Radix Salviae, can attenuate cardiac remodeling and clarified the underlying mechanisms. Using the spontaneously hypertensive rat (SHR) as a cardiac remodeling model, DA ameliorated blood pressure, cardiac injury, and myocardial collagen volume and improved cardiac function. Bioinformatics analysis revealed that DA might attenuate cardiac remodeling through modulating mitochondrial dysfunction and reactive oxygen species. DA repaired the structure/function of the mitochondria, alleviated oxidative stress in the myocardium, and restored apoptosis of cardiomyocytes induced by angiotensin II. Besides, DA inhibited mitochondrial redox signaling pathways in both the myocardium and cardiomyocytes. Thus, our study suggested that DA attenuates cardiac remodeling induced by hypertension through modulating mitochondrial dysfunction and reactive oxygen species.Current therapeutic approaches have a limited effect on cardiac remodeling, which is characteristic of cardiac fibrosis and myocardial hypertrophy. In this study, we examined whether Danshenol A (DA), an active ingredient extracted from the traditional Chinese medicine  Cardiovascular disease is still a serious threat to senior citizens in aging countries . CardiacEvidence has been increasing that oxidative stress serves as an important mechanism for myocardial remodeling and cardiac failure , 6. As aRadix Salviae (Danshen) is a kind of traditional Chinese herb applied extensively in the treatment of cardiovascular diseases in China. As a drug for cardiovascular diseases, the compound Danshen dropping pill is currently undergoing phase III trials in clinical centers over nine countries. It is expected to become the first Chinese medicine authenticated by the Food and Drug Administration [Radix Salviae. In contrast with the well-known tanshinone form of Radix Salviae, for instance, tanshinone I, tanshinone IIA, and cryptotanshinone, few studies have reported the biological effects of Danshenol. Previous reports indicated that the anti-inflammatory properties of DA are superior to those of tanshinone IIA, thereby prompting that DA has potential efficacy for atherosclerosis [Over the past decades, natural plant medicines have been extensively applied and popularized in numerous countries. The World Health Organization encourages the utilization of natural medicine as a promising adjuvant treatment strategy , 11. Radstration . Danshenclerosis . Anotherclerosis . PrelimiOur manuscript is intended at investigating the protective effects of DA on cardiac remodeling induced by hypertension and identifying whether the underlying mechanisms are associated with the oxidative stress pathway.DA  was purchased from EMMX Biotechnology LLC . Captopril tablets (12.5 mg) as the positive control were provided by Bristol-Myers Squibb Co. Ltd. (lot 1209031).The biochemical kits for the detection of lactate dehydrogenase , creatine kinase , CK-MB (batch number: H197), alanine aminotransferase , aspartate aminotransferase , creatinine , blood urea nitrogen , and ROS (batch number: E004) were obtained from Nanjing Jiancheng Biotech Co. Ltd. . Biochemical kits of MitoCheck Complex I activity (lot 700930), MitoCheck Complex II activity assay kit (lot 700940), MitoCheck Complex II/III activity assay kit (lot 700950), and citrate synthase activity (lot 700990) were obtained from Cayman Chemical Company .Animal feeding and experimental procedures were conformed to institutional animal ethics committee guidelines, which acted in accordance with the Care and Use of Laboratory Animals published by the United States National Institutes of Health .Forty male spontaneously hypertensive rats (SHR) and eight male Wistar-Kyoto (WKY) rats at the age of 16 weeks were bought from Vital River Laboratory Animal Technology Co. Ltd. The rats were conventionally raised for 5 days in a SPF laboratory animal room at first, where the environment was set procedurally at 24°C ± 2°C and 35% ± 5% humidity under a regular 12 h/12 h light/dark schedule. All rats have free access to sterile water and forage.n = 8), CAP group , DAL group , DAM group , and DAH group . The WKY group  consisted of male Wistar-Kyoto rats. DA or captopril was orally administrated daily for 12 weeks, while the WKY and SHR groups received the equal volume of normal saline.Using a randomized complete control study, the forty SHR were assigned into five groups: SHR group , diastolic blood pressure (DBP), and mean blood pressure (MBP) were determined using noninvasive tail pressure equipment . In brief, animals were preheated at 38°C for 8 minutes in a thermostat pad, and three stable consecutive measurements of blood pressure including SBP and DBP were recorded. Also, ejection fraction (EF) and fractional shortening (FS) were detected via color Doppler ultrasound diagnostic instrument in M-mode with a 10 MHz probe (S40 Exp).After treatment for 12 consecutive weeks, the blood samples were gathered from the caudal vein of rats and centrifugated at 3000 rpm/min for 12 min. Then, the serum was collected carefully from the supernatant and stored at −80°C. Myocardial injury was evaluated with the serum concentration of LDH, CK, and CK-MB, whereas liver and renal functions were examined using ALT, AST, Cr, and BUN.Executed with narcotic overdose, hearts from the rats were separated and weighed to determine the HW/BW index (the rate of heart weight to body weight). The left ventricle tissue was dissociated partially and fixed with 4% polyformaldehyde for 48 h. Then, the tissue was embedded in paraffin, sliced at 5 mm, and stained with Masson's trichrome  to visualize fibrillar collagen. The extent of fibrosis was observed in 8 random fields of vision each sample and quantitated as the collagen volume fraction (CVF) using a light microscope  at 40x magnification and ImageJ software .http://lsp.nwu.edu.cn/index.php) [http://bionet.ncpsb.org/batman-tcm) [http://lilab.ecust.edu.cn/pharmmapper/) [https://cytoscape.org) [P < 0.05, min overlap ≥ 3).To clarify the molecular mechanisms of DA on cardiac remodeling, molecular targets of DA were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform   and Bioiman-tcm) . The tarmapper/) . Subsequape.org)  to show 4-buffered solution (pH 7.4) was used to postfix the tissue samples for 1 h. The resins were embedded and sectioned through the EM Ultramicrotome LKB-2088 and stained using 1% toluidine blue solution. The ultrathin sections were then stained with uranyl acetate and lead citrate twice. Eventually, the morphology of mitochondria in the myocardium was observed using an electron microscope (Hitachi H-7500).Anterior walls of the left ventricle were transferred into 2.5% glutaraldehyde and 1% paraformaldehyde for 24 h. After washing at least twice with 0.1 M PBS at 4°C, 1% OsOMitochondria in heart tissue were extracted as described above, and the activity of the mitochondrial complex was examined by corresponding biochemical kits. In brief, complex I activity was measured as the oxidation extent of nicotinamide adenine dinucleotide. Complex II activity was measured as the diminution in artificial electron acceptor L2 6-dichlorophenolindophenol. Complex III activity was determined as the reduction of cytochrome c. Complex IV was determined by the reduction in acetyl-CoA, which was measured as the content of oxaloacetate.4 , then homogenized directly and transferred into a centrifuge at 10000 r/min for 15 min. The supernatant was neutralized and filtered after centrifugation under the same conditions. The test solution was completely separated with isocratic elution via 96% 0.05 M KH2PO4 (pH 6.5) and 4% methanol for 20 min in high-performance liquid chromatography (HPLC) with a Waters C18 column . Content of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was measured at 254 nm via an external standard method for quantification. The energy charge was calculated as ((ATP + ADP)/2)/(ATP + ADP + AMP) [The heart tissue and cardiomyocytes were collected and immersed in 0.6 M HClOP + AMP) .After treatment for 12 consecutive weeks, myocardium samples were collected as mentioned above. The level of oxidative stress was determined by measuring the concentrations of ROS, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) in the myocardium of rats.5 cells/cm2 and plated into an incubator where the environment was maintained at 37°C with 5% CO2.Primary cultures of rat cardiomyocytes were conducted in accordance with previous studies. In brief, neonatal Sprague-Dawley rats were sacrificed. The hearts were rapidly separated from the rats and washed using phosphate-buffered saline. Then, cardiac tissues were minced via amicrobic scissors and digested using Hanks' solution with 0.1% trypsin at 37°C for 3 min. Cells were isolated by digestion for 8–10 times and transferred to Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS). The cardiomyocytes were then cultivated in culture flasks with a density of 1 × 10μmol/L) and captopril (5 μmol/L) for 35 min, followed by 0.1 μmol/L angiotensin II (Ang II) for another 48 h. After incubation, cells were cleaned with PBS and resuspended. Then, fluorescein-conjugated annexin V (5 mL) and propidium iodide reagent (5 mL) were added to cell suspensions. The mixture above was incubated for 15 min protected from light. Finally, the rate of apoptosis was determined and quantified by FACScan flow cytometry (Beckman Coulter).The apoptosis rate of cardiomyocytes was determined via an Annexin V-FITC Apoptosis Detection Kit and a flow cytometer. Cardiomyocytes were cultivated in six-well culture plates with Dulbecco's modified Eagle medium with 10% FBS for 48 h. The medium was replaced with serum-free medium and cultivated for 24 h. Then, cells were pretreated with DA  membrane via a gel transfer device. Then, PVDF membranes were incubated via primary antibodies against Bax, Bcl-2, (GTP)p-Ras, Ras, p-Raf, Raf, p-Mek, Mek, p-Erk, Erk, Ask1, p-Jnk, Jnk, p-p38, p38, and GAPDH overnight at 4°C. After that, the primary antibodies were cleaned with Tween20/TBS solution, and then secondary antibodies were incubated for another 1 h. Using developing solution, the expression of proteins was measured via an Odyssey infrared imaging system (LI-COR Biosciences) and normalized to the GAPDH protein level.P value less than 0.05 was defined by having the difference of statistics. Statistical analysis was determined via GraphPad Prism 5.01 for Windows.Results are presented as the mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA and Dunnett's test. Dose-related alternations in SBP, DBP, and MBP for the six groups are exhibited in Figures EF and FS were used to evaluate the impairment of cardiac function, and the impairment of EF and FS suggested cardiac functional insufficiency. As shown in Figures P < 0.001), indicating that hypertension induced heart damage , liver function , and kidney function  were detected in the present study. As shown in t damage . With trConsistent with the alternations in blood pressure and cardiac function, significant deterioration of cardiac remodeling under histological analysis was determined in the SHR compared with the WKY group , as evidMasson's trichrome-stained sections  and CVF Using the TCMSP database and BATMAN-TCM, we obtained 25 potential molecular targets of DA . Moleculz line compared with that in the WKY group. By contrast, DA treatment restored the damage to mitochondrial morphology and cardium structure.Electron microscopy observations on the cardium showed obvious morphological changes in the SHR group compared with the WKY group . The denMaximal activities of complexes I–VI were determined in cardiac mitochondria to evaluate the function of cardiac mitochondria. Mitochondrial complex enzyme activities decreased in the SHR group than in the WKY group, whereas DA treatment enhanced the activities, which showed a dose-dependent manner. .The measurement of adenine nucleotide variants was conducted to evaluate the energy production in myocardial mitochondria. As a result, DA treatments increased ATP and ADP content in the myocardium, while ATP and ADP content in the SHR group was obviously lower than that in the WKY group . Besidesμmol/L) for 48 h enhanced the apoptotic rate of cardiomyocytes  obviously decreased the apoptotic rate of cardiomyocytes incubated by Ang II, which was accompanied with the decreased expression of Bax and increased expression of Bcl-2 (Incubation with Ang II (0.1  Figures  with incof Bcl-2 .Mitochondrial redox signaling pathways are mainly regulated by Ras, Raf, Mek, Erk, Ask1, Jnk, and p38. The result of Western blot showed that the protein levels of p-Ras, p-Raf, p-Mek, p-Erk, Ask1, p-Jnk, and p-p38 were raised in the SHR group than in the WKY group, while pretreatment with DA decreased those . A similCardiac remodeling, an adaptive response of the heart to pressure overload, is a result from physiological or pathological stimuli , which pSHR is a mature hypertensive model that was introduced in 1963, in which myocardial damage occurs and eventually leads to cardiac remodeling after 16 weeks . Hence, The present study indicated that DA treatment significantly decreased SBP and DBP in SHR, but the effects were inferior to captopril. EF and FS are the fractions of outbound blood pumped from the heart in each cardiac cycle. EF and FS, which are measured by an echocardiogram, are general indicators of cardiac function . As a reTo explore the specific mechanism of DA on cardiac remodeling, bioinformatics technology was used in this study. TCMSP is an advanced platform of network pharmacology for Chinese herbal medicine which contains the correlations among drugs, targets, and diseases . BATMAN-Accordingly, the analysis determined that the molecular targets were significantly enriched in functions associated with “protein targeting to mitochondrion/regulation of response to reactive oxygen species,” “positive regulation of epithelial cell proliferation,” “protein maturation,” and “amoebiasis.” Among them, “protein targeting to mitochondrion/regulation of response to reactive oxygen species” was highly enriched with a target level of 6 and considered one of the most essential pathways modulating the mechanism of DA. On the basis of the annotation data of gene ontology , the defGrowing evidence indicates that ROS is essential for the pathogenesis of cardiac remodeling, which may also exert an obvious effect in the progression from pathological remodeling to heart failure , 27. ForMitochondrial dysfunction will amplify apoptotic signals, and cardiomyocyte apoptosis plays a crucial role in cardiac remodeling and heart failure , 36. PreExtensive reports implicated that ROS generated from mitochondrial dysfunction can result in the release of remodeling factors including Raf, Ras, Mek, Erk1/2, Ask1, JNK, and p38. Ask1 is intensively provoked by ROS, and it then activates MAPKs p38 and JNK. The deletion of Ask1 decreases p38 and JNK activation and improves cardiac remodeling induced by Ang II, which were validated in our results. Downstream signaling pathways mediated by ROS were activated under the pathological conditions of ventricular remodeling in vivo and in vitro. The downstream signaling pathways included the MAPK classical pathway, ASK1/JNK pathway, and p38 MAPK pathway, and the expression of proteins in those pathways significantly increased. All of these responses were significantly reversed by DA treatment in primary cardiomyocytes and in the myocardium of SHRs. Thus, DA may improve cardiac remodeling by modulating mitochondrial redox signaling pathways.In summary, our study demonstrated that DA could improve cardiac function, ameliorate cardiac fibrosis, restore mitochondrial structure/function in SHR, and decrease the apoptosis of cardiomyocytes. These functions may be completed by promoting mitochondrial dysfunction and reducing reactive oxygen species. We also proved that the mechanism may involve inhabitation of mitochondrial redox signaling pathways in cardiomyocytes and myocardium.


Extra Corporeal Membrane Oxygenation (ECMO) has become an accepted treatment option for severely ill patients. Due to a limited availability of ECMO support therapy, patients must often be transported to a specialised centre before or after cannulation. According to the ELSO guidelines, an ECMO specialist should be present for such interventions. Here we describe the safety and efficacy of a reduced team approach involving one anaesthesiologist, experienced in specialised intensive care medicine, and a specialised critical care nurse.This study is a 10 years retrospective, single institution analysis of all data collected between January 2007 and December 2016 from the medical records at the University Hospital Bonn, Germany.The Bonner mobile ECMO team was deployed in 170 cases for on-site evaluation for ECMO support therapy. 4 (2.4%) patients died prior to arrival or during the implementation of ECMO support. Of the remaining 166 patients, 126 were cannulated at the referring site, 40 were transported without ECMO. Of those, 21 were subsequently cannulated out our centre. 19 patients never received ECMO treatment. The primary indication for ECMO treatment was ARDS (159/166 patients). Veno-venous ECMO was initiated in 137, whilst 10 patients received veno-arterial ECMO treatment. Mean transportation time was 75 ± 36 min, and mean transport distance was 56 ± 57 km. In total, 26 complications were observed, three being directly transport-related. The overall survival was 55%.Initiation of extracorporeal membrane oxygenation and subsequent transport can be safely and efficiently performed by a two-man team with good outcome.The online version of this article (10.1186/s12871-019-0687-9) contains supplementary material, which is available to authorized users. Patients affected by acute respiratory distress syndrome (ARDS) may require the use of extracorporeal membrane oxygenation (ECMO) as an emergency life-saving therapy . DespiteThis paper aims to describe the safety and efficacy of a reduced team approach for performing primary transports of ECMO patients over a ten-year time frame.Data from our hospital  were retrospectively analysed regarding ECMO indication, transport mode, duration and distance.Type of study: retrospective cohort study. For further study details and study limitations we followed the “Strengthening The Reporting of Observational Studies in Epidemiology-STROBE” criteria . The STRCohort of interest: All patients undergoing evaluation for ECMO initiation and primary ECMO transport by our ECMO retrieval team.The Respiratory ECMO survival prediction (RESP) score  and survData for 10 consecutive years (January 2007 – December 2016) are presented. Statistical and graphical data handling was performed using GraphPad Prism 5.0d (GraphPad Software) and Microsoft Excel for Mac 2011.Student’s paired t-test was used for statistical comparison of groups where applicable  is given, if the observed mortality does not fall into the 95% CI of the predicted mortality.For comparison of observed mortality rates with the predicted mortality rates using RESP/SAVE scores, we used the Standardized Mortality Ratio (SMR) , 12. TheSequential Organ Failure Assessment (SOFA) score [Simplified Acute Physiology Score (SAPS) II [and Acute Physiology And Chronic Health Evaluation (APACHE) II score [Therapeutic Intervention Scoring System (TISS-28) [24 h post admission to our hospital, standard disease severity classification systems were used to calculate each patient’s A) score , SimplifSAPS) II , and AcuII score . NursingTISS-28)  and lungTISS-28) .In compliance with the ELSO General Guidelines, veno-venous-(VV) ECMO support was indicated in all patients with conventional mechanical ventilation, as a rescue therapy to treat hypoxaemia and hypercapnia. All decisions for implantation were made following consensus between two experienced members of the ECMO team. In patients with refractory cardiogenic shock, a veno-arterial-(VA) ECMO support was established. Refractory cardiogenic shock was defined as an acute or acute on chronic heart failure with hypotension (systolic blood pressure ≤ 90 mmHg) unresponsive to fluid rescucitation requiring increasing inotropic therapy or physiologic evidence of visceral hypoperfusion. The decision regarding the mode of ECMO support and its implementation technique was left to discretion of the ECMO physician on site.Depending on the underlying type of organ failure, either VV-ECMO or VA-ECMO support was implemented.VV-ECMO support was performed by inserting either two or three cannulas, usually into both femoral veins (17–24 Ch.) and/or the right jugular vein (15–21 Ch.) after ultrasound guided percutaneous blood vessel puncture. Patients with refractory cardiogenic shock received peripheral VA-ECMO treatment through percutaneous cannulation, usually via the femoral artery/vein.The Bonner ECMO team is comprised of 9 ECMO physicians  and 9 ECMO nurse specialists. All team members have long-term experience in intensive care medicine and a minimum of three years experience in the treatment of ARDS and ECMO patients, as well as the general transport of critical ill patients. This staffing allows a 24/7, 365 day availability for ECMO implantation and retrieval at external sites via an organised on call-service.The retrieval team consists of one ECMO physician and one ECMO nurse specialist who, upon arrival at the referring hospital, re-evaluate the patient on-site and decide whether initiation of ECMO is necessary. Then, the decision regarding what type of ECMO support and what cannulation approach (VV/VA) is needed. The team performs all steps of the cannulation itself.Following cannulation and stabilisation, a timely departure and safe transport to the University Hospital Bonn is vital.To maximize patient and team safety throughout the whole high risk process of initiating ECMO therapy at an external site and the subsequent intensive care transportation of the critically ill patient, standardised checklists  supply, informing next-of-kin/legal guardian) and potential optimizations of therapy  is given. Availability of ultrasound and/or on site radiologist is requested. The response time for activating the ECMO-Team and the required airborne and/or ground-based transport vehicles is 30 to 90 min following a standard operating procedure, depending on the time of the day and availability of personnel on duty.For prompt departure and minimal time of preparation, all necessary equipment is prepacked in transport bags, sealed and stored on ICU. For safety reasons, certain equipment  are packed in duplicate. This redundancy allows safe operation and potential re-initiation of ECMO in case of adverse events. The ECMO circuit is primed on site by the ECMO team in the referring hospital, after the final on-site decision for ECMO support is made.2, SpO2 and optional parameters as needed. For ground based transport the patient was placed on a Performance Pro XT or Power Pro XT stretcher  especially modified for intensive care transportation. Until 2013 a modified equipment platform was used to mount the Levitronix, and later the Cardiohelp, console to fit different types of stretcher systems used in airborne transport. Since then the certified mobile transport carrier by Maquet is used in ground based and airborne transport.During the study period, CentriMag consoles and centrifugal pumps  were used in combination with ELS and PLS Oxygenators and tubing  until February 2011. Since 2012, transports are carried out with the Cardiohelp System and HLS Set Advanced . For peripheral VV or VA cannulation Fem-Flex II single-lumen cannulas  and Maquet HLS single-lumen cannulas were used. For ventilation during transport Oxylog 3000 or 3000plus  were utilised. In the case of airborne transport Hamilton T1  respirators were used. Except for a few long-term transports no blood gas analyses were assessed during the transport phase due to unavailability during transport. No heater units were used during transport, but efficient conventional measures to avoid accidental cooling were taken. Vital signs were monitored using Propaq encore , Corpuls3  and X-Series CCT  monitors, measuring continuously and standardised ECG, invasive blood pressure, etCODuring the ten years observation period, the Bonner ECMO team was deployed 170 times. The overall number of requests from other hospitals was not consistently recorded and thus cannot be reported in sufficient detail. Three patients died prior to arrival of the mobile ECMO team  received VV-ECMO, whilst ten patients (4%) received VA-ECMO Fig. b. One paDecisions regarding transport considering several factors like weather, time of day, distance, and availability of a dedicated transport vehicle (airborne or ground).Whilst 24 patients were transported using a helicopter, the majority, 138 patients, were transported on ground. The remaining patients´ mode of transport was irretrievable from available records   years. 94 patients were male, 73 female Fig. e. AveragPrimary indication for ECMO referral was ARDS with different underlying aetiologies. In the majority of cases, ARDS was secondary to pneumonia with associated hypoxic and/or hypercapnic respiratory failure Table . AmongstThe average SOFA score on admission was 12 ± 3 and SAPS II score was 40 ± 13. The mean nursing workload, estimated using TISS-28, was 30 ± 8. Mean APACHE II score was 23 ± 7 and mean Lung injury score was 3 ± 1 .ECMO efficiency was assessed 30–60 min post implantation. Markers of efficiency included improved patient blood gas parameters, improved pH, reduction of FiOers Fig. a-c, whenSurvival is reported as in-hospital survival, i.e. discharge from our hospital or transfer to another hospital.Out of 147 patients who received an ECMO support, 81 patients survived (55%). In detail, of the ten patients initially receiving VA-ECMO treatment, 3 (30%) survived. Predicted survival for VA-ECMO, determined using the SAVE-Score, was 33.5%. Of the VV-ECMO cohort, 78 (56.9%) patients survived. Predicted survival for VV-ECMO, determined using the RESP-Score, was 59.6% . However, in their small cohort, SOFA score was not associated with outcome . BrechotICU length of stay observed in our cohort was 31 ± 29 days. This is similar to ICU LOS reported by Lucchini et al., who reported a mean ICU LOS of 27 ± 28 days for all patients, and 27 ± 27 days for patients transported with ECMO .In their description of 221 patients under ECMO Bryner et al. point out the immense strain on the involved personnel. At times, 5 staff members were deployed for one transport for a time frame of 8 to 12 h , efficieHence, our experience with performing primary ECMO transports with a reduced team comprising only two members, may help address this problem.The study by Brechot et al. was also performed with a two-member team . In this regard, it is the only comparable study to our 2-member approach. They did not report a mean mission time, but rather a deployment to ECMO initiation time of 192 min. This is considerably shorter than our mean mission time of 339 min, however their transport times (35 min) and distances (15 km) are well below those reported in our study (74 min and 48 km), which might have contributed to the different times. This indicates, that a two-member approach is time efficient, thus helping reducing the personnel burden for ECMO centres.Most recently, Heuer et al. reported a study of 75 patients transported under ECMO . This stWe observed a total of 26 major incidents (15.6%). The majority of incidents were related to ECMO implantation or critical condition of the patient. This finding is in accordance with the observed rate of critical events in the DACAPO study, where 59 (13.6%) critical events occurred, with 42 being patient related . The obsEven in teams consisting of more personnel, including a cannulating surgeon, complication rates are higher than in our cohort. One of the largest of these studies, consisting of 282 primary ECMO transports, Broman et al., observed a total complication rate of 27.3%, with 22% being patient related and 5.3% being technical failure . The stuMain limitation of this study is its retrospective nature and single centre experience. Due to the non-comparability of mean survival rates, no direct comparison from our study with survival rates of other studies could be performed. Thus comparison is based on indirect standardisation and not direct comparison. Furthermore, no causality between mortality and transport can be concluded.Interfacility ECMO transport by a anaesthesiology-led two-man team is feasible, safe and efficient. We did not observe increased mortality beyond the already high-risk of ECMO itself. Our reduced team approach can safely extend lifesaving therapy to patients in other institutions, whose available treatment options have been exhausted. This without increasing observed adverse events during/related to transport of the critically ill. ICU LOS and survival to hospital discharge of transported patients with ECMO is comparable to other studies with larger ECMO retrieval teams , 21. We Additional file 1:Checklist – On site evaluation prior to ECMO. ECMO therapy protocol. ECMO debriefing protocol. STROBE Checklist for cohort studies (PDF 899 kb)


Homeless persons have a high risk for tuberculosis. The prevalence of latent tuberculosis infection and the risk for a progression to active tuberculosis is higher in the homeless than in the general population. The objective was to assess the prevalence and risk factors of tuberculosis/latent tuberculosis infection in a homeless population in Germany.Homeless individuals (n = 150) were enrolled in a cross-sectional study at three shelters in Münster, Germany (October 2017–July 2018). All participants were screened using an ELISPOT interferon-γ release assay (IGRA). Those participants tested positive/borderline by IGRA provided three sputa for microbiological analysis  and underwent a chest X-ray to screen for active pulmonary TB. Risk factors for tuberculosis/latent tuberculosis infection were analysed using a standardized questionnaire.Of the 142 evaluable IGRA, 21 (15%) were positive and two (1%) were borderline. No participant with a positive/borderline IGRA had an active tuberculosis as assessed by chest X-ray and microbiology. A negative IGRA was associated with a citizenship of a low-incidence country for tuberculosis , low-incidence country of birth (p<0.001) or main residence in a low-incidence country in the past five years (p = 0.002).The prevalence of latent tuberculosis infection (diagnosed by a positive/borderline IGRA) was 16%; no active tuberculosis was detected. The highest risk for latent tuberculosis infection was found in patients from high-incidence countries. This population at risk should be either treated for latent tuberculosis infection or need to be monitored to early detect a progression into active disease. Mycobacterium tuberculosis, respectively  [The incidence of tuberculosis (TB) decreased in Germany in the past decades, but increased again between 2013 and 2016  with 69% of notified patients being non-German citizens [“A state of persistent immune response to stimulation by M. tuberculosis antigens with no evidence of clinically manifest active TB” [One major source sustaining the global TB epidemic are latent TB infections (LTBI) . LTBI istive TB” . The avetive TB” . The mostive TB” . Howevertive TB” .The diagnostic tools to detect LTBI are imperfect . HoweverExposure to communicable diseases is especially high in the homeless population due to close contacts to large and fluctuating communities in shelters . ConsequLTBI is found more often in homeless living under crowded conditions than in those sleeping rough (33–45% vs. 23%) . BesidesEthical approval was obtained from the ethical committee of the Medical Association of Westfalen-Lippe and the University of Münster (2017-349-f-S).All participants gave a written informed consent prior to any study related activities. All procedures were conducted in cooperation with the local Public Health Agency .Considering an expected prevalence of 36% of LTBI among homeless persons (as shown in a comparable population in Poland) , a 95% cParticipants were recruited between October 2017 and July 2018 at three shelters for homeless persons in Münster, Germany (“Haus der Wohnungslosenhilfe” (accommodation and support for men), “Gertrudenhaus” (accommodation and support for women), “Treffpunkt an der Clemenskirche” ). To improve compliance, we worked closely with the existing social and health care services. Criteria for inclusion were (i) written informed consent, (ii) use of assistance for homeless people provided by one of the three shelters mentioned above, (iii) an age of ≥18 years and (iv) proficiency in one of the languages, which were used for the informed consent and questionnaire , medical history , as well as TB-specific information . All symptoms were recorded as reported by the participants and assessed by a physician.M. tuberculosis was detected in sputum samples or if chest X-ray showed any typical signs for active TB in IGRA positive individuals . Based on the difference of the number of spots, test results were interpreted as negative (≤4 spots), borderline (5–7 spots) or positive (≥8 spots). In case of positive or borderline results, further diagnostics were initiated to identify potential cases of active pulmonary tuberculosis . Specimens were also cultured both in liquid media  for maximum six weeks and on solid media  for maximum eight weeks [Blood samples (Lithium-Heparin-Tube) were stored at room temperature (18–25°C) for a maximum of 32 hours until the IGRA  was performed and interpreted according to the manufacturer’s instructions; “T-cell Xtend” solution was used if the samples were not processed within eight hours after sampling. The number of spots in the nil control was subtracted from the number of spots following the stimulation of interferon-γ secretion with rculosis . A chestht weeks .All participants with positive findings in chest X-ray and/or sputum were referred to a specialist to undergo treatment . Accordi2 test or Fisher’s exact test, where appropriate. Multivariable analysis was done with logistic regressions. Continuous values were tested for normal distribution by Shapiro-Wilk test; normally distributed variables were compared with Student’s t-test. Non-normal data were compared with Wilcoxon rank-sum test.Calculations were performed with “R” . The significance level was set at α = 0.05. Categorical variables were compared between IGRA positive and IGRA negative individuals using chi2  in three cases and in five cases the IGRA yielded non-interpretable results . These persons were excluded from the final analysis (n = 8). The per protocol population (n = 142) had a mean (SD) age of 42 years (12) and an imbalanced ration of females to males 14:128, . The cal2 5.0; .Of 142 volunteers, 21  had positive and two (1%) had a borderline IGRA (5 and 7 spots). Patients with a positive or borderline IGRA were grouped together as the risk for a positive IGRA in re-tests can be up to 37% in high-risk groups [The proportion of German citizens was higher in the IGRA negative group  compared to the IGRA positive/borderline group  and high-incidence countries  . A negatThe proportion of participants with a health insurance was higher in the IGRA negative compared to the IGRA positive group (87% (103/119) vs. 70% (16/23), OR = 2.8, 95%CI: 1−7.9, p = 0.04, IGRA negative participants had a longer school education  compared to IGRA positive participants  was significantly associated with a positive IGRA (24% (28/119) vs. 44% (10/23), OR = 0.4, 95%CI: 0.2−1, p = 0.05, Haemophilus influenzae pneumonia. None of the IGRA positive participants consented a preventive chemotherapy for LTBI.Of the 23 participants with a positive/borderline IGRA, five were lost to follow-up for X-ray 22%) and seven were lost to follow-up for sputum analysis (30%). Of the remaining 18 participants, all underwent a chest X-ray to rule out active pulmonary TB. Except for one participant, no radiological signs of TB were seen . All samples were negative by culture, microscopy and line-probe assay.M. tuberculosis, it mirrors a lasting immune response to M. tuberculosis, and is considered a useful tool to detect LTBI (together with tuberculin skin test) [We performed a cross sectional study on the prevalence of TB/LTBI among homeless people and found 23 IGRA positive/borderline participants 16%). This proportion is lower than the overall estimation for LTBI prevalence in the world  [%. This pEven though 27% of homeless persons in Germany are female, our sample only included 14 (10%) women . The difThe prevalence of positive IGRA among homeless people was 36.7% in Poland, 12–32% in the USA, 50.6% in Japan and 75.9% in South Korea . CompareOur analysis showed that a citizenship of a low-incidence country, a place of birth or residence in a low-incidence country is associated with a negative IGRA . This isScreening for active TB in homeless in Frankfurt (Germany) found no difference in terms of the geographical origin of participants .Presence of a health insurance was another item that showed a significant association with a negative IGRA. All German citizens are health insured by law. A health insurance can therefore be considered as a confounder of an association with a positive/borderline IGRA.Our data suggest that lower educational levels (e.g. years of school attendance) increase the risk of a positive/borderline IGRA. This has already been described as risk factor for active tuberculosis in homeless . It highM. tuberculosis) [M. tuberculosis).Five participants reported a known TB infection in the past . This coculosis) . TherefoSome limitations of our study need to be addressed. First, we only screened for active pulmonary TB in patients with positive/borderline IGRA. As 25% of TB cases are extrapulmonary, we might have missed these cases in our screening approach . Second,In conclusion, the prevalence of LTBI (diagnosed by a positive/borderline IGRA) was 16% in our population of homeless persons; no active TB was detected. The highest risk for LTBI had patients from high-incidence countries. This population at risk should be treated if LTBI is detected.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This study aimed to compare the clinical outcomes of stereotactic ablative radiotherapy (SABR) and conventionally fractionated radiotherapy (CFRT) in hepatocellular carcinoma (HCC) patients with portal vein invasion (PVI).HCC patients with PVI treated with radiotherapy from 2007 to 2016 were analysed. CFRT was administered at a median dose of 51.5 Gy  with 1.8–3 Gy per fraction. SABR was administered at a median dose of 45 Gy  with 6–12.5 Gy per fraction. Treatment efficacy, toxicity, and associated predictors were assessed.p = 0.003). The 1-year overall survival (OS) rate  and in-field progression-free survival (IFPS) rate  were also significantly higher in the SABR vs. CFRT group. All 3 rates remained higher in the SABR group after propensity score matching. Multivariable analysis identified SABR and a biologically effective dose ≥65 Gy as favourable predicators of OS. There was no difference between treatment groups in the incidence of radiation-induced liver disease or increase of Child-Pugh score ≥ 2 within 3 months of radiotherapy.Among the 104 evaluable patients (45 in the SABR group and 59 in the CFRT group), the overall response rate  was significantly higher in the SABR group than the CFRT group (62.2% vs. 33.8%, SABR was superior to CFRT in terms of ORR, OS, and IFPS. We suggest that SABR should be the preferred technique for HCC patients with PVI. Portal vein invasion (PVI) frequently develops in patients with advanced hepatocellular carcinoma (HCC), and has an estimated incidence rate of 34 to 80% , 2. WithFor various locoregional modalities including surgical resection, transarterial chemoembolization (TACE), transarterial radioembolization, hepatic artery infusion chemotherapy attempted in patients with PVI, only carefully selected patients are amendable. Radiotherapy (RT) presents the only noninvasive alternative which is not dependent on vasculature to access the tumor, and is therefore not associated with a risk of hepatic ischemia. Over the past few decades, the development of the three-dimensional conformal technique has allowed for partial liver irradiation. Studies of conventionally fractionated radiotherapy (CFRT) in PVI have shown improved outcomes, with 1-year overall survival (OS) of 16.7–40.2% and overall response rates (ORR) of 23.5–45% –12.Stereotactic ablative radiotherapy (SABR) is an emerging technique, and can achieve tumorcidal doses in limited fractions, with significant normal tissue sparing. Several studies have reported favorable results with SABR for treating different cancers , 14. We The aim of this study was to present a single-institutional experience with a relatively large number of patients, and to compare the difference in clinical outcomes between the two RT techniques.After obtaining waiver of consent for this retrospective research from the institutional review board of Tri-Service General Hospital , we identified HCC patients with PVI undergoing RT from January 2007 to December 2016. Patients with Vp3/Vp4 invasion , an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2 and a Child-Pugh (CP) class of A or B were eligible. Any prior interventions were allowed, except for previous RT. Those patients were considered unsuitable for standard locoregional treatment via multidisciplinary committee discussion. Given increasing evidence of the benefits of SABR for HCC patients in the past decades, our hospital practice naturally shifted from use of CFRT to use of SABR. Because SABR was not reimbursed until 2015 in Taiwan, the choice of RT technique was determined partly by the patients’ financial resources.Diagnosis of HCC was made either by biopsy or by radiologic investigation based on characteristic imaging findings . PVI wasFor better delineation of the upper gastrointestinal tract, most patients were asked to take oral contrast medium before simulation. A non-contrast CT simulation with a 3 mm slice thickness was performed. During the scanning procedure, all patients were supine, and were immobilized with a vacuum cushion, with arms raised overhead. Motion management with four-dimensional CT was conducted in some patients. Breath-holding was not mandatory. Either three dimensional conformal radiotherapy (3DCRT) or intensity-modulated radiotherapy was designated, depending on the physicians’ discretion, using the Nucletron Plato RTS v2.6.3 planning system, which was replaced by Philips Pinnacle v9.0 planning system in 2009. Instead of defining a gross target volume, the clinical target volume (CTV) was contoured directly. Contrast images registration was used to assist CTV delineation. The CTV was defined as a margin of 3–5 mm around the detectable PVI. Partial hepatic tumor was included in the CTV if the primary hepatic tumor was near to the detectable PVI or if the portal vein was invaded by the tumor directly. An example of target delineation is shown in Fig. SABR was administered using the CyberKnife radiosurgery system , delivering 6 MV photons. At least one week before CT simulation, 4–6 fiducial markers were placed within or around the tumor under sonographic or CT guidance. An individually shaped vacuum pillow allowed patient immobilization in the supine position, with a vest for synchrony tracking and with abdominal compression devices to reduce respiratory motion. A contrast CT scan with a 1 mm slice thickness was obtained for treatment planning with or without MRI scan registration. The definition of CTV in SABR was the same as in the CFRT technique. The PTV was defined as the volume with a margin of 0–3 mm added to CTV for patients with fiducial implantation. If fiducial implantation failed or was unsuitable, the PTV margin was expanded to 8–20 mm craniocaudally, based on the liver motion. Margin modification was permitted for respecting normal tissue tolerance. Prior to August 2009, the MultiPlan CyberKnife treatment planning system version 1.7.0 was employed for treatment planning, and was updated to version 2.1.0 thereafter. The median total dose was 45 Gy, at 6–12.5 Gy per fraction, with 4 to 5 fractions administered on consecutive working days. The detailed dose-limiting organs and their constraints are described in our previous publication .All patients were seen at least once per week during RT, 1–2 months for the first six months after RT, and every three months thereafter. Image evaluation with CT or MRI was obtained every 1–3 months. Modified Response Evaluation Criteria in Solid Tumors was used for evaluation of the PVI response .Hepatic toxicity assessment consisted of Radiation-induced liver disease (RILD) and increase of CP score ≥ 2. Only patients with either adequate follow-up of three months or death and/or occurrence of toxicity within 3 months were included in the analysis. Physical examinations and blood tests conducted at every visit, were used for toxicity assessment. RILD was defined as either classic or non-classic, without intrahepatic tumor progression noted within three months after RT. Classic RILD manifested as the presence of nonmalignant ascites and the elevation of anicteric alkaline phosphatase level to at least twice the upper normal values. Non-classic RILD manifested as the elevation of transaminase levels to at least five times the upper limit of the normal or pre-treatment values.p < 0.05 was designated as statistically significant for all tests except when variables with p < 0.10 in the univariable Cox model were entered into a multivariable Cox model. SPSS  version 22 and R statistical software version 3.4.3 ) were used for data analysis.Doses were converted to biologically effective doses (BED) with an α/β ratio of 10 for analysis. Between-group comparisons were conducted by chi-square tests or by Student’s t-tests as appropriate. OS was measured from the first day of RT until the date of death from any cause or last follow-up. In-field progression-free survival (IFPS) was measured from the first day of RT until the date of tumor recurrence or progression in irradiated field or last follow-up and patients who have received local intervention  were censored at the date of procedure. Kaplan Meier curves were constructed for OS and IFPS, and the difference was compared using the log-rank test. The Cox Proportional Hazards model was applied to identify potential predictors of survival. A receiver operating characteristic (ROC) curve was used to define optimal cut-off points for continuous variables based on the Youden index. Given the imbalance of potential confounders between groups, propensity score-matching was used. Propensity scores were estimated from a logistic regression model that included treatment period, sex, age, prior treatment, virus infection type, performance, tumor size, tumor number, extrahepatic metastasis, PVI location, AFP level, CP class, and sorafenib use. A nearest neighbor method with a caliper width of 0.2 was used to create matched cohorts. A 2-tailed Initially, 186 patients were identified by chart review. Forty-six patients were excluded owing to ineligibility, missing data or loss of follow-up after RT Fig. . Finallyp = 0.052). Among evaluable patients, five complete response, 23 partial response, 15 stable disease, and two progressive disease cases were observed in the SABR group, and five complete response, 15 partial response, 27 stable disease, and 12 progressive disease cases were observed in the CFRT group. The ORR  was significantly higher in the SABR group than it was in the CFRT group . Of all patients, 18 in the SABR group and 13 in the CFRT group were able to achieve either complete or partial recanalization of the invaded vein ; subsequent TACE was conducted in 9 patients of the SABR group and in 11 patients of the CFRT group .Follow-up images were available for 45 SABR patients and for 59 CFRT patients , these variables were analyzed by two different Cox models to avoid collinearity. The presence of SABR and BED ≥65 Gy correlated significantly with superior OS in separate multivariable analysis models (Table p = 0.005)  in the SABR group, and grade 3 diarrhea (n = 2) in the CFRT group were recorded. One patient in CFRT group presented with a treatment-related grade 5 duodenal ulcer.Acute toxicities observed in the groups are shown in Table p = 0.646). There were no RILD–related deaths. After excluding 23 patients with missing follow-up CP scores, 10 patients (22.2%) in the SABR group and 19 patients (26.4%) in the CFRT group experienced an increase of CP score ≥ 2, which was not statistically different between groups (p = 0.612).Seven patients (13%) in the SABR group and 11 patients (12.8%) in the CFRT group experienced non-classic RILD, while two patients (3.7%) in the SABR group and six patients (7%) in the CFRT group experienced classic RILD. The incidences of RILD were not different between groups, even after pooling RILD types ; similar trends were observed with local control and tumor response. In the meta-analysis performed by Rim et al. [Until now, direct head-to-head comparisons between different RT techniques in HCC with PVI have remained rare and controversial. In a cohort of patients with either PVT or inferior vena cava tumor thrombosis, Matsuo et al.  treated m et al. , SABR dim et al. , in whicp = 0.034). Toya et al. [Some studies reported a higher prescribed dose or BED was correlated with better LC or survival –26. Accoa et al.  have alsa et al.  reportedHistorically, RT has not been widely used for liver tumors because of a significant risk of RILD, though modern RT techniques have reduced this risk. A RILD incidence of 8–19% for CFRT , 30 and The high possibility of failure outside the radiation field implies the necessities for combining regional or systemic treatments with RT. Numerous combinations have been reported, though most were retrospective and poorly-evidenced. A recently-published randomized-controlled trial conducted by Yoon et al.  demonstrThe primary limitation of this study is the retrospective, single-institutional design. Even though propensity score matching minimizes the bias related to treatment assignment, unmeasured confounding may have existed. Further prospective studies are warranted. We note that issues regarding state-of-the-art technology may have affected treatment outcomes. However, we grouped patients into 2 treatment periods in the statistical analysis, which may eliminate this concern.In summary, we demonstrated that compared to CFRT, SABR led to superior ORR, OS, and IFPS in propensity score-matched PVI patients. SABR delivers higher BED without increasing hepatic toxicities, and hence is a suitable RT modality for PVI patients. Further studies are required to validate our results.


The Chinese believe that the meat of pigs reared in the past with free range tastes better than that of the pigs reared indoor on a large scale today. Gastrointestinal microflora is closely associated with the main factor of meat flavour, including fibre characteristics and lipid metabolism. Our method in this study involved different raising patterns within the semi free-grazing farm (FF) or indoor feeding farm (DF), the measurement of fat deposition and myofiber type by paraffin section and reverse transcription polymerase chain reaction and the identification of microbiome and functional capacities associated with meat quality through metagenomic sequencing.Oscillibacter, in the feces of the FF group was higher than that in DF group, and the relative abundance of some bacteria with probiotic function, including g_Lactobacillus and g_Clostridium, was lower than that in DF group. The abundance of g_Clostridium was significantly positively correlated with the intramuscular fat area, whereas health-related bacteria, such as g_Butyricicoccus, g_Eubacterium, g_Phascolarctobacterium and g_Oribacterium, was significantly negatively correlated with abdominal fat area, myofiber density and adipose triglyceride lipase (ATGL) mRNA expression. KEGG analysis showed that pigs raised in semi free-grazing farm can activate the pathway of inosine monophosphate (IMP) biosynthesis, glycolysis/gluconeogenesis and alanine, aspartate and glutamate metabolism.Results showed that the fat area in muscle and adipose tissue and the myofiber density significantly increased in the pigs of the FF group. The relative abundance of bacteria associated with lipid metabolism, such as g_Free range feeding improves meat quality by changing the fibre type, myofiber density and metabolic pathways related to flavour amino acids, IMP or glycolysis/gluconeogenesis in muscle. However, prolonged feeding cycle increases fat deposition and associated microbial communities.The online version of this article (10.1186/s12866-019-1556-x) contains supplementary material, which is available to authorized users. Except for the influences of nutrition, feeding management and slaughter mode, muscle fibre and intramuscular fat are the main factors affecting muscle quality, such as tenderness, juiciness and flavour. The number and volume of muscle fibres mainly determine the meat quantity, and the type of muscle fibre is closely related to the pH change, colour, and water-holding capacity of post-mortem muscles –3. The cIntestinal flora and its metabolites are closely associated with the immune system, bone, cardiovascular system and nervous system and thus affect the physical and mental health , 6. MostAlmost all the Chinese believe that pork raised in the past with free range tastes much better than pork raised indoor on a large scale today. On June 18, 2016, the famous international media New York Times published large-scale reports on Linan Sun Commune and China’s rural construction. They followed past pig raising model, where pigs were running in the yard, swimming in the pool, rolling in the mud, and using their mouths to arch the fruit tree fences. Hence, in this study, pigs in the same litter were raised separately. One part of the pigs was raised in traditional indoor farm, while the others were raised above Linan Sun Commune with the semi free-grazing model. The main factors affecting meat flavour, including fibre characteristics and lipid metabolism, and the correlation between porcine gut microbiome and muscle fibre or fat deposition would be tested, which will provide a theoretical basis for regulating pork quality through intestinal microflora and its metabolites.p < 0.01) bigger than that in the indoor feeding farm (DF) group  higher than that in the DF group  higher than that in the DF group  group were significantly p < 0.0 bigger tMetagenome sequencing by using HiSeq Illumina platform produced 64,035.32 Mbp of raw data from 10 faecal samples , Clostridium (6.05%), Oscillibacter (3.74%), Lactobacillus (3.28%), Ruminococcus (3.03%), Bacteroides (2.15%), Treponema (1.84%), Streptococcus (1.44%), Alistipes (1.24%) and Methanobrevibacter (0.68%), while those in the FF group included Prevotella (8.64%), Oscillibacter (6.81%), Clostridium (4.72%), Ruminococcus (2.90%), Bacteroides (2.65%), Streptococcus (1.73%), Treponema (1.25%), Alistipes (0.60%), Lactobacillus (0.48%) and Methanobrevibacter (0.10%) ) and FF (2.84%). The species were followed in abundances by Lactobacillus johnsonii (1.19%), Prevotella sp. P5–92 (0.89%) and Prevotella sp. P2–180 (0.74%) in the DF group and by Ruminococcus sp. CAG:177 (1.65%), Oscillibacter sp. PC13 (1.35%) and Clostridium sp. CAG:138 (1.34%) in the FF group  and FF .84%. TheR = 0.373, p < 0.05) in the species level  and ANOSIM distances were utilised to visualise the differences in taxa composition between groups. The PCA cluster showed an obvious separation between the DF and FF group Fig. b, and thvel Fig. c, whereivel Fig. c. The bap < 0.05), back fat area (p = 0.1) and MyHC2b (p = 0.1) had the greatest effect on microbial community composition, followed by myofiber density, leaf fat area, intramuscular fat area, ATGL mRNA, SOSC3 mRNA and MyOD1 mRNA. After applying CCA, all significant correlation between the selected phenotypic data and gut microbial by using the spearman rank correlation at the genus level are shown as a heat map in Fig. We used the canonical correlation analysis (CCA) to detect the correlation between the eight selected environmental variables and the overall microbial community Fig. a. CCA ang_Clostridium , and a significant negative correlation was observed in the intramuscular fat area of g_Streptococcus   and g_Succinatimonas , whereas a significant negative correlation was observed in myofiber density and abdominal fat area of g_Oribacterium , g_Phascolarctobacterium , g_Eubacterium  and g_Butyricicoccus . A significant negative correlation was observed in ATGL mRNA expression of g_Oribacterium , g_Butyricicoccus , g_Faecalibacterium  and g_Butyrivibrio    and a significant negative correlation in leaf  and back fat area  were observed on g_Angelakisella  expression and a significant negative correlation in leaf  and abdominal fat area  were observed on g_Eubacterium  revealed a predominance of pathways related to metabolism , genetic information processing (translation) and environmental information processing (membrane transport) as shown in Additional file The histological characteristics of muscle fibre are closely related to meat quality . Rearing conditions lead to substantial changes in the MyHC transformation as evidenced by the different proportions of myofiber types and differences in their myofiber numbers. Muscle tissue is composed of myofibers that can be subdivided into four interchangeable types, marked by the expression of four myosin heavy chain (MyHC) isotypes: one slow (MyHC I) and three fast MyHC isoforms  . The shoIn comparison with the DF group, percentage changes of MyHCs in the FF group were only found between oxidative myofibers: I ← IIa → IIx, whereas no changes were observed in the percentage of MyHC IIb. The main reason for the increased percentage of slow-twitch type I myofibers in the FF group in our study may be explained by the different raising condition. As a comprehensive factor, raising pattern had a substantial influence on muscle fibre composition. In our study, pigs in the DF group were kept under indoor breeding conditions and fed with a conventional commercial diet ad libitum, while the pigs in FF group were raised in a large yard and were highly physically active daily. This result was consistent with the report of Fazarinc et al. 2017) , wherein , whereiThe composition of skeletal muscle fibre type was related to meat quality, lipid content and distribution in muscle, which directly affects muscle quality . The smaIntramuscular accumulation of lipids and myofiber type of skeletal muscles is the result of interactions between genetical background and environmental factors. Other explanation for increasing IMF and levels of type I fiber in longissimus dorsi muscles of animals in FF group maybe influence by some bioactive compounds in diet, the longer feeding cycle. For example, the bioactive compounds in mulberry leaves may play a regulatory role in fat deposition and muscle fiber types, like resveratrol, anthocyanin or 1-deoxynojirimycin . A decreFirmicutes/Bacteroidetes (F/B) was associated with the energy harvest and fat deposition of host [Oscillibacter was the major determinant of obese or normal status [Bacteroides and Faecalibacterium were equally abundant. As a conditional pathogenic bacterium, Oscillibacter was positively associated with obesity-related metabolic pathways [g_Oscillibacter in feces was relative higher in higher fat deposition group. In the top 10 genera, the relative abundance of g_Lactobacillus was relative higher in the DF group. Linear discriminant analysis effect size (LEfSe) analysis found that s_Lactobacillus_johnsonii and s_Lactobacillus_reuteri were significantly higher bacterial species in the DF group. Lactobacillus is recognised probiotics in animal husbandry, which compete for nutrients with existing gut microbiota, which reduce body weight and fat mass [g_Lactobacillus and obesity are controversial. Among the bacterial genera, Lactobacillus increased after a weight loss program in adolescents [s_Lactobacillus_reuteri was associated with obesity in adults. These findings suggest a possible role of Lactobacillus at the species level in body weight and obesity. From the LEfSe analysis at the species level, significantly higher bacterial species was found in the FF group s_Firmicutes_bacterium_CAG_176, s_Oscillibacter_sp_CAG_241_62_21 and s_Firmicutes_bacterium_CAG_83, which all belong to Clostridium cluster IV. Clostridium cluster IV is a dominant bacterial group known as butyrate-producing bacteria [Clostridium cluster IV decreases in vegetarians [s_Ruminococcus_flavefaciens was another higher bacterial species in the DF group. Ransom-Jones et al. (2012) [R. flavefaciens and Clostridium cluster IV degraded dietary fibre. Above all, bacteria related probiotic function, including g_Lactobacillus and Clostridium cluster IV abound in DF group, and the relative abundance of g_Oscillibacter, which was associated with obesity, was higher in the FF group.The effect of feeding pattern on intestinal microorganisms was studied. Prolonged of feeding cycle of pigs in the FF group increased fat deposition. The ratio of  of host , and the of host , which sl status , when Bapathways . Consistfat mass . The rellescents , but it lescents . Millionlescents  demonstrbacteria , which hbacteria . The abuetarians . s_Rumin. (2012)  found thRoseburia and Blautia, were higher in obese Rongchang pigs and its mouse recipients [g_Butyricicoccus, g__Eubacterium, g_Phascolarctobacterium and g_Oribacterium had significant negative correlation with abdominal fat area, myofiber density and ATGL mRNA expression. Butyricicoccus is a butyrate-producing clostridial cluster IV genus. The abundance of Butyricicoccus increased significantly with age [Eubacterium of the gut microbiota belongs to Clostridium clusters XIVa, and are known for polysaccharide fermentation and bile acids dehydroxylation [g_Oribacterium is a health-associated genus [Phascolarctobacterium may exert a beneficial role in the human gastrointestinal tract and can produce short-chain fatty acids, including acetate and propionate [Phascolarctobacterium spp. specialise in the utilisation of succinate produced by other bacteria. The abundance of Parabacteroides, which is a major producer of succinate, was increased by high fat diet and was positively correlated with body weight [Phascolarctobacterium faecium-like bacteria in younger individuals were maintained at a high level with a gradual increase with increasing ages (between 1 and 60 years old) but with a decrease in elderly individuals (> 60 years old) [Reports about the relationship between faecal microbial and muscle fibre types or fat deposition were rare. Yan et al. (2016) demonstrated abundance levels of major bacteria that produce butyrate and acetic acid, such as species from cipients . In our with age . Genera xylation . Patientxylation . Many Clxylation , 40. Cloxylation . Clostrixylation , 42. Thexylation , and lowxylation . g_Oribaed genus , which dopionate , 46. Besy weight . Phascolars old) . In brieSixteen functional KO were detected to be differentially abundant in at least one of the two raising patterns. Ten of these KO were significantly enriched in FF group compared with the DF group. Interestingly, two metabolic KO (K01952 and K00764) were involved in the modules of IMP biosynthesis M00048), which is the pathway module for phosphoribosyl pyrophosphate + glutamine = > IMP [, which iClostridium butyricum -treated group enriched alanine, aspartate and glutamate metabolism provided supporting evidence for the increased meat quality in breast muscle of ducks [C. utilis were up-regulated according to KEGG pathway modules analysis [O. flexuosa transcripts than from the nearly-complete genome of B.malayi [24h [Glu, Asp and Ala are important flavour amino acids, which can act on the precursor amino acid as a fundamental substance forming delicate flavour of meat; especially, Glu is the most pivotal amino acid affecting meat flavour and acid-base buffering capacity , 50. By of ducks , 52. Sigof ducks . IMP is of ducks –56. IMP of ducks . IMP andof ducks . The genanalysis . More seB.malayi . GlycolyB.malayi . Drip loB.malayi  found thayi [24h . A reporayi [24h . They hyayi [24h . NotablyIn conclusion, pigs reared in semi free-grazing farm increased myofiber density, MyHC I proportion and intramuscular fat area. It also activated the metabolic pathways related to meat flavour, including flavour amino acid metabolism, IMP biosynthesis and glycolysis/gluconeogenesis. These findings provide evidence for the good taste of free range pork. Prolonged feeding cycle of pigs in the FF group increased the conditional pathogenic bacteria associated with obesity.3 back fat or abdominal fat was collected from the junction of the sixth and seventh thoracic vertebra. Approximately 1 m3 leaf fat was collected from perirenal fat. Exactly 5-cm-thick longissimus dorsi muscle from the last third or fourth rib backwards was collected from the carcass within 30 min after exsanguination. One piece of longissimus dorsi muscle was placed in the RNA locker immediately for RNA isolation. The other piece of longissimus dorsi muscles, back fat, abdominal fat and leaf fat were fixed in 10% formalin for histology.Animal studies were conducted in accordance with the guidelines of the Zhejiang Farm Animal Welfare Council of China and approved by the ethics committee of Zhejiang Academy of Agricultural Sciences. Twenty Bajiazhe black piglets from Jicheng Animal Husbandry Co., Ltd. were divided into two groups of 10 at 3 months of age with equal representation of littermates and sex. Piglets in the group of the indoor feeding farm (DF) was fed with general compound feed  for 12–24 h and then dehydrated and subjected to routine haematoxylin eosin staining. The muscle tissue was randomly selected at 5–10 visual fields at 100× (intramuscular fat area measurement) or 200× magnification (muscle fibre number and single muscle fibre area measurement) visual field. Then, the intramuscular fat area and single muscle fibre area were measured using Image-ProPlus 5 image analysis software. The adipose tissue was randomly selected at five visual fields at a 50× magnification visual field. Then, the Image-ProPlus 5 image analysis software was used to measure the adipocyte diameter of each sample (15 cells per specimen), as well as the area of the single fat.− 6 dilution series template. The amplification efficiency ranged from 90%~ 110%. Therefore, the relative level of gene expression between different groups can be calculated using the 2-△△Ct method.Total RNA was extracted according to the RNAiso Plus instructions. According to the PrimeScript® RT-reagent Kit manual, the total RNA was used for first strand cDNA synthesis. The primers used are shown in Table S5. Real-time PCR was carried out with ABI plus one  by using SYBR Premix Ex TaqTM. Two-step PCR amplification was performed under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 62 °C for 34 s. Melting curve analysis was performed to verify the single-product generation at the end of the assay. Standard curves were generated based on the data obtained from the standards of the 2~2Total genomic DNA from was extracted using the hexadecyltrimethylammonium bromide method. The concentration of the extracted DNA was measured using 1% agarose gel electrophoresis. DNA purity was checked using Nano Photometer® spectrophotometer . OD260/OD280 ≈ 1.8 was a qualified DNA sample. Then, the sample was diluted to 1 ng/μL by using sterile water.Exactly 1 μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM DNA Library Prep Kit for Illumina  following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, the DNA sample was fragmented by sonication to a size of 350 bp. Then, DNA fragments were end-polished, A-tailed and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. At last, PCR products were purified (AMPure XP system), and libraries were analysed for size distribution in an Agilent2100 Bioanalyzer and quantified using real-time PCR. Index-coded samples were clustered on a cBot Cluster Generation System according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform, and paired-end reads were generated.https://github.com/cjfields/readfq). To prevent host DNA contamination, it needs to be compared with the host database to filter out the reads that may originate from the host by using SoapAligner software  with parameters [Clean data were obtained from raw data through quality control by using Readfq  with parameters [De novo assembly of the short reads. Clean data were assembled using the SOAPdenovo software   was used to predict ORFs in scaftigs from the single or mixed sample (> = 500 bp) [http://www.bioinformatics.org/cd-hit/) from predicted ORFs with parameters ‘-c 0.95, -G 0, -aS 0.9, -g 1, -d 0’ [MetaGeneMark . Informa1, -d 0’ . SoapAli1, -d 0’ . UnigeneCore-pan gene analysis, correlation analysis and Venn diagram of gene numbers were employed based on the gene abundance in each sample.https://www.ncbi.nlm.nih.gov/) of NCBI were used as reference database, and DIAMOND software  [https://en.wikipedia.org/wiki/Lowest_common_ancestor) was used to identify species annotation of the sequence. Then, the species abundance and gene numbers of the six-level taxonomic classification from phylum to species of each sample were obtained. Then, Krnoa analysis [p value of Metastat statistical analysis was obtained using permutation test of each classification between groups; then, the q value was obtained by the correcting p value by using the Benjamini and Hochberg false discovery rate method [Bacteria, fungi, archaea and viruses in the non-redundant (NR) library   was usediamond/) . To avoianalysis , relativanalysis  and NMDSanalysis . Dimensie method . LEfSe se method .http://www.kegg.jp/kegg/). Best blast hit (one HSP > 60 bits) from above blast was selected for subsequent analysis. The relative abundance of different function levels (five levels in KEGG database) was counted. Based on the abundance of different function levels, the numbers of annotated gene, relative abundance profile and heat map of abundance clustering were obtained, and PCA and NMDS dimension reduction analysis, ANOSIM analysis, metabolic pathway analysis and Metastat and LEfSe statistical analysis were carried out.DIAMOND software (V0.7.9) was used to blast unigenes with function databases with parameters ‘blastp, -e 1e-5’. The function databases included the KEGG database Additional file 2:Figure S2. Relative abundances of the bacteria in the top 10 species level taxa of the DF and FF groups. (TIF 275 kb)Additional file 3:Figure S3. Functional classification of annotated NR genes by using KEGG analysis. (TIF 304 kb)Additional file 4:Table S1. Total statistic information of the metagenome sequencing by using HiSeq Illumina platform from 10 faecal samples. (XLS 57 kb)Additional file 5:Table S2. Absolute abundance of unigenes in species level. (XLS 1105 kb)Additional file 6:Table S3. Composition of the basal diets in the traditional feeding farm. Table S4. Composition of the basal diets the semi free-grazing farm. Table S5. Primer sequences (5′to 3′) used for the quantitative polymerase chain reaction. (DOC 59 kb)


Mycoplasma capricolum subspecies capripneumoniae (Mccp). The disease is characterized by severe serofibrinous pleuropneumonia, very high morbidity (∼100%), and mortality (80–100%). CCPP affects goats in more than 40 countries of the world thereby posing a serious threat to goat farming around the globe. The characteristic clinical signs of CCPP are severe respiratory distress associated with sero-mucoid nasal discharge, coughing, dyspnea, pyrexia, pleurodynia, and general malaise. In later stages, severe lobar fibrinous pleuropneumonia, profuse fluid accumulation in pleural cavity, severe congestion of lungs and adhesion formation is observed. Mycoplasmal antigen interactions with host immune system and its role in CCPP pathogenesis are not clearly understood. CCPP is not a zoonotic disease. Diagnosis has overcome cumbersome and lengthy conventional tests involving culture, isolation, and identification by advanced serological  or gene-based amplification of DNA  and sequencing. The latex agglutination test (LAT) is rapid, simple, and better test for field and real-time diagnosis applicable to whole blood or serum and is more sensitive than the CFT and easier than the cELISA. Moreover, the studies on antibiotic sensitivity and exploration of novel antibiotics  can help in better therapeutic management besides preventing menace of antibiotic resistance. Re-visiting conventional prophylactic measures focussing on developing novel strain-based or recombinant vaccines using specific antigens (capsular or cellular) should be the most important strategy for controlling the disease worldwide.Contagious caprine pleuropneumonia (CCPP) is a serious disease of goats, occasionally sheep and wild ruminants, caused by  Considering the importance of this disease, worldwide measures are being taken for understanding this disease for better diagnosis, prevention, and control. Periodically, the disease dynamics are reviewed by researchers and agencies of national and international mandate and repute for keeping vigil on this transboundary emerging threat that endangers naive goat populations in various disease-free countries from the surrounding infected herds of neighboring and mutually cooperative countries. The chronological reviews on CCPP by Rurangirwa et al. (1987), Thiaucourt et al. , ThiaucoOur effort in this regard has been to revive the study of the disease dynamics including epidemiology, pathophysiology, current status, and future prospects with emphasis on diagnostics, therapeutics, and prophylactics and future challenges or threats.1.1.CCPP is considered as one of the most severe and highly infectious disease of goats. It results in heavy economic losses to countries involved in goat farming especially in Africa, Asia and the Middle East , Mycoplasma mycoides subsp. mycoides small colony strains (MmmSC), Mycoplasma sp. bovine group 7 of Leach (Mbg7), Mycoplasma capricolum subsp. capricolum (Mcc), and Mycoplasma mycoides subsp. capri (Mmc)  are considered naturally susceptible domestic species, and sheep can also be affected -Pashmina goat interaction could not predict any effect on the occurrence of CCPP although improvement in diagnostic aids may help in the detection of CCPP in wildlife  in the environment and longer in affected animals (weeks to months) , persistence of infection may be 5–10 days if the animal survives or until recovery in chronic stages or development of sterile immunity. However, exact data on persistence of infection are not published yet  or pasteurellosis infection to which CCPP is often confused in many parts of the world also cause variability.In most countries, meagre data on CCPP prevalence have been produced and where available figures are diverse. One of the reasons for variability is type of diagnostic test used and laboratory protocol followed. Time of observation also affects prevalence outcome of the study for a particular area. Frequent infection of goats with other mycoplasmas of the Risk factors may also affect prevalence , especially when no therapeutics are used. Mortality rate may be up to 90% and CFR ranges from 60 to 100% MacOwan  but usua2.9.CCPP is not a zoonotic disease AU-IBAR . There i3.CCPP is characterized by clinical signs of respiratory system involvement aggravated by mycoplasma induced systemic serofibrinous and inflammatory cascade that affect the lower respiratory tract (including the lungs), pleura and pleural cavity, and associated organs (heart), sometimes the upper respiratory tract, and rarely the eyes, joints, udder, liver and kidneys. The disease may be manifested in peracute, acute or chronic forms. Severe forms of the disease like peracute and acute forms usually result when fully susceptible herds are exposed to the pathogen, whereas chronic forms occur in endemically affected areas. In peracute form, death is sudden usually within 24–72 h and by definition without premonitoring (respiratory) signs MacOwan . In lessOther respiratory signs include inspiratory dyspnea accompanied by grunting and snoring due to inflammatory lesions or exudations. Cough is generally painful, moist and productive and exacerbates on exercise. Continuous nasal discharge, which is initially serofibrinous straw coloured exudate followed by thick mucoid or purulent and rust coloured, might be observed  in CCPP on pathology often unilateral, followed by alveolar exudation and pleural fluid accumulation (91%) and pleural adhesion (73%) when the microscopic lesions mainly comprise of septal peribronchiolar fibrosis (82%), along with fibrinous pleuritis (64%) and peribronchiolar cuffing of inflammatory mononuclear cells (55%) in pulmonary tissue   activate the immune system and stimulate the inflammatory and oxidative cascade , pancreatic digest of casein (7.0 g), beef extract (3.0 g), yeast extract (3.0 g), sodium chloride (5.0 g), agar (14.0 g) enriched by horse serum (20.0 ml), yeast extract (fresh autolysate) (10.0 ml) and thallium acetate (50.0 mg). Such media are generally supplied commercially.2, sometimes with 95% N2  for up to 10–14 days . Agglutination tests (non-specific and specific), immunoassays (enzyme-linked immunosorbent assay [ELISA]), fluorescent antibody assay (FAT), complement fixation assay (CFT), passive or indirect haemagglutination test (IHT), amplification tests  are the currently employed diagnostic tests with latex agglutination, cELISA, and PCR being of prime importance and routinely used in global studies. However, the field level availability or even absence in research facilities in most of the countries is a major limitation. Moreover, cost involvement is another binding on use in limited spheres  and CFT were reported as diagnostic tests for CCPP by Muthomi and Rurangirwa  in 0.03 ml of serum. Simultaneously, serological investigations were carried out utilizing these serological tests in Ethiopia by Sharew et al.  was by MacOwan and Minette  for diagrangirwa . These trangirwa . Refinemrangirwa . Furtherrangirwa  reportedrangirwa . Lefèvrerangirwa  mentionerangirwa  in the drangirwa  to Mycopt et al.  in theirt et al.  for the w et al. . Furtherw et al.  developew et al. . Mekuriaw et al.  used CFTw et al.  used CIEw et al.  and is bw et al. . Hence, w et al.  and has w et al.  as it isw et al. . Using tw et al.  studied w et al.  and LAT w et al. , Sheikh w et al. , and Thaw et al. . Bahir ew et al.  recentlyw et al. . From thHowever, the main limitations of these serological tests have been non-specificity , complementary deoxyribonucleic acid to mycoplasmal ribosomal ribonucleic acids , synthetic 16S rRNA oligonucleotide sequences, or cloned mycoplasmal protein genes Razin . The DNAMycoplasma mycoides cluster and noted that Mccp can be easily identified by three specific nucleotide positions or by sequencing the 1298 bp long fragment. Improvising PCR techniques for convenience and identifying novel and specific DNA segments lead to emergence of new PCR protocols for Mccp. Another specific PCR system was developed by Woubit et al.  was used by He et al. .As a result, these PCR-based diagnostic systems proved effective both at field and laboratory level diagnosis. However, with passage of time need for exploring full genome of Mccp and strain-specific differentiation among Mccp strains arose. Chu et al.  studied Thus, the molecular gene-based techniques including PCR, DNA hybridization, and sequence analysis can become the tools of choice for evaluating phylogenic relationships among mycoides cluster . This creates air bronchogram which are characteristic sign of consolidation. Other X-ray findings in consolidated lungs are poorly defined uniform or homogeneous opacity obscuring blood vessels, formation of silhouette, loss of lung or soft tissue areas or interface, air-bronchogram extension to the pleura or fissure, but usually not crossing it, and no volume loss. Mycoplasmal pneumonia starts perihilar and can become confluent and/or patchy and no air bronchograms as disease progresses. On X-ray there is opacity with pleural effusion, airspace opacity, lobar consolidation, or interstitial opacities.6.M. bovis) included oxytetracycline (>32), enrofloxacin (2.5–32), tylosin (8–1024), and in some Mycoplasma mycoides cluster field isolates antibiotics tested were florfenicol (2.3–6), tylosin (1.3–6.6), ceftiofur (0.8–1.2), sulphamonomethoxine (2.3–7.3), erythromycin (1.7–6.7), lomefloxacin (3.3–6.7), and ofloxacin (6.7–9.3) , Middle East , Turkey and China. However, concerns have been raised regarding the quality of these vaccines when considering the European Pharmacopoeia guidelines  for effective treatment and minimizing risk of antibiotic resistance. Prevention and control are better achieved through prophylaxis. Vaccination needs advancement from conventional inactivated vaccines to subunit and recombinant vaccines exploring specific local isolates with most potent antigens (capsular glycolipids or proteins or cellular enzymes or proteins) for better immune response and requirement for single vaccination at the most not less than annually. Global efforts are required for combating this emerging and severe threat to goat farming globally over by devising appropriate diagnostic, prophylactic and therapeutic regimes for prevention and control of CCPP and OIE, FAO, AU-IBAR, EFSA AHAW and national or regional research stations have important role to play in future also.


Little is known about the correlation between the clinicopathological features, postoperative treatment, and prognosis of multiple gastric cancers (MGCs). In this study, we aimed to investigate the correlation between these features and the impact of postoperative adjuvant chemotherapy on the long-term survival of patients with MGC.The clinical and pathological data of patients diagnosed with gastric adenocarcinoma who had radical gastrectomy from January 2007 to December 2016 were analyzed. Using propensity score matching, the prognostic differences, and the impact of postoperative adjuvant chemotherapy between those with MGC and solitary gastric cancers (SGC) were compared.P = 0.013). Multivariate analysis revealed that MGC, age, pT stage, pN stage, and adjuvant chemotherapy were independent predictors of OS . Stratified analyses demonstrated that for the cohort of advanced gastric cancer (AGC) patients who did not had adjuvant chemotherapy, the 5-year OS rate of advanced cases of MGC was inferior than that of SGC patients  but there were no significant difference in the 5-year OS rate between advanced MGC and SGC patients who had adjuvant chemotherapy . Further, we found that the 5-year OS rate of advanced MGC who had adjuvant chemotherapy was significantly higher than those who did not had adjuvant chemotherapy .Among the 4107 patients investigated, the incidence of MGC was 3.2% (133/4107). Before matching, patients with MGC and SGC had disparities in the type of gastrectomy, pathological tumor stage (pT), pathological node stage (pN), and pathological tumor-node-metastasis stage (pTNM). After a 1:4 ratio matching, the clinical data of 133 cases of MGC and 532 cases of SGC were found to be comparable. The 5-year overall survival (OS) rate was 56.6% in the entire matched cohort, 48.1% in the MGC group, and 58.7% in the SGC group (Patients with advanced MGC was identified as having a poorer survival as to SGC patients, but the implementation of postoperative adjuvant chemotherapy showed that it had the potential to significantly improve the long-term prognoses of MGC patients. Gastric cancer (GC) is the 4th most common malignant tumor and the 3rd leading cause of cancer-related mortality worldwide . Multipln = 74); (2) a history of previous gastric surgery (n = 77); (3) treatment with neoadjuvant chemotherapy (n = 140); and (4) pathological T4b stage tumor (n = 95); and (5) incomplete clinical and pathological data (n = 120). All patients’ clinical and pathological data were collected using the Gastric Cancer Clinical Data Management and Analysis System  [The clinicopathological data of 4613 patients diagnosed with gastric adenocarcinoma who had radical gastrectomy at the Fujian Medical University Union Hospital  from January 2007 to December 2016 were retrospectively analyzed. The inclusion criteria were as follows: (1) preoperatively confirmed diagnosis of GC; (2) no evidence of distant metastasis before surgery; (3) radical gastrectomy (R0 resection); and (4) no records of other additional adjuvant treatments other than adjuvant chemotherapy. The exclusion criteria were as follows: (1) a history of previous malignancy  . Each reThe definition of MGC complied with the criteria proposed by Moertel et al. , which a2, based on the World Health Organization (WHO) classification standards. Family history for the first- and second-degree relatives was queried and was categorized as absence of family history, family history of GC, and family history of other malignancy. The comorbidities analyzed in this study comprised of hypertension, diabetic mellitus, heart disease , pulmonary disease , central nervous system disease , liver disease , renal disease , anemia, hypoalbuminemia, and hyperthyroidism. The postoperative pathological stage of the tumor was determined according to the eighth edition of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) staging manual [2. Capecitabine/S-1 was administered orally twice daily from day 1 to 14 , followed by 1-week rest; after which the next cycle was resumed.Variables included in this study were age, sex, body mass index (BMI), family history, tobacco and alcohol use, the American Society of Anesthesiologists (ASA) score, comorbidities, clinical tumor stage (cT stage), clinical node stage (cN stage), surgical approach, type of gastrectomy, number of eLNs, pathological tumor stage (pT stage), pathological node stage (pN stage), pathological tumor-node-metastasis stage (pTNM stage), tumor size, tumor histological type, postoperative complications, and adjuvant chemotherapy. The patients’ BMI were classified into these following categories: < 18.5, 18.5–24.9 and ≥ 25 kg/mg manual . The typg manual . Based og manual . Most adg manual , 14. AllFollow-up evaluation after surgery generally consisted of clinic visits, serologic chemistry profiles, and computed tomography (CT) scans repeated every 3–6 months for the first 2 years then every 6–12 months for the following 3–5 years, and annually afterwards. The overall survival (OS) time was recorded as from the date of surgery to the last follow-up date, date of death, or date until the end of follow-up (such as loss to follow-up or death due to other diseases). The last date of follow-up was June 2017.t-test or the Mann–Whitney U test. The propensity score matching ratio was set to 1:4 ratio to minimize the differences between MGC and SGC due to age, sex, BMI, ASA score, comorbidities, clinical stage, type of gastrectomy, and pathological stage with the nearest neighbor method using R software  [P < 0.05) in the univariate analysis were computed into the multivariate analysis to identify the independent prognostic factors. Statistical analyses were performed using the IBM SPSS software  and the R software, all statistical tests were two-sided, and a P value < 0.05 was considered statistically significant.Categorical variables were analyzed using the Chi square test or Fisher’s exact test, whereas continuous variables were analyzed using the student’s ect.org) , 16. TheA total of 4107 patients were included in this study, comprising of 133 3.2%) patients with MGC and 3974 (96.8%) patients with SGC , pT stage (P = 0.033), pN stage (P = 0.035) and pTNM stage (P = 0.041) between the two groups, but no significant differences were observed in the preoperative data, namely, age, sex, BMI, family history, tobacco and alcohol use, ASA score, comorbidities, cT stage, or cN stage   Tables .Table 1PP > 0.05 between all variables) , and the 5-year OS rate was 56.6%. For the MGC and SGC groups, the 5-year OS rates were 48.1% and 58.7%, respectively  and SGC  were found (P = 0.014), age (P = 0.029), cT stage (P < 0.001), cN stage (P < 0.001), surgical approach (P < 0.001), type of gastrectomy (P < 0.001), pT stage (P < 0.001), pN stage (P < 0.001), tumor size (P < 0.001), histological subtype (P = 0.009), and adjuvant chemotherapy (P = 0.032) were correlated with OS. However, on multivariate analysis only MGC (P = 0.039), age (P = 0.043), pT stage (P < 0.001), pN stage (P < 0.001), and adjuvant chemotherapy (P = 0.014) were identified as independent risk factors for OS  patients who did not had adjuvant chemotherapy after surgery was found to be 43.4%, of which the 5-year OS rates of the MGC and SGC groups were 34.0% and 46.1%, respectively. This difference in 5-year OS was found to be statistically significant (P = 0.013) and that MGC was an independent risk factor for survival . Further, our results identified that postoperative adjuvant chemotherapy could improve the 5-year OS rate of patients with advanced MGC.Our study demonstrated that the 5-year OS rate of patients with MGC was 48.1%, which was significantly poorer than those with SGC (58.7%) , while for those who had adjuvant chemotherapy, the 5-year OS rates of patients with MGC and SGC were comparable . Further, we found that the 5-year OS rate of advanced MGC patients with adjuvant chemotherapy was significantly higher than those without adjuvant chemotherapy . The efficacy of adjuvant chemotherapy for AGC has been confirmed by multiple prospective studies. In multicenter studies from Japan and South Korea, the 5-year OS rate of patients receiving adjuvant chemotherapy after surgery was significantly higher than that of patients undergoing radical surgery alone [As shown in Fig. ry alone , 24, andDespite demonstrating the impact of adjuvant chemotherapy on the long-term survival of patients with MGC, the present study has several limitations that need to be addressed. First, the retrospective and non-randomized nature of this study makes it subjective to certain selection bias. Second, since our database only recorded whether the enrolled patients had or did not have adjuvant chemotherapy, data regarding the specific number of chemotherapy cycles were incomplete, and we believe this may have had some impacts on the results obtained, to some extent. Third, since we were unable to obtain accurate data on the type of cancer relapse  from our database, we could not analyze if there was a difference in the type of relapse between MGC and SGC. Fourth, due to the limited cases of MGC in our study, we did not further classify N3 stage as N3a and N3b. Despite these limitations, this study provided a reliable insight on the outcomes of patients with MGC and could be used as a preliminary basis for prospective multicenter studies on the treatment selection for this category of patients.In this study, by using propensity score matching analysis to balance the differences between cofounding variables, we have found that MGCs were an independent factor for survival and demonstrated worsen postoperative prognoses as compared to those with SGCs. However, the implementation of postoperative adjuvant chemotherapy to advanced MGC cases demonstrated significant improvement in their long-term survival and should be highly recommended.