Little is known about the human lung lipidome, its variability in different physiological states, its alterations during carcinogenesis and the development of pulmonary emphysema. We investigated how health status might be mirrored in the lung lipidome. Tissues were sampled for both lipidomic and histological analysis. Using a screening approach, we characterised lipidomes of lung cancer tissues and corresponding tumour-free alveolar tissues. We quantified 311 lipids from 11 classes in 43 tissue samples from 26 patients. Tumour tissues exhibited elevated levels of triacylglycerols and cholesteryl esters, as well as a significantly lower abundance of phosphatidylglycerols, which are typical lung surfactant components. Adenocarcinomas and squamous cell carcinomas were distinguished with high specificity based on lipid panels. Lipidomes of tumour biopsy samples showed clear changes depending on their histology and, in particular, their proportion of active tumour cells and stroma. Partial least squares regression showed correlations between lipid profiles of tumour-free alveolar tissues and the degree of emphysema, inflammation status, and the age of patients. Unsaturated long-chain phosphatidylserines and phosphatidylinositols showed a positive correlation with a worsened emphysema status and ageing. This work provides a resource for the human lung lipidome and a systematic data analysis strategy to link clinical characteristics and histology. It comprises a complex mixture of mainly phospholipids and surfactant proteins. The main components are saturated phosphatidylcholine (PC) species such as PC 16:0/16:0, PC 16:0/14:0 and PC 16:0/16:12. Phosphatidylglycerol (PG) is, with 7 to 15% of total phospholipids3, the second most abundant surfactant lipid class and plays a crucial role as immune regulator5.The anatomical organisation of the human lung enables the exchange of gases between blood and air. Alveoli represent the basic units for gas exchange within the lung. Pulmonary surfactant, which lines the inner surface of the lung, prevents alveolar collapse at the end of expirationDrosophila melanogaster6 and Mus musculus7. A recent study showed that widespread alterations of phospholipid profiles occurred in lung cancer tissues8. These alterations especially affect pulmonary surfactant components, but also sphingomyelin (SM)8. Further investigation by mass spectrometric imaging showed that lipidomes of cancer cells comprise specific alterations within surfactant lipids.The functional connection between the lipidome and tissues, as well as cell types, has been investigated by using biological model organisms such as 10. In addition to the abundant major phospholipid classes PC, phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and SM, we included the neutral lipids diacylglycerol (DAG), triacylglycerol (TAG) and cholesteryl ester (CE). We also included the sphingolipids ceramides (Cer) and hexosylceramides and the glycerophospholipids including PG and phosphatidic acid (PA). Clinical characteristics such as age, gender, body mass index (BMI), history of smoking, and quantitative scores for histological phenotypes were also assessed. Using this information, we systematically evaluated the multivariate statistical methods suitable for relating lipidomics data with individual clinical and histological characteristics. Omics data were explored by hierarchical clustering, principal component analysis (PCA) and partial least squares (PLS) regression14.In this study, we analysed tissues from non-small cell lung cancer (NSCLC) and corresponding tumour-free alveolar control tissues by applying shotgun lipidomics screens15. Therefore, it is possible to evaluate several data interpretation strategies in terms of the physiological impact of a phenotype. From this perspective, we expected to find the most prominent alterations in the comparison between lipidomes of tumour and tumour-free control tissue, because of the profound metabolic reprogramming of cancer cells. On the other hand, we expected the influence of ageing on the lipidomes of alveolar tissues to be much more elusive. Hence, we followed this logic to test the application range of certain data analysis strategies. These range from objectively large histopathological changes that affect the composition of the lipidome itself, to subtly nuanced alterations in clinical parameters such as age, BMI, and gender. This study enabled us to compile a resource for the human lung lipidome that shows how histological diversity and clinical data are reflected in distinct molecular features.It is known that cells undergo a significant reprogramming of their metabolic networks during carcinogenesis, which is mirrored in systematic lipidome changes17. We devised sample preparation and extraction procedures that had small sample size requirements and could be performed in a typical laboratory. Mass spectrometric conditions for negative ionisation were optimised to improve sensitivity for negatively charged lipids such as PI, PG, PA and PS  and ageing (Q2 = 0.14) on the lipidome was significant  and GOLD stage—no further trends were observed . All procedures were carried out in accordance with respective guidelines and regulations. The clinical parameters of the 26 lung cancer patients are summarised in Table 36.When available, representative biopsy samples of tumour-containing and tumour-free lung parenchyma were collected in the range of 0.5 to 1 g wet-weight. All samples were divided into two approximately equal-sized parts for processing for histopathology analyses and shotgun lipidomics. For shotgun lipidomics, samples were shock-frozen in liquid nitrogen and stored at −80 °C until analysis. For histological characterisation, tissue samples were fixed using the HOPE technique (HEPES-glutamic acid buffer-mediated organic solvent protection effect)et al.37 in grades between 0 (no emphysema) and 10 . Tumour-containing tissues were characterised by their relative content of vital tumour, stroma and necrosis, defined as tissue fraction given in percentage. Additionally, inflammation was scored in stages between 0 and 3. Tissue scans were recorded with a Plustek OpticsLab H850 scanner and the pictures can be found in Supplement Tissue sections of 1 µm were prepared from paraffin-embedded blocks and stained with haematoxylin and eosin. Slices were characterised by light-microscopy. Phenotypic features were scored according to the specific tissue type. For tumour-free tissues, emphysema, inflammation, alveolar macrophages and fibrosis were scored on a scale from 0 to 3. A score of 0 would represent no inflammation/fibrosis/macrophages and a score of 3 would indicate a high degree of inflammation/fibrosis/macrophages. The emphysema grade was determined, according to the method by Nagai 38 . A constant ratio between tissue wet-weight and buffer volume of 1:20 (wt/vol) was applied for all samples. Accordingly, 300 μL tissue homogenate, containing 14.4 mg tissue, was extracted using the MTBE-based lipid extraction procedure10 were performed using an Apex Qe Fourier Transform Ion Cyclotron Resonance mass spectrometer  equipped with a TriVersa NanoMate  as autosampler and ion source. Mass spectrometric acquisitions of 5 minutes’ time were separately recorded for the positive and negative ion mode  using 3.7 mM ammonium acetate as additive for positive ion mode and 0.05 mM ammonium chloride for negative ion mode. Lipidomics screens39, using deuterated cholesterol as internal standard , PS, lysophosphatidylglycerol (LPG), PG, lysophosphatidylethanolamine (LPE), PE, PE-O, PA, lysophosphatidylcholine (LPC), PC, PC-O, lysophosphatidylinositol (LPI), PI, cardiolipin, CE, Cer and hexosylceramide—using peak lists as input . Hierarchical clustering was computed applying Euclidean distance and complete linkage using Gene Cluster 3.043 and visualised by Java Treeview-1.1.6r444 To determine the lipid panel for differentiation of alveolar control tissues and tumour tissues, only lipid species that were detected in all 43 samples were included. As a result, 20 lipids were chosen that were significantly changed between tumour and alveolar control tissues with p > 0.01 (two-tailed t-test) and comprised a log2-fold change of <−1 or >1 (Supplement (ii)For discriminating ADC and SCC tissues, a lipid panel was chosen from lipids present in each tumour tissue sample. A panel of 7 lipid species was chosen with p < 0.05 (two-tailed t-test), which showed a log2-fold change of <−1 or >1 between the two tumour types .et al. and Geladi and Kowalski14. PLS regression was calculated using the R package plsdepot45 using the function plsreg2. Prior to PLS regression, clinical data and histopathology scores  and all lipid quantities  were transformed to consist only of values between 0 and 1. Categorical variables, like gender or tumour diagnosis, were also scaled in a range of 0 to 1.To calculate a mathematical model that correlates histological and clinical data to the lipidome, we used PLS regression, as described by Wold For the PLS regression model of tumour tissues presented in Fig. The PLS regression model of alveolar lipidomes shown in Fig. leave-out-one algorithm of the R function plsreg2 of the plsdepot R package13. Briefly, the dataset is split randomly into ten equally sized segments consisting of individual patients’ data. Nine of these segments are used as training set to compute the model, while the remaining segment is used for validation. This process is repeated so that all ten segments are once utilised for validation. The results of the cross-validation are expressed by the Q2 value. A model for a response variable is considered to be significant when at least one Q2 value for one principal component is ≥0.097545.Cross-validation was performed using the default 18. The source code to calculate LUX scores can be downloaded from the website http://lux.fz-borstel.de. The overall fatty acid composition of the lung lipidome was determined by tandem mass spectrometry, performed in negative ion mode on a Q Exactive Plus Orbitrap mass spectrometer . We used the resulting list of 35 fatty acids to infer isomeric species for all identified lipids and to choose a representative molecule  and standard deviation (SD) of (1) t = 0.005, SD = 0.002 and (2) t = 0.003, SD = 0.001.Chemical space model and LUX scores were calculated as described previouslySupplement 1Supplement 2Supplement 3Supplement 4Supplement 5Supplement 6Supplement 7Supplement 8Supplement 9Supplement 10Supplement 11


Functional dyspepsia (FD) is considered a bio-psychosocial disorder. The role of psychosocial factors in FD pathogenesis remains unclear.This study evaluated sleep quality and mood symptoms in patients with FD, assessing the associations of FD severity, disordered sleep, and psychological symptoms. One-hundred-and-fifteen adult patients with typical FD symptoms were enrolled alongside 61 healthy volunteers. Rome III criteria were used to evaluate FD symptoms; sleep disorder was assessed with the Pittsburgh Sleep Quality Index (PSQI), and Symptom Checklist-90-Revised (SCL-90R) was utilized to determine the status of depression, anxiety and other psychological symptoms.PSQI scores and nine symptomatic dimensions of SCL-90R were significantly higher in FD patients than in controls. Multiple logistic regression indicated that lower BMI, lower level of education, and sleep disturbance were independently associated with FD and FD subgroups. Hostility and phobic anxiety were independent risk factors for FD. Further analysis showed that hostility was an independent risk factor for both FD subgroups, and somatization and additional psychiatric symptoms for epigastric pain syndrome.We found that FD was associated with sleep disorder and psychopathological factors. These findings suggest that implementing sleeping and/or psychological therapies may help reduce FD symptoms. Helicobacter pylori infection, heightened visceral sensitivity, central nervous system disturbance, and unhealthy lifestyle [Helicobacter pylori infection prevalence, the symptoms, epidemiology, pathogenetic factors, and management differ between Asian and Western FD patients [Functional dyspepsia (FD), one of the most common functional gastrointestinal disorders (FGIDs), is defined as pain and/or discomfort centered in the upper abdomen in the absence of obvious organic abnormalities . The Romifestyle . Due to patients .Mounting evidence suggests that psychological distress is associated with FD; however, very few studies have assessed psychological factors that contribute to FD development. A Swedish 10-year-follow-up study of FD patients applying the Rome III definition showed an association of anxiety but not depression with FD ; meanwhiBesides psychological factors, sleep disorder is a common phenomenon in FD patients and may influence the severity of dyspepsia symptoms . In addiIn the present study, we used an integrated questionnaire consisting of the major dyspepsia symptom scale, Symptom Checklist-90-Revised (SCL-90R), and Pittsburgh Sleep Quality Index (PSQI) to assess FD severity, psychological health and sleeping condition in patients meeting Rome III criteria for FD. The study aimed to explore the associations of psychological distress or sleep disturbance with FD and FD subgroups based on Rome III criteria.Adult outpatients from the digestive and psychological departments of First Affiliated Hospital of Nanjing Medical University meeting ROME III criteria for FD between July 2015 and May 2016 were recruited for this study. The included patients have undergone an upper endoscopic test to exclude organic digestive diseases that might have explained the observed symptoms. Local adult volunteers without known gastrointestinal or psychological disorders, who underwent gastric endoscopy as regular medical examination, were recruited as healthy controls. All patients and controls underwent detailed history taking. All subjects with the following conditions were excluded: (a) a history of upper gastrointestinal tract surgery, (b) a history of ulcerative disease or malignancy, (c) current treatment with non-steroidal anti-inflammatory drugs (NSAIDs), (d) use of monoamine oxidase inhibitors or selective serotonin reuptake inhibitors, (e) pregnancy for women, (f) pancreatitis or hepatobiliary disorders, and (g) physical or psychological impairments that prevented from completing the questionnaires. Patients with concomitant irritable bowel syndrome (IBS) symptoms but not meeting the ROME III criteria for IBS were not excluded.FD was defined as the presence of postprandial fullness/early satiation or pain/burning more than once per week, in the last 3 months according to the Rome III criteria. To diagnose FD and evaluate the severity of dyspepsia related symptoms, the original Rome III functional dyspepsia module  was tranA Chinese version of the PSQI questionnaire was used to assess the patient’s recent history of sleep quality. The PSQI questionnaire consisted of 19 self-rating items that can be categorized into seven components, including subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbance, use of sleep medication, and daytime dysfunction. The score of each component score was rated from 0 to 3. The sum of these seven component scores provided a global PSQI score, which ranged from 0 to 21. Higher scores indicate poorer sleep. The Chinese Version of PSQI was translated and validated by Liu et al. . In thatPsychiatric distress was estimated using the Chinese version of SCL-90R . The SCLt test and one way analysis of variance (ANOVA) were used to statistically assess demographic variables such as weight, height, and BMI. Various sleep disorder parameters and psychological factors between FD patients and healthy controls were also assessed by Student t test. The association of total PSQI score and FD symptom severity score was assessed by Spearman correlation analysis. Risk factors for dyspepsia were initially explored by univariate analysis. Those with P < 0.05 were then entered into a multivariate analysis. Independent risk factors for dyspepsia were subsequently determined by backward elimination logistic regression. Data were expressed as odds ratio (OR) with 95% confidence interval (95% CI). P < 0.05 was considered statistically significant.Data were assessed with Statistical Package for the Social Sciences (SPSS) for Windows, version 13.0. Measurement data are mean ± standard deviation (SD). Student Of the 263 subjects eligible for inclusion, a total of 224 (85.1%) individuals agreed to participate in the study. However, 109 individuals did not complete the questionnaires because of low education level or desire to withdraw. Therefore, a total of 115 FD outpatients completed questionnaires, yielding a response rate of 43.7%. Meanwhile, 61 healthy controls were enrolled. The mean age of the control population was 41.36 ± 12.32 years; 80.3% of control individuals were female.Table Mean duration of FD symptoms was 52 ± 50 months, and the patients reported that FD symptoms were present 4.1 ± 2.3 days per week. Among the 115 FD participants, 87 (75.7%) had symptoms consistent with EPS  and 54 (47.0%) conformed to PDS (including 28 with pure PDS symptoms); 26 patients (22.6%) reported dyspeptic symptoms compatible with both EPS and PDS.P < 0.05). Occasional sleep problems were common in both FD and control groups; indeed, 67.2 and 58.6% of FD patients and control participants, respectively, reported having sleep abnormality 1 to 3 days a month. Significantly higher PSQI scores were found in FD patients compared with controls . In addition, as shown in Table Average sleep durations in FD patients and healthy controls were 6.24 ± 1.37 and 6.87 ± 1.05 h, respectively . A total of 61 FD patients had a score above 11, with 5 having values > 15, which reflects severe sleep disturbance in all domains. Moreover, there was a consistent, stepwise increase of FD symptom scores with increasing sleep scores  and phobic anxiety  were independent risk factors for dyspepsia. However, no significant associations of symptomatic dimension scores with the FD symptom score were found.To further identify which psychological factors were independent risk factors for FD, a multivariate logistic regression model was employed Table . HostiliP < 0.05), lower education levels , and higher PSQI score  were also independent risk factors for FD  sleep and slow wave sleep (SWS) interruption could decrease mechanical pain thresholds in healthy volunteers .The role of adverse psychological factors in the development of functional gastrointestinal disorders (FGIDs) attracts increasing attention . In a reWith respect to clinical application, whether treating FD patients with sleep or psychotherapeutic medications improves dyspeptic symptoms remains unclear. Related data are limited, as only few randomized controlled trials have been conducted for FD psychotherapy , 22. A sThis is a cross-disciplinary research spanning gastroenterology and psychology. We focused on evaluating sleep quality and mood symptoms in Chinese patients with FD, with a view to compare with other racial population in future. It is helpful for clinicians to pay more attention to the sleep disturbance and psychological factors of FD patients and to provide more effective treatments for them.The present study had several limitations. For example, no physiological measurements were used in this study, although the diagnosis of sleep disturbance is still largely dependent on patient’s subjective feelings and symptoms.In conclusion, this study demonstrated that sleep disturbance and negative emotions were common in patients with FD. Impaired sleep was an independent risk factor for FD and both FD subgroups, and may play a role in symptom expression. Hostility and phobic anxiety were independently associated with FD development. We further demonstrated that hostility was an independent risk factor for both FD subgroups, while somatization and additional psychiatric symptoms predicted EPS. Further exploration of the mechanisms linking sleep disturbance and negative emotions with FD may yield new approaches to interventions that reduce FD symptoms.


Qingyihuaji formula (QYHJ), confirmed efficacious in a series of clinical trials, has been applied to human pancreatic carcinoma treatment in Shanghai Cancer Center for years. Recent evidence highlighted that pluripotent stem cells transcription factor Nanog plays a pivotal role in carcinogenesis. However, there is little published information regarding the underlying clinical significance and mechanisms of transcription factor Nanog in pancreatic cancer. In this study, our results indicated that Nanog is overexpressed in human pancreatic cancer stem cells and downregulated by QYHJ, which may contribute to explain the clinical effectiveness of QYHJ and provide advanced pancreatic cancer patients with a new therapeutic option, supporting our hypothesis that the degradation pathway is another mechanism by which QYHJ affects Nanog expression. Pancreatic cancer is a devastating neoplasm and ranks the fourth among the most common causes of cancer deaths worldwide . AlthougQingyihuaji formula (QYHJ), composed of traditional Chinese herbs, has been applied to human pancreatic cancer treatment for many years. Our clinical study indicates that the treatment with QYHJ combined with chemotherapy has prolonged survival time of advanced pancreatic cancer patients , 6. Howe in vitro and in vivo. Since aberrant Nanog expression occurs in tumorigenesis, therapeutics targeted Nanog may improve the outcomes of patients with pancreatic cancer, thus suggesting the use of QYHJ to further improve preventive and therapeutic approaches in patients with this devastating disease.The transcription factor Nanog controls stemness acting as a key determinant of both embryonic stem cells self-renewal and differentiated somatic cells reprogramming to pluripotency. Nanog is a member of ANTP class NK family genes and plays a key role in stem cell self-renewal and pluripotency differentiation . In addiThus, the aim of the study is to evaluate Nanog expression in human pancreatic cancer tissue and the effects of QYHJ on transcription factor Nanog in human pancreatic cancer stem cell.Pancreatic cancer cell line PANC-1 was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences  and grown in complete growth medium as recommended by the manufacturer. CD24+/CD44+/ESA+ subpopulation cells were isolated from the pancreatic cancer cell lines PANC-1 as the previous description . The cul Amorphophallus konjac, herba scutellariae barbatae, Coix seed, akebia stem, fiveleaf gynostemma herb, and java amomum fruit), was prepared as previously described [Db = Da × (Rb/Ra)×(Wb/Wa)2/3, where D, R, and W represent dosage, shape coefficient, and body weight, respectively, and a and b represent human and mouse, respectively. Gemcitabine (Eli Lilly) was dissolved in sterile PBS, 25 mg/kg [μm) was purchased from Corning Incorporated.QYHJ formula, comprised of seven Chinese herb medicines .A total of 5 × 10For cell cycle analysis of cells, cells growing at low density (40%) in normal growth medium were treated with QYHJ (32 mg/mL) or vehicle. After 24 h, cells were fixed with 1% paraformaldehyde in PBS  for 15 minutes and refixed with 70% ethanol. The cells were then treated following the standardized protocol, and cell cycle analyses were done by flow cytometry as described previously .μL free-serum culture medium. Culture medium containing 10% FBS (600 μL) was added in the lower chamber as a chemoattractant. After incubation for 24 h at 37°C, all of the noninvaded cells were removed from the upper face of the membrane with a cotton swab. The invaded cells on the lower face of membrane were then fixed for 10 min with 4% paraformaldehyde  and stained with 0.2% crystal violet for 15 min. Cells that had invaded through the membrane were counted using the inverted microscope. The general procedure of the migration assay was similar to that of the invasion assay, with the exception that the membranes in each chamber were not coated with Matrigel and the number of cells seeded in the upper chamber was 5 × 104 cells/well.Transwell chambers were coated with Matrigel for 3 h to form a basement membrane prior to use. Following treatment with QYHJ (32 mg/mL) for 24 h, the CD24+/CD44+/ESA+ PCSCs were harvested and seeded in the upper chamber at a density of 30,000 cells/well in 200 For colony formation, CD24+/CD44+/ESA+ PCSCs were seeded (500 cells/well) in six-well plates overnight. After 14 days, the culture medium was removed, and cells were briefly rinsed with PBS. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, and colonies were counted by visual inspection.μL reaction mixture containing SYBR Green reaction mix  and 0.5 mM of primer and analyzed using a Roche Light-Cycler . The relative gene expression in cells was determined using the comparative delta-delta CT method (2−ΔΔCt) and the fold change in gene expression of tissues was calculated using the standard ΔΔCT method. The primer sequences of Nanog are sense: 5′-GCAATGGTGTGACGCAGAAGGC-3′, antisense: 5′-TGGGTCTGGTTGCTCCAGGTTG-3′; GAPDH sense: 5′-CAAGGTCATCCATGACAACTTTG-3′, antisense: 5′-GTCCACCACCCTGTTGCTGTAG-3′.Total RNA was extracted from frozen plasma or cultured cell lines using TRIzol reagent  according to the manufacturer's instructions. With GAPDH as an internal control, qRT-PCR was performed for Nanog. Total RNA was then converted to cDNA by reverse transcription using oligo-dT primers and SuperScript II reverse transcriptase (Invitrogen). For qRT-PCR, three replicates of each sample were amplified in a 20 CD24+/CD44+/ESA+ PCSCs were washed in PBS and lysed with RIPA buffer (Invitrogen), and a bicinchoninic acid protein assay kit  was used to calculate the protein concentration of each sample. Equivalent amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes for immunoblotting. The membranes were blocked in 5% fat-free milk for 2 hours at room temperature, washed three times, then incubated with the following primary antibodies: rabbit anti-human Nanog antibody  and rabbit anti-human GAPDH antibody . GAPDH was used as a loading control. Horseradish peroxidase conjugated secondary antibodies  and an ECL chemiluminescence kit were used to detect bound antibody. Western blot was performed according to standard protocols [ ad libitum. The study protocol was approved by the Shanghai Medical Experimental Animal Care Committee. For QYHJ treatment, an appropriate concentration (36 g/kg) of QYHJ and gemcitabine 25 mg/kg, twice per week by i.p. injection, were fed to nude mice.Female BALB/c-nu/nu nude mice, 4 to 6 weeks old, were obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences , and housed in laminar flow cabinets under specific pathogen-free conditions with food and waterμm) were stained with primary antibodies for Nanog (1 : 200) and then stained with HRP conjugated secondary antibodies. Sections were developed in diaminobenzidine and counterstained with hematoxylin. Images were taken, and five fields (200x) selected randomly from each section were examined. For the data quantification, integrated optical density (IOD) of positive expression was measured by Image Pro Plus 6.0 software.Paraffin-embedded sections  treated with QYHJ was significantly lower than control group (p < 0.01, p < 0.01, p < 0.01, Using a cell proliferation assay, we observed that thep < 0.01,  in vitro proliferation and invasive ability of PCSCs.At the same time, the soft agar colony formation assay results showed that the colony formation rate of PCSCs treated with QYHJ in soft agar was significantly lower than control group (Due to the poor prognosis, there is an urgent need to prolong pancreatic cancer patients' lifetime. Previous studies have shown only limited benefit with current treatments. The prognosis of PDAC is extremely dismal and the disease is obviously resistant to current therapies .Traditional Chinese medicine has had an advantage in cancer therapy for thousands of years. QYHJ has shown promising outcomes in treatment for patients with pancreatic cancer. In QYHJ treatment group, the survival rate of 5 years was 8.4%, and the median survival time was 7.6 months, obviously longer than that of control group [Little is known about the clinical and biological function of transcription factor Nanog thus far. To our knowledge, the findings of our current study provide the first evidence of the potential clinical utility of Nanog expression as a prognostic factor in pancreatic carcinoma. Our current works indicate that Nanog overexpression correlates with TNM stage and prognosis of patients with pancreatic cancer. Strong evidences have suggested that overexpression of Nanog is closely related to tumorigenesis, tumor metastasis, and distant recurrence after chemoradiotherapy , 18. NanIn a previous study, we demonstrated that QYHJ exerted an inhibitory effect on the growth of human pancreatic cancer cell and liver metastasis, perhaps by targeting VEGF and Cyr61 . However in vitro and in vivo by QYHJ. QYHJ inhibited the proliferation and invasion of human pancreatic cancer stem cell PANC-1, partly through downregulation of transcription factor Nanog expression. Nanog acts as a therapeutic target of QYHJ in the treatment of pancreatic cancer stem cells, and its expression status mediates different responses to QYHJ treatment of pancreatic cancer stem cells.In conclusion, we found that Nanog is overexpressed in pancreatic cancer tissues and its expression is downregulated


Univariate analysis revealed that a tumor size >3 cm was significantly associated with worse cancer-specific survival (p = 0.002) and IVR (p = 0.011). However, the influence was not statistically significant after controlling for other factors in the multivariate analysis . In conclusion, UTUC patients with a larger tumor present aggressive biological characteristics and tend to have a worse prognosis. Previous studies have reached diverse conclusions about the influence of tumor size on the oncologic outcomes in patients with upper tract urothelial carcinoma (UTUC). In this study, we retrospectively analyzed the records of 687 patients and evaluated how tumor size affected the prognosis of patients with UTUC after surgery. Clinicopathologic characteristics and oncological outcomes were compared according to tumor size (≤3 cm versus >3 cm). During a median follow-up period of 65 months (range 3–144 months), 225 patients (32.8%) died from UTUC and 228 patients (33.2%) experienced intravesical recurrence (IVR). Patients with a larger tumor size tended to have a significantly higher percentage of being male ( Upper tract urothelial carcinomas (UTUCs) are rare and account for only 5% to 10% of urothelial carcinomas , 3, and Tumor size has been recognized as a risk factor for poor oncologic outcomes in urothelial carcinoma of the bladder, while the prognostic influence of the tumor size in UTUC has not been fully addressed. A few studies –13 aboutThe aim of this high-volume study is to assess the association between tumor size and biological characteristics and validate the impact of tumor size on the oncological outcomes of UTUC patients treated with radical nephroureterectomy (RNU).Between Aug 1, 1999, and Dec 31, 2011, a total of 820 patients underwent operations for clinically localized UTUC. Seventy-six patients with concomitant or previous bladder tumors, 22 patients with metastatic disease, and 35 patients with incomplete follow-up data were excluded from this study. After obtaining institutional review board approval, the clinical and pathological data for the remaining 687 patients were retrospectively analyzed.In this cohort, all patients received RNU with the excision of the ipsilateral bladder cuff. The definitions of routine lymph node dissection, tumor stage, tumor grade, and tumor size were described in our previous study . In thisDuring the follow-up period, cystoscopy, laboratory tests, chest X-ray, and urological ultrasound were performed every 3 months during the first 2 years, which extended to a semiannual period for the next 2 years and once a year thereafter. Abdominal and pelvic computed tomography or magnetic resonance imaging scans were carried out annually. Follow-ups were censored until their last visit or death.p < 0.05 was considered significant. Chi-squared and Mann–Whitney U tests were used for the categorical variables. Kaplan-Meier method with the log-rank test was used to assess recurrence-free survival and CSS. Multivariate analyses were conducted using Cox's proportional hazard model.SPSS version 20.0  was used for all statistical analyses and a p = 0.011) and had a significantly higher percentage of tobacco consumption , lack of preoperative ureteroscopy history , renal pelvic location , and tumor necrosis . Significantly higher rates of advanced pathologic T stage (p < 0.001), tumor grade (p = 0.003), and positive lymph node (p = 0.018) were present in the cohort of patients with a tumor >3 cm.The clinicopathologic demographics of the patients are listed in p = 0.002; The median follow-up time was 65 months (range 3–144 months). In this cohort, 225 patients (32.8%) died from UTUC, including 112 patients with a tumor ≤3 cm and 113 patients with a tumor >3 cm, and bladder recurrence was found in 74 (10.8%) of this cohort of patients. The five-year CSS was 74.8% for patients with a tumor ≤3 cm and 62.6% for patients with a tumor >3 cm (p = 0.002) and several other variables were significant risk factors for a worse CSS . In the multivariate analysis, however, after adjusting for other clinicopathologic characteristics, tumor size was not a significant independent predictor of worse CSS . Rerunning the database with 4 cm or 5 cm as the cut-off value did not change the results. Older age , male gender , presence of hydronephrosis , advanced tumor stage , and positive lymph nodes  were independent risk factors for cancer-related mortality , lower tumor grade , cN0 or pN0 status , and tumor multifocality  remained as prognostic factors for recurrence ; only preoperative ureteroscopic history and tumor multifocality remained independent prognostic factors for bladder recurrence .If we excluded 151 patients who died before developing bladder recurrence during the follow-up period, smaller tumor size was not a risk factor for increased bladder recurrence by univariate analysis  [In this cohort, patients with a tumor size >3 cm had a higher rate of having biological aggressive characteristics, including advanced tumor stage, higher tumor grade, lymph node metastasis, and tumor necrosis. Zigeuner et al. confirmed that extensive tumor necrosis was independently associated with disease recurrence and survival  . Simone , resp.)  reportedThe rate of IVR was 33.2%, in accordance with previous studies –8. The mp = 0.139. In our research, the impact of tumor size on IVR was not supported by multivariate analysis. Although the impact of tumor size on IVR is controversial, we still suggest that a stringent surveillance plan for postoperative IVR is needed for patients with larger tumors. A more personalized risk-based follow-up scheme and potential adjuvant treatment might be possible after consideration of tumor size. And we would be closer to precise patients' risk-stratification with more understanding of these risk factors.The different finding requires convincing explanation. One reason for this inverse impact on oncologic outcome may be that larger tumor size is correlated with a high cancer-specific mortality and patients may die before the development of a bladder tumor. Patients with smaller tumor size may have a longer lifespan so that more IVR can be found. After excluding 151 patients who died without bladder recurrence, the impact of tumor size was not statistically significant, with RNU with the excision of an ipsilateral bladder cuff is the gold-standard treatment for UTUC . The useOur study has some limitations. First, this is a retrospective and single-center study. Second, none of the patients, especially those with advanced tumor stage and grade, received neoadjuvant chemotherapy. Finally, the lack of information on postoperative chemotherapy and radiotherapy would influence patient survival. Nonetheless the current study is still one of the largest single-center cohorts of UTUC patients, and the detailed analysis with regard to each clinical and pathological feature based on this large sample could provide more convincing information to clinicians.Patients with a tumor size >3 cm showed a higher rate of aggressive biological characteristics and tended to have worse CSS. A prospective and large-scale study is urgently needed to define the impact of tumor size and find a more satisfying cut-off value for tumor size.


Selected cell-cycle regulators and extracellular matrix proteins were found to play roles in malignant peripheral nerve sheath tumor (MPNST) biology. We aimed to analyze whether initial tumor tissue expressions of survivin, p53, cyclin D1, osteopontin (OPN) and fibronectin (FN) correlate with the response to neo-adjuvant CHT (naCHT) in children with advanced inoperable MPNST.M/F 14/12, median age 130 months) treated in Polish centers of pediatric oncology between 1992 and 2013. Tissue expression of markers was studied immunohistochemically in the manually performed tissue microarrays and assessed semi-quantitatively as low and high, based on the rate of positive cells and staining intensity.The study included 26 children with MPNST (Good response to naCHT was noted in 47.6%, while poor—in 52.4% of patients. The response to naCHT was influenced negatively by the presence of neurofibromatosis NF1 and high initial tumor tissue expression of OPN, survivin, p53 and cyclin D1. Patients with high tumor expression of either OPN, survivin or p53 and those with simultaneous high expression of ≥ 3 of the markers, responded significantly worse to naCHT, than patients, in whom expression of ≤ 2 markers were detected at diagnosis. Nearly, 85% of patients expressing ≥ 3 markers, responded poor to CHT; while 87.5% of children, expressing ≤ 2 markers, were good responders.The initial tumor tissue expression of OPN, survivin, p53 and cyclin D1 may serve as markers to predict response to naCHT in pediatric advanced MPNST. Future studies in more numerous group of patients are needed to confirm these preliminary results. Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas (STS) of neurogenic origin. Most cases arise in adulthood, however, 10–20% of MPNST occur in the first decades of life. MPNST may arise from pre-existing neurofibromas, particularly in patients with neurofibromatosis type 1 (NF1), or de novo  is recommended for local control in patients in whom incomplete resection was performed. Nevertheless, the prognosis in such patients remains unsatisfactory—with the 5-year-overall survival (5-y-OS) rate of approximately 34–44% for children and adults  in patients with initially inoperable MPNST is uncertain. Some authors have regarded MPNST as a chemoresistant malignancy  family, survivin regulates cell division at G2 phase. Its accumulation interferes with the activation of caspase activity during programmed cell death and thus promotes cell growth  protein and activate transcription factors involved in the transition from G1 to S phase in the cell cycle , registered in the Polish Pediatric STS Registry. All MPNST diagnoses were performed by pathologists from two independent institutions. For the purpose of this study, the tumor specimens were reviewed again by one of the authors (E.I-S.). The stages of MPNST were defined according to both clinical tumor nodes metastases (TNM) pre-treatment staging system and the Intergroup Rhabdomyosarcoma Study (IRS) post-surgical grouping system.All children were treated between III’1992 and XI’2013, using a multi-modality therapeutic CWS (Cooperative Weichteilsarkom Study Group) protocols, including surgery, CHT, and RTX. The surgical tumor resection performed as an initial measure was termed primary excision (PE). Microscopically complete surgery was termed R0 (IRS I), microscopically incomplete—R1 (IRS II), excision with macroscopic residues and biopsy only—R2 (IRS III). IRS IV meant metastatic disease at diagnosis. RTX with the use of external beam irradiation was administered to patients considered at risk of local recurrence. Chemotherapeutic schemes did not change substantially over the years and comprised cycles composed mostly of: vincristine, ifosfamide, dactinomycin, anthracyclines (doxorubicin or epirubicin), etoposide and carboplatin. The combination of drugs depended on the risk group classification and response to initial CHT.Response to naCHT was evaluated after three courses of CHT (between 9th and 10th weeks of treatment). It was based on the reduction in volume of all measurable lesions, using the following criteria: complete response (CR) = complete disappearance of disease; good response (GR)—tumor reduction ≥ 67% <100%; partial response (PR) = tumor reduction ≥ 33 < 67%; stable disease (SD) = tumor reduction < 33%; progression of disease (PD) = increase in tumor size or new lesion detection. “Good response” to CHT comprised CR, GR and PR, while SD and PD were termed “poor response” to CHT.The study was carried out in 26 archival histological samples of fresh-frozen paraffin-embedded (FFPE) primary, chemo-naive MPNST tumors, obtained at diagnosis of pediatric patients diagnosed and treated in Polish oncologic centers between 1992 and 2013. The samples were marked in a way preventing identification by other persons.Pathological analyses and IHC testing were performed in the Department of Pathology and Neuropathology Medical University of Gdansk (MUG). From the representative sections, the tissue microarrays (TMA) was constructed, using the commercially available Tissue -Tek® Quick-Ray ™ TMA System kit, of the Sakura Finetek USA, Inc. The IHC stainings were performed on TMA, using monoclonal antibodies to detect analyzed markers. The appropriate positive and negative controls were introduced. The qualitative and quantitative analysis of protein expression in MPNST tissue was performed using the digital image analyzer. The immunophenotyping was made by the visualization system Dako, Denmark.The expressions of analyzed markers in FFPE tumors were assessed using a semiquantitative method, based on the intensity of color reaction , and the rate of immunopositive cells . Based on these two variables, a numerical ratio of the markers’ expression was estimated and used for further analyses. Its value determined the expression of markers as low or high. Figure The study was approved by the Independent Bioethical Committee of the MUG, Poland (No. NKBBN/449/2013).p < 0.05 was considered statistically significant.The results were subjected to statistical study, using computer statistical software package EPIINFO Ver. 7.1.1.14 (02-07-2013). The clinical characteristics of the study group and the results of the tumor expressions of analyzed markers are presented in Table n = 16; IRS III) or metastatic . The primary tumors were located in unfavorable sites, including: head and neck parameningeal region, bladder and prostate, inner organs, retroperitoneum, extremities and other in 24 (92%) patients. Most of the tumors were large , invasive (T2 feature) in 88% of patients and deeply seated . In ten patients (38%), NF1 was diagnosed. The 5-year-event free survival (EFS) for the whole study group was 35.6 ± 9.7% and 5-y-OS—62.2 ± 10.2%. Median EFS was 19.8 months and median OS was 51 months.Over 80% (21/26) of patients were diagnosed in highly advanced stages of disease—inoperable (Response to naCHT was assessable in 21 children (81%). Five patients, who were not assessable, included: four children with no macroscopic tumor residue after PE and one child after R2 PE, who died of complications soon after treatment introduction. Ten patients (47.6%) achieved good response to naCHT , while 11 patients (52.4%)—poor response to naCHT (SD in six and PD in five). The response to naCHT and prognosis were significantly worse in NF1 patients. Among ten patients with NF1, only three achieved good response to naCHT. Within 16 patients without NF1 good response to naCHT was stated in 7 children (43.8%). All of them are alive and without evidence of disease , OPN and p53 (p = 0.023) and cyclin D1 and FN (p = 0.032).Survivin was expressed in 16/21 (76%) patients, OPN in 14 (67%), FN in 13 (62%), cyclin D1 in 11 (52%) and p53 in 11 (52%). Figure n = 5) responded well to naCHT . In the univariate analysis, it was shown that high expressions of survivin, p53, and OPN were significantly associated with a poor response to naCHT . The tumor expressions of cyclin D1 and of FN were found higher in poor responders, than in good responders, however, the differences between the groups were insignificant .Patients with poor response to naCHT demonstrated high expressions of analyzed markers more frequently than patients with good response to naCHT. In poor responders, survivin was expressed in all patients, OPN in 81.8%, cyclin D1 in 72.7%, p53 in 81.8%, and FN in 81.8%. In good responders, the expression of analyzed markers was less frequent . All patients expressing negatively for survivin , two achieved PR, one CR and one SD. All these children survived. The only patient in our study to achieve CR was found to express FN alone.Among 13 patients, who expressed three or more markers, 11 children (84.6%) responded poor to naCHT and only two (15.4%) achieved PR to naCHT. To the contrary, among eight children expressing no more than two markers, there were seven good responders (87.5%) and only one poor responder . Six children, who expressed all five analyzed markers, had poor response to naCHT. To the contrary, four of five patients who did not express any of the markers or expressed only one marker, responded well to naCHT.p = 0.026). Similarly, poor response to CHT was significantly more frequent in children with MPNST with simultaneous high expressions of p53 and cyclin D1, and of OPN and p53, in comparison to patients not expressing any of these markers . Due to low number of cases, it was impossible to assess the statistical difference between subgroups of patients with and without high expressions of survivin and OPN, survivin and p53, survivin and FN, and OPN and FN. However, in all subgroups of patients with high expressions of these pairs of markers, poor response to naCHT predominated. It was noted that simultaneous high expressions of OPN, p53 and cyclin D1 was significantly more common in poor responders than no expression of the markers (p = 0.005). In addition, high simultaneous expressions of survivin, OPN and p53 were associated with significantly worse response to naCHT, than high expression of a maximum one of these markers (p = 0.009). Patients, in whom high tumor tissue immunoreactivity towards OPN, survivin, p53 and cyclin D1, or just three of the four markers were stated at diagnosis, responded significantly worse to CHT than patients, who expressed no more than two of these markers (p = 0.009).It was observed that simultaneous high expression of particular markers in one tumor sample was associated with worse response to naCHT Table . For exaThe aim of the study was to assess whether selected proteins regulating the cell-cycle, tumor cells apoptosis and tumor invasion, i.e.: survivin, p53, cyclin D1, OPN and FN, may become potential biomarkers to predict the response to naCHT in advanced pediatric MPNST. Identification of such biomarkers would allow for more individualized treatment based on optimal selection of candidates for multidrug naCHT. This would diminish the risk of unnecessary CHT-related complications in those, who are not likely to benefit from CHT.It is indisputable that complete surgical tumor excision with negative (wide) margins offers the best outcome, both in children and adults . However, in their study, much better outcome was noted in patients treated with naCHT-containing ifosfamide , than in those receiving regimens including cyclophosphamide  or cisplatin and etoposide (20%) . It was found in univariate analysis, that high tumor expressions of survivin, p53, and OPN correlated significantly with poor response to naCHT. The tumor expressions of cyclin D1 and of FN were also higher in poor responders, than in good responders, however, the differences between the groups were insignificant. Our findings suggest that overexpression of the markers involved in tumor cells apoptosis, cell-cycle regulation and cancer invasiveness, may contribute to the resistance to CHT in childhood MPNST.The influence of survivin, an anti-apoptotic protein, on the response to CHT and RTX is uncertain. In head and neck cancer, higher tumor survivin expression was associated with a better response to RTX and longer survival. Accordingly, survivin silencing in cell lines led to decreased sensitivity to radiation . It was concluded that tumor expression of cyclin D1 may serve as a predictive biomarker in selecting patients with HNSCC, who may benefit from naCHT . Generally, the more markers were expressed simultaneously in a tumor tissue, the worse was the response to naCHT. Patients expressing all five markers responded poor to CHT, while four of five patients with no markers or only one marker expressed—responded well to CHT. Interestingly, the tumor expression of FN, either alone, or together with other markers, did not correlate with the response to naCHT in children with MPNST. Several previous studies have confirmed the role of FN in the pathogenesis of RMS and the invasiveness of adults RMS FN has been also suggested to be essential for lung metastasis in STS (Ito et al. The response to naCHT in advanced pediatric MPNST is influenced by the NF1 status and initial tumor tissue expression of OPN, survivin, p53 and cyclin D1.Patients with high tumor expression of either OPN, survivin or p53 and those with simultaneous high expression of ≥ 3 of the analyzed markers, responded significantly worse to naCHT, than patients, in whom expression of ≤ 2 markers were detected at diagnosis.The role of FN in predicting response to naCHT—as a sole analyzed marker and together with others—was not confirmed.Future studies in more numerous groups of patients are needed to confirm these preliminary results.


Daphnia magna Straus, 1820 (Crustacea: Cladocera), based on partial mitochondrial COI sequences and using specimens from populations spread longitudinally from westernmost Europe to easternmost Asia, with many samples from previously strongly understudied regions in Siberia and Eastern Asia. The results confirm the previously suspected deep split between Eastern and Western mitochondrial haplotype super-clades. We find a narrow contact zone between these two super-clades in the eastern part of Western Siberia, with proven co-occurrence in a single lake in the Novosibirsk region. However, at present there is no evidence suggesting that the two mitochondrial super-clades represent cryptic species. Rather, they may be explained by secondary contact after expansion from different refugia. Interestingly, Central Siberia has previously been found to be an important contact zone also in other cladoceran species, and may thus be a crucial area for understanding the Eurasian phylogeography of freshwater invertebrates. Together, our study provides an unprecedented complete, while still not global, picture of the phylogeography of this important model species.Species with a large geographic distributions present a challenge for phylogeographic studies due to logistic difficulties of obtaining adequate sampling. For instance, in most species with a Holarctic distribution, the majority of studies has concentrated on the European or North American part of the distribution, with the Eastern Palearctic region being notably understudied. Here, we study the phylogeography of the freshwater cladoceran  Planktonic water fleas (Crustacea: Cladocera) are attractive models for phylogeographic investigations –4. IndeeMany cladocerans are thought to be distributed across the entire Holarctic region, with populations being common throughout the range, which would potentially make them excellent models for Pan-Holarctic phylogeographic studies. However, our conclusions on their large distribution ranges may be too preliminary, because each taxon may in fact consist of several “cryptic” species with more local distribution ranges –12. One Moina and Chydorus), we demonstrated the existence of two faunistic super-complexes: (1) a European-Western Siberian super-complex and (2) a Eastern Siberian-Far Eastern super-complex, with a transition zone in the Yenisey basin, approximately near the boundary between Western and Eastern Siberia [Most previous phylogeographic studies of cladocerans concentrated on North America  and Euro Siberia . We also Siberia . The genDaphnia (Ctenodaphnia) magna Straus, 1820, is the object of hundreds of scientific publications per year. Besides being used for evolutionary, ecological, and physiological studies [D. magna have concentrated on Europe [D. magna is a common species also in the Middle East, Central Asia, several regions of Western and Eastern Siberia, China, and Japan , and is also found in North Africa, South Africa, and North America [COI sequences have been reported from some of these regions [Among numerous taxa of the cladocerans, one particular species,  studies ,35, it i studies ,37, and n Europe ,38,39 an America ,41,9. Se regions ,43, thouDaphnia magna in the Palaearctic, with special reference to the Eastern Palearctic region. Through concentrating on this region we also aimed at increasing our knowledge of potentially general phylogeographic patterns among cladocerans (and perhaps other freshwater-inhabiting taxa) in the Palaearctic. We use partial COI sequences of a large number of specimens from many populations spread across the Northern part of the region, with fewer specimens also from other parts of the region  and from North America. These samples are complemented with already published data on specimens of known origin.The aim of our study is to address these taxonomic, biogeographic, and phylogenetic knowledge gaps for Daphnia magna, three specimens from a single population of D. similis, and 17 specimens from 14 populations of D. sinensis  or rectangular dip nets  in different types of freshwater habitats and preserved in 90–96% ethanol. Before DNA extraction, each specimen was preliminarily identified to species level (D. magna or other species of the subgenus Ctenodaphnia), based on its morphology according to existing identification keys [We collected a total of 174 specimens from 67 populations of sinensis . The lation keys ,41.Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit  according to the manufacturer’s instructions, and fragments were amplified with primers listed in Polymerase chain reactions (PCR) were carried out in a total volume of 25 μl, consisting of 2 μl of genomic DNA, 8.5 μl of bi-distilled water, 1 μl of each primer (10 mM) and 5 μl PCR 5x Taq ScreenMix-HS . The PCR conditions for the amplification followed those described in the papers mentioned in D. magna sequences [COI Sequence JF821194 from Turkey was excluded from the analysis because a preliminary tree suggested that this sequence belongs to a separate clade, which, however, could be an artifact and needs further confirmation. Sequences were edited and assembled in uGene v.1.26 [COI flanking regions in Crustacea, no universal primers for barcoding exist, in contrast, for example, to fishes [COI sequences were cropped to the minimal overlapping length of 563 bp.The authenticity of the sequences was verified by BLAST comparisons with published equences . COI Seqe v.1.26 . The orie v.1.26 ,58,59,60e v.1.26  with defo fishes . As a coTo analyze the genetic variation among samples, we estimated the following parameters for each gene fragment and for each species separately: number of haplotypes (Nh), number of variable (polymorphic) sites (Nv), number of parsimony informative sites (Np), haplotype diversity (Hd), nucleotide diversity (Pi), and average number of nucleotide differences (k) ,64. We aBayesian information criterion [p-distance [D. similis and D. sinensis as outgroup species. A maximum likelihood (ML) phylogenetic reconstruction was performed using the IQ-TREE web server [The best-fitting model of nucleotide substitution was selected using the ModelFinder web application , based oriterion . Within-distance . Phylogeb server  and ultrb server  resampleb server . A heurib server . Six indb server .http://www.fluxus-technology.com) was used to construct a median-joining haplotype network with Steiner maximum parsimony post-processing [p-distances [To establish whether there was any evidence for cryptic species, we performed a Bayesian generalized mixed Yule coalescent (bGMYC) analysis  with a tocessing , puttingocessing  to reducocessing  to assesistances  with 100D tests [FS tests [The reduced-complexity network was also used for a nested clade analysis, which identifies significant phylogroups, and to test for historical demographic processes within the major phylogroups. The nested clade phylogeographic analysis (NCPA) was performed in the program ANeCA v.1.2  with intD tests  and Fu’sFS tests  were perFS tests  with 100FS tests  in DnaSPFS tests . For theFS tests . The demDapnia/Ctenodaphnia split (145 MYA), which suggests that the speed of nucleotide substitutions is significantly lower than suggested by [Divergence times were estimated using a relaxed molecular clock approach with uncorrelated lognormal distributions of branch rates in the program BEST v.1.6.1, following methods and calibrations of . A seconested by .COI sequences of three Daphnia species  from different regions of the Palaearctic  than in super-clade B (mainly Asian populations), possibly explained by the wider geographic distribution of super-clade A. However, nucleotide diversity and the number of polymorphic sites were almost identical between the two clades (The genetic diversity at o clades  Tables. COI data set (our sequences and GenBank sequences combined) was HKY with four categories of gamma  and a proportion of invariable sites  with a relative BIC weight of 0.3648. Based on the cumulative relative weight, two other models were also within the 95% confidence interval. One best ML tree was found, with an overall likelihood score of -ln L = -2775.1574. These analyses revealed the existence of two highly divergent and well-supported main super-clades within Dapnia magna , the Middle East, Turkey and Caucasus, as well as in a few populations in Western Siberia. Within this clade, there are several sequences from the Genbank labeled as originating from North America , but in reality most of these may have originated from laboratory cultures, presumable of European origin . Few othThe super-clade B includes several Asian and North American lineages and co-occurs with clade A1 in a single lake in the Novosibirsk Area (Western Siberia) (COI haplotypes (The TCS network consists of the 93 different plotypes . Each ofSuper-clade A is again clearly subdivided into a diverse clade A1 and a distant clade A2 (North American samples). Within the A1 clade, we found several sub-networks with a star-like topology, suggesting recent population expansion. The central haplotype of sub-network A1_1  was founD and mismatch distribution analyses , but not penetrating to the west of Lake Baikal. The clade B1 is separated from others by five mutations. All other clades  form a remarkable monophyletic group interconnected through a hypothetical (extinct or un-sampled) haplotype. Tajima's analyses  were conCOI clades of D. magna , Group 2 –Eastern Siberian clades B2, B4, B5 , Group 3—Western Siberian clade B1 , Group 4 –Canadian clade B6 , Group 5 –North American clade A2 , and Group 6 –European clade A1.The ABDG test based on haplotype groups (s_numbers) according to the star contraction algorithm  revealedmagna and the outgroup species. However, using the additional calibration of the sub-genera Ctenodaphnia / Daphnia split, which was estimated at 145 MYA [D. magna and other Ctenodaphnia species  and the Eastern Siberian-Far Eastern-Chinese-"Beringian" = East Asian ones (B1-B5). Sampling locations were distributed almost evenly from Western Europe to Easternmost Asia, and the transition between the two groups appears to be rather abrupt. The network structure suggests that this suture zone  is a is aD. me . Interestingly, the clade B1, which is the clade that occurs in the contact zone between A and B super-clades, has an intermediate position between the two super-clades in the haplotype network . The interpretation of this observation needs further investigation, but again highlights the special interest of the boundary between Western and Eastern Siberia.The individual clades within super-clade B are more distinct, though the existence of intermediate haplotypes in non-sampled populations remains a realistic possibility. Several clades  were found only in a small region or even only at a single location, either at the boundary between Eastern and Western Siberia  or in Northeastern Siberia (B5). All aforementioned B clades may be relicts of a previous, hypothetical "pan-Beringian" population , in contrast to the more common northward expansion that is assumed for most postglacial colonization events .D. magna in North America only very briefly here, as it is clear that too few samples from that continent were available for our study to reach more detailed conclusions. Furthermore, the precise sampling locations of this material are unclear (see above). Nonetheless, the observation that both super-clades seem to be present in North America, with haplotypes that are clearly distinct from the haplotype clades found in Europe and Asia, suggests the possibility that North America may historically have been colonized from both sides .Two clades A2 and B6 were found exclusively in North America. We will discuss the phylogeography of D. magna a good fit with the RGF (restricted gene flow) model, which is in agreement with the cladoceran biology (existence of isolated populations with a large number of generations per season). For the high-ranking super-clades (A and B) the finding of correspondence to the RGF model is a useful conclusion, as it suggests long-term  isolation.We have used two common approaches to separate evolutionary significant groups, the Nested Clade Phylogeographical Analysis ,83 and aCOI polymorphism in D. magna allowed us to use this method here, with apparently more adequate conclusions concerning larger clades. In the literature, methods of statistical analysis of phylogeographic patterns appear to be chosen somewhat haphazardly, rather than according to the advantages and limits of each method for a given data set . Mor. MorCOI set , several lines of evidence strongly suggest that they represent two divergent super-clades within a single species. First, the genetic distance between the two super clades is much smaller than between D. similis, D. sinensis, and D. inopinata, which are good, though very closely related biological species with reproductive isolation strongly supported by differences in male morphology [D. magna.Many previous genetic studies have advocated the existence of species complexes instead of single species among different species groups of nia see ,12). Alt. AltDaphrphology . Second,rphology . TherefoDaphnia, are very popular laboratory animals and are often used as food in aquaculture. Moreover, their resting stages are long-lived, which favors dispersal by human activities . Many Culations . Howeverulations  is very D. magna does not seems to be a species with a strong invasive potential–in contrast to D. lumholtzi, D. cf. pulex, and D. galeata [Daphnia and many other organisms, and require a critical interpretation of the obtained results.In general,  galeata –103. ButD. magna from its sister species, as well as of the super-clades and clades within the species. Currently the issue remains unresolved, and its resolution will have to take into account Frey's [The two molecular clock scenarios used yielded strongly different results on the timing of divergence of t Frey's ,7 paradit Frey's , as wellt Frey's ,106. In t Frey's .S1 FigNumbers identify star groups of haplotypes see . Circle size is proportional to the frequency of the haplotypes. Small dark circles indicate unsampled or extinct haplotypes. Geographic regions for star groups are as follows: s1 –Western, Central Europe, Mediterranean region, Middle East; s2 –Western Europe; s3 –Scandinavian Peninsula and Western Europe; s4-s8 –Mediterranean region; s9 –Mediterranean region and Near East; s10 –Western Europe; s11 –European Russia; s12—Near East; s13-s14 –European Russia; s15-s16 –Mediterranean region; s17-s18 –North America; s19 –European Russia s20 –Mediterranean region; s21 –Western Europe; s22 –Western and Central Europe; s23 –Central and Eastern Europe; s24-s25 –Western Europe; s26 –Western Europe and Scandinavian Peninsula; s27 –different regions of Europe and laboratory cultures; s28 –Mediterranean region; s29 –Western and Central Europe; s30 –Scandinavian Peninsula; s31-s33 –European Russia; s34-s36 –Western Siberia; s37 –Eastern Siberia; s38-s39 –Western Siberia; s40-s42 –North America; s43-s45 –Eastern Siberia; s46 –Far East of Asia.(TIF)Click here for additional data file.S2 FigClades highlighted in red represent the maximum likelihood limits. The colored lines correspond to a sequence-by-sequence matrix, with lines colored according to the posterior probability that the sequences are conspecific, which allows visualizing uncertainty in group limits.(TIF)Click here for additional data file.S3 FigAll original samples have prefix zh, while the Genbank samples have no this prefix. GenBank accession numbers for original samples are given in (TIF)Click here for additional data file.S4 FigAll original samples have prefix zh, while the Genbank samples do not have this prefix. GenBank accession numbers for original samples are given in (PDF)Click here for additional data file.S1 TableCOI clade designations as defined in (XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Tablen—sample size, Nh—number of haplotypes, Nv—number of variable (polymorphic) sites, Np—number of parsimony informative sites, Hd—haplotype diversity, Pi—nucleotide diversity, k—average number of nucleotide differences.(DOC)Click here for additional data file.S4 Tablen—sample size, Nh—number of haplotypes, Nv—number of variable (polymorphic) sites, Np—number of parsimony informative sites, Hd—haplotype diversity, Pi—nucleotide diversity, k—average number of nucleotide differences; R2 population size expansion test and results of Tajima’s D, Fu’s FS and mismatch distributions: tau-parameter, SSD (sum of squares deviation) and Harpending's Raggedness index including associated p-values. Biological processes are : RGF–restricted gene flow; D–dispersal; LDD–long-distance dispersal; IBD–isolation by distance; AF–allopatric fragmentation; PF–past fragmentation; LDC–long-distance colonization.(DOC)Click here for additional data file.S5 Tableet al., 2013), second digit–“slow clock” from .In diagonal, bold–estimates of average evolutionary divergence over sequence pairs within groups. Below diagonal–estimates of evolutionary divergence over sequence pairs between groups. The presence of n/c in the results denotes cases in which it was not possible to estimate evolutionary distances. Above diagonal–time divergence, in MYA; first digit–“fast clock” from (Schwentner (DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


In the last decade, scrub typhus (ST) has been emerging or re-emerging in some areas of Asia, including Guangzhou, one of the most affected endemic areas of ST in China.Based on the data on all cases reported in Guangzhou from 2006 to 2014, we characterized the epidemiological features, and identified environmental determinants for the spatial distribution of ST using a panel negative binomial model.A total of 4821 scrub typhus cases were reported in Guangzhou during 2006―2014. The annual incidence increased noticeably and the increase was relatively high and rapid in rural townships and among elderly females. The majority of cases (86.8%) occurred during May―October, and farmers constituted the majority of the cases, accounting for 33.9% in urban and 61.6% in rural areas. The number of housekeeper patients had a rapid increment in both rural and urban areas during the study period. Atmospheric pressure and relative humidity with lags of 1 or 2 months, distributions of broadleaved forest and rural township were identified as determinants for the spatiotemporal distribution of scrub typhus.Our results indicate that surveillance and public education need to be focused on the elderly farmers in rural areas covered with broadleaf forest in southern China.The online version of this article (doi:10.1186/s12879-016-2153-3) contains supplementary material, which is available to authorized users. Orientia tsutsugamushi (O. tsutsugamushi), is characterized by fever, eschar or ulcer, rash, lymphadenopathy, hepatosplenomegaly. Severe complications or even death may occur. O. tsutsugamushi is transmitted occasionally to humans by the bites of infected chiggers  [Scrub typhus, a bacterial zoonosis caused by d mites) , 2. Roded mites) . Recentld mites) –7. It wad mites) , 8.Human cases of scrub typhus were reported in 1948 in Guangzhou. Constantly troubled by the disease, Guangzhou has listed it as one of the local reportable infectious diseases since 1995 . Scrub t2 (http://data.gzstats.gov.cn/gzStat1/chaxun/njsj.jsp). It features a subtropical monsoon climate with annual average temperatures of 21–23 °C and annual average precipitations around 1900 mm. The landscape is characterized by mountains and hills in north or northeast, basin and alluvial plain in south or southwest.The study area of Guangzhou prefecture , a political subdivision of a province, is the capital of Guangdong Province in southern China. Located at the Pearl River Delta, Guangzhou harbors around 13 million residents in its 12 counties with a total land area of nearly 7500 kmhttp://www.chinacdc.cn/tzgg/200901/t20090105_40316.htm). The clinical diagnosis of patients were mainly based on some of the following clinical manifestations and signs: (i) Epidemiological exposure history within 3 weeks prior to the onset, (ii) fever, (iii) lymphadenopathy, (iv) skin rash, and (v) specific eschar or ulcer. A clinically diagnosed cases are defined by meeting at least above “i, ii, and v” or “ii, iii, iv, and v” after excluding other diseases with similar clinical manifestations. A laboratory-confirmed case is defined as a clinically diagnosed patient with at least one of the following laboratory results: (vi) An agglutination titer ≥ 1:160 in the Weil-Felix test using the OXK strain of Proteus mirabilis, (vii) a fourfold or greater rise in serum IgG antibody titers between acute and convalescent sera detected by using indirect immunofluorescence antibody assay (IFA), (viii) detection of O. tsutsugamushi by polymerase chain reaction (PCR) in clinical specimens, or (ix) isolation of O. tsutsugamushi from clinical specimens. A patient meeting above “i, ii, iii or iv” or “ii, iii, iv” after excluding other diseases with similar clinical manifestations, and at least one of above “vii, viii, ix” is also diagnosed as a laboratory-confirmed case according to the Technical Guides for Prevention and Control of Scrub Typhus (available at: http://www.chinacdc.cn/tzgg/200901/t20090105_40316.htm.).All clinically diagnosed and laboratory-confirmed cases of scrub typhus are reported to the China Information System for Disease Control and Prevention (CISDCP) since 2006. The diagnosis of scrub typhus is based on the national guide lines issued by the Chinese Center for Disease Control and Prevention (http://data.cma.cn/) : average monthly atmospheric pressure, average monthly temperature, average monthly relative humidity, aggregate monthly precipitation, and average monthly wind velocity. Land cover data were derived from a raster version of the “GlobCover 2009 land cover map” provided by the European Space Agency (available at http://due.esrin.esa.int/page_globcover.php). Elevation raster with a spatial resolution of 1 *1 km2 was obtained from the Global Digital Elevation Data Products (http://www.gscloud.cn). Population data at the township level were obtained from the National Bureau of Statistics of China, which is based on the Sixth National Census in 2010. These variables were extracted for each township of Guangzhou in ArcMap version 9.3.To explore factors influencing the spatiotemporal distribution of scrub typhus cases in Guangzhou, data on meteorological, environmental and ecological factors were collected. The following meteorological data were obtained from China Meteorological Data Sharing Service System  in response to the change of each variable by a given amount . All 12 counties of Guangzhou and 98.8% of the townships (159/161) were affected. Cases were mainly distributed in areas with high densities of built-up lands and croplands Fig. . The aveThe majority of cases (86.8%) occurred during May―October, and the incidence peaked in either June/July (most years from 2006―2014) or October (2007―2008) with dual peaks in some years. The annual incidence increased slowly during Period I (2006―2008) and grew steadily during Period II (2009―2011), then fluctuated and remained high in Period III (2012―2014). Rural areas had 2–4 times higher incidences and a more rapid increase of the incidence than urban areas during the study period Fig. . FarmersThe senior population older than 60 years had the highest average annual incidence, especially the senior females, which have also shown a quicker rise of average annual incidences in both urban and rural areas during the three periods, rather than other age groups. The age difference in average annual incidence seemed greater in recent period . Four variables, including average atmospheric pressure with 1-month lag, average relative humidity with 2-month lags, percentage coverage of broadleaved forest and type of township, were shown to be independent predictors for the spatiotemporal distribution of the disease in the multivariate regression model. Each one hundred Pa increase of average atmospheric pressure was associated with an 11% (95% CI: 10–12%) decrease in the incidence of scrub typhus in the next month, while a 10% rise in monthly average relative humidity corresponded to an 4% (95% CI: 3–5%) increase in the scrub typhus incidence in the month after the next. For every 10% increase in the percentage coverage of broadleaved forest, the incidence of the disease went up by 5% (95% CI: 3–6%). On average, a rural township had an 81% (95% CI: 43–129%) higher risk than an urban township  had higher incidence than elderly males, whereas an opposite pattern was observed among the younger generations, regardless of urban or rural townships . Additional experimental studies and the identification of confirmed cases should further explore the epidemic dynamics and influencing factors of ST.Our study highlighted the urgent need for prevention programs to contain the spread of scrub typhus in Guangzhou. A set of preventive strategies including public education and personal protection equipment shall be promoted in high-risk populations such as the elderly, farmers and housekeepers. Surveillance and early diagnosis should be reinforced in suburb and rural areas, especially places covered by broadleaf forests. We recommend future studies collect behavioral factors so that risk assessment can be adjusted for individual exposure levels.


Objective. Panax ginseng is widely used for treatment of cardiovascular disorders in China. Ginsenoside Re is the main chemical component of Panax ginseng. This study aimed to investigate the protective effect of Ginsenoside Re on isoproterenol-induced myocardial injury in rats. Methods. Male Wistar rats were orally given Ginsenoside Re  daily for 7 days. Isoproterenol was subcutaneously injected into the rats for two consecutive days at a dosage of 20 mg/kg/day (on 6th and 7th day). Six hours after the last isoproterenol injection, troponin T level and creatine kinase-MB (CK-MB) activity were assayed. Histopathological examination of heart tissues was performed. The levels of malondialdehyde (MDA) and glutathione (GSH) in heart tissues were measured. The nuclear factor erythroid 2-related factor 2 (Nrf2) content in nucleus and the proteins of glutathione cysteine ligase catalytic subunit (GCLC) and glutathione cysteine ligase modulatory subunit (GCLM) in heart tissues were assayed by western blotting method. Results. Treatment with Ginsenoside Re at dose of 5, 20 mg/kg reduced troponin T level and CK-MB activity of rats subjected to isoproterenol. The cardioprotective effect of Ginsenoside Re was further confirmed by histopathological examination which showed that Ginsenoside Re attenuated the necrosis and inflammatory cells infiltration. Ginsenoside Re inhibited the increase of MDA content and the decrease of GSH in heart tissues. Moreover, the Nrf2 content in nucleus and the expressions of GCLC and GCLM were significantly increased in the animals treated with Ginsenoside Re. Conclusion. These findings suggested that Ginsenoside Re possesses the property to attenuate isoproterenol-induced myocardial ischemic injury by regulating the antioxidation function in cardiomyocytes. Myocardial injury is the common presentation of the ischemic heart disease. Ischemic heart disease affects a high proportion of the population in both developed and developing countries. Despite the improved clinical care and the availability of modern medicines, previous report predicted that cardiovascular disease will be a major cause of death worldwide by the year 2020 .Reactive oxygen species (ROS) such as free radicals, singlet oxygens, and peroxides are generated during aerobic metabolism as by-products and are tightly controlled by antioxidants. Excess production of ROS or depletion of antioxidants can lead to a state of oxidative stress that can inflict damage to lipids, proteins, and DNA. ROS generation during ischemia induces a peroxidation of the components of myocardium . Followi Panax ginseng is a perennial herb which belongs to the Araliaceae family and is distributed in 35 countries, particularly in China and South Korea. Panax ginseng has been considered as a traditional medicine in Asian countries for thousands of years for its health promotion in various disease conditions. Panax ginseng has various effects, such as antifatigue, antiaging, improving immune function [ Panax ginseng has cardioprotective and neuroprotective properties [ Panax ginseng, ginsenosides have been shown to be the major pharmacological ingredient in Panax ginseng. Since the first isolation of six ginsenosides from Panax ginseng in the 1960s, plenty of ginsenosides have been isolated and identified from the Panax ginseng. Depending on their structures, ginsenosides are divided into three groups: the panaxatriol group including Re, Rf, Rg1, Rg2, and Rh1, the panaxadiol group including Rb1, Rb2, Rb3, Rc, Rd, Rg3, and Rh2, and the oleanolic acid group [In recent years, Traditional Chinese Medicine (TCM) has been widely used in China, Korea, Japan, and other Asian countries in the treatment of cardiovascular diseases. Furthermore, an increasing number of studies have confirmed the efficacy of TCM for treating ischemic heart disease . Panax gfunction . It is aoperties . Among tThe experiments were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals  and were approved by the Ethics Committee of Jilin University. Wistar rats weighing 240 to 270 g were provided by the Experiment Animal Center of Norman Bethune University of Medical Science . The animals were housed in diurnal lighting conditions (12 h/12 h) and allowed free access to food and water for 7 days before the experiment. Panax ginseng were collected from Tonghua country of Changchun city in Jilin Province during June 2015. Its identity was confirmed by Professor Yanping Chen with authentic specimens retained at the herbarium, Jilin University. A voucher specimen was deposited in Department of Medicinal Chemistry, Jilin University. Panax ginseng (5 kg) was pounded into powder and then was soaked with 95% ethanol  for 12 h. After being filtrated, the extracts were added to nonpolar macroporous resin  column and the impurities were washed out by water. Then total saponin was eluted by 75% ethanol. The eluent was freeze-dried finally giving amorphous powder. The residue was dissolved with 75% ethanol again and the suspension was filtrated. The precipitate was subjected to a silica gel column chromatography (2 cm × 20 cm) and eluted with a stepwise gradient of trichloromethane : methanol : acetic ether : H2O (15 : 22 : 40 : 23) and then underwent further chromatography on silica gel columns, employing the same eluent systems, to give a Ginsenoside Re. The yield of the extract as a dried material was approximately 0.45% by weight of the original material. Further analysis by HPLC showed that the content of Ginsenoside Re in the resultant extract was 98.6% . CK-MB kit was obtained from Biosino Biotechnology Company Ltd. . Isoproterenol and GSH were purchased from Sigma Chemical Company . Carboxymethyl cellulose sodium salt (CMC-Na) was from Shanghai Jinshan Chemical Co., Ltd. . Other reagents were of commercially analytical grade.Fifty-six rats were randomly divided into four groups with 14 animals in each group: control, isoproterenol, and Ginsenoside Re (5 and 20 mg/kg) groups. Rats in control and isoproterenol groups were orally given 0.5% CMC-Na daily for 7 days. While animals in Ginsenoside Re groups were intragastrically treated with Ginsenoside Re at doses of 5 or 20 mg/kg, respectively, daily for 7 days. Isoproterenol (20 mg/kg) was dissolved in normal saline and subcutaneously injected to rats at an interval of 24 h for 2 days (on 6th and 7th day) to induce myocardial ischemic injury . Isoprot g) at 4°C for 10 min. Then the serum was harvested and stored at −80°C for biochemical assay. According to the manufacturer's instructions, troponin T level was assayed using an ELISA kit. The activity of CK-MB was analyzed using commercial kits by employing an automatic biochemical analyzer .The animals were anaesthetized with isoflurane and blood samples were collected in dry test tubes without anticoagulant at 24 h after the last isoproterenol injection. The samples were centrifuged . The MDA content in heart tissues was assayed according to the previous method [After blood collection, eight animals of each group were sacrificed by decapitation. The hearts were homogenized in 4 volumes of 0.1 mol/L ice phosphate buffer (pH 7.4) and then were centrifuged at 10,000s method . GSH wass method .μg) and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (Millipore Corporation) for 1.5 h at 100 V. Blocked with 5% nonfat milk for 1 h, the membranes were incubated with the primary antibodies: rabbit anti-Nrf2 , anti-GCLC , anti-GCLM , anti-Lamin B1 , or rabbit anti-β-Actin  overnight at 4°C. The membranes were processed with the respective horseradish peroxidase-labeled secondary antibody (1 : 3000). Bands were visualized using the ECL detection reagents .The nuclear and cytoplasmic protein extracts were prepared using nuclear and cytoplasmic protein extraction kit (heart tissues of three rats in each group). Proteins were loaded . Compared with the isoproterenol group, the troponin T level in the Ginsenoside Re (5 and 20 mg/kg) groups was significantly reduced , (Compared with the control group (4.2 ± 0.82 ng/ml), the troponin T level was significantly increased in isoproterenol group , .P < 0.01). Compared with isoproterenol group, treatment with Ginsenoside Re at doses of 5 and 20 mg/kg significantly inhibited the augment of the CK-MB activity in isoproterenol-induced myocardial ischemic rats (P < 0.01).As shown in In control group, myocardium showed clear integrity of myocardial cell membrane and no inflammatory cells infiltration was observed. Heart tissues from isoproterenol-induced myocardial injury rats showed widespread myocardial structure disorder and subendocardial necrosis, which was related to inflammatory cells infiltration and myofibrillar fracture. The rats in Ginsenoside Re (5 mg/kg) group exhibited mild necrosis and less infiltration of inflammatory cells. The heart tissues of animals in Ginsenoside Re at dose of 20 mg/kg showed nearly normal myofibrillar structure with few neutrophil granulocytes infiltration .P < 0.01). Treatment with Ginsenoside Re at a doses of 5, 20 mg/kg attenuated the increase of MDA content , MDA content was significantly augmented after isoproterenol injection  as compared to the control group. Compared with the isoproterenol group, the Ginsenoside Re  groups showed a significant increase of GSH level  in the heart tissues  observed in isoproterenol-induced myocardial ischemic rats (P < 0.01). Ginsenoside Re treatment increased the Nrf2 content in nucleus of cardiomyocytes . However, after treatment with Ginsenoside Re at doses of 5, 20 mg/kg, the expressions of GCLC and GCLM increased significantly (P < 0.05 or P < 0.01).β-adrenergic agonist. It has acute positive inotropic and chronotropic effects on the heart. The strong inotropic and chronotropic actions of isoproterenol will induce necrosis, inflammatory cell infiltration, hypertrophy, and fibrosis [Isoproterenol is a fibrosis . HistoloTroponin T, a regulatory protein controlling the calcium-mediated interaction of actin and myosin contractile protein, is a highly specific and sensitive marker in the diagnosis of human and animal model of myocardial ischemic injury . ElevatiOxidative stress is well documented in the pathogenesis of humans as well as isoproterenol-induced myocardial ischemic injury. In rats, isoproterenol had been reported to increase oxygen demand, deplete ATP levels, cause calcium overload, and undergo autoxidation leading to formation of free radicals . CardiomIn summary, this study indicated that Ginsenoside Re possessed cardioprotective effects in isoproterenol-induced myocardial injury rats. The pharmacological action of Ginsenoside Re was related to regulating the antioxidation function in cardiomyocytes.