless, we conclude that cluster formation of EGFP-HSET, whether by addition of tubulin or binding to QDots, leads to aster formation from preformed MTs.When we introduced EGFP-HSET-QDots to preformed MTs, asters formed over ~20 min Fig. . We dire35 to generate a HeLa cell line that inducibly overexpresses EGFP-HSET. By immunoblotting, we observed that EGFP-HSET reached a maximum expression level of ~4-fold overexpression relative to endogenous HSET after 3 days’ treatment with doxycycline (compare to ~2.7-fold overexpression in 1 day)  tubulin in cells. We investigated this hypothesis by changing the relative concentrations of both HSET and soluble (non-MT) tubulin during mitosis. To increase HSET protein levels, we used recombination-mediated cassette exchangeion Fig. .Fig. 6In36, as endogenous aster-promoting factors are present in these cells. Finally, we used a live-imaging approach to observe EGFP-HSET during aster formation over time. Following the addition of nocodazole to cells overexpressing EGFP-HSET, we observed a conversion of bipolar spindles to multiple individual asters over 15–30 min  tubulin concentrations, we used nonsaturating amounts (500 nM) of nocodazole to partially depolymerize MTs. We treated cells either containing endogenous Fig. , top or min Fig. . These e27. This result is also consistent with a recently published study where HSET was shown to diffuse bidirectionally on single MTs29. In spite of these unremarkable single-molecule properties, we have shown that kinesin-14s can assemble and remodel the two basic MT architectures employed in higher-order organization: bundles and asters. Our data suggest that aster formation occurs in the presence of soluble (non-MT) tubulin, which causes the clustering of HSET to promote motor processivity. We thus propose that the activity of HSET is context-dependent, being influenced directly by the availability of soluble (non-MT) tubulin vs. MT polymer, and therefore inextricably linked to the dynamic rearrangement of the cytoskeleton during cell division.In this study, we have shown that single HSET motors are minimally processive on MTs, consistent with previous reports on the fly kinesin-14 Ncd34. Three pieces of evidence suggest that HSET is not regulated in this way: (i) tail deletions do not result in activation of either Ncd25 or HSET (this study), (ii) Ncd shows nonprocessive behavior whether bound to single MTs or to MT overlaps where the tail is engaged24, and (iii) HSET adopts an open conformation when visualized by negative stain EM37. Rather, our data demonstrate that HSET's processivity is promoted by motor multimerization upon binding soluble (non-MT) tubulin to form clusters. It is likely that cluster formation occurs in solution, rather than on the MT surface, as we did not observe fluorescence intensity increasing within clusters over time  is unknown. Here, we identify soluble tubulin  as a physiologically relevant clustering agent for HSET.The observation that soluble (non-MT) tubulin promotes HSET's processivity suggests a distinct mechanism of kinesin motor activation. Many processive kinesins are regulated by an autoinhibition mechanism, where cargo binding to the tail domain overcomes an inhibitory tail–motor interactionime Fig. . After b41. This population is relatively rare and requires ultra-sensitive techniques for its detection 40. Analogous to conditions where we observed HSET's activation by tubulin  possess an enhanced affinity for rare tubulin oligomers in solution, or (ii) HSET induces the formation of tubulin oligomers. The order of events in the formation of HSET-tubulin clusters is an important issue that will be addressed in future studies.What is the population of tubulin that activates HSET's processivity? Because tubulin activates HSET's processivity under conditions that are not compatible with MT polymer formation  tubulin (this work)  tubulin is present in sufficient quantities  tubulin promote HSET’s aster formation activity? Soluble tubulin has previously been shown to regulate the MT-severing protein katanin by disrupting its ability to bind MT polymerrk) Fig. . In the rk) Fig. , top andrk) Fig. , bottom,ies Fig. , the taires Fig. . Thus, oWhat is the molecular nature of HSET-tubulin clusters? Based on our fluorescence intensity measurements, these clusters appear to be heterogeneous in size, composition, and stoichiometry. Our AUC results suggest that either cluster heterogeneity obscures peak detection, or only a small number of clusters is sufficient to drive aster organization. Because these clusters are diffraction-limited, the geometrical nature of these clusters is also yet to be determined. Regardless, we have determined a number of key features for these clusters: (i) they contain multiple HSET motors (possible range of 2–6) and multiple tubulin dimers (possible range of 3–26), (ii) cluster formation does not depend on GTP-driven tubulin assembly, and (iii) clusters are likely not the result of an in vitro aggregation artifact. Importantly, we have shown that HSET's activity in cells is modulated by the intracellular levels of soluble (non-MT) tubulin, providing evidence that our in vitro observations are relevant within the cell.9. Detailed structural investigations of these tail-mediated interactions thus present an exciting direction for future study.Our observation that QDots bound to tailless HSET do not form either bundles or asters points to two additional, critical roles for the HSET tail domain in higher-order MT organization. First, it is currently unclear how cargo MTs are engaged by HSET-tubulin clusters to initiate transport Fig. , bottom.α/β is a demonstrated regulator of HSET activity in cells47. By binding to HSET’s tail, importin α/β could act as a global on/off switch for HSET activity, thereby limiting the pool of tail domains available to bind either MT polymer or soluble (non-MT) tubulin. Additionally, HSET has been shown to bind EB1 and track the tips of growing MTs in vitro48. Thus, +TIP proteins may play an additional role in tuning HSET function at growing MT ends. Finally, eliminating the activity of HSET is sufficient to block the conversion of multipolar spindles to bipolar structures in cells with amplified centrosomes11. This has also been observed for dynein49, another motor capable of forming MT asters in vitro. How a motor’s ability to form MT asters in vitro translates to centrosome clustering in cells is currently unknown. Future studies determining the precise mechanisms of supernumerary centrosome clustering will be essential to understanding how cancer cells overcome this obstacle to continue their rapid cell division.In summary, our data demonstrate how the interplay between tubulin and HSET can lead to the organization of morphologically distinct higher-order MT architectures in vitro. In cells, additional molecular factors play a role in modulating HSET activation and regulation. For instance, importin All constructs were prepared using an HSET cDNA corresponding to Gen Bank Accession Number BC121041. For motor domain (amino acids 305–673)-containing constructs, plasmids were prepared in the baculovirus vector pFASTBAC-HTa (Life Technologies) for use with the insect cell expression system, whereas all noncatalytic constructs  were cloned into the bacterial expression vector pET15b (Novagen).KIFC1 (Homo sapiens kinesin-14) were determined by sequence alignment to Ncd (Drosophila melanogaster kinesin-14) using24 as a guide, and used to design pET15b-HTa-EGFP-HSETΔMotor and pFASTBAC-HTa-EGFP-HSETΔTail. The pET15b-HTa-EGFP-HSETDoubleTail sequence was designed by sequence alignment to XCTK2 (X. laevis kinesin-14) using19 as a guide. Where desired, a flexible amino acid linker (GGSGGS) was inserted to ensure rotational freedom between domains.Amino acid sequences of the tail, stalk, and motor domains of HSET/Each construct contains the corresponding amino acid sequence, which was verified by DNA sequencing:6-YDIPTTENLYFQGAMDPEF-EGFP(aa 1–239)-HSET(aa 1–673).pFASTBAC-HTa-EGFP-HSET: MSYY-His6-SSGLVPRGSH-EGFP(aa 1–239)-SGLRSRAQASNS-HSET(aa 1–304).pET15b-HTa-EGFP-HSETΔMotor: MGSS-His6-SSGLVPRGSH-EGFP(aa 1–239)-SGLRSRAQASNS-HSET(aa 1–307)-GGSGGS-HSET(aa 1–137).pET15b-HTa-EGFP-HSETDoubleTail: MGSS-His6-YDIPTTENLYFQGAMDPEF-EGFP(aa 1–239)-GGSGGS-HSET(aa 139–673).pFASTBAC-HTa-EGFP-HSETΔTail: MSYY-His6-DYDIPTTENLYFQGAMDPEF-HSET(aa 1–673).pFASTBAC-HTa-HSET: MSYY-HispEM791-EGFP-HSET: EGFP(aa 1-239)-HSET(aa1-673).50. PCR fragments consisting of HSET amino acids 1–673 , 1–304 (pET15b-HTa-EGFP-HSETΔMotor), 139–673 (pFASTBAC-HTa-EGFP-HSETΔTail), and 1–307 + 1–137 (pET15b-HTa-EGFP-HSETDoubleTail) were generated by amplification of the HSET ORF (using pEGFP-C1-HSET as a DNA template37) using Phusion DNA polymerase (New England Biolabs #M0530S). These fragments were then assembled into their expression vectors cut using the indicated restriction enzymes: pFASTBAC-HTa-EGFP-HSET: EcoRI/XhoI. pFASTBAC-HTa-EGFP-HSETΔTail and pFASTBAC-HTa-HSET: EcoRI/KpnI. pET15b-HTa-EGFP-HSETΔMotor and pET15b-HTa-EGFP-HSETDoubleTail: NdeI/XhoI. pEM791-EGFP-HSET: BsrGI/BglII. Constructs in the pFASTBAC-HTa vector were used with the Bac-to-Bac system (Invitrogen) per manufacturers’ protocol to create a baculovirus expressing the indicated protein.All constructs  were prepared by isothermal assembly37. For other constructs, the following primers were used for isothermal assembly:The cloning of pFASTBAC-HTa-EGFP-HSET was described previouslypET15b-HTa-EGFP-HSETΔMotor Forward Primer: “15b-_Cherry_IA_5”:5′-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTGAGCAAGGGCGAGG-3′pET15b-HTa-EGFP-HSETΔMotor Reverse Primer: “HSET304REV”:5′–CGGGCTTTGTTAGCAGCCGGATCCTCGAGTCACAGCTGGTTGTGCAGTCG-3′pET15b-HTa-EGFP-HSETDoubleTail Forward Primer 1: “15b-_Cherry_IA_5”:5′-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTGAGCAAGGGCGAGG-3′pET15b-HTa-EGFP-HSETDoubleTail Reverse Primer 1: “H137-GGS2-H307REV”:5′-GACCTCTGCGGATCCATTGATCCTCCTGATCCTCCGAGTTCCTGCAGCTGGTT-3′pET15b-HTa-EGFP-HSETDoubleTail Forward Primer 2: “H307-GGS2-H137FOR”:5′-AACCAGCTGCAGGAACTCGGAGGATCAGGAGGATCAATGGATCCGCAGAGGTC-3′pET15b-HTa-EGFP-HSETDoubleTail Reverse Primer 2: “15b-HSET137-REV”:5′-CGGGCTTTGTTAGCAGCCGGATCCTCGAGTCACCAGGCTGGACGTTTGC-3′pFASTBAC-HTa-EGFP-HSETΔTail Forward Primer 1: “pFB-EGFP-FOR”:5′-GTATTTTCAGGGCGCCATGGATCCGGAATTCATGGTGAGCAAGGGCGAGG-3’pFASTBAC-HTa-EGFP-HSETΔTail Reverse Primer 1: “H139-GGS2-EGFP-REV”:5′-GTCACATAACTGACCCTTTAATGATCCTCCTGATCCTCCCTTGTACAGCTCGTCCATG-3′pFASTBAC-HTa-EGFP-HSETΔTail Forward Primer 2: “H139-GGS2-H673-FOR”5′-CATGGACGAGCTGTACAAGGGAGGATCAGGAGGATCATTAAAGGGTCAGTTATGTGAC-3′pFASTBAC-HTa-EGFP-HSETΔTail Reverse Primer 2: “pFB-HSET_UnlREV”:5′-TCCTCTAGTACTTCTCGACAAGCTTGGTACCTCACTTCCTGTTGGCCTG-3′pFASTBAC-HTa-HSET Forward Primer: “pFB-HSET_UnlFOR”:5′-GTATTTTCAGGGCGCCATGGATCCGGAATTCATGGATCCGCAGAGGTC-3′pFASTBAC-HTa-HSET Reverse Primer: “pFB-HSET_UnlREV”:5′-TCCTCTAGTACTTCTCGACAAGCTTGGTACCTCACTTCCTGTTGGCCTG-3′pEM791-EGFP-HSET Forward Primer: “EM791_GFP-HSET_5′”:5′-CACTCTCGGCATGGACGAGCTGTACAAGATGGATCCGCAGAGGTC-3′pEM791-EGFP-HSET Reverse Primer: “EM791_GFP-HSET_3′”:5′-CATAATTTTTGGCAGAGGGAAAAAGATCTTCACTTCCTGTTGGCCTG-3′Sf9 cells (Invitrogen) by infecting them with the corresponding baculovirus for 72 h. Constructs in the pET15b vector were expressed in BL21DE3 cells (Stratagene) with 0.4 mM IPTG for 16 h at 16 °C. Each protein was purified via His6-affinity chromatography, followed by size exclusion chromatography, as follows: cells were pelleted and resuspended in PNI buffer  with 5 mM β-mercaptoethanol (β-ME), 1% NP40, and protease inhibitors . Bacterial cells were incubated on ice with 1 mg/mL lysozyme (Sigma) for 30 min to remove bacterial cell walls; this step was omitted for Sf9 purifications. Cells were then lysed by sonication and clarified by centrifugation at 4 °C at 35 K RPM  for 1 h in a Ti-45 rotor (Beckman). 4 mL of Ni++-NTA agarose (Qiagen) were incubated with the supernatant for 1–2 h, then washed extensively with wash buffer  for 3–4 h. For proteins containing the catalytic motor domain, this wash buffer was supplemented with 50 µM MgATP to ensure proper nucleotide binding to the motor. The proteins were then eluted from the Ni++-NTA agarose column in PNI with 5 mM β-ME and 180 mM imidazole (supplemented with 100 µM MgATP for proteins containing the motor domain). Peak fractions  were then subjected to size exclusion chromatography on a Hiload 16/60 Superdex 200 preparatory grade column  in gel filtration buffer . Protein concentrations were determined in mg/mL using Bradford assays (BioRad) and converted to molar units assuming dimer formation. Powdered sucrose was added to 20% w/v, then each protein was aliquoted, snap frozen in liquid nitrogen, and stored at −80 °C.Constructs in the pFASTBAC-HTa vector were expressed in 51. Briefly, brain homogenates were cycled twice in high-molarity PIPES buffer  to remove MT-associated proteins, resuspended in BRB80 buffer , diluted to 20 mg/mL, snap frozen in liquid nitrogen, and stored at −80 °C. To generate fluorescently labeled or biotinylated tubulin, NHS-Alexa594, NHS-Cy5, and NHS-Biotin  were conjugated to cycled tubulin using succinimidyl ester chemistry essentially as described52. Briefly, 10 mg/mL tubulin was polymerized in BRB80 supplemented with 50% glycerol and 1 mM GTP for 30 min at 37 °C, then spun over a high pH (pH 8.6) glycerol cushion at 40 K RPM  for 45 min in a Ti-50.2 rotor (Beckman) at 37 °C. Polymerized tubulin was washed 2×, the dye (indicated above) was added at 20× molar excess, and incubated at 37 °C for 40 min. The reaction was quenched in 50 mM K-glutamate for 5 min, then pelleted by spinning over a low-pH (pH 6.9) glycerol cushion at 80 K RPM  for 20 min in a TLA 100.3 rotor (Beckman) at 37 °C. Labeled pellets were depolymerized by resuspension in ice-cold 50 mM K-glutamate +0.5 mM MgCl2 (pH 7.0) and subjected to continuous dounce homogenization for 25 min on ice. The labeled soluble tubulin was clarified by spinning at 80 K RPM  for 10 min in a TLA 100.3 rotor (Beckman) at 4 °C. The clarified supernatant was then repolymerized in BRB80 supplemented with 50% glycerol and 1 mM GTP at 37 °C for 30 min. The polymerized, labeled mixture was spun again over a low-pH glycerol cushion and depolymerized as above. After a final clarifying spin (see above), the supernatant was recovered and flash frozen in 2 μL aliquots for subsequent use. In TIRF experiments with Cy5-labeled tubulin, 100% labeled tubulin was used.Bovine brain tubulin was purified according to Castoldi and Papov19. Experiments were performed in narrow (~10 µL volume) flow cells prepared by attaching a clean #1.5 coverslip (Fisherbrand) to a glass slide (Thermo) with double-sided tape (Scotch). For all experiments, the experimental mixtures were assembled at room temperature (rather than on ice) to prevent GMPCPP-MT depolymerization. Tubulin, GMPCPP-MTs, and the indicated HSET constructs were added to a 4× self-organization buffer, which was then diluted to 1× with water to the following final concentrations: 20 mM K-PIPES, pH 6.8, 1 mM EGTA, 7.5 mM MgCl2, 5 mM ATP, 1.5 mM GTP, 50 mM KCl, 200 mM sucrose, 2 µM taxol, 250 µg/mL casein, and an oxygen scavenging mix . Tubulin and HSET were added at the concentrations indicated in figure legends. GMPCPP-MTs were prepared as follows: 20 µM bovine brain tubulin (10% Alexa594-labeled), 1 mM GMPCPP, and 1 mM DTT were mixed in 1× BRB80 and incubated on ice for 5 min, then transferred to 37 °C for 1 h. When present, 1 µL GMPCPP-MTs were used in a total volume of 20 µL experimental volume for a theoretical polymer concentration of 1 µM. In all experiments, HSET was added last to minimize premature cross-linking of MTs. The chamber was loaded with self-organization mix, sealed with VALAP, and immediately (<1 min) mounted to a preheated microscope chamber at 37 °C.Self-organization experiments were performed similar to previous studiesImaging was performed at 37 °C on an inverted DeltaVision Elite  microscope equipped with a WeatherStation environmental chamber (Applied Precision), a 20× lens (NA = 0.75) (Olympus), and a CoolSnapHQ2 CCD camera (Roper), and controlled by SoftWorx image acquisition software . Time-lapse wide-field fluorescence images were acquired using standard TRITC and FITC filters at 15 s intervals for a total duration of 30 min and the focus was fine-tuned as necessary. Contrast was defined as the standard deviation of the entire field of view measured in the tubulin channel, and was calculated using ImageJ.2) containing 1 mM ATP, 0.5 mg/mL casein, an oxygen-scavenging mix (see above), and EGFP-HSET at the concentrations indicated in the figure legends. For all other experiments studying motility behavior, the final experimental mix was assembled in BRB80 buffer containing 50 mM KCl, 1 mM ATP, 1 mM MgCl2, 0.5 mg/mL casein, an oxygen-scavenging mix (see above), and HSET/tubulin at the concentrations indicated in the figure legends. The chamber was loaded with experimental mix, sealed with VALAP, and immediately (<1 min) mounted to the TIRF microscope with objective heater (Tokai Hit) set to 35 °C.All TIRF assays were performed in narrow (~10 µL volume) flow cells prepared by attaching a clean #1.5 coverslip (Fisherbrand) to a glass slide (Thermo) with double-sided tape (Scotch). Flow chambers were processed at room temperature by infusing with biotinylated bovine serum albumin (BSA) (Thermo Fisher Pierce #29130) at 2 mg/mL in BRB80 for 10 min, washing twice in BRB80 supplemented with 0.5 mg/mL casein (BRB80/casein), infusing with NeutrAvidin (Invitrogen #31000) at 1.67 mg/mL in BRB80 for 10 min, washing twice in BRB80/casein, and blocking with 1% Pluronic F-127 (Sigma #P2443) in BRB80 for 20 min. After two additional washes in BRB80/casein, fluorescent, biotinylated GMPCPP-MTs  were introduced at a 1:40 dilution in BRB80 and incubated for 2 min to adhere to the surface, and the flow chambers were washed twice in BRB80/casein. For single-molecule diffusion experiments, the final experimental mix was assembled in P12 buffer  equipped with an H-TIRF module, Perfect Focus, 100× Apochromat total internal reflection fluorescence (TIRF) objective  with a 1.5× tube lens, EMCCD detector , a LuNA solid state laser system with four laser lines  combined into a single fiber and rapidly controlled with an acousto-optic tunable filter . Using Nikon Elements image acquisition software, images were acquired continuously with 100 ms exposures for 1 min (single-molecule diffusion experiments) or 1 s exposures for 10–15 min (processive motility experiments), and image acquisition was controlled by Nikon Elements software. For near-simultaneous two-color imaging of HSET and tubulin, a quad-band filter turret was used (C-FL TIRF Ultra Hi S/N 405/488/561/638 Quad Cube), and the AOTF was used to rapidly switch between 488 nm and 640 nm laser excitation with 100 ms exposures in each channel.53 [http://bigwww.epfl.ch/sage/soft/spottracker] was modified to batch-process motility data, and the frame-by-frame centroid position of each particle was determined as described54. Briefly, particles above a brightness threshold were detected automatically and the centroid position was determined by fit to a 2D Gaussian function to determine the frame-by-frame position with subpixel resolution. Manual detection was used when photoblinking or nearby particle(s) obstructed automatic detection. Particles persisting at least 5 frames (500 ms) were considered, and particle tracking ended when particles intersected a neighboring particle or disappeared completely (either by photobleaching or unbinding from the MT). Diffusion coefficients were calculated using the MSD analyzer MATLAB plugin55, where the first 5 s were used for MSD analysis.For single-molecule particle tracking, the SpotTracker plugin for ImageJProcessive motility properties  were analyzed by kymograph. Maximum intensity projections were generated to determine the location of MTs in the channel of interest, and kymographs were generated using the Multiple Kymograph plugin in ImageJ (width = 3 pixels). End dwell time was defined as the vertical component of the kymograph, which is the time spent at the end of the MT, in seconds. For each end dwell event in the green channel, the particle was compared to the MT location in the red channel to verify its location at the end of the MT rather than along the lattice. For a particle to be considered for analysis, it: (i) must persist at least 5 frames (5 s), (ii) could not coincide with other particles at the end of the MT, and (iii) must arrive and detach at the end of the MT during image acquisition. Run length was defined as the horizontal component of the kymograph, which is the distance traveled along the MT in µm, and included all pauses. For a particle to be considered for run length analysis, it must: (i) persist at least 5 frames (5 s), (ii) begin its run during image acquisition, and (iii) must not be bound to the MT at the end of image acquisition. Events that reached the end of the MT were counted in the analysis but treated separately (as indicated in the figure legends). Velocity was defined as the run length  divided by time  in µm/s. Only moving segments were considered, i.e., if no movement was observed in a segment at least 5 frames (5 s) long, this portion was excluded from the analysis. For a particle to be included for velocity analysis, it must: (i) persist at least 5 frames (5 s), (ii) begin its run during image acquisition, and (iii) must not be bound to the MT at the end of image acquisition. For event frequency measurements (Events/(min*µm)), time was defined as the image acquisition time (min), MT length was determined from the red channel (µm), and events were determined by kymograph analysis, defined as moving (nonstationary) particles persisting at least 5 frames (5 s).2, and 0.5 mg/mL casein. A 10 µL of triply washed anti-GFP Binding Magnetic Beads  were then added and the solution was allowed to rotate overnight at 4 °C. Beads were pelleted by a 5 min incubation a magnetic rack, washed twice in 500 µL BRB80, resuspended to 20 µL, diluted 1:1 in 2× sample buffer , and boiled. Samples were then resolved by SDS-PAGE on a polyacrylamide (10%) gel and transferred to a nitrocellulose membrane for immunoblotting. Immunoblots were analyzed by blotting with a monoclonal mouse antibody to anti-His6 at a 1:1500 dilution  and a polyclonal rabbit antibody to alpha-tubulin at a 1:5000 dilution (Abcam #18251). Inputs were loaded at 15% of the total molar amount of the immunoprecipitated material.For co-immunoprecipitation, purified components (concentrations: 50 nM for the indicated EGFP-HSET truncation and 250 nM for tubulin) were mixed to a total volume of 190 µL on ice in BRB80 buffer containing 50 mM KCl, 1 mM ATP, 1 mM GTP, 2 mM MgCl30. Samples were diluted in BRB80 supplemented with 0.5 mg/mL casein for 2 min. Flow cells were then washed twice with 50 µL BRB80 supplemented with 0.5 mg/mL casein to remove nonadsorbed fluorescent proteins, and imaged by TIRF (see above). These particles were compared to the first frame of moving fluorescent particles in TIRF-based motility assays on the same day using identical imaging and analysis parameters.To determine single-molecule fluorescence intensities for EGFP-HSET and Cy5-tubulin, 50 pM of material was introduced to an unblocked narrow flow cell (~10 µL volume) to nonspecifically adhere single molecules to the glass surface similar to previous studieshttp://www.sussex.ac.uk/gdsc/intranet/microscopy/imagej/gdsc_plugins#install), local maxima were detected automatically, and the reported Gaussian intensity values (area under the curve) were reported after local background correction. For time-lapse intensity traces showing stepwise photobleaching, background-corrected intensity values were obtained by implementing the 2D Gaussian fitting routine on the same x–y position in time. The intensity was reported as zero when the intensity fell below the required threshold for the fitting routine.To obtain first frame intensity values, a 2D Gaussian fitting routine was implemented in ImageJ  and σ :µ over the resampled datasets was taken as the standard error for each fitted quantity, and the data were reported as the mean ± the 95% CI, where the 95% CI was defined as two times the standard error.Wherever mean ± 95% CI was reported, cumulative distribution functions (CDFs) of distributions were generated in MATLAB and mean values were obtained similar to previous studiesx0 = 0.3 µm were fit to the hypothetical CDF for an exponential distribution with the free parameter t:Similar to the normal distributions above, run length CDFs above a minimum t. End dwell CDFs were fit to the same hypothetical exponential CDF without the minimum correction. Errors for these values were then determined as above. For display, Gaussian or exponential functions using the determined parameters were overlayed to the binned data in Excel.The mean run length was then determined by adding the minimum run length to g) at 4 °C, where absorbance at 280 nm was monitored. Samples were loaded into double-sector cells (path length of 1.2 cm) with charcoal-filled Epon centerpieces and sapphire windows. Sedfit (version 15.0) was used to analyze velocity scans using every scan from a total of between 250 and 300 scans57. Approximate size distributions were determined for a confidence level of p = 0.95, a resolution of n = 300, and sedimentation coefficients between 0 and 30 S. For tubulin and tubulin with EGFP-HSET, the frictional ratio was allowed to float.Purified tubulin (5 µM) with or without EGFP-HSET (200 nM) in BRB80 was analyzed in an Optima XLI ultracentrifuge  equipped with a four hole An-60 Ti rotor at 42,000 RPM  was mixed with streptavidin-coated QDots  in BRB80 and incubated overnight at 4 °C to create anti-His QDots. The following day, anti-His QDots  were mixed with the indicated HSET construct  and incubated for at least 30 min on ice to form HSET/QDot assemblies at a 3:1 ratio. For TIRF experiments where biotinylated GMPCPP-MT seeds were used, any unbound streptavidin on the QDot surface was quenched by incubating HSET/QDot assemblies  with d-Biotin (ACROS Organics #230095000) in BRB80 for at least 10 min on ice. All experiments were then performed as described above using the indicated concentration of HSET/QDot assemblies.For experiments using QDots, HSET/QDot assemblies were constructed as follows. A biotinylated mouse monoclonal anti-His30) were transfected in six-well plates with 1 µg/µL of pEM791-EGFP-HSET. One day after transfection, cells were cultured in the presence of 1 µg/mL puromycin for 48 h and then incubated in media containing 2 µg/mL puromycin until puromycin-sensitive cells were eliminated. Puromycin-resistant cells were then expanded in media containing 1 µg/mL puromycin, pooled, and tested negative for mycoplasma contamination. Cells were cultured in DMEM containing 10% FBS, 1 µg/mL puromycin, penicillin/streptomycin, and either 2 µg/mL doxycycline (to induce EGFP-HSET expression) or DMSO for 1–3 d.To generate HeLa-Kyoto cells (Naoki Watanabe) that express EGFP-HSET in a doxycycline-inducible manner, HeLa acceptor cells  freshly supplemented with 1 mM ATP and protease inhibitors . After the lysate was clarified by centrifugation at 16,000×g at 4 °C, samples were loaded directly onto a 10% polyacrylamide gel at 1/2 well per sample. Samples were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblotting.Doxycycline/DMSO-treated cells were cultured in six-well plates until reaching ~80% confluency. Cells were then trypsinized and harvested by low-speed centrifugation at 1500×For immunoblotting, nitrocellulose membranes were blocked in 5% milk diluted in Tris-buffered saline plus 0.1% Triton X-100 (TBST). Rinses were performed in TBST. Primary antibodies were diluted in 5% milk/TBST and probed for 1 h. The following primary antibodies were used for immunoblotting: mouse anti-tubulin , 1:2000, rabbit anti-GFP (Invitrogen #A11122), 1:1000, and rabbit anti-HSET , 1:2000. Species-specific secondary antibodies conjugated to Alexa Fluor 700/800 (Invitrogen) were used at 1:5000 for 45 min. Bound antibodies were detected using an Odyssey fluorescence detection system (Mandel Scientific). Protein levels were quantified with Fiji using tubulin as a loading control.58), 1:1500. DNA was stained with 5 µg/mL Hoechst 33342. Coverslips were mounted in Prolong Gold for imaging.Doxycycline/DMSO-treated cells were cultured in six-well plates until reaching ~80% confluency, then treated with 500 nM nocodazole  or DMSO for 15 or 30 min. Immediately following nocodazole treatment, cells were fixed with methanol at −20 °C for 10 min. Cells were rehydrated with 3 × 5 min washes with TBST. Coverslips were blocked with AbDil (TBST + 2% BSA [Sigma]) for 10 min, probed with primary antibodies diluted in AbdDil for 1 h, rinsed, probed with secondary antibodies diluted in AbDil for 1 h, and rinsed. The following primary antibodies were used for immunostaining: mouse anti-tubulin , 1:500, rabbit anticentrin  were acquired at 37 °C on an inverted DeltaVision Elite  microscope equipped with a WeatherStation environmental chamber (Applied Precision), a 60× objective (NA = 1.4) (Olympus), and a CoolSnapHQ2 CCD camera (Roper). Z-sections spaced at 200 nm apart were acquired and deconvolved with SoftWorx . Images were subsequently processed with Fiji .To determine the fraction of mitotic cells that had ≥1 acentrosomal aster after nocodazole treatment, metaphase cells were selected for imaging and analysis using the blue channel (DNA). For every cell, each discrete mass of tubulin was manually evaluated for colocalization with centrin using deconvolved Z sections of tubulin and centrin. An acentrosomal aster was classified as a discrete polymer of tubulin lacking a centrin marker.d-lysine coated dishes (MatTek) 3 d before imaging. Cells were imaged at 37 °C (5% CO2) in L-15 medium without phenol red supplemented with 10% FBS and 7 mM HEPES, pH 7.7, using the aforementioned DeltaVision Elite system. Metaphase cells were identified in the EGFP-HSET channel, whereupon 500 nM nocodazole was added to partially depolymerize MTs. Immediately (~10 s) after nocodazole addition, Z-sections spaced at 200 nM apart were acquired in the EGFP-HSET channel, and Z-stacks were acquired at 3 min intervals for 30 min.For live-cell imaging of aster formation, doxycycline/DMSO was added to EGFP-HSET cells as they were plated onto glass bottom poly-Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9


Cardiovascular disease is the major cause of death in patients with diabetes. Current treatment strategies for diabetes rely on lifestyle changes and glucose control to prevent angiopathy and organ failure. Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is used as an add-on therapy to insulin treatment. Exenatide also has multiple beneficial effects in addition to its hypoglycemic effects, such as preventing hepatic steatosis and protecting against cardiac injury from doxorubicin-induced cardiotoxicity or ischemic reperfusion. However, the mechanisms underlying the cardioprotective effects of exenatide in diabetes have not been fully clarified. To address this issue, we investigated the cardioprotective effects of exenatide in type 1 and type 2 diabetic mice. We found that exenatide simultaneously attenuated reactive oxidative species (ROS) production through increases in the antioxidant enzymes manganese dependent superoxide dismutase (MnSOD) and catalase. Moreover, exenatide decreased tumor protein P53 (p53) expression and prevented cell apoptosis in H9c2 cells. The presence of the catalase inhibitor 3-AT attenuated the effects of exenatide. Overall, the results strongly indicate that exenatide treatment may be protective against the development of diabetic cardiomyopathy. Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. Cardiovascular disease is the major cause of death in diabetic patients. Patients with diabetes are at least 10 times more likely to suffer from heart dysfunction than their non-diabetic counterparts . CurrentGLP-1 is an insulinotropic hormone released from intestinal L cells in response to nutrient ingestion, and it modulates glucose metabolism by affecting pancreatic islet secretions. However, it can be rapidly inactivated by the enzyme dipeptidyl peptidase IV in the circulation. Exenatide is a kind of GLP-1 analog that can evade rapid clearance by dipeptidyl peptidase IV; thus, it has a long half-life when injected subcutaneously. Exenatide was approved for type 2 diabetic treatment in 2005. Previous studies have shown that in addition to the hypoglycemic effect, exenatide reduced weight gain and alleviated hepatic steatosis in diabetic mice , 5. In aIn vivo and in vitro studies were performed to assess cardiac function, glucose tolerance, antioxidative stress capacity and cell apoptosis. The effects of exenatide on ROS production and apoptosis were evaluated in H9c2 cardiomyoblasts. Furthermore, the underlying mechanism of exenatide-induced protective effects was investigated. Our study provides a new mechanism underlying the cardioprotective actions of exenatide.The pathological mechanisms underlying diabetic cardiomyopathy, including inflammation, mitochondria dysfunction, lipotoxicity, and oxidative stress, are complex and multifactorial. The molecular mechanisms underlying the cardioprotective effects of exenatide remain unclear. It has been reported that exenatide prevents cardiomyocyte apoptosis by enhancing autophagy and reducing lipotoxic ceramide accumulation , 13, as Six-week-old male C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology. The mice were housed in an environmentally controlled breeding room . All mice had free access to tap water. All procedures were approved by the Ethics Committee for the Use of Experimental Animals of Qingdao University.n = 8) or a high-fat diet . After a 20-week dietary intervention, the HFD group was then randomly subdivided to receive either exenatide  or normal saline as a control  by intraperitoneal injection during the light cycle while continuing the HFD feeding for 4 weeks.For the high-fat diet-induced type 2 diabetic mouse study, mice were randomly distributed into two initial groups fed a control diet  or normal saline as a control  by intraperitoneal injection during the light cycle while continuing feeding with the chow diet for 4 weeks.Type 1 diabetes was induced by treating the mice with a high dose of streptozotocin (STZ)  as previously described . Brieflyn = 8 in each group) was measured only in type 2 diabetic mice. Briefly, the IPGTT was conducted after an overnight fast (12–16 h). Mice were injected with 40% glucose . Blood glucose was measured from the tail tip using a glucose meter  at 0, 30, 60, 90, and 120 min.After 24 weeks, body weights were measured in all groups. However, the glucose tolerance test (n = 8 in each group) was measured using a commercial ELISA kit . Homeostasis model assessment (HOMA-IR) was calculated by fasting plasma glucose  × fasting plasma insulin /22.5.Plasma insulin . Transthoracic two-dimensional M-mode echocardiography and pulsed wave Doppler spectral tracings were obtained using a Vevo 2100 Imaging System  with a 30-MHz transducer for each mouse. The percentages of ejection fraction (EF%), left ventricular end-systolic diameter (LVSD), and left ventricular end-diastolic diameter (LVDD) were measured using M-mode tracings. The percentage of fractional shortening (FS%) was calculated according to the following formula [(LVDD-LVSD)/LVDD] × 100%. The ratio of early ventricular filling peak velocity (E wave) and late filling velocity (A wave) (E/A) were measured by pulsed wave Doppler spectral tracings to determine the diastolic function.H9c2 cardiomyoblasts were purchased from Cobioer . H9c2 cells were maintained in high-glucose Dulbecco's modified Eagle's medium  with 10% fetal bovine serum (FBS), 2% L-glutamine, 10% sodium bicarbonate, 10% sodium pyruvate, 5% HEPES, 1% penicillin/streptomycin, and 1% gentamycin in an incubator . For high-glucose treatment, H9c2 cells were incubated for 24 h in high-glucose medium (40 mM) with or without exenatide present .The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was carried out according to the manufacturer's protocol with a one-step TUNEL apoptosis assay kit . The results were scored semiquantitatively by averaging the numbers of TUNEL-positive cells per low-power field for five fields in each experiment.H9c2 cells in the presence of control medium or high-glucose medium (40 mM) were treated with or without exenatide (25 or 50 nM) in the dark with 10 μM DCFH-DA  for 20 min. To observe DCF fluorescence during illumination, cells were mounted on a microscope slide and illuminated and visualized with a 20 × objective on a Nikon-TI SR microscope. The number of positive cells was counted in at least three random pictures in each group.To further quantify ROS production, H9c2 cells were seeded in 96-well plates in the presence of high glucose and treated with or without exenatide (25 or 50 nM). After 10 μM DCFH-DA labeling, the fluorescence intensity of the cells was recorded by a fluorescence microplate reader at 488/525 nm.Hearts from each group of animals were homogenized, and protein samples were prepared with ice-cold RIPA lysis buffer. Protein concentrations were measured using the Bradford assay (Bio-Rad protein assay kit). For western blotting detection of p53, catalase, MnSOD, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression, 80 μg of protein was used. All proteins were separated by 12% SDS polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes (PVDF). Membranes were blocked with 5% (w/v) skim milk for 2 h at room temperature and then incubated with primary antibodies with gentle agitation overnight at 4°C. The membranes were washed 3 times for 10 min each with 15 ml of TBST  Tween-20 and then incubated with secondary antibody at room temperature for 2 h.in vitro western blotting experiments, upon completion of the experiments, H9c2 cells were harvested using ice-cold RIPA lysis buffer. Protein concentrations were measured using the Bradford assay (Bio-Rad protein assay kit). For western blotting detection of P53, catalase and MnSOD protein expression, the method used is the same as that described above.For in vivo and in vitro experiments were visualized with enhanced chemiluminescence solution and images generated with a GENE Imaging system. The images were quantified using Image Analysis Software (Quantity One). Primary antibodies for p53 , catalase , and MnSOD  were purchased from Proteintech . GAPDH and HRP-conjugated secondary antibodies were purchased from Yesen Biotechnology .Proteins for t-test was used to determine significant differences between groups. For comparison of more than two groups, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was performed. Adjusted two-sided P-values were calculated, and P < 0.05 was considered indicative of statistical significance.Data are presented as the mean ± SEM. An unpaired two-tailed Student's The mean body weights of the CON, T2DM, T2DM+EXE and T1DM, T1DM+EXE animals were measured. The body weight of T2DM mice was increased compared to that of the CON mice, and treatment of T2DM animals with exenatide (24 nmol/kg/d) decreased body weight significantly. T1DM mice had significantly reduced body weight compared to the CON mice, and treatment of T1DM animals with exenatide (24 nmol/kg/d) slightly increased body weight, although the difference was not significant . In addiTo assess whether exenatide protects against the development of diabetic cardiomyopathy in diabetic mice, echocardiography was performed on CON, T2DM, T2DM+EXE and T1DM, T1DM+EXE animals one week prior to sacrifice. EF%, LVSD, and LVDD were used to determine systolic function, and FS% and E/A were used to determine diastolic function. Systolic and diastolic dysfunction developed in T2DM and T1DM animals in comparison to CON mice, and exenatide treatment of T2DM animals attenuated this dysfunction . LVSD waIt was previously demonstrated that exenatide treatment attenuated myocardial infarction-induced cardiac injury through reduced ROS production . MnSOD aP53 is known to initiate apoptosis upon DNA damage. We examined cardiac p53 levels in CON, T2DM, T2DM+EXE, T1DM, and T1DM+EXE animals. The level of p53 protein was significantly increased in T2DM and T1DM animals compared to CON mice. Exenatide-treated T2DM and T1DM animals exhibited significantly decreased p53 levels compared to diabetic animals . Thus, ein vitro , and cytotoxicity was examined. Incubation of cells with 0–50 nM exenatide was not cytotoxic .P < 0.05). Exenatide at concentrations of 25 and 50 nM for 24 h decreased the ROS level  in the presence of high glucose (35 mM) in H9c2 cells. ROS levels were significantly increased by high-glucose stimulation compared with those in the control group (OS level . The resOS level . Rosup wP < 0.05). Exenatide treatment at concentrations of 25 and 50 nM for 24 h increased the MnSOD (P < 0.05) and catalase levels (P < 0.05)  treatment in the presence of high glucose (35 mM). MnSOD and catalase levels in the high-glucose group were significantly decreased compared with those in the control group ( < 0.05) . These rWe next examined cell apoptosis in high glucose-treated cells. H9c2 cells were incubated in the absence or presence of 35 mM glucose in combination with exenatide or the catalase inhibitor 3-AT or both. H9c2 cells incubated with 35 mM glucose exhibited in a significant increase in cell apoptosis compared to the control cells . TreatmeSince high glucose-induced cell apoptosis results from an increase in ROS, we examined whether inhibiting antioxidant capacity could reverse this process. H9c2 cells were incubated in the absence or presence of 35 mM glucose in combination with exenatide or the catalase inhibitor 3-AT or both. As shown in In the present study, we found that exenatide protected cardiac function in both type 1 and type 2 diabetic mice. In addition, exenatide simultaneously attenuated ROS production through increased antioxidant enzyme levels, which were observed both in mouse hearts and H9c2 cardiomyocytes. Moreover, inhibition of catalase by a catalase inhibitor (3-AT) diminished exenatide's protective effect on cell survival. These observations suggested that regulation of ROS production may be involved in the exenatide-mediated improvement of cardiac function in diabetic mouse hearts.The T1DM and T2DM mouse models we used in the current study are two well-developed diabetic mouse models and have been used extensively in studies related to diabetes –25. T1DMThe doses of exenatide we chose in this study are based on previous studies. In humans, clinical administration of exenatide (10 μg) twice daily or exenatide (2 mg) once weekly as an add-on therapy to type 2 diabetic patients was associated with greater improvements in several cardiovascular risk markers , 31–33. 2) to superoxide (O2O2), which is then reduced to H2O by catalase or glutathione peroxidase. In this case, the intracellular antioxidative ability to increase ROS levels is largely determined by the expression of the enzymes SOD, catalase, and glutathione peroxidase (GSH-Px). It has been reported that exenatide treatment inhibits ROS production and attenuates fibrotic islet destruction in apoptotic pancreatic beta cells induced by fatty acids (Exenatide was reported to protect cardiac function by anti-inflammation or autophagy upregulation . Additioty acids . In our P53 is closely related to cardiac cell apoptosis. Activation of p53 reduces the expression of genes opposing cell death, such as Bcl-2, and upregulates genes promoting apoptosis, such as Bax, therefore promoting cell apoptosis . In diabIn summary, our current findings demonstrate that exenatide treatment improves cardiac dysfunction in diabetic mice and attenuates apoptosis in high glucose-induced H9c2 cardiomyoblasts. The mechanisms underlying these beneficial effects are a reduction in ROS production via increased activation of MnSOD and catalase and reduced P53 activation. We hypothesize that exenatide may be a candidate drug for the treatment of diabetic-related cardiovascular disease.This study was carried out in accordance with the recommendations of the tips in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee, China. The protocol was approved by the Ethics Committee for the Use of Experimental Animals of Qingdao University.JW and XZ designed the study. WD, WC, and JW drafted and wrote the manuscript. WD, WC, DX, XG, YL, and DD performed the experiments and statistical analyses. All authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.