Poria cocos  is commonly used in traditional Chinese medicine and its diuretic effect was confirmed in rat. Ultra performance liquid chromatography/quadrupole time-of-flight high-sensitivity mass spectrometry and a novel mass spectrometryElevated Energy data collection technique was employed to investigate metabonomic characteristics of chronic kidney disease (CKD) induced from adenine excess and the protective effects of FLP. Multiple metabolites are detected in the CKD and are correlated with progressive renal injury. Among these biomarkers, lysoPC(18∶0), tetracosahexaenoic acid, lysoPC(18∶2), creatinine, lysoPC (16∶0) and lysoPE(22∶0/0∶0) in the FLP-treated group were completely reversed to levels in the control group which lacked CKD. Combined with biochemistry and histopathology results, the changes in serum metabolites indicate that the perturbations of phospholipids metabolism, energy metabolism and amino acid metabolism are related to adenine-induced CKD and to the interventions of FLP on all the three metabolic pathways. FLP may regulate the metabolism of these biomarkers, especially their efficient utilization within the context of CKD. Furthermore, these biomarkers might serve as characteristics to explain the mechanisms of FLP.The surface layer of  Kidney diseases are a serious and prevalent health problem, and manifestation includes changes in renal detoxification capacity, deregulation of salt and water balance and altered endocrine functions -overall, exerting a significant impact on the patient’s short- or long-term survival. The prevalence of chronic kidney disease (CKD) continues to increase, mainly due to an increase in secondary disease as a consequence of diabetes and hypertension.et al introduced MSE (where E represents collision energy) technique for the first time E can provide parallel alternating scans for acquisition at either low collision energy to obtain precursor ion information, or ramping of high collision energy to obtain full-scan accurate mass fragment, precursor ion and neutral loss information Metabonomics is a part of systems biology which refers to a holistic analytical approach to the low molecular mass organic endogenous metabolites in the tissue or bio-fluids An adenine-induced CKD model provides valuable information of pathological mechanism for various complications in a persistent uremic state. In mammalian metabolism, excess adenine becomes a significant substrate for xanthine dehydrogenase, which oxidizes adenine to 2,8-dihydroxyadenine (DHA) via an 8-hydroxyadenine intermediate Poria cocos (Schw.) Wolf (Polyporaceae) is a well-known traditional East-Asian medicinal plant that grows around the roots of pine trees in China, Japan, Korea and North America Poria cocosPoria cocos Wolf as Fuling in China and Hoelen in Japan. The inner parts of the sclerotia of P. cocos, called “Fu-Ling” in Chinese, are used to treat chronic gastritis, acute gastroenteric catarrh, gastric atony, edema, nephrosis, dizziness, nausea and emesis Poria cocos mainly include triterpenes, polysaccharides and steroids Poria cocos presented a remarkable diuretic effect As reported previously, the chemical constituents of Elevated Energy (MSE) data collection technique was applied to investigate the serum metabolite profiling of the renoprotective effect of FLP and its action mechanism. Potential biomarkers related with CKD were identified, and their metabolic pathways were also discussed. Furthermore, clinical biochemistry study were also carried out to ensure the success of the CKD model and to investigate the renoprotective effect of FLP. Therefore, metabonomics could be a promising scientific platform for therapeutic evaluation and action mechanism study of TCM.In the current study, metabonomics study based on ultra performance liquid chromatography/quadrupole time-of-flight high-sensitivity mass spectrometry (UPLC-Q-TOF/HSMS) and a novel mass spectrometryAdenine  and formic acid solution  were purchased from Sigma-Aldrich . Creatinine  was obtained from the National Institutes for Food and Drug Control . L-tryptophan  and Valine  were purchased from Amresco Company . LC-grade methanol and acetonitrile were purchased from the Baker Company . Ultra high purity water was prepared using a Milli-Q water purification system . Other chemicals were of analytical grade and their purity was above 99.5%.FLP was collected from Shaanxi Province in March 2012, and was identified by Prof. Y. Z. Wang . A voucher specimen (120304) was deposited at Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Northwest University, Xi’an, Shaanxi. FLP was ground to powder (about 20 meshes) by a disintegrator, and the powder (2 kg) was extracted three times with 15 L 95% ethanol for 0.5 h by ultrasonic method. The extracts were combined together and filtrated, and then the filtrate was concentrated in vacuum using a rotary evaporator to give dried powder.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the State Committee of Science and Technology of the People’s Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Northwest University (Permit Number: SYXK 2010-004). All surgery was performed under uretane anesthesia, and all efforts were made to minimize suffering. All procedures and care of the rats were in accordance with the institutional guidelines for animal use in research. Male Sprague-Dawley rats were obtained from the Central Animal Breeding House of the Fourth Military Medical University . The rats were maintained at a constant humidity (ca. 60%) and temperature (ca. 23°C) with a light/dark cycle of 12 h.Male rats underwent an adaptation period of several days, during which they were fed a commercial feed. Rats weighing 200 to 220 g were divided into 3 groups (n = 8/group): group 1(control group), group 2 (CKD model group) and group 3 (FLP-treated group with CKD). Groups 2 and 3 then were then given 200 mg/kg body weight of adenine dissolved in 1% (w/v) gum acacia solution by oral gavage once everyday continuously for 2 weeks, which produced experimental renal failure in the animal for two weeks. Group 1 was similarly provided an equal volume of gum acacia solution. During the adenine gastric gavage after 3 h, Group 3 was administered FLP (60 mg/ml) by gastric irrigation. The control group and model group were only administered by oral gavage with the 1% (w/v) gum acacia solution. Body weights were recorded daily.After two weeks, all the rats were anesthetized with 10% urethane, and blood samples were obtained by carotid artery cannula. Blood was centrifuged at 3000 rpm for 10 min and the supernatant was collected and stored at –80°C. The rats were killed and Kidneys were collected immediately after blood was drawn and the tissues were washed with saline buffer.At the end of the experiment, rats were housed individually in metabolic cages for 24 h urinary collection and body weight was measured. Kidneys were weighed to determine the organ indexes .The levels of Serum creatinine (SCr), blood urea nitrogen (BUN), cholesterol and triglyceride were determined by Olympus AU640 automatic analyzer and the levels of white blood cell count (WBC), red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) were determined by HF-3800 Routine blood analyzer.A portion of kidney tissue was immersed in 10% neutral buffered formaldehyde solution, the tissues were dehydrated, embedded in paraffin, cut at 5 micrometer thickness and stained with hematoxylin and eosin (H&E) for histopathological examination.Prior to the analysis, the serum samples were thawed at room temperature. Acetonitrile (400 µl) was added to serum (200 µl) and vortex-mixed  vigorously for 3 min. The mixture was settled at room temperature for 10 min, and then centrifuged  at 13000 rpm for 10 min at 4°C. The supernatant (400 µl) were pipetted out and lyophilized.The UPLC analysis was performed with a Waters Acquity™ Ultra Performance LC system  equipped with a Waters Xevo™ G2 QTof MS . Chromatographic separation was carried out at 45°C on an ACQUITY UPLC HSS (high strength silica) T3 column . The mobile phase consisted of water (A) and acetonitrile (B), each containing 0.1% formic acid. The optimized UPLC elution conditions were: 0–0.5 min, 1% B; 0.5–3.5 min, 1–35% B; 3.5–7.0 min, 35–99% B; 7.0–8.0 min, 99% B and 8.0–10.0 min, 99.0–1.0% B. The flow rate was 0.45 ml/min. The autosampler was maintained at 4°C. The lyophilized serum samples were dissolved in 100 µL of distilled acetonitrile/water (4∶1). Every 2 µL sample solution was injected for each run.Mass spectrometry was performed on a Xevo™ G2 QTof , a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer. The scan range was from 50 to 1200 m/z. For both positive and negative electrospray modes, the capillary and cone voltage were set at 3.0 kV and 30 V, respectively. The desolvation gas was set to 800 L/h at a temperature of 450°C; the cone gas was set to 20 L/h and the source temperature was set to 150°C. The mass spectrometry was operated in W optics mode with 12,000 resolution using dynamic range extension. The data acquisition rate was set to 0.1 s, with a 0.1 s interscan delay. All analyses were acquired using the lockspray to ensure accuracy and reproducibility. Leucine–enkephalin was used as the lockmass at a concentration of 300 ng/mL and flow rate of 5 µL/min. Data were collected in continuum mode, the lockspray frequency was set at 10 s, and data were averaged over 10 scans. All the acquisition and analysis of data were controlled by Waters MassLynx v4.1 software.E analysis or interpreted with available biochemical databases, such as HMDB (http://www.hmdb.ca/), Chemspider (http://www.chemspider.com) and KEGG (http://www.kegg.com/). Other statistical analyses were used by one-way analysis of variance (ANOVA) and independent sample t-test. They were performed with SPSS 11.0 . Significant differences were considered significant when test P values were less than 0.05.The mass data acquired were imported to Markerlynx XS  within the Masslynx software for peak detection and alignment. All of the data were normalized to the summed total ion intensity per chromatogram, and the resultant data matrices were introduced to the EZinfo 2.0 software for principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) analyses. Metabolite peaks were assigned by MSP<0.01), but decrease of urinary volume was revealed in the FLP-treated group.P<0.01). These results demonstrate that the rat model exhibited typical pathologic features associated with CKD. Levels of BUN and Scr were significantly intervened by FLP (P<0.01).After day 14, blood samples were collected and BUN, Scr, cholesterol and triglyceride were determined. The results are given in P<0.01). These blood parameters showed that adenine can cause anemia symptom. FLP could increase RBC, HGB and HCT to the same extent although they could not increase RBC, HGB and HCT to the normal levels. These results demonstrate that the CKD was being prevented and alleviated, exhibiting a recovery via similarity to the healthy control group after taking FLP.The results of blood routine are given in High reproducibility is crucial for any analytical protocols, especially for metabonomics study which requires handling many samples. The precision and repeatability of the UPLC-MS method were validated by the reduplicate analysis of six injections of the same quality control samples and six parallel samples prepared using the same preparation method, respectively. The relative standard deviations (RSD) of retention time and peak area are below 0.45% and 3.4%, respectively. The resulting data showed that the precision and repeatability of the proposed method were satisfactory for metabonomic analysis. Metabolic profiling of serum samples was acquired using UPLC Q-TOF/MS system in the positive ion mode. The base peak intensity (BPI) chromatograms of serum samples from healthy control group, CKD group and FLP-treated group are shown in To determine whether the FLP was possibly to influence metabolic pattern of adenine-induced CKD rats and to find the metabolites with a significant concentration change , PLS-DA was carried out on the UPLC-MS data of serum samples. A PLS-DA model was built by 2112 variables. http://www.hmdb.ca/), Chemspider (http://www.chemspider.com/) and KEGG (http://www.kegg.com/) by masses and MSE data, and due to the possible fragment mechanisms, items without the given mass fragment information were removed from the candidate list and only the most probable items survived. MassLynx i-FIT algorithm is used to screen suggested elemental compositions by the likelihood that the isotopic pattern of the elemental composition matches a cluster of peaks in the spectrum, increasing confidence in identified compounds and simplifying results. The lower the i-FIT value, the better the fit. By comparing the retention times and mass spectra to the authentic chemicals, a part of the CKD-related metabolites were structurally confirmed.In the score plot, scattered points of various samples were classified into three groups, which suggested that proper CKD-related patterns could be revealed by the proposed PLS-DA model and serum metabolic pattern significantly changed after the treatment of FLP. The loading plot displayed 20 potential CKD-related metabolites according to their VIP (Variable Importance in the Projection) values. To identify these metabolites, we first searched candidates from the databases of HMDB , creatinine and lysoPE(22∶0/0∶0) were up-regulated, but palmitic acid, phytosphingosine, PC(16∶0/18∶2), tryptophan, lysoPC(20∶4), lysoPC(16∶1), lysoPC (16∶0) and valine were down-regulated compared with control rats. To evaluate protective effects of FLP, the intensity level of 14 biomarkers in the different groups was also analyzed  and valine, mean level of the key biomarkers was reversed to control at different degrees after oral administration , tetracosahexaenoic acid, lysoPC(18∶2), creatinine, lysoPC (16∶0) and lysoPE(22∶0/0∶0) have been identified. Combined with biochemistry and histopathology results, the changes in serum metabolites indicated pharmacological effects of FLP are related to phospholipids metabolism, energy metabolism and amino acids metabolism. The work shows that the metabonomics method is a valuable tool in drug mechanism research.


Campylobacter fetus bacteremia is rare and occurs mainly in patients with immunosuppression. This infection, which often involves secondary localizations has already been reported in some primary humoral immune deficiencies. We describe three cases of severe infection due to C. fetus with cellulitis at presentation, but without any gastrointestinal symptoms, occurring in patients with acquired hypogammaglobulinemia. Campylobacter fetus represent only a small proportion of Campylobacter infections, usually responsible for acute diarrhea. C. fetus bacteremia is relatively rare and occurs mainly against a background of immunosuppression  [Infections due to atients) , 2. Thisatients) , 4. C. fetus occurring in immunosuppressed patients with acquired hypogammaglobulinemia.We describe three cases of severe infection due to C. fetus was isolated from blood cultures, but not from the fecal culture. Azithromycin was initiated for a total duration of 2 weeks. One month later, the patient presented with a novel septic state, this time associated with abdominal cutaneous cellulitis. C. fetus was again recovered from blood cultures (5/5 bottles). Initial antibiotherapy (piperacillin-tazobactam) was again replaced by azithromycin for a duration of 4 weeks. Substitution treatment with monthly intravenous immunoglobulins (IVIg) was initiated, and a residual level of 6 g/L was obtained. No relapse was noted after more than 12 months of followup.A 77-year-old diabetic man, with chronic lymphocytic leukemia (not treated), was hospitalized for sepsis without any obvious clinical source, notably no diarrhea syndrome. He received empiric antibiotherapy with a combination of ceftriaxone and metronidazole. Apart from known hyperlymphocytosis (24 giga/L), the rest of his hemogram was unremarkable. There was clear hypogammaglobulinemia: IgG = 3.2 g/L  and IgA = 0.4 g/L . C. fetus. The infection responded rapidly to combined antibiotherapy (piperacillin-tazobactam and azithromycin) for a duration of 6 weeks. No relapse was noted after a followup of 19 months.A 58-year-old man with SHARP syndrome (mixed connective tissue disease with predominant autoimmune myositis) and hypogammaglobulinemia secondary to immunosuppressive treatment  was hospitalized for sepsis associated with cellulitis of the left upper arm, without any obvious clinical source. His hemogram was unremarkable (neutrophils = 16 giga/L). The patient had already received monthly IVIg substitution for 4 years and had an IgG level of 6 g/L and IgA of 1.05 g/L at the time of appearance of the infection. Blood cultures (5/5 bottles) were positive for C. fetus (2/2 bottles). She was successfully treated with ceftriaxone 2 g/day followed by oral ciprofloxacin (1000 mg/day) for a total duration of 3 weeks. Treatment with IVIg was continued with the aim of achieving a residual level of >6 g/L, but at 2 months she presented with a new episode of severe sepsis due to Escherichia coli leading to death.A 71-year-old woman, followed for several years with mixed cryoglobulinemia complicating low-grade lymphoma, was hospitalized for sepsis at 1 month after her first course of chemotherapy (rituximab-cyclophosphamide and dexamethasone). The chemotherapy was rapidly followed by the development of infectious complications including pneumopathy and then otitis due to Pneumococcus. Severe hypogammaglobulinemia (IgG = 2.89 g/L and IgA = 0.64 g/L) led to substitution treatment with IVIg. One month later, in the absence of neutropenia, she presented with cellulitis of the left leg with reactional arthritis of the homolateral knee (IgG = 5.25 g/L at that time). Blood cultures were positive for C. fetus bacteremia developing in the setting of acquired immunosuppression with hypogammaglobulinemia. These “opportunistic” infections are a real concern for the clinician since Campylobacter bacteremia often involves extraintestinal localizations [C. fetus, which is not the most common Campylobacter species identified in the general population, is more often found in immunosuppressed patients where it represents more than one-half of cases [Campylobacter) [We report three cases of severe izations , 2, 5. CC. fetus postthymectomy [Campylobacter species [Campylobacter infections, and measurement of immunoglobulins in these patients revealed undetectable levels of IgA and IgM [C. fetus infections due to the important role of these immunoglobulins in the anti-infectious defense of the body at the level of the digestive mucous membranes. Since the introduction of tritherapy, the number of cases reported in connection with HIV has been superseded by those occurring in other types of immunosuppression, particularly in subjects with humoral immune deficiencies. The first case reported in the literature concerned a 50-year-old patient with Good's syndrome who presented with bacteremia due to ymectomy . Other s species . More re and IgM . This hyC. fetus, yet digestive symptoms such as diarrhea are not always present (less than one patient in two). This may divert the clinician away from the diagnosis and explains in part why the diagnosis is most often made from blood cultures and not from fecal cultures [C. fetus infection reported in the literature [Campylobacter species) [The digestive tract is the usual route of contamination by cultures , 5. Thisterature , 5. In tspecies) . These dC. fetus is often resistant to quinolones and macrolides, and some authors recommend the use of imipenems as first-line treatment [C. fetus was resistant to imipenems, while 32% were resistant to fluoroquinolones, and no patient on imipenem had died at 30-day followup [Campylobacter species [The evolution of the patients in terms of mortality depends on the underlying disease and also on the initial choice (adapted or not) of antibiotherapy. reatment , 2. In tC. fetus readily recurs with atypical symptoms and requires prolonged antibiotherapy [Bacteremia due to otherapy , 5. Multotherapy . Some stC. fetus infections in the context of hematological or rheumatologic conditions with acquired hypogammaglobulinemia. Actually, C. Fetus bacteremia should be suspected among any immunocompromised patients with gastrointestinal symptoms, as well as among those presenting with cellulitis but no diarrhea.Our three cases illustrate both the atypical presentation with absence of gastrointestinal symptoms and the potential risk of severe


A) is measured by the dilution of an inert gas during a single breath-hold maneuver. Such measurement is sensitive to ventilatory disturbances. We conducted a prospective, observational study to establish the prognostic value of VA in systolic HF.Ventilatory impairment is known to occur in patients with heart failure (HF). Alveolar volume (VCO) by the single-breath method. We also measured the cardiothoracic ratio on frontal chest radiographs, and the circulating levels of N-terminal pro-hormone of B-type natriuretic peptide (NT-proBNP). The hazard ratio (HR) of death was estimated with Cox regression, and the percentiles of survival time with Laplace regression. For survival analysis, VA was categorized as < 80% (n = 135), or ≥ 80% of the predicted value (n = 125).We studied 260 consecutive patients who were hospitalized for systolic HF. All patients were evaluated under stable clinical conditions, before hospital discharge. Lung function studies included spirometry and determination of the lung diffusing capacity for carbon monoxide . After adjusting for age, New York Heart Association class III-IV, cardiothoracic ratio >0.5, NT-proBNP, persistent atrial fibrillation, DLCO, COPD comorbidity, use of beta-blockers and angiotensin converting enzyme inhibitors, the HR decreased to 1.9 but remained statistically significant (p = 0.039). Two percent of the patients with VA < 80% died about 0.9 years earlier than those with VA ≥ 80% (p = 0.033). The difference in survival time at the 20th percentile was 0.8 years.Follow-up had a median duration of 2.7 years . The crude mortality rate was 27% in the whole sample, 36% in patients with VA is a significant, independent predictor of reduced survival in patients with systolic HF.V Heart failure (HF) is a common clinical disorder carrying significant morbidity and mortality,2. VentiAs of now, most of the studies on the prognostic impact of respiratory dysfunction in HF focused on simple spirometry-14, withA) is the volume of air in the lung available for gas exchange, and is currently measured by the dilution of an inert gas during a single breath-hold maneuver[A equals total lung capacity (TLC) measured by multiple-breath helium dilution. In subjects with ventilatory impairment, VA is often much lower than TLC because of the insufficient mixing of gas in alveolar spaces[Alveolar volume . Before entering the study, the subjects provided an informed written consent.The study sample comprised 260 consecutive patients with an established diagnosis of systolic HF. They were hospitalized at the CNR Institute of Clinical Physiology and "G. Monasterio" Foundation, Pisa, Italy, between January 1, 2000 and December 31, 2007. The diagnosis of HF was based on the Framingham criteria, and the1), and total lung capacity (TLC). At least three spirometric measurements were obtained and the highest values were chosen. Spirometry was performed by experienced technologists in conformity with the ATS/ERS standards[1/SVC below the 5th percentile of predicted value)[1 was > 80%, between 80 and 50%, and < 50% of predicted, respectively. Ventilatory restriction was diagnosed if FEV1/SVC ratio was normal, and TLC below the 5th percentile of predicted[CO) was determined using the single-breath method in conformity with ATS/ERS recommendations[A was measured during the single-breath maneuver using helium as the inert gas[All the patients were evaluated under stable clinical conditions, shortly before hospital discharge. Lung function studies included the measurement of slow (SVC) and forced vital capacity (FVC), forced expiratory volume in one second . The degredicted. The difndations. VA was nert gas.Postero-anterior and lateral digital chest radiographs were obtained in all the subjects at the time of lung function testing, and were examined by two of the authors  for the presence of heart, pulmonary, and pleural abnormalities. On the postero-anterior view, we also measured the cardiothoracic ratio. This was regarded as abnormal if > 0.5. We measured the circulating levels of hemoglobin, creatinine, and N-terminal pro-hormone of B-type natriuretic peptide (NT-proBNP). The latter was measured by an electrochemiluminescent sandwich immonoassay using Elecsys 2010 analyser . MedicatThe 260 patients were followed up until death or March 31, 2009, whichever occurred first. All the subjects were seen periodically at the outpatient clinic of our institution. Whenever required, their family physicians were also contacted. The cause of death was established by reviewing medical records, autopsy findings, or death certificates.A and survival was evaluated with two methods: Cox proportional hazard regression to estimate hazard ratio (HR) for the risk of death, and Laplace regression to estimate the percentiles of survival time[A which was categorized as < 80% or ≥ 80% of the predicted value. Based on univariate analysis, the following potential confounders were considered: tertiles of age , New York Heart Association (NYHA) class III-IV, cardiothoracic ratio > 0.5, DLCO as % predicted , tertiles of NT-proBNP , persistent atrial fibrillation, COPD comorbidity, use of beta-blockers, and of angiotensin converting enzyme (ACE) inhibitors. The crude and adjusted percentiles, and the hazard ratios and associated 95% confidence intervals (CI) are reported. The proportionality of the hazard in the Cox regression was tested based on the Schoenfeld residuals for each model separately. Two-tailed p-values < 0.05 were considered statistically significant throughout. The statistical analysis was performed with Stata version 11 .Differences between groups were assessed by Fisher’s exact test for the categorical variables, and by Mood’s median test for the continuous variables. Continuous variables in the text and in the tables are reported as median and interquartile range (IQR). The association between Vval time. Survivaval time. The maiA was 79% of predicted . There was a highly significant, inverse correlation of VA with NT-proBNP  and cardiothoracic ratio , and a weaker borderline significant correlation with LVEF .The baseline characteristics of the study sample are given in Table A category. As compared with patients having VA ≥ 80%, those with VA < 80% featured the following statistically significant differences: (a) older age; (b) higher prevalence of NYHA class III-IV, abnormal cardiothoracic ratio, persistent atrial fibrillation, COPD comorbidity, and ventilatory restriction; (c) lower FEV1 and DLCO; (d) higher levels of NT-proBNP. As regards medical treatment, significantly less patients with VA < 80% were prescribed beta-blockers and ACE-inhibitors, and significantly more were on oral anticoagulants than those with VA ≥ 80%.Table A < 80% and in 3% of those with VA ≥ 80% (p < 0.001). Similarly, small pleural effusions prevailed significantly in the lower VA category than in the other , whereas no statistically significant difference was observed as regards the prevalence of radiologic signs of emphysema . None of the sampled patients had evidence of overt lung fibrosis.Radiologic signs of interstitial lung edema were detected in 16% of the patients with VA < 80%, and 16% (20/125) in those with VA ≥ 80%. The causes of death were: heart failure (n = 44), cancer (n = 9), respiratory failure (n = 5), sudden death (n = 4), liver failure (n = 2), sepsis (n = 2), multiple trauma (n = 2), and renal failure (n = 1).Follow-up was completed in all the patients, and had a median duration of 2.7 years . The crude mortality rate was 27% (69/260) in the whole sample, 36% (49/135) in patients with VA < 80% than in the others (p = 0.032). The mortality curves are adjusted for age, NYHA class III-IV, cardiothoracic ratio >0.5, NT-proBNP, persistent atrial fibrillation, DLCO as % predicted, and COPD comorbidity.Figure A. The unadjusted HR of death in patients with VA < 80% was 2.3-fold higher than in those with VA ≥ 80% (p = 0.002). After adjusting for potential confounders, the HR of death decreased to 1.9 but remained statistically significant (p = 0.039).Table A category are displayed in Figure CO as % predicted, and COPD comorbidity. Two percent of the patients with VA < 80% died about 0.9 years earlier than those with VA ≥ 80% (p = 0.033). The difference at the 20th percentile was 0.8 years. So, the frailest were the most affected individuals by reduced VA.Laplace regression estimates of the percentiles of survival by VA, as percent of the predicted value, is a prognostic indicator in systolic HF. Our results can be summarized thus: (a) the cumulative incidence of death is significantly higher in patients with VA < 80% predicted than in those with VA ≥ 80%; (b) the estimated HR of death is about two-fold greater among the patients in the lower VA category, and remains significantly higher after adjusting for relevant confounding variables; (c) as indicated by Laplace regression, reduced VA is strongly associated with early deaths.The present study was designed to establish whether VA and cardiothoracic ratio. This suggests that heart enlargement is a major determinant of reduced VA in chronic HF. Generalized stiffness of the lung parenchyma may also contribute to diminish VA. Such reduced lung distensibility is due to chronic interstitial edema, and pulmonary vascular remodeling that are known to occur in long-standing HF[We found a significant, inverse correlation between Vnding HF.A is significantly correlated with the circulating levels of NT-proBNP, an established prognostic biomarker of HF[A remains statistically significant so indicating that VA is an independent predictor of survival.We also observed that Ver of HF. After aA number of studies investigated on simple spirometry as a potential indicator of future cardiovascular events. In the setting of the Framingham Study, Kannel et al. observed that FVC is a significant, independent predictor of cardiovascular morbidity and mortality in allegedly healthy subjects. Later, 1 < 50% of predicted value) appeared to be a predictor of reduced survival [1 has independent prognostic value [In recent years, there has been increasing interest in evaluating the prognostic impact of COPD comorbidity in patients with chronic HF. Mascarenhas et al. conducteto 4.22). In 527 < 0.001). By cont< 0.001). The thr1 – be it due to airflow obstruction or ventilatory restriction – is a significant, independent predictor of reduced survival in 439 patients with systolic HF[1 <80% predicted as compared with those having FEV1 ≥80%[Recently, Miniati et al. reported that a reduction of FEVtolic HF. In thatEV1 ≥80%.CO at two different concentrations of oxygen in the inspired test bolus[As of now, limited data is available on the prognostic relevance of lung function tests other than spirometry. It is well established that chronic HF brings about extensive remodeling of the pulmonary intra-alveolar vessels and interstitium, which mst bolus,16. ThisA represents an estimate of the lung gas volume into which CO is distributed and, therefore, is critical in the measurement of DLCO with the single-breath approach[A primarily reflects the volume of the airspaces into which the tracer gas rapidly equilibrates during the breath-hold time[A <80% predicted. So, we believe that measuring VA may add valuable information as regards lung function assessment in patients with systolic HF.Vapproach. In the old time. The estA affect the lung diffusing capacity, including the physical properties of the alveolar membrane, the density of alveolar capillaries, and the hemoglobin concentration[CO among the potential confounding variables. After adjustment, the HR of death associated with the lower VA category remained statistically significant.Undoubtedly, factors other than Vntration. Thus, iA in HF. Third, we obtained a single determination of VA. So, it would of interest to evaluate the effect of cardiovascular and pulmonary medications on VA size over time, and to test whether improvement in VA is associated with a better oucome in patients with chronic HF.First, we only studied patients with systolic HF, so our findings may not apply to patients with isolated diastolic heart dysfunction. Second, our data originated from a single referral center, and included a relatively small number of cases. Broader multicenter prospective studies are needed to firmly establish the prognostic value of VA, as percent of the predicted value, is a significant, independent predictor of reduced survival in patients with systolic HF.The present study indicates that VThe authors declare that they have no competing interests.Study design (MM). Data collection . Data analysis . Drafting the manuscript (MM). Approval of the final version of the manuscript .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/69/prepub


Hypoxic tumors are refractory to radiation and chemotherapy. High expression of biomarkers related to hypoxia in head and neck cancer is associated with a poorer prognosis. The present study aimed to evaluate the clinicopathological significance of erythropoietin receptor (EPOR) expression in oral squamous cell carcinoma (OSCC).The study included 256 patients who underwent primary surgical resection between October 1996 and August 2005 for treatment of OSCC without previous radiotherapy and/or chemotherapy. Clinicopathological information including gender, age, T classification, N classification, and TNM stage was obtained from clinical records and pathology reports. The mRNA and protein expression levels of EPOR in OSCC specimens were evaluated by Q-RT-PCR, Western blotting and immunohistochemistry assays.p < 0.001), advanced TNM stage (p < 0.001), and positive N classification (p = 0.001). Furthermore, the univariate analysis revealed that patients with high tumor EPOR expression had a lower 5-year overall survival rate (p = 0.0011) and 5-year disease-specific survival rate (p = 0.0017) than patients who had low tumor levels of EPOR. However, the multivariate analysis using Cox’s regression model revealed that only the T and N classifications were independent prognostic factors for the 5-year overall survival and 5-year disease-specific survival rates.We found that EPOR were overexpressed in OSCC tissues. The study included 17 women and 239 men with an average age of 50.9 years . The mean follow-up period was 67 months . High EPOR expression was significantly correlated with advanced T classification (High EPOR expression in OSCC is associated with an aggressive tumor behavior and poorer prognosis in the univariate analysis among patients with OSCC. Thus, EPOR expression may serve as a treatment target for OSCC in the future. In 2007, 22,560 new cases of oral cancer were reported in the United States, with approximately 5,370 deaths attributed to this disease . The protein concentration in each sample was estimated using a Bio-Rad Protein Assay . Immunoblotting was performed according to standard procedures. The antibodies included polyclonal antibodies against EPOR and β-actin . The first antibodies were detected by incubation with secondary antibodies conjugated to HRP  and developed using Western Lighting chemiluminescence reagent . X-ray films were used to detect the proteins.Tissue protein extraction was carried out in frozen samples that were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer [50 m2O2 for 30 min to block endogenous peroxidase activity. Next, the sections were microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. After antigen retrieval, the sections were incubated with diluted anti-EPOR antibody  for 1 h followed by washing with PBS. Horseradish peroxidase/Fab polymer conjugate  was then applied to the sections for 30 min followed by washing with PBS. Finally, the sections were incubated in diaminobenzidine for 5 min to develop the signals. A negative control that omitted the primary antibody was run simultaneously. Positive immunostaining was defined as cytoplasmic and/or membrane immunoreactivity. The level of reactivity in the immunostained tissues was evaluated independently by two pathologists who were blinded to the subjects’ clinical information, and scored into four groups according to the percentage and intensity of cytoplasmic and membrane staining. Specimens in which >30% of the cells were stained were scored as strongly positive (+++), specimens in which 10–30% of the cells were stained were scored as moderately positive (++), and those in which <10% of the cells were stained were scored as weakly positive (+). Specimens with no staining were scored as negative (−) [Adjacent noncancerous and OSCC tissue samples were selected by a pathologist based on diagnosis and microscopic morphology. The tissues were fixed in 10% buffered formalin, then embedded in paraffin and decalcified in 10% EDTA solution. Representative blocks of the formalin-fixed, paraffin-embedded tissues were cut into 4-mm sections and deparaffinized with xylene and rehydrated in a series of ethanol washes . Slides were washed with phosphate-buffered saline (PBS) and treated with 3% Htive (−) . A high p-value less than 0.05 was deemed to be statistically significant. The clinicopathological variables and the expression of EPOR were taken into account for the analysis of survival based on the Kaplan-Meier method; statistical significance, defined as a p < 0.05, was assessed using the log-rank test. To determine the effect of particular prognostic factors on survival, a multivariate analysis was performed using Cox’s regression model.Several clinicopathological factors were evaluated, including gender, age (59 versus 60 years), T classification , N classification (negative versus positive), and TNM stage . Fisher’s exact test was used to evaluate the correlation between the clinicopathological variables and EPOR expression. A The study included 239 men and 17 women with an average age of 50.9 years . Of the 256 patients, 39 were classified as T1, 55 as T2, 64 as T3, and 98 as T4. The N classification was N0 in 153 patients (59.8%), N1 in 38 (14.8%), N2b in 48 (18.7%), N2c in 13 (5.1%), and N3 in 4 (1.6%) patients. The cancer stage was classified as I in 32 patients (12.5%), II in 34 (13.3%), III in 63 (24.6%), and as stage IV in 127 (49.6%) patients (Table p < 0.001), positive N classification (p = 0.001), and an advanced TNM stage (p < 0.001).EPOR immunoreactivity was observed in the neoplastic epithelial cells of the OSCC Figure . Late-stEPOR expression was measured in 12 paired samples of tumor tissue and adjacent non-tumor oral tissue (from early to late stage) using Q-RT-PCR. EPOR mRNA was overexpressed in all OSCC tissue samples, indicating a higher level of EPOR in the tumor tissue than in the adjacent non-tumor oral tissue after normalization using GAPDH Figure . To confp = 0.0011; Figure p = 0.0017; Figure p < 0.001; Tables p = 0.015) and 5-year disease-specific survival rate .The TNM staging system is used to evaluate and predict the prognosis of patients with head and neck SCC (HNSCC). Clinicians develop therapeutic strategies according to the TNM stage of patients. Biomarkers that could reliably predict tumor behavior would be useful in the clinical setting. The clinical value of biomarkers related to angiogenesis and hypoxia in patients with HNSCC had been established in recent years -36. ThesHigh EPOR expression in patients with OSCC was associated with tumor progression in the present comprehensive study. These results suggest that EPOR expression is an important prognostic factor for OSCC. Thus, EPOR expression may serve as a treatment target for OSCC in the future.The authors declare that they have no competing interests.YTL, HCC, CHC, GLA, and CYC: collected the clinical data of patients, conceived the study design, carried out and coordinated immunohistochemical examinations of tumor specimens, and drafted the manuscript. HKC and CCH: participated in the interpretation of data and conducted immunohistochemistry analysis. CHC: performed the IHC staining and biochemical experiments. FMF: performed statistical data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/194/prepub


Diabetes mellitus (DM) and intermittent claudication (IC) are frequently associated health conditions. Our hypothesis is that the nature, severity and quality of life (QoL) of patients with IC and DM are worse than those of claudicant patients without diabetes.An observational, cross-sectional and multicentre study of 920 patients with IC, divided into two groups: diabetic (n = 477) and non-diabetic (n = 443). For each group, we examined clinical and biological characteristics (including levels of glucose and lipids), the ankle-brachial index (ABI), responses to the Walking Impairment Questionnaire (WIQ) and the European Quality of Life-5 Dimensions (EQ-5D) questionnaire.Compared with claudicant patients without diabetes, claudicants with diabetes were older (p < 0.001), more likely to be female (p = 0.006), with a higher BMI (p < 0.001), more likely to have a sedentary lifestyle (p < 0.001) and to be a non-smoker (p < 0.001). Claudicant patients with diabetes also had significantly more cardiovascular risk factors (p < 0.001), more frequent ischaemic cardiopathy (p = 0.023) and chronic renal failure (p = 0.002), and fewer prior ictus events (p = 0.003). No significant differences between groups were found with respect to blood pressure, levels of cholesterol or triglycerides. The mean ABI of diabetic-IC patients was slightly lower than IC patients without diabetes (p = 0.016). All WIQ subdomains scores were significantly lower (p < 0.001), indicating poorer walking ability, in claudicant and diabetic patients with compared with those without diabetes. The mean E5-QD global scores and the mean EQ-5D visual analogue scale in the whole series were 0.58 (SD = 0.21) and 55.04 (SD = 21.30), respectively. Both E5-QD scores were significantly lower, indicating poorer QoL, in claudicant patients with diabetes than claudicant patients without diabetes (p < 0.001).Patients with IC and DM had more risk cardiovascular factors, cardiovascular conditions, disability and worse haemodynamic status and QoL than claudicant patients without diabetes. Peripheral arterial disease (PAD) of the legs has a range of different clinical presentations, from pain with walking  to gangrene. IC is always accompanied by different levels of incapacity and morbidity-mortality (cardiovascular disease-associated) that significantly impair patients’ quality of life (QoL). The prest century”. The prevalence of DM for all age groups worldwide was estimated to be 2.8% (171 million) in 2000, a figure that was predicted to double over the subsequent two decades[Diabetes mellitus (DM) is considered the “epidemic of the 21 decades. DM is a decades. Accordi decades.Despite these dramatic figures, there is surprisingly little information about diabetic claudicants, especially if we compare this with other phases  or the process of PAD (diabetic foot) Table . In the An observational, prospective, cross-sectional and multicentre study (covering the entire country) was undertaken between May and December 2011. The study was approved by the Scientific and Ethics Committee of the Clinical Hospital, Barcelona, Spain. The study was carried out by vascular surgeons, members of the Spanish Society for Angiology and Vascular Surgery. Each participating investigator included 3–5 consecutive patients with IC (with or without associated DM).ad hoc committee. All VS participants are active members of the Spanish Society of Vascular Angiology and Surgery and were selected according to predefined selection criteria: a) involvement in our Society, and b) even geographical distribution. None of the VS invited refused to participate in the study.1,192 patients with IC at least one year before the start of study were recruited from the entire Spanish territory by 356 vascular surgeons (VS). VS participants were invited, via a letter, by an The IC diagnosis was ensured by medical history (including Edinburgh questionnaire), physical exploration and Ankle-brachial Index. All incFor each patient, the investigator completed a Clinical Questionnaire (CQ) covering three areas: 1) demographics and clinical data; 2) blood pressure and laboratory tests (glycaemia and lipids levels); and 3) ankle-brachial index (ABI). Additionally, each patient included in the study filled in two questionnaires: the Walking Impairment Questionnaire (WIQ) and the European Quality of Life-5 Dimensions (EQ-5D) questionnaire.A portable Doppler apparatus was used (8 MHz probe). Techniques were applied according to the recommendations of the American Heart Association[WIQ is a brief, easy-to-complete, disease-specific questionnaire specifically designed for assessing the consequences of IC. WIQ hasEQ-5D was designed by a European group. It is a2 or Fisher´s exact test. Fisher’s exact test was performed when expected percentages were less than 5.All data were anonymously documented to ensure confidentiality, and collated in a PASW version 18 . Continuous variables were summarised as the mean and standard deviation (SD); discrete variables were expressed as percentages. Continuous variables with a Gaussian distribution were compared using unpaired t-tests, while those with a non-Gaussian distribution were compared using the Mann–Whitney test. Discrete variables were compared by the χOf the 1192 patients with IC included in the study, 272 were removed (148 and 124 in the diabetic and non-diabetic groups respectively) because their data were incomplete . The sample was divided into two groups: with and without diabetes mellitus   and chronic renal failure . Other cardiovascular diseases  were similarly less frequent in both groups.The most common cardiovascular disease associated with IC was ischaemic cardiopathy . Compared with non-diabetic claudicants, those with diabetes more frequently exhibited ischaemic cardiopathy .vs. 35.7%, p < 0.001). Lumbar pathology and other osteoarticular comorbidities  occurred at similarly lower frequencies in the two groups.The most common osteoarticular comorbid conditions were arthrosis (45.8%) and lumbar pathology (16.5%). Arthrosis was significantly more frequent in IC with than without diabetes .No significant differences between the groups were found with regard to blood pressure, levels of cholesterol and triglycerides. The mean ABI of the total series was 0.63 (SD = 0.19). The ABI was significantly lower in claudicant patients with than without diabetes . An ABI less than 0.50 was detected in 170 claudicant patients, such values being more frequent amongst those with diabetes than those without .WIQ and EQ-5D scores are shown in Table The mean E5-QD global score and the mean EQ-5D visual analogue scale in the overall series were 0.58 (SD = 0.21) and 55.04 (SD = 21.30), respectively. Both E5-QD scores were significantly lower, indicating poorer QoL, in claudicant patients with diabetes than in those without diabetes (p < 0.001).Our results indicate that claudicant patients with diabetes have some distinct clinical characteristics as well as more severe disease and poorer QoL than claudicant patients without diabetes.The medical-social and economic consequences of PAD and IC are very important, as is apparent from the extensive bibliography. PAD is a major public health problem in western countries. IC is a common, chronic disease that can cause injuries throughout a substantial portion of a patient’s life. The true prevalence of PAD in people with diabetes has been difficult to determine because most patients are asymptomatic. Population-based studies have revealed a prevalence of PAD in people with diabetes of up to 30%. PAD is The characteristics of our series are quite similar to those of other series examined throughout the world,17. DiabAs other authors have observed,20, we fThe mean ABI in our total series was 0.63 (SD = 0.19). Similar mean ABIs have been found in previous studies -24. The However, the most outstanding feature of ABI in the diabetic population is its diagnostic and prognostic value. ABI thrWalking deterioration measured by the WIQ in our series was reasonably consistent with the results of previous studies,9,22,28.It is well know that PAD is associated with a significant reduction in quality of life. Specifically, IC represents a deterioration in QoL compared with controls. AccordiAtherosclerotic disease, specifically angina pectoris, causes a greater deterioration of QoL in IC. As our Two systematic reviews of QoL and IC revealed that deterioration in QoL affected the physical domain more than the psychological and social domains,37. LoweOur study had several characteristics that differentiate it from other studies with similar aims: a) the large number of patients (n = 920 claudicants), which increases the reliability of our results; b) our study covered the whole country, thereby encompassing cultural variability; and c) the scoring of several parameters. Finally, the biggest potential limitation of our study was the lack of the treadmill test. However, PAD and WIQ are well correlated.In conclusion, patients with IC and diabetes had more risk cardiovascular factors, cardiovascular conditions, disability and a poorer haemodynamic status and QoL than claudicant patients without diabetes.The authors declare that they have no competing interests.Conception and design: FSL, JRM, EC, JML. Data collection: Grupo Saned. Statistical analysis of results: Grupo Saned, FSL. Analysis and interpretation of results: FSL, JRM, EC, JML. Writing up article : FSL, JRGP. Final approval of the article: FSL, JRM, EC, JRGP, JML, ER.


Facial cleft deformities, including cleft lip with or without cleft palate (CL/P) and cleft palate (CP), are common congenital birth anomalies, especially in Asia. This study aimed to analyze the prevalence of CL/P and CP and to identify associated factors in Taiwan.This population-based epidemiological study retrospectively analyzed birth data obtained from the Department of Health in Taiwan for years 2002–2009. Frequency distribution, percentages and related predictors were investigated, and findings were presented by types of cleft deformities. Logistic regression analysis was performed to identify factors associated with cleft deformities.Overall prevalence of cleft deformities among 1,705,192 births was 0.1% for CL/P and 0.04% for CP over the 8-year study period. Higher prevalence of CL/P or CP was observed with multiple pregnancies, being male for CL/P, being female for CP, gestational age ≤37 weeks and lower birth weight (<1.5 kg). Both CL/P and CP were significantly associated with gestational age <37 weeks and birth weight<1.5 kg . CL/P was significantly associated with multiple parities (P = 0.0004–0.002). Male newborns and female newborns were significantly associated with CL/P and CP, respectively (both P<0.0001).Overall prevalence for congenital cleft deformities in study subjects was 0.1%, in keeping with high rates in Asia. Results suggest the need for awareness and early identification of those at high risk for cleft deformities, including newborns with gestational age <37 weeks, weighing <1.5 kg at birth and women with multiple parities, as a potential strategy to counter long-term adverse effects on speech and language in this population. Facial cleft deformities, including cleft lip with or without cleft palate (CL/P) and cleft palate (CP), are among the most common congenital birth anomalies. While the worldwide prevalence of such deformities is about 1.5 per 1,000 live births, the rate varies six-fold for CL/P and three-fold for CP Even when newborns with cleft deformities receive appropriate treatment, some still have facial deformity and speech impairment We conducted a population-based epidemiologic study utilizing retrospective secondary database analysis of national data collected by the Taiwan Bureau of Health Promotion of the Department of Health, Executive Yuan from 2002 to 2009. The Executive Yuan collects health information from all hospitals, as well as other providers, to help guide the administration of public health policy in Taiwan. For this analysis, we used data from the Birth Registration Database Statistical analyses was done to examine the correlation of demographic characteristics of mothers and newborns with CL/P and CL. To examine the effect over time, the entire duration was divided into earlier and later periods (2002–2005 and 2006–2009), and the distribution of CL/P and CP was compared between the two groups using Chi-square test; data analyzed included maternal age , parity , newborn gender, gestational age  and birth weight . Count data were described using frequency and percentage. Logistic regression analysis was applied to identify factors associated with CL/P and CP. Statistical analysis was performed with SPSS version 18.0 software . A p value of <0.05 was established as statistical significance.The demographic and clinical characteristics of mothers and newborns are presented in We then categorized the information into two periods, 2002–2005 and 2006–2009, to determine the demographic characteristics related to prevalence of CL/P and CP, including mothers’ age, parity, gender, gestational age, and birth weight. Results from analysis are shown in Logistic regression analysis of the correlation between CL/P, CP and cohort demographic characteristics  showed tOur study characterizes the current national birth prevalence of CL/P and CP in Taiwan. We found overall birth prevalence of 0.1% for CL/P and 0.04% for CP over this 8-year period. Previous studies showed a worldwide prevalence of 0.1% for CL/P In our study, CL/P was more frequently found in and associated with male newborns. In addition to higher prevalence of cleft deformities, boys suffer greater psychosocial disruption from facial cleft deformities than girls In the present study, mothers with age ≥34 years were associated with an increased risk of having infants with a facial cleft deformity, but the association did not reach statistical significance. Other studies report similar results involving older mothers or parents We found that gestational age <37 weeks was associated with greater birth prevalence of facial cleft deformity, especially in the earlier cohort. Association of facial cleft disorders with low birth weight, premature birth or other developmental anomalies has led to calls for future research in prevention intervention and identifying genetic risk and predisposition In Taiwan, low birth weight of <1.5 kg was also significantly associated with facial cleft deformities. While birth weight is often not recorded in these studies, many find associated pregnancy complications  or other genetic anomalies in these infants, perhaps diagnosed prenatally Our study has certain limitations. The database did not provide information on maternal health or peri-conceptual conditions, which may play a role in development of facial cleft deformities In conclusion, overall prevalence for congenital cleft deformities in Taiwan was 0.1%, in keeping with high rates in Asia. Results of this study suggest the need for increased physician awareness, early identification of and possibleearly intervention for high risk mothers aged ≥34 years, those with multiple parities and those whose newborn weighed <1.5 kg at birth, which may be a potential strategy to counter long-term adverse effects on speech and language in this population.


Loxodonta africana), in relation to local and regional rainfall patterns.The identification of temporal thresholds or shifts in animal movement informs ecologists of changes in an animal’s behaviour, which contributes to an understanding of species’ responses in different environments. In African savannas, rainfall, temperature and primary productivity influence the movements of large herbivores and drive changes at different scales. Here, we developed a novel approach to define seasonal shifts in movement behaviour by examining the movements of a highly mobile herbivore . Piecewise regression models provided the best fit for elephant movement, which exhibited a segmented, waveform pattern over time. Major breakpoints in speed occurred at the end of the dry and wet seasons of each year. During the dry season, female elephant are constrained by limited forage and thus the distances they cover are shorter and less variable. Despite the inter-annual variability of rainfall, speed breakpoints were strongly correlated with both local and regional rainfall breakpoints across all three years. Thus, at a multi-year scale, rainfall patterns significantly affect the movements of elephant. The variability of both speed and rainfall breakpoints across different years highlights the need for an objective definition of seasonal boundaries.By using objective criteria to determine behavioural shifts, we identified a biologically meaningful indicator of major changes in animal behaviour in different years. We recommend the use of such criteria, from an animal’s perspective, for delineating seasons or other extrinsic shifts in ecological studies, rather than arbitrarily fixed definitions based on convention or common practice. The study of animal movement patterns allows ecologists to determine the distribution of species both in space and time, and the factors that influence their movements in different environments Cervus elaphus) Alces alces) Rangifer tarandus) Loxodonta africana) Recent advances in animal-mounted GPS technology has increased the availability of fine-scale animal movement data, thus enhancing our ability to better understand patterns in animal movement behaviour Large herbivores are highly mobile and able to cover large distances across the landscape. Since they encounter forage resources at various levels of spatial and temporal scale In this study, we focus on defining broad-scale temporal changes in the movement behaviour of female elephant. The African elephant is considered to be a keystone species in African savanna systems, since elephant foraging behaviour affects various ecosystem processes Because of the broad-scale effects of rainfall on both water availability and vegetation phenology and biomass Elephant capture & handling was conducted in strict accordance with ethical standards. Specific approval for this particular research project was obtained through the University of KwaZulu-Natal Animal Ethics sub-committee . This research also forms part of a registered and approved SANParks project, in association with Kruger National Park and Scientific Services (Ref: BIRPJ743).2, in the north-eastern Lowveld region of the South Africa. Our study area covers the southern, central and western regions of KNP and includes the associated private reserves, since elephant are able to move freely between these areas  and associated private reserves along the western boundary , extend across an area of approximately 21,281 kmse areas . VegetatThe elephant population in KNP was estimated to be ∼ 14,000 individuals during 2010 . From 2006 to 2010, we collected geographical location data, downloaded from GPS/GSM Collars , fitted to 17 elephant cows from different herds. To ensure the independence of sampling, a single female in each herd was selected and collared. The movements of these collared females are thus assumed to represent the movement behaviour of the breeding herd to which they belong To examine the temporal scale over which elephant movement behaviour changed, we calculated the mean daily speed (km/h) and the variance (standard deviation) associated with the speed. To calculate these variables, we computed step-lengths at 30 min intervals using Hawth’s Tools tY is the random variable representing the elephant movement at day t, t]E[Y is its expected value to be expressed as a segmented function of day with parameters ψ and b. More specifically, ψ represents the breakpoints, i.e. the days where the elephant movement changes, and b regulates the slopes in the different time periods. tZ is the generic numeric covariate representing the time index, which has a piecewise linear relationship with the response variable, t.YWe analysed the daily time series for each elephant separately using a piece-wise linear or segmented regression model (hereafter PRM), since this provides a useful method for determining ecological thresholds t-value, .The analysis of elephant movement patterns revealed that in all cases, the Piecewise Regression Model (PRM) provided a better fit than the Generalised Linear Model (GLM). The movement patterns of female elephant exhibited a distinct waveform trend over the three-year period, with behavioural changes occurring at the transition between both the wet and dry season, and the dry and wet season. Thus we obtained dry-wet season breakpoints at the troughs within the model, (hereafter referred to as ‘lower’ speed breakpoints) and wet-dry season breakpoints at the peaks (hereafter referred to as ‘upper’ speed breakpoints) see . Althougth of June and the 7th of July, this elephant increased her average speed on 11 of the 23 days (>0.5km/h). Examination of AM108’s movement pattern during this period indicated that she was in close association with AM106 within the south of Kruger from the 15th until the 19th of June, where after she began moving rapidly and directly, across Sabi Sand Reserve, and into the central region of Kruger, covering a distance of approx. 50 km in 3 days (19th–21st June). From this point, she moved further into the northern section of her range. AM108’s increased movements may be the result of a disturbance event In all years, the relationship between both speed and variance of speed, and day of year was negative during the period between the wet and dry season, followed by a breakpoint at the dry-wet season boundary. Beyond this, as the wet season commenced, the relationship was positive, up until a breakpoint at the end of the wet season  in 2007 (95% CI ∼ 128 days), day 614 (approx. 5th September) in 2008 (95% CI ∼ 160 days), and day 950 (approx. 7th August) in 2009 (%95 CI ∼ 138 days) (th November) in 2007 (95% CI ∼ 41 days). There were only 2 breakpoints in 2008, for collars AM306 and AM307)  (95% CI ∼ 1 day). In 2009, breakpoints occurred on day 754 (approx. 23rd January) (95% CI ∼ 16 days)  . Upper b16 days) .th August) in 2007 (95% CI ∼ 93 days), day 543 (approx. 26th June) in 2008 (95% CI ∼ 115 days), and day 956 (approx.13th August) in 2009 (95% CI ∼ 121 days) (th December) in 2007 (95% CI ∼ 90 days). There was only one breakpoint in 2008, for the collar AM306 . In 2009, breakpoints occurred on day 764 (approx. 2nd February) (95% CI ∼ 41 days)  and wet-dry season breakpoints at the peaks  see . Major l21 days) . Local rays) see .th August) in 2007 (95% CI ∼ 102 days); day 561 (approx. 14th July) in 2008 (95% CI ∼ 43 days); and day 962 (approx. 19th August) in 2009 (95% CI ∼ 60 days), (th November) in 2007 (95% CI ∼ 83 days), no upper breakpoints for regional rainfall occurred in 2008; and in 2009, breakpoints occurred on day 742 (approx. 11th January) (95% CI ∼ 8 days)  occurred, on average, on day 222 (approx. 100 days), . Major r 8 days) .We found that, overall, 35% of elephant increased their speed before the rainfall breakpoint, while 65% increased their speed after this breakpoint. When viewed separately, upper and lower breakpoints showed different trends: Lower breakpoints (dry to wet season transition): 29% of elephant increased their speed before lower rainfall breakpoints, 71% after see ; Upper bBivariate correlations revealed a strong positive relationship between speed, and both local  and regional  rainfall breakpoints. However, when we used ‘year’ and ‘cluster’ as controls in a partial regression analysis, we found slightly lower, but still significant correlations: speed and local rainfall breakpoints; r = 0.752, P<0.01 and speed and regional rainfall breakpoints; r = 0.828, P<0.01. For upper breakpoints (wet to dry season transition), we found very little effect of either ‘year’ or ‘cluster’: speed and local rainfall: r = 0.913; speed and regional rainfall: r = 0.972. However, for lower breakpoints (dry to wet season transition), ‘year’ had a confounding effect on speed and rainfall breakpoints . ‘Cluster’, as a second control, did not appear to affect the breakpoints .It is widely acknowledged that, within savanna environments, elephant movements are affected by seasonal changes in rainfall In general, elephant in KNP increased their speed during the wet season (summer months) up until a maximum threshold point at the onset of the dry season, following which their speed reduced, until a minimum threshold point at the end of the dry season. Beyond this point, at the onset of the wet season, their speed increased once more see . The lowThere is a strong inter-annual variability in rainfall patterns in savannas, where cycles of above- and below-average rainfall occur in different years Rainfall directly affects primary productivity in savanna systems Although abiotic factors such as rainfall, snowfall or temperature may be informative when examined alongside movement patterns e.g. , in isolThis study provides a unique method for identifying appropriate temporal breakpoints over broad scales, which can be applied to other species, in particular large mammals capable of carrying GPS collars. Within this framework, variables that may influence the movement of an animal  can be examined alongside movement patterns, in order to examine potential interactions. Although we have used a broad-scale approach, other temporal scales , or a multi-scale approach may be applied. Thus the methods used in this study provide useful tools that can be used to extract a comprehensive description of the animal’s movement behaviour, in association with important variables.Appendix S1Table 1 Results for speed, local rainfall and regional rainfall breakpoints from all collars, obtained using multi-year piecewise regression models.(DOC)Click here for additional data file.Appendix S2Table 1 Average speed, local rainfall and regional rainfall breakpoints for all collars, obtained using multiyear piecewise regression models.(DOC)Click here for additional data file.


Intensity modulated radiotherapy for head and neck cancer necessitates accurate definition of organs at risk (OAR) and clinical target volumes (CTV). This crucial step is time consuming and prone to inter- and intra-observer variations. Automatic segmentation by atlas deformable registration may help to reduce time and variations. We aim to test a new commercial atlas algorithm for automatic segmentation of OAR and CTV in both ideal and clinical conditions.The updated Brainlab automatic head and neck atlas segmentation was tested on 20 patients: 10 cN0-stages  and 10 unselected N-stages . Following manual delineation of OAR and CTV, automatic segmentation of the same set of structures was performed and afterwards manually corrected. Dice Similarity Coefficient (DSC), Average Surface Distance (ASD) and Maximal Surface Distance (MSD) were calculated for “manual to automatic” and “manual to corrected” volumes comparisons.In both groups, automatic segmentation saved about 40% of the corresponding manual segmentation time. This effect was more pronounced for OAR than for CTV. The edition of the automatically obtained contours significantly improved DSC, ASD and MSD. Large distortions of normal anatomy or lack of iodine contrast were the limiting factors.The updated Brainlab atlas-based automatic segmentation tool for head and neck Cancer patients is timesaving but still necessitates review and corrections by an expert. Over the past fifteen years, head and neck cancer (HNC) radiotherapy treatment has quickly shifted from classical two-dimensional 2D) radiotherapy to Intensity Modulated Radiotherapy (IMRT), resulting in better Organs at Risk (OAR) sparing and quality of life preservation . IMRT plD radiothmean and more than 20% of the brainstem Dmax[A tedious delineation is time-consuming, particularly in the complex head and neck region, where complete segmentation times of up to 180 minutes are reported, in contrast with the mere 20 minutes needed to create a simple 2D plan . Delineastem Dmax. In anotstem Dmax, signifistem Dmax. The mosstem Dmax, neck nostem Dmax.The concept of atlas-based automatic segmentation is appealing since it could help save significant delineation time while potentially reducing the inherent inter-observer variability. Different deformation registration strategies were developed, based on either individual patient data, averaged patient generation, multiple patient data  or, moreAn updated version of the automatic head and neck atlas was developed for an upcoming Brainlab treatment planning solution . It is based on the deformation registration of an atlas patient followed by active post-processing. The aim of the current study is to validate both time gain and accuracy of this software for the various OAR and CTVs that we use in clinical practice for unoperated HNC patients in two subpopulations: one “ideal” population  and another “clinical” population .Two groups of un-operated HNC patients were defined, all referred to our Radiotherapy Department for definitive radiotherapy with or without concomitant chemotherapy. All patients were simulated head first supine with their head blocked by an anatomical neck cushion and an individual five-pin thermoplastic mask . Without contra-indication, iodine contrast was injected in two phases: first 45 cc at 1.5 cc/sec, then after a rest of 120 sec by 40 cc at 2.0 cc/sec. CT acquisition started directly after the end of the second phase with a GE Lightspeed RT CT-scanner ; 2.5 mm thick slices were reconstructed from the upper orbita down to the lower part of the clavicula. Before processing, all data were imported into the Brainlab treatment planning software  and anonymized with a number.Group A. Ten patients (numbers 1 to 10) with at least one hemi-neck showing no pathological node (cN0). These patients were retrospectively selected from our database to test the algorithm without the interference of pathological nodes introducing a variation in the normal anatomy. All but one received iodine contrast injection. OAR selection was voluntarily restricted to spinal cord (SC), brainstem (BS) and homolateral parotid gland (PG). All possible neck node levels were individually selected in the cN0 hemi-neck: 1a, 1b, 2, 3, 4, 5, 6, retrostyloid (RST), retropharyngeal (RP) and retroclavicular (RCL).Group B. Ten patients numbers 11 to 20) with various N-stages to test the algorithm in real clinical conditions. To avoid any selection bias, these patients were the 10 first patients of another prospective study on adaptive radiotherapy  proposed to patients on a consecutive scheme (i.e. without any other selection criteria than definitive radiotherapy and informed consent). All but one received iodine contrast injection. OAR were selected according to our clinical protocol: SC, BS, both PG, mandibula (M) and submandibular glands (SMG) only if level Ib was excluded from CTV. CTV selection , cartilages , muscles (sterno-cleido-mastoid), glands  and nervous structures . Available nodal CTV levels are those described in the guidelines: 1a, 1b to 6, RST, RP and RCL. An independent expert who never worked with the first author performed all segmentations.The reference atlas is built on the CT of a patient in standard position (head first supine with thermoplastic mask) with iodine contrast injection, from the vertex down to the 6Our approach entails a fully automatic segmentation algorithm using the most common atlas-based registration technique with various standard and model based support algorithms Figure . It consFirst author performed all segmentations. Reference segmentation was first performed manually (MAN volumes) and slice-by-slice for each patient. A few days later, MAN volumes were blinded and atlas-based automatic segmentation was applied (AUTO volumes). Last, AUTO volumes corrected if necessary (CORR volumes).Generated volumes were compared to each other in two ways. The first one was a comparison between AUTO and MAN volumes to measure the accuracy of the atlas-based segmentation. Secondly, a comparison between CORR and MAN volumes allowed measuring both the time needed to improve AUTO volumes and the intra-observer variability.The human time needed to perform manual segmentation and necessary corrections was recorded in minutes, as well as the computer time to generate AUTO volumes.A,B = (2 |A ∩ B|) / (|A| + |B|) [Dice Similarity Coefficient (DSC) and average and maximal surface distances  were used for comparisons. DSC is a statistical parameter investigating the intersection volume of two objects by normalizing the intersection volume to a value between 0 (no overlap) and 1 (perfect overlap) and is defined as DSC| + |B|) . ASD and| + |B|) ,16, smalStatistical analyses of the paired comparisons were performed using a double-sided T-test in Excel for Mac 2008  with level of significance set at 0.05.P < 0.05). This gain was relatively larger for OAR  than for nodal levels . Of note, CORR time was equivalent to MAN time for levels 1a, 1b, 4, RP and RST.For the “ideal” patients in group A   Figure e, which ) Figure c. Most ovs 34.5 min, P < 0.05). Like for group A, most of the gain was obtained for OAR . Again, SC did not need any correction, neither M except in one patient with teeth filling artefacts. Regarding the CTV, results are mixed, with patient 13 requiring an additional 2 min correction time, while 13 min were gained for patient 14. The average time gain for all patients in this series was 4.7 min. No significant time gain was observed for patients 13 (no iodine contrast injection), 16 (large N3) and 17 (large N2a) who demonstrated large anatomical deviations compared to the atlas . Time ranged from 25 to 50 sec, with an average of 39 sec. In group B, automatic segmentation was performed on a standalone workstation. For this group, time ranged from 90 to 155 sec, with an average of 116.5 sec.The present study demonstrates the advantages and limitations of an automatic segmentation tool for OAR and nodal CTV to help radiation oncologists in their daily work, presently for the HNC treatments. It clearly helps saving time, particularly for the tedious OAR segmentation. We found a steady 40% reduction in both groups, independently of the number of delineated OAR. Though remaining significant, time gain was less prominent for the nodal CTVs, mainly because of individual anatomical variations. In the group without pathological node (group A), we could test the algorithm in ideal conditions and determine its limitations level by level. Some levels were easy to segment automatically and hence, needed very few corrections. This was particularly true for levels 2, 3, 5 and 6. On the other hand, levels 1a, 1b, RS and RP anatomical borders were more difficult to be determined automatically, mainly because of the lack of spontaneous contrast between the anatomical key structures . Regarding level 4, a systematic inclusion of the scalenus muscles posteriorly relates to the lymph nodes mask that is voluntarily enlarged to take account of the matching uncertainties of the atlas. A tighter mask in this region could help solve the problem. Last, RCL level suffers large interpretations of its anatomical boundaries definition, resulting into large intra-observer variations, though delineation time can be saved. The study in true clinical conditions without patient recruitment bias (group B) also shed light on intrinsic limitations of automatic segmentation due to large deformations of normal anatomy or lack of iodine contrast injection. Finally and as a consequence of all these limitations, it was clearly demonstrated that -except for spinal cord and mandibula without teeth fillings- any automatically obtained volume should always be checked and corrected where necessary by an expert before planning.Automatic segmentation accuracy is highly dependent on similarity between the underlying atlas and the patient . DiffereThe comparison of all these different approaches to our one is limited by variations in the selected volumes , methodological differences and the multiple experts involved. Most groups reported their results by DSC metric, using the same methodology of MAN segmentation compared to AUTO, thus providing a reasonable basis for comparison. For BS, mean DSC of 0.83, 0.78, 0.58, 0.91 and 0.76 were observed for IND , MUL 1515, AVG , AVG-AC Whatever method used, none produces automatic volumes directly usable for planning. It was nicely demonstrated with a MUL algorithm applied to nine patients that significant underdosage is observed in reference PTV when planning is performed based on a PTV generated from automatic contours. Underdosage of 11 Gy may be observed even for very good metrics like DSC = 0.8 and ASD < 1 mm. Of note, this had no impact on OAR . CorrectUsing an atlas may also improve inter- and intra-observer variability. In a study where five different HNC radiation oncologists each delineated the CTV for five patients, it was convincingly demonstrated that the corrected atlas-based contours showed less inter-observer variability than manual segmentations. Corrected contours were on average larger than the manual ones, highlighting the potential bias induced by automatic contours . The samLast, some points were not directly addressed and could be further studied.● Inter-observer variability could be checked on our patients by repeating the study on a blinded multicentre frame.● All our patients were unoperated; the algorithm may be evaluated against a post-operative population, which has never been tested. However, since anatomy would even be more deformed than in our population B, we think that more “intelligent” algorithms should be developed to overcome this limitation.● Some more OAR could be included in the atlas  and make the purpose of a new validation. Anyway, since different guidelines are available -27, one An updated commercial HNC atlas was validated for both OAR and prophylactic CTV delineation, proving its interest as help in daily clinical work. It was clearly demonstrated as time-saving, even though a physician should always review generated contours. Potential small improvements were highlighted, e.g. use of MUL atlas, improved correction tools or introduction of new OAR in the atlas .Written informed consent was obtained from the patient for the publication of this report and any accompanying images.2D: Two-dimensional; AC: Active contour; ASD: Average Surface Distance; AUTO: Automatic; AVG: Averaged; BS: Brainstem; CORR: Corrected; CT: Computed tomography; CTV: Clinical target volume; DSC: Dice Similarity Coefficient; GTV: Gross Tumour Volume; HNC: Head and neck cancer; HP: Hypopharynx; IMRT: Intensity modulated radiotherapy; IND: Individual; L: Left; M: Mandibula; MAN: Manual; MRI: Magnetic Resonance Imaging; MSD: Maximal surface distance; MUL: Multiple; OAR: Organs at risk; OC: Oral cavity; OP: Oropharynx; PG: Parotid gland; PTV: Planning Target Volume; R: Right; RCL: Retroclavicular; RP: Retropharyngeal; RST: Retrostyloid; SC: Spinal cord; SMG: Submandibular gland; TV: Target volume.Financial: JFD received speaker fees from Brainlab; AB is a Brainlab employee; Radiotherapy Department received free software as compensation.Non-financial: none to declare.JFD designed the study, acquired the data, performed the analyses and wrote most of the article. AB developed and designed the software and wrote the technical part. All authors read and approved the final manuscript.JFD was PhD fellow 1999 – 2002) and clinical fellow (2002–2003 and 2005–2006) of Professor Vincent Grégoire in Université Catholique de Louvain, main author of the international guidelines [002 and cNodal levels selection for CTV 50 Gy of patients in group B.Click here for fileAverage segmentation times in group A.Click here for file


To investigate the relationship between health-related quality of life (HRQOL) and survival in Chinese patients with chronic liver disease (CLD).HRQOL was measured with the Chinese version of Short Form 36 (SF-36). SF-36 scores, demographic and clinical data were collected at baseline and after 18 months follow-up. Kaplan-Meier and Cox Proportional Hazard Regression survival analyses were used for interpretation of data. Surviving patients were censored in the analyses.A total of 415 Chinese patients with CLD and 86 healthy controls were enrolled. During the follow-up period 50 patients died. SF-36 scores in healthy controls and surviving patients were higher compared with those in deceased patients. Scores of physical component summary (PCS) in healthy controls, surviving and deceased patients were 54.1 ± 5.2, 48.9 ± 7.7 and 33.5 ± 8.2 respectively (p < 0.001). Scores of mental component summary (MCS) in healthy controls, surviving and deceased patients were 56.6 ± 8.2, 53.0 ± 5.6 and 37.1 ± 12.1 (p < 0.001) respectively. Survival was significantly associated with PCS and MCS scores, and the presence of ascites.HRQOL was associated with survival in patients with CLD. PCS and MCS scores were predictors of survival. Chronic liver disease (CLD) is known to cause significant morbidity and mortality, typically due to a number of complications that include ascites, hepatic encephalopathy, variceal hemorrhage and hepatorenal syndrome ,2. CLD nThe study received ethics approval from the Ethics Board of Beijing Chao-Yang Hospital, Capital Medical University and all participants gave written informed consent.Between September 2009 and September 2011, eligible patients with CLD, aged 18 to 80 years from the Digestive Department of Beijing Chao-Yang Hospital, Capital Medical University were approached to participate in the study. Patients with other chronic active medical diseases , psychiatric conditions, malignancy, liver transplantation and those unable to communicate or who declined to participate were excluded. Patients with hepatitis B virus (HBV) infection and chronic hepatitis C virus (HCV) infection or who received interferon therapy in the previous 3 months were also excluded. The healthy controls consisted of individuals without chronic disease, aged 18 to 80 years, male or female and who regularly undertook health screening in our hospital’s health examination center. Healthy controls were used to exclude the influence of unpredictable events, social and environmental factors.Each patient had an established diagnosis confirmed by a Hepatologist. The diagnosis of chronic HBV infection was based on the presence of hepatitis B surface antigen for more than six months, elevated serum alanine aminotransferase levels, with or without HBV DNA as detected by the hybridization method . The diaOn admission, patients gave written informed consent and completed the self-administered HRQOL questionnaire. The Medical Outcomes Study of Short Form36 (SF-36 v2 Chinese version) is a widely used and validated generic HRQOL questionnaire. Extensive demographic and clinical data were collected on admission and marital status was dichotomized into single and paired; single was extended to include unmarried persons, divorced or deceased couples.Laboratory data included alanine and aspartate aminotransferases, alkaline phosphatase, γ-glutamyl transferase, total bilirubin, serum albumin, serum creatine, prothrombin time, serum potassium and sodium, hemoglobin, white blood cell and platelet counts .The SF-36 v2 Chinese Version  consists of 36 items divided into eight domains that are aggregated into two summary scores, a mental component summary (MCS) and a physical component summary (PCS). These domains range from reflecting predominantly physical wellbeing, that include physical function (PF), the ability to perform expected physical roles (RP), the degree of bodily pain (BP) and overall sense of general health (GH) to those reflecting predominantly social and emotional well-being that include overall sense of vitality (VT), ability to function in social roles (SF), ability to perform expected emotional and social roles (RE) and overall sense of mental health (MH) -25.We followed all participants longitudinally and terminated the follow-up at the time of the patient’s death or March 1st, 2013, whichever occurred first.After the follow-up period, patients were divided into survival and deceased groups.t test, one-way analysis of variance (ANOVA) or Chi-square test. Univariate Cox Proportional Hazard Regression analysis was conducted for PCS, MCS, demographic and clinical variables. Variables with a p value <0.1 in univariate Cox Proportional Hazard Regression analysis were entered into multivariate Cox Proportional Hazard Regression analysis. Survival curves were estimated with the Kaplan-Meier method across the quartiles of PCS and MCS scores. A p value <0.05 was considered as statistically significant. All data were analyzed with SAS 9.1.Categorical data were described as the number and continuous data as mean ± SE. Data were analyzed using independent sampled A total of 415 Chinese patients with CLD and 86 healthy controls were enrolled and 18 months of follow-up was completed. A total of 50 patients died and two patients were treated with liver transplantation, one in our hospital and one in a different hospital. Both patients died within one month and were therefore excluded from the analysis. The duration of follow-up for surviving and deceased patients was 30.7 ± 10.0 months and 12.1 ± 5.8 months respectively. A further 22 patients were lost to follow up and were also excluded from the analysis. There were no significant difference between the groups for health control and CLD, on age, gender, marital status or educational level . Ascites usually becomes refractory to diuretic control with progression of CLD, where survival rates approach 50% at one year .HRQOL measures, such as the SF-36, a generic questionnaire, is used to measure HRQOL in the general population and in patients with chronic diseases including CLD. Many research centers have used HRQOL to compare CLD patients with healthy controls and have generally reported impairment of HRQOL. Additionally, measurement of HRQOL can facilitate integration of biomedical and psychosocial models of health. This integrated approach in the study of patients with CLD will capture the impact of these diseases on patient’s health and well-being -30. SeveOur study has a number of limitations. All subjects were recruited from one hospital in one city. Our hospital is a public general affiliated hospital of Capital Medical University and three levels of first-class or China’s most senior hospital in Beijing. Our department has 42 beds including an endoscopy suite. We serve over 150,000 outpatients per year, over 1,800 in-patients per year and over 20000 endoscopies per year that might introduce selection bias. Our sample consisted mostly of patients with health insurance who have better access to healthcare. Therefore, our patient sample excludes patients without health insurance with limited access to care. Our study included patients with CLD with different etiologies and severity. We are addressing these issues in ongoing studies by assessing a larger group of patients in multiple centers.HRQOL was associated with survival in patients with CLD. PCS and MCS scores were predictors of survival.The authors declare that they have no competing interests.FG participated in the study design, access to data, the data analysis and interpretation, and drafted the manuscript. RG participated in the study design, the data analysis, and important content changes. GL participated in the study design and access to data. ZMS participated in the study design and the data analysis. JYH participated in the study design, the data analysis and interpretation, and important content changes. All authors read and approved the final manuscript.