The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components  and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses. Fluorescence imaging has become an essential tool for research and education in biological sciences and bioengineering. However, instrumentation for fluorescence imaging is expensive and access to software is often restricted, which imposes a high barrier for democratizing its usage outside academic labs and wealthy research institutions. Open hardware and free/libre and open source software (FLOSS) provides the tools for fabricating these devices at a much lower cost and with better adaptability. The advent of easy-to-use microcontrollers, off-the-shelf electronics and customizable manufacturing technologies such as 3D printers and laser-cutting machines, has given rise to a diverse community of open hardware developers, users and tinkerers. This ethos has facilitated the development of inexpensive scientific devices and laboratory equipment –4; such A broad series of open source digital microscopes and imaging devices have been created e.g. –20). Som. Som20])Here, we combined low-cost color cameras, off-the-shelf electronics, inexpensive acrylic filters, open-source software, easy-to-use genetic resources, and recently developed long Stokes shift fluorescent proteins to create an integrated imaging system for multiple fluorescence signals from a single excitation wavelength . The devhttps://osf.io/dy6p2/.All vectors were constructed by Golden Gate using BsaI (NEB) and T4 ligase (NEB). The acceptor vectors for Golden Gate assembly were constructed by Gibson Assembly. Nine level 0 vectors and one acceptor vector compatible with the CIDAR MoClo Toolkit (Addgene) were constructed by Gibson Assembly. The CIDAR MoClo Parts Kit was a gift from Douglas Densmore (Addgene kit # 1000000059). The PCR fragments were amplified using Phusion High-Fidelity ADN Polymerase (NEB) and purified using Wizard SV Gel and PCR Clean-Up System (Promega). The purification of all plasmids was performed using Wizard Plus SV Miniprep DNA Purification System (Promega). All the assembled vectors were checked by colony PCR and later sequenced commercially (Macrogen Inc.). See E.coli Top10 (Invitrogen) grown at 37°C in LB medium. Media were supplemented with 1.5% m/v agar for assays on solid media. For inducer applications, we prepared a stock solution of 0.1 M IPTG, sterilized by 0.22 mm filter. Antibiotics were used as solutions of kanamycin (50 ug/ml) and carbenicillin (100 μg/ml).All tests were carried out on Different fluorescent proteins were tested by streaking transformed cells onto petri dishes from glycerol stocks and growing them for 24 hrs at 37°C. Expression was induced with 30 μl of 0.1M IPTG.E.coli Top10 (Invitrogen) . Cells stored at -80°C were thawed on ice for 20 minutes. For single transformations, a 1 μl of plasmid stock (concentration ~ 1ng/μl) was added to 50 μl of chemocompetent Top10 E. coli. In the case of co-transformations, 100 ng total of each plasmid were mixed with deionized water to obtain a final volume of 6 μl, which were then added to 50 μl of chemocompetent Top10 E. coli. The cells were incubated for 25 minutes and heat-shocked at 42°C for 1 minute, after which they were incubated on ice for an additional 2 minutes.The chemocompetent cells used for all the transformations were made by the CCMB80 method in A 250 μl volume of LB medium was added to each transformation and incubated with shaking (250 rpm) at 37°C. The incubation time for the single transformation was 1 hour, after which a volume of 25 μl of transformed cells was plated in solid LB medium supplemented with antibiotics. In the case of co-transformations, the incubation time was 1.5 hours, after which 35 μl of transformed cells were plated on LB-agar supplemented with antibiotics. Plates were incubated at 37°C overnight (~ 12–16 hours) for colony formation.Cultures of the three TOP10 cells containing the different vectors were grown overnight and mixed proportionally. The culture mixture was diluted 1:100 or 1:10,000 and a 0.5 μl drop was placed in the center of a petri dish. Plates were then incubated at 37°C for 12 hours and imaged every 12 hours for 7 days at room temperature.dx.doi.org/10.17504/protocols.io.jsecnbe).We have created a protocol online describing this experimental procedure in more details (dx.doi.org/10.17504/protocols.io.jsfcnbn).An ISO-GRID membrane filter  was placed on top of LB agar plates, with the hydrophobic grid facing up. Bacteria co-transformed with the sender/receiver plasmids were grown at 37°C in LB liquid media to OD600 0.03 (~5 hours) and 0.5 μl of cell culture was pipetted within each well following arrangements that depend on the assays. We have created a protocol online describing this experimental procedure in more details (dx.doi.org/10.17504/protocols.io.jsucnew).Time lapses and experiments shown in Figs For and raspistill -t 4000 -ss 50000 -ISO 300 -awbg 1,1 -co 30 -oTime lapses and experiments shown in Figs To allow for full customization, the mainframe was developed as enclosures made of editable laser-cut 3mm acrylic pieces . These cThe top module supports the Raspberry Pi 3 computer for camera control  S2 FileS2 File. http://docubricks.com/viewer.jsp?id=701517893260717056 for detailed assembly instructions and full description of the pieces.The bottom module, inspired by open source blue transilluminators used for DNA electrophoresis gels , holds tEscherichia coli grown in the same plate , developed in this work were created as modular parts standardized for fast, combinatorial and efficient fabrication of genetic constructs. In particular, we domesticated these components as “level 0” components following the CIDAR MoClo standards for Golden Gate assembly DNA assembly  Fig 2C)Fig 2C). In order to demonstrate the range of possible applications of our system, we tested different experimental setups using the Raspberry Pi camera software (RPI foundation). First, we imaged DNA electrophoresis gels loaded with different volumes of 1 kb DNA ladder; obtaining signal detection in the range of volumes often used in DNA gel electrophoresis (e.g. 4–6 μl) . Next, wIn order to provide resources that permit full flexibility in the construction of cells that produce and/or respond to these signals, we created level 0 parts for: i) LuxI and LasI enzymes, in charge of the synthesis of C6 and C12 homoserine lactones, respectively. ii) LuxR and LasR genes, in charge of binding and responding to C6 and C12 HSLs, respectively. and iii) pLux and pLas promoters, from which transcription is induced by LuxR/LasR in response to C6-HSL or C12-HSL respectively. With these tools, we created strong and weak senders and receivers of both signals and registered their behaviour in time . The imaNext, we evaluated an experimental design used in microbial ecology studies, colony-sectoring assay, in which a mix of bacterial strains is inoculated as a single drop on a petri dish and the dynamics of their growth registered in time lapse , 49 Fig. Strainshttps://github.com/SynBioUC/FluoPi; see folder “Hardware_control”). We also created an extensive Python module (fluopi) with functions for analyzing and visualizing these image sequences. We made extensive use of existing open-source projects Numpy, Scipy, Matplotlib, and Scikit-image, as well as the Raspberry Pi foundations python modules.We developed simple python code to operate hardware, run time-lapse experiments and store resulting image sequences e.g.  Movies, Jupyter provides an intuitive and interactive way to run and develop Python code while immediately viewing the results. As such, these notebooks are ideal for teaching classes or as an introductory tutorial for those students new to programming.Separately, we provide example Jupyter (IPython) notebooks containing code for analysis of time-lapse image series that contain detailed explanation of the analysis process. A simple tutorial notebook introduces the concepts used in loading images and processing them to extract biologically meaningful information . We also provide a simple tutorial notebook describing how to control the imaging hardware from Python . Below we show how this open source software is used to find the location of colonies, estimate their size, growth-rate, and fluorescence and from these data extract the rate of gene expression.Timelapse image sequences of bacterial colonies expressing fluorescent proteins contain valuable data about growth, gene expression and possible inter-colony interactions. The first step in extracting this information is to process images to generate separate signals for each colony’s size and fluorescence.The images are composed of three channels—red (R), green (G), blue (B) -, each of which can contain different information about the cells and is managed separately. We created a Jupyter notebook to analyze timelapse images of growing colonies constitutively expressing different fluorescent proteins  as an exskimage is used to find the position of each colony in the plate and assign labels to them  detector from  to them . With th to them  and alloFrom the data extracted from images as described above, it is possible to estimate the growth rate of each colony and the average constitutive gene expression rate of a fluorescent reporter, considering multiple colonies on a single plate. To get growth information—size (area) and growth rate (relative change in area) -, it is possible to approximate the colony size by the region expressing fluorescent signal. This size can be obtained by applying the gaussian blob detector to each SROI in all frames (i.e. time points), obtaining the colony radius/area over time . The resThen, as growth rate is defined by Eq , we are On the other hand, we show see  that theThen, we need to compute the mean intensity signal per unit of area (I/A). To achieve this, we get the colony fluorescent intensity in each frame by summing the three channel values for each pixel inside its computed area. Then dividing by the total number of pixels within this area it is possible to get the mean fluorescent intensity in each time step . These vAn interesting aspect of being able to detect diverse fluorescent proteins is the possibility to label and identify different strains expressing them. To do this we first extract colony fluorescence signals as described above. With this data it is possible to get the characteristics of each fluorescent protein and classify the colonies according to strain.These signals are in three channels, nominally labelled “red”, “green” and “blue” and depend on the filters and sensitivity of the camera. Each fluorescent protein has a characteristic spectrum which overlaps with these three channel spectral sensitivities. If we assume a linear relation between fluorescent protein expression and intensity of signal, it is possible to find a characteristic linear relation between the intensities in the  channels of each fluorescent protein see . That isTo test this, we performed independent timelapse experiments of strains expressing sfGFP, CyOFP and BeRFP  Movies, Following the impact of FOSS in software development, open hardware aims to democratize and accelerate hardware development in a similar manner. The scientific community has started to embrace this approach; with hundreds of labs around the globe designing and making their own equipment (e.g. ). Often http://docubricks.com/viewer.jsp?id=701517893260717056) and operational code (github.com/synbiouc/fluopi). Good documentation is critical for crowdsourced design improvements. It also permits adaptations to local needs and restrictions, for example due to availability of parts, and facilitates the incorporation of new parts adapted from other open source projects. For instance the use of an adjustable focus RPI camera, instead of a fixed lens mounted on the Raspberry PI V2 camera, was suggested to us by another group working on similar developments . To facThe imaging device is fully modular , permittOur project integrates software, hardware and wetware to achieve single-excitation multiple-emission fluorescence microscopy. The vast repertoire of available fluorescent proteins  permitteIn this project, we declare that we have no intentions to exert protection over the genetic resources used and aim to distribute them under an open material transfer agreement that is currently being drafted . Free anBuilding hardware, software and genetics from scratch leads to a better understanding of technologies and their integration. New educational modalities based on “tinkering” with biology, hardware and software for STEM training and capacity building have recently emerged Click here for additional data file.S2 FigGel loaded with 20, 15, 10, 5, 4,3,2 and 1 l of 1 Kb Ladder (NEB) labelled with SYBR-Safe. and imaged using raspistill command: -t 5000 -ss 240000 -ISO 300 -awbg 1,1 -co 30. (B) using raspistill command: -t 5000 -ss 170000 -ISO 300 -awbg 1,1 -co 0.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.S3 Movie(AVI)Click here for additional data file.S4 Movie(AVI)Click here for additional data file.S5 Movie(AVI)Click here for additional data file.S6 Movie(AVI)Click here for additional data file.S7 Movie(AVI)Click here for additional data file.S8 Movie(AVI)Click here for additional data file.


Hepatitis C virus (HCV) infection is an important cause of chronic liver disease which has been affected 3% of world’s population. Some studies have shown that adding Sofosbuvir (SOF), an HCV polymerase inhibitor to the conventional therapy of Pegylated-interferon (PegIFN) plus Ribavirin (RBV) can increase the rate of sustained virologic response (SVR) among HCV-infected patients. This study was conducted to determine the effect of combination therapy with PegIFN and RBV plus SOF for chronic hepatitis C genotype 1 infection using systematic review with meta-analysis.In this study, electronic databases including PubMed, Scopus, Science Direct, and Web of Science were comprehensively searched using appropriate strategies containing all related keywords of “hepatitis C”, “PegIFN”, “RBV” and “SOF”. Studies assessed the efficacy of combination therapy with PegIFN and RBV plus SOF for chronic hepatitis C genotype 1 infection were included in the meta-analysis.χ2 = 5.29, P = 0.26 and I2 = 24.4%), pooled SVR rate for treatment regimen of PegIFN and RBV plus SOF was calculated as 88.54% (95% CI = 85.77%–91.32%).After screening of 757 records, we included five articles with total sample size of 411 to the meta-analysis. Based on the fixed-effect model (Combination therapy with PegIFN and RBV plus SOF results in high treatment response in patients with HCV genotype 1 infection.The online version of this article (doi:10.1186/s40199-017-0177-x) contains supplementary material, which is available to authorized users. Hepatitis C infection is the major cause of acute and chronic hepatitis globally. Based on the World Health Organization (WHO) estimation, about 3% of world population is infected with hepatitis C virus (HCV) and the worldwide prevalence of chronic hepatitis C is more than 180 million who are at the predisposing to cirrhosis and/or liver cancer [Treatment of chronic hepatitis C comprises several components including, reduction of inflammation, prevention of fibrosis, cirrhosis and hepatocellular carcinoma and virus eradication. Currently, sustained virologic response (SVR) functions as the best indicator of effective treatment , 5. AlthSofosbuvir (SOF) is a HCV NS5B polymerase inhibitor that results in suppression of HCV replication and life cycle. Sofosbuvir as a new direct-acting antiviral agent (DAA) was approved for treatment of chronic HCV genotypes 1 to 4 infections. HCV genotype 1-infected patients should receive PegIFN, RBV and SOF for 12 weeks. From the data of trials, the latter combination therapy results in SVR12 rates of 50%–90% .This study set out with the aim of assessing efficacy of 12-week combination of PegIFN, RBV and SOF for treatment of patients with chronic hepatitis C infection caused by HCV genotype 1.In this systematic review and meta-analysis, electronic databases including PubMed, Scopus, Science Direct, and Web of Science were comprehensively searched using exact and sensitive search strategies .The PRISMA guideline for reporting of systematic review was used . Two revIFNL3 (rs12979860), and HCV subtype 1a/1b.Following parameters were extracted from the included studies; author’s first name, history of previous treatment, publication year, country, sample size, mean/median age, gender, body mass index (BMI), HCV RNA level before treatment, rate of cirrhotic patients, polymorphism near For evaluating risk of biases in each included clinical trial, Cochrane’s assessment tool was used . These bFor assessing quality of the included non-randomized studies, Newcastle-Ottawa Scale (NOS) was used . This toP value less than 0.1 was considered statistically significant for chi-squared. We evaluated publication bias by Begg’s and Egger’s tests. Based on the presence or absence of heterogeneity, random- or fixed-effect model was employed for calculation of pooled SVR12 rate and 95% confidence interval (CI). All data analyses were performed using STATA 10.We used chi-square and I-squared (lies from 0 to 100%) for evaluating heterogeneity across studies’ results. A total of 757 papers were found through database searching after removing duplications. In title screening, 519 irrelevant titles and in the abstract screening, 230 irrelevant abstracts were excluded. Then, eight full-text articles were assessed for eligibility and finally five articles with total sample size of 411 were included in our quantitative synthesis  and cohort (N = 1) studies. We found three eligible studies related to 2013 and two other related to 2015. Four of our finally included studies were from the United States and another one was from Germany. Furthermore, in three of them, included patients were treatment naïve and in two other studies, included both treatment naïve and treatment experienced participants.Table Using Cochrane’s risk of assessment tool four clinical trials were evaluated and all of them scored more than 6 (low risk). Furthermore, using NOS, one cohort study was evaluated and this study achieved 6 out of 8 possible stars. Therefore, no study categorized as low quality and also none of them were excluded based on this assessment Table .Table 2R2 = 5.29, df = 4, P = 0.26) and I-squared . Therefore, we used fixed-effect model and the pooled rate of SVR for HCV treatment regimen PegIFN, RBV plus SOF for 12 weeks was 88.54% (95% CI = 85.77%–91.32%) , and Egger’s (P = 0.28) tests.We found no publication bias according to the Begg’s (This study showed that combination therapy with PegIFN, RBV plus SOF with 88.5% treatment success is an effective antiviral therapy for treatment of patients with HCV genotype 1 infection. Before 2011, the standard of care for therapy of HCV genotype 1 infection was combination regimen of PegIFN and RBV for 24–72 weeks with 40–60% success and many complications –16. IntrThe PegIFN, RBV plus SOF regimen was superior to PegIFN and RBV combination therapy in terms of higher efficacy, shorter treatment duration, and fewer side-effects. Furthermore, fewer host and virus parameters affect the treatment success in comparison with PegIFN and RBV regimen . Among hIt has been proved that combination of PegIFN, RBV and SOF can lead to complete elimination of HCV RNA in about 95% of HCV-infected patients at week 4 of treatment , 19, 20.Ledipasvir/Sofosbuvir is the first IFN-free treatment regimen for HCV which was approved by FDA in 2014. It provides >95% SVR rate among non-cirrhotic HCV genotype 1 patients –23. AlsoThe future treatments of HCV will be IFN-free regimens with high-resistance barrier which can be applied for treatment of special group patients and can clear the virus in nearly 100% of the HCV-infected patients. With knowing that the ultimate HCV treatment will be available, there is a great concern to clear HCV infection until 2030 globally. However, there are major steps toward eradication of HCV which should be addressed in the program of HCV eradication including development of HCV vaccine and other preventive strategies, availability and affordability of high efficacious regimens in developing countries, nation-wide screening programs for finding the patients with HCV infection, and paying special attention to special patient groups such as thalassemia, kidney- and liver-transplant patients and HIV/HCV co-infected patients , 30.In conclusion, PegIFN, RBV plus SOF regimen is a highly effective therapy for treatment of HCV genotype 1 infection. While there are more expensive HCV antiviral treatments with higher efficacy than that of obtained by PegIFN, RBV plus SOF, the low cost of SOF and availability of this medication in many developing countries make the PegIFN, RBV plus SOF regimen as the recommended regimen for treatment of HCV genotype 1 infection especially when the patients cannot afford IFN-free regimens.


The immune response was measured by circulating levels of natural killer (NK) cell activity and cytokines. Both groups were comparable with regards to age, sex distribution, smoking and drinking status, and body mass index. When compared to the MHO group, the MUO group showed higher systolic and diastolic blood pressure, serum levels of triglyceride, glucose, glucose-related markers, and lower levels of HDL cholesterol. Compared to the MHO group, the MUO group showed 39% lower interferon-γ levels (not significant) and 41% lower interleukin (IL)-12 levels (significant). The MUO group also showed lower NK cell activity at E:T ratios of 10:1, 5:1, 2.5:1, and 1.25:1  than the MHO group. This study indicates that individuals displaying the MUO phenotype present an unfavorable immune system with lower NK cell activities under all assay conditions and lower serum levels of IL-12 than the activities and levels in similarly overweight MHO individuals. This result suggests that the immune system may be altered in overweight individuals who are at risk for overweight/obesity-related comorbidities.The purpose of this study was to determine whether the immune system is involved in the different metabolic circumstances in healthy and unhealthy overweight individuals. We examined the metabolic and immune characteristics of 117 overweight individuals. Subjects were classified as metabolically healthy overweight (MHO,  The prevalence of overweight and obesity, which are defined as excessive fat accumulation, has increased rapidly worldwide. More than half (53.8%) of the adult in The Organization for Economic Co-operation and Development countries are overweight or obese . Even soAn increased body mass index (BMI) is crucial for up to 30% of various cancers, particularly pancreatic, colorectal cancer, etc. , and it The immunometabolic interaction in overweight/obese individuals is currently considered a key factor in chronic low-grade inflammation . For exa2, World Health Organization definitions) and aged 40–69 years were enrolled. The exclusion criteria included cardiovascular disease, cancer, immune disease, liver disease, renal disease, pregnancy, and regular dietary supplement use. All participants provided written informed consent and the Institutional Review Board of Yonsei University approved the study protocol, which complied with the Declaration of Helsinki. Sample size was determined and calculated using R software v.3.4.1 with package “pwr.” In an exploratory pilot study, the NK cell activity at an E:T ratio of 10:1 in the MUO group was 20.57 ± 19.74% (mean ± standard deviation) lower than that in the MHO group (31.69 ± 18.08%). The sample size was determined via a two-sample t-test power calculation with effect size (d = 0.596), power of 0.8, and level of significance (α = 0.05). The result indicated that a minimum of 45 subjects per group were needed, and thus, we selected an MUO:MHO ratio of 1:1.6 to increase statistical power for the test.Study subjects were recruited through advertisements by the Clinical Nutrigenetics/Nutrigenomics Laboratory at Yonsei University from March 2016 to February 2017. Volunteers who agreed to participate were screened to measure BMI and personal history of any diseases. After screening, subjects who were overweight or obese (BMI ≥ 25 kg/mn = 72) or MUO (n = 45) based on the definition of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). In detail, the definition of “metabolically unhealthy” was described in previous published study  levels via a modified Sanz method, blood urea nitrogen (BUN) via a kinetic UV assay for urea/urea nitrogen, creatinine levels via the creatinine Jaffe method, serum albumin concentrations via the bromocresol green method, and serum high-sensitivity C-reactive protein (hs-CRP) levels via a latex-agglutination turbidimetric immunoassay. Plasma adiponectin concentrations were measured by an enzyme immunoassay . The serum level of cytokines including interleukin (IL)-6, IL-1β, TNF-α, interferon (IFN)-γ, and IL-12 were measured using kits .To assess the lipid profile, serum TG, total cholesterol, and HDL cholesterol were measured using kits and auto analyzer and LDL cholesterol was calculated by the Friedewald formula. Fasting serum glucose and glucose-related markers are also measured including insulin and homeostatic model assessment of IR (HOMA-IR) index. These laboratory assessments including lipid profile and glucose-related markers are previously described in detail . A HitacDetailed descriptions are provided in previous published study  and desc4 K-562 cells  with the four serial dilutions . The following formula was used to obtain% cytotoxicity for respective E;T ratio:Detailed descriptions are provided in previous published study  describet-test, chi-square test, Pearson’s correlation coefficient. Skewed variables were log transformed. Overall statistical analysis was reported at significant at two-tailed P value < 0.05.SPSS statistics 24.0 software  was used for overall test; independent Table P = 0.013), 5:1 (P = 0.043), 2.5:1 (P = 0.040), and 1.25:1 (P = 0.012) than the MHO group , and positive correlation trends were shown between IFN-γ levels and NK cell activity at an E:T ratio of 5:1 . Although a positive correlation was also shown between IFN-γ and IL-12 levels , our results did not show any correlation between IL-12 and NK cell activity at any E:T ratio.There was a positive correlation between IFN-γ and NK cell activity at an E:T ratio of 2.5:1  increases with age, and estimates indicate that in the age category of over 50 years, MS influences more than 40 and 30% of the population in US and Europe, respectively –20. WhenNatural killer cells represent a key element of innate immunity, as these cells secrete different cytokines  to stimulate other immune cells and can directly destroy virus-infected or malignant-transformed cells , 23. In r = 0.241, P = 0.023), but there was no relevant correlation between IL-12 levels and NK cell activity. Although there was no direct correlation between NK cell activity and IFN-γ or NK cell activity and IL-12 in the present study, the levels of IFN-γ, IL-12, and NK cell activity were lower in the MUO group, and this effect might be due to IL-12-induced IFN-γ production mediated by T cells. We could not prove the exact mechanism involved in the reduced levels of NK cell activity and IL-12 in the MUO group; nevertheless, our results suggested that the immune system may be altered in overweight subjects with unfavorable metabolic phenotype.Interleukin-12 induces increased proliferation; enhanced cytotoxicity and expression of cytotoxic mediators; and increased production of cytokines, especially IFN-γ, in NK cells, T cells, and natural killer T (NKT) cells . However3−)CD56bright cells were mainly considered to be cytokine producers, whereas (CD3−)CD56dim cells were considered to be cytotoxic effector cells 56, CD16, and CD3 and by expressing different degrees of lytic activity , 31. Peror cells . Upon stce IFN-γ . CD56dimNK cells , 34, of NK cells  reportedNK cells . AlthougExcessive body fat mass elevates circulating adipokine levels, which affect immune responses and functions , 36. NK Chronic inhalation of cigarette smoke changes immunological responses . The infDespite several intriguing findings that explain the differences between the MUO and MHO groups, the current study has limitations. First, although we matched for several potential confounding factors such as age, sex, and BMI, we had a moderate sample size. Second, we could not determine any causations between MS and abnormal system or other inflammatory risk factors through this cross-sectional study design. Third, the phenotype of NK cells, which express differing degrees of lytic activity, and markers of NK cell activation were not assessed in this study. Lastly, the underlying mechanism of the relationship between MS and an altered immune system was not clarified in this study. Individual components of MS are related risk factors for an abnormal immune system, but how the clustering of these components is connected with the development of immune dysfunction remains unknown. It seems obvious that a condition characterized by multiple risk factors, such as MS, resulted in a greater risk for adverse effects than a single risk factor.In conclusion, the results of the current study indicate that individuals displaying the MUO phenotype present an unfavorable immune system with lower NK cell activities under all assay conditions and lower levels of IL-12 compared to similarly overweight MHO individuals. These lower levels of NK cell activity and IL-12 expression in MUO individuals suggest that the immune system may be altered in overweight population who are at risk for overweight/obesity-related comorbidities.The aim of this study was carefully explained to all participants, and each participant provided written informed consent. The Institutional Review Board of Yonsei University approved the study protocol, which complied with the Declaration of Helsinki.MinkyungK and HY contributed to the acquisition, analysis, and interpretation of the data and helped draft the manuscript. MinjooK and JL contributed to the conception and design of the research and the analysis and interpretation of the data and drafted the manuscript. All authors critically revised the manuscript, read and approved the final manuscript, and agreed to be fully accountable for ensuring the integrity and accuracy of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a conclusion, ACA displays a specific ANA staining pattern with a bimodal distribution, and ACA-positive RA may constitute a distinct subset with specific clinical features.Anti-centromere antibody (ACA) is one of the classical anti-nuclear antibody (ANA) staining patterns. However, characteristics of ACA in comparison with the other ANA patterns and clinical features of ACA-positive subjects have not been elucidated. Here, we examined all ANA patterns by indirect immunofluorescence for 859 rheumatoid arthritis (RA) patients. Together with the ANA data of 9,575 healthy volunteers, we compared distributions of the ANA levels. ACA was the only ANA that demonstrated a definite bimodal distribution of levels. ACA showed significantly higher levels than the other ANA staining patterns in both RA and healthy population (p < 0.0001). ACA-positivity was associated with old age and was observed more in females. We further recruited another cohort of 3,353 RA patients and confirmed the findings. ACA was also associated with Raynaud’s phenomenon (p = 6.8 × 10 Although ACA has been considered specific for limited cutaneous systemic sclerosis (SSc)2, it is occasionally detected in patients with primary biliary cirrhosis (PBC) and primary Sjögren’s syndrome (SS) with various range of positivity 5. In some reports, a subset of patients with rheumatoid arthritis (RA) were also positive for ACA7, as well as healthy subjects8.Anti-centromere antibody (ACA) is a type of anti-nuclear antibody (ANA) detected by indirect immunofluorescence displaying a centromeric pattern (discrete speckled pattern)8. ACA was associated with old age and was observed more in females in the healthy subjects. After reporting these findings, we noticed that for other ANA staining patterns most of Ab-positive subjects were enriched by less than 1:160, but subjects positive for ACA were enriched at higher titer levels, by more than 1:640. Thus, we hypothesized that ACA might exhibit a specific distribution where most ACA-positive subjects had high titer levels. Some previous studies have reported that patients with rheumatic diseases might have high levels of ACA9, but they had no comprehensive data for ANA and failed to reach a clear conclusion supported by statistical significance.We previously quantified ANA in 9,575 healthy subjects without connective tissue diseases and reported that 87 individuals (0.91%) of healthy subjects were positive for ACAIn this study, we compared the distribution level of ACA with that of the other staining patterns, namely, speckled, homogeneous, nucleolar and cytoplasmic patterns in healthy subjects, and in RA patients. We also analyzed correlates and clinical features of ACA-positivity in patients with RA.We took advantage of the ANA data of 9,575 subjects from our previous report and analyzed the distribution of ANA levels in each staining pattern. In the 4,332 healthy subjects who were positive for ANA Fig. , only thWe confirmed this finding in patients with RA using data of 859 RA patients in the Kyoto University Rheumatoid Arthritis Management Alliance (KURAMA) cohort without SSc p < 0.0001, Fig. , Fig. 1.We analyzed the clinical features of ACA-positive patients with RA Fig. . The ACA−5 and p = 0.0089, respectively, Table We analyzed 3,353 RA patients without SSc whose ANA data were available from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort to confirm the associations. 61 subjects of the 3,353 patients (1.8%) were positive for ACA. The association between ACA-positivity and old age and females were replicated  and 7.37 (2.02–21.5), respectively, Table We further investigated the associations between ACA-positivity and complications observed in RA. Raynaud’s phenomenon and secondary SS were significantly associated with ACA-positivity , a common allelic group showing associations with RA susceptibility, did not show a significant difference  to ACA-positivity.The data collection and statistical analyses in this study were approved by the ethic committee in Kyoto University  and Tokyo Women’s University  and adhered to the ethical guidelines for clinical studies in Japan.8.A total of 9,575 Japanese healthy volunteers in our previous report in which we had excluded subjects suspected of having connective tissue diseases were recruited for analyzing the distribution of the levels of each ANA staining patternWe screened 1,187 RA patients from the KURAMA cohort of Kyoto University for the availability of ANA data. 880 patients had ANA data, but we excluded 21 subjects since they fulfilled American college of Rheumatology (ACR) criteria for SSc in 1980 and ACR/European League Against Rheumatism (EULAR) criteria for SSc in 2013. As a result, 859 RA patients were included in the analysis  for patients in the KURAMA cohort, and two other experienced readers (SY and KY) for patients in the IORRA cohort, according to the Sharp/van der Heijde method with a total score range of 0 to 44828 were available for 360 patients among 859 patients with RA. In the IORRA cohort, HLA-DRB1 genotyping data by a sequencing-based typing method using the AlleleSEQR HLA-DRB1 typing kit  were available for the 481 patients. DRB1*01:01, *04:01, *04:04, *04:05, *04:10, *10:01, *14:02 and *14:06 were classified as SE which is a five amino acid sequence motif in residues at amino acid position 70–74 of the HLA-DRβ chain associated with RA onset and severe joint destruction.In the KURAMA cohort, data of HLA-DRB-1 typed with the WALKFlow system2(level/40) + 1) and their distributions were compared by Wilcoxon rank sum test between ACA group and each of the other groups based on staining patterns in the healthy volunteers or the RA patients who were positive for ANA. We compared clinical and genetic characteristics between ACA-positive and -negative RA patients. We performed Wilcoxon signed-rank test for quantitative data, and Pearson’s chi-square test or Fisher’s exact test for binary data. Linear regression analysis with response variate of TSS and an independent variable of ACA with covariates of RA duration, gender, age and RF was performed by standard least squares. Odds ratios were also calculated with 95% confidence intervals.The ANA levels were log-transformed (log29.The analyses were separately performed for RA patients in each cohort. We also performed the analyses in the combined study. Since TSS is influenced by various factors whose distribution was different between the cohorts, we put an indicator variable of cohort on TSS analysis in the combined study. We performed power calculation of the current study for the HLA analyses with use of ‘Genetics Design’ package of R softwareStatistical analyses were performed using JMP pro version 12 or R statistical software. Significant level was determined by Bonferroni’s correction. When clinical information were available in both the KURAMA and IORRA cohorts, we regarded an association as significant only when it showed a consistent association pattern with p-value less than 0.05 in each cohort and overall p-value in the combined study less than the significant level.supplementary


The aim was to determine the prevalence and impact of an occluded “culprit” artery (OCA) in patients with non-ST segment elevation myocardial infarction (NSTEMI).We searched PubMed, EMBASE, and Web of Science, with no language restrictions, up to 1 Jul. 2016. Observational cohorts or clinical trials of adult NSTEMI were eligible for inclusion to determine the prevalence if the proportion of OCA on coronary angiography was reported. Studies were further eligible for inclusion to determine the outcome if the association between OCA and clinical endpoints was reported.p = 0.007), and this was associated with lower left ventricular ejection fraction , higher peak enzyme level , and higher risk for cardiogenic shock , compared with patients with a non-occlusive culprit artery. Death rate  and recurrent myocardial infarction  were also higher in patients with OCA, compared with patients with a non-occlusive culprit artery.Among the 60,898 patients with NSTEMI enrolled in 25 studies, 17,212 were found to have OCA. The average proportion of OCA in NSTEMI was 34% (95% CI 30–37%). Patients with OCA were more likely to have left circumflex artery as their culprit artery (odds ratio (OR) 1.65, 95% CI 1.15–2.37, Patients with OCA comprised a substantial portion of the NSTEMI population. These patients present with more severe symptoms and worse clinical outcome. Whether these patients should be treated with more aggressive strategy warrants further study.The online version of this article (10.1186/s13054-018-1944-x) contains supplementary material, which is available to authorized users. Acute coronary syndrome (ACS) has been categorized into ST segment elevation myocardial infarction (STEMI) and non-ST segment elevation ACS (NSTEACS) based on the results of initial 12-lead electrocardiography (ECG). In patients with STEMI, early reperfusion therapy of the culprit artery is a class I indication in current guidelines .STEMI results from acute total or nearly total occlusion of a coronary artery . HoweverThe clinical implication of OCA in NSTE-acute coronary syndrome (ACS) is still controversial, and how patients with NSTEACS with OCA should be treated is also unknown. Some studies report worse outcome for patients with OCA , 7–9, whThis systemic review and meta-analysis was performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines . A literStudies included in the analysis were prospective or retrospective cohorts or randomized controlled trials. The exclusion criteria were (1) abstracts, review articles, case-control studies and case series with the number of patients fewer than 20; (2) studies on myocardial infarction that only reported Q wave or non-Q wave MI; (3) studies on patients with unstable angina, stable coronary artery disease (CAD), or STEMI; (4) studies selecting patients by angiography results ; (5) studies that did not specify the result of cardiac necrosis biomarkers; (6) animal studies; and (7) duplicate reports.The inclusion criteria were (1) adults aged 18 or above; (2) patients with chest pain; and (3) patients with elevated cardiac necrosis biomarkers (troponin or creatine kinase-MB isoenzyme (CK-MB)) according to the criteria specified in individual studies. Patients with STEMI or patients with NSTEACS and negative cardiac necrosis markers were excluded from analyses.The determination of the culprit artery and the definition of OCA were based on the protocol of the individual study. Sensitivity analysis will be performed to include only studies defining OCA as Thrombolysis In Myocardial Infarction (TIMI)-flow grade 0 or 1 in the culprit artery (the most commonly used definition in these studies).Outcomes were assessed as all-cause mortality and recurrent MI. Endpoints were taken at 1 year or the longest follow up available (if the follow-up period was less than 1 year) in each study.Extraction of data on study characteristics and outcomes was performed independently by two reviewers (CSH and YHC). Discrepancies between reviewers were resolved by consensus. The following data were extracted from each study enrolled in our analysis: the first author’s last name, year of publication, country of study performed, biomarkers measured, total number of patients, numbers of patients with OCA, mean age, gender, conventional risk factors for CAD  and outcome (recurrent MI or death). The study quality was judged using the Newcast-Ottawa scale (NOS) for observational studies . This scI2 statistic to assess heterogeneity of the event rates across studies [p values were two-sided, and those below 0.05 were regarded as statistically significant.We use the random-effects method because of its conservative summary estimate and because it incorporates between-study and within-study variance. We used the  studies . A funne studies . SubgrouThe initial search identified 1432 citations. After critical assessment of these papers, 25 studies fulfilled the inclusion criteria and were used to estimate the pooled proportion of OCA among NSTEMI patients. Ten of these 25 papers fulfilled the criteria and were used to analyse the impact of OCA on outcome , European (n = 8), Asia-Pacific (n = 10), or multinational regions (n = 2). The number of patients varied from 42 to 30,386, with mean age from 58 to 69 years, and 60 to 78.6% were male. Presence of hypertension was reported in 35 to 85.5% of patients, diabetes in 11.5 to 53.9%, hyperlipidaemia in 13 to 69.2%, and smoking in 21.3 to 80.3% of patients. Coronary artery bypass grafting (CABG) was excluded in five studies [Characteristics of the 25 included studies are presented in Table pean n = , Asia-Pa studies –20. The ific n = 0, or mul studies , 21–23 rX2 = 1644.9, degrees of freedom (df) = 24, p < 0.001, I2 = 98.54%). After excluding studies with OCA definition other than TIMI flow of 0 or 1, the average proportion of OCA in the remaining 20 studies was 0.35    0.30–0.38)  . Two studies reported a substantially lower proportion of patients with multivessel disease compared with other studies (13.6% in Pride 2010 ). After I2 = 92.3%) in low NOS studies, 0.35  in moderate NOS studies, 0.36  in high NOS studies. These results showed that subgroup analysis by different NOS did not reduce the heterogeneity .The leave-one-out sensitivity analyses could not identify studies with major impact on the between-study heterogeneity. The univariate meta-regression with the covariate of NOS, year of publication, age, proportion of patients with diabetes mellitus, hypertension, hyperlipidaemia and single-vessel disease, showed no significant impact on between-study heterogeneity    in patients with OCA, compared with patients with a non-occlusive culprit artery  score) were reported in 3 studies , 11, 12.p = 0.007; X2 = 38.5, df = 5, p < 0.001)  for patients with OCA, compared with patients with a non-occlusive culprit artery    and 80-lead body surface potential mapping  among these possible predictors [Among the 10 studies used for analysis of outcome, 5 –11, 23 rRisk stratification scores had been reported in four studies, with TIMI score reported in three , 10, 21 p < 0.001; X2 = 6.34, df = 7, p = 0.501) for patients with OCA compared with patients with a non-occlusive culprit artery    for patients with OCA compared with patients with a non-occlusive culprit artery   . The small-study effect persisted even when excluding studies with a less strict OCA flow definition and studies with time to angiography more than 1 week (Egger test p < 0.001). The funnel plot for studies reporting on outcome suggested publication bias (Egger p < 0.001)  were asymmetrical Fig. . The Egg01) Fig. .Fig. 5FuThis meta-analysis showed that (1) the overall estimated proportion of OCA in NSTEMI was 34%; (2) NSTEMI with OCA was associated with more LCx as the culprit artery, higher peak enzyme level, lower LVEF, and more cardiogenic shock; and (3) NSTEMI with OCA was associated with a higher rate of recurrent MI and death. These important findings remained the same if we limited our analysis to studies using TIMI flow 0–1 as the definition of OCA and timely diagnostic angiography after onset.There is substantial heterogeneity in our analysis, with a wide range of proportion of OCA reported in the studies included 0.15–0.75). The explanations for this variation include differences in the study geographic region, study design and OCA definition, patient population, and timing of angiography. The standard initial ECG evaluation protocol is often unavailable in retrospective studies, and subtle ST change, especially in the posterior leads, may be missed. Some of the studies excluded patients with prior coronary artery bypass grafting (CABG) –0.75. Th, 18–20. Our analysis suggested that OCA represents a substantial portion of NSTEMI, and carries a worse clinical outcome. Some researchers proposed that this condition should be considered as “STEMI equivalent” and treated as such , 7. HoweConsidering the large number of patients with NSTEMI worldwide , it is iOur analysis has several strengths. First, we enrolled only patients with elevated cardiac necrosis markers. Focusing our biomarker-positive NSTEACS population helps us to decrease the ambiguity, targeting only patients with objective myocardial injury. More importantly, patients with biomarker-positive NSTEACS may benefit from an early invasive strategy, compared with biomarker-negative patients . Second,Our analysis does have several limitations. First, the ECG protocol used was not reported in detail in most of the studies. Whether posterior leads have been applied in individual studies was unclear. Second, patients with contraindication for angiography or percutaneous coronary intervention were excluded by the analysis design. Therefore our results cannot be generalized in this population. Third, angiography in the included studies was done within one week after symptom onset. Hence the true OCA incidence at the time of onset could not be deducted from the present analysis. Fourth, a certain proportion of patients with posterior STEMI may be classified as having NSTEMI. This misclassification may be improved in the future by including routine posterior leads to detect posterior STEMI in patients with chest pain. Fifth, because the outcomes reported in the studies had not been stratified by some important moderators including the procedures and the number of diseased vessels, further analysis on the impact of OCA according to these moderators cannot be performed. Finally, given the asymmetry of funnel plot, we cannot exclude the potential publication bias against studies finding no significant difference in the outcomes of NSTEMI with or without OCA.Approximately one third of patients with NSTEMI have OCA. These patients present with more severe clinical symptoms and have a worse outcome, compared with those with a non-occlusive culprit artery. There is as yet no reliable tool to identify this group of patients before performing angiography. Whether timely reperfusion will benefit this group of patients warrants further studies.Additional file 1:Supplementary figures and table. (DOCX 3836 kb)


The aim of the study was to evaluate whether the risk factors for cardiovascular disease (CVD) are similar in the northern and southern regions of Cameroon.The participants answered a questionnaire concerning their lifestyle. Anthropometric and blood pressure measurements were evaluated in 192 individuals and biochemical parameters in 50 randomly selected volunteers.Northerners displayed low alcohol and tobacco consumption, little practice of sport but physically demanding professions, and consumption of soybean, refined palm and other polyunsaturated oils. Southerners consumed alcohol, practiced sport, had intellectually based professions, and consumed crude and refined palm oils. Waist circumference and body mass index were higher in the southerners compared to the northerners. Blood glucose levels, and systolic and diastolic blood pressures were higher among the northerners than the southerners. Among the southerners, there were positive correlations between total cholesterol levels and systolic or diastolic blood pressure, low-density lipoprotein cholesterol and blood glucose levels or diastolic blood pressure, triglyceride levels and systolic blood pressure.Providing region-adapted, health-related advice for northern and southern Cameroonians would contribute to reducing risk factors for CVD. Cardiovascular disease (CVD) remains the leading cause of death worldwide, accounting for around 17 million deaths per year.3Monitoring of CVD is based on the evaluation of non-modifiable risk factors , behavioural risk factors , and modifiable risk factors .4In developing countries, relationships between malnutrition and CVD have been poorly explored.The various ethnic groups distributed across 10 regions, including rural and urban zones, are characterised by distinct eating habits. The second Cameroonian household survey assessed the average energy intake per capita to be 3 241, 2 721 and 2 887 kcal/day for far north, far south and the whole country, respectively.In Cameroon, crude palm oil represents 10.6%, cottonseed oil 2.1%, refined palm oil 0.9% and peanut oil 0.4% of the total energy intake.9Plasma omega-3 fatty acid levels were correlated to the eating habits in the three European countries investigated.13In this context, the impact of dietary lipids on the development of CVD represents the focus of current health concerns, not only in economically developed countries but also in developing countries. While eating behaviours differ between the north and south of Cameroon, the energy intake remains comparable between the two regions, 3 241 kcal/day in the north and 2 721 kcal/day in the south.This study was performed on 192 volunteers, composed of 101 women and 91 men aged 35 to 65 years. They were distributed between 89 volunteers from the north (45 men and 44 women) and 103 volunteers from the south (46 men and 57 women). The volunteers answered a questionnaire concerning their lifestyle. In addition, their anthropometrics and biochemical parameters were measured.This study (P2013/164) was approved by the ethics committee of the Université Libre de Bruxelles, Erasme University Hospital and was conducted in compliance with the Declaration of Helsinki. All volunteers gave written informed consent before participation.2 × p × (1–p)/m2] where n is the minimum sample size to obtain significant results for an event and a fixed level of risk; t is the confidence level ; p is the estimated prevalence of CVD, based on the literature  and m is the margin of error .The sample size was calculated using the following standard formula: [n = tDue to logistical and financial reasons, the biochemical parameters were measured in 50 randomly selected volunteers . Anthropometric and blood pressure measurements were performed on all 192 volunteers.All volunteers were living in the urban zone from different cities of the south (S): Douala, Yaoundé, Bafang and Dschang; and the north (N): Garoua and Ngaoudéré. All volunteers belonged to the working and middle social classes. The volunteers were randomly recruited in their home or work place.Basic information was collected and included the following: gender: female (F) and male (M); age: volunteers were grouped by age: 35–50 and 51–65 years; tobacco consumption: defined as never smoked (no); former smoker or smoker (yes); alcohol consumption: defined as no consumption (none); consumption once or twice a week ; consumption more than twice a week (regularly); physical activity  was defined as sport: never practice (no); practice intense or moderate activity once or more than once a week (yes); for profession, domestic workers, farmers and traders were considered physically active, whereas office workers were considered intellectual; oil consumption: the oils mostly used for cooking were recorded.2 (m) and BMI categories were defined as underweight with BMI ≤ 18.5 kg/m2; normal was BMI 18.5–24.9 kg/m2; overweight was BMI 25–29.9 kg/m2; and obese was BMI ≥ 30 kg/m2.15Various anthropometric parameters were collected for all volunteers. Height was collected from the ID card and weight was measured using an electronic scale. Body mass index (BMI) was calculated using the formula: weight (kg)/heightSystolic and diastolic blood pressure measurements were performed on all volunteers in a seated position at rest and monitored for at least five minutes using an electronic medical wrist blood pressure monitor . Two measurements were performed on each subject and the mean value was used. Blood pressure ranges were for systolic blood pressure (SBP): normal < 140 mmHg, and high ≥ 140 mmHg; for diastolic blood pressure (DBP): normal < 90 mmHg, and high ≥ 90 mmHg.16Biochemical parameters such as blood glucose (BG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG), were measured in 50 randomly selected volunteers, using an equal number of men and women from different cities, who were advised to fast prior to and up to the time of measurement .Disposable test strips were used with a Cardio Chek Analyser . Low-density lipoprotein cholesterol (LDL-C) was calculated using Fridewald’s formula: LDL-C = TC – (HDL-C + TG/5).Blood glucose values were defined as normal < 100 mg/dl (5.55 mmol/l); limit 100–125 mg/dl (5.55–6.94 mmol/l); diabetic > 125 mg/dl (> 6.94 mmol/l).TC ranges were defined for women as normal: 155–255 mg/dl (4.01–6.6 mmol/l); hypocholesterolaemia: < 155 mg/dl (< 4.01 mmol/l) and hypercholesterolaemia: 255 mg/dl (> 6.6 mmol/l). TC ranges were defined for men as normal: 130–250 mg/dl (3.37–6.48 mmol/l); hypocholesterolaemia: < 130 mg/dl (< 3.37 mmol/l) and hypercholesterolaemia: > 250 mg/dl (> 6.48 mmol/l).HDL-C ranges were defined for women as normal: 50–92 mg/dl (1.3–2.38 mmol/l); hypocholesterolaemia: < 50 mg/dl (< 1.3 mmol/l), and hypercholesterolaemia: > 92 mg/dl (> 2.38 mmol/l). HDL-C ranges were defined for men as normal: 37–65 mg/dl (0.96–1.68 mmol/l); hypocholesterolaemia: < 37 mg/dl (0.96 mmol/l) and hypercholesterolaemia: > 65 mg/dl (1.68 mmol/l).LDL-C ranges were defined for women as normal: 100–150 mg/dl (2.59–3.89 mmol/l), hypocholesterolaemia: < 100 mg/ dl (< 2.59 mmol/l) and hypercholesterolaemia: > 150 mg/dl (> 3.89 mmol/l). LDL-C ranges were defined for men as normal: 110–160 mg/dl (2.85–4.14 mmol/l), hypocholesterolaemia: < 110 mg/dl (< 2.85 mmol/l) and hypercholesterolaemia: > 160 mg/dl (> 4.14 mmol/l).Triglyceride ranges were defined for women as normal: 35–140 mg/dl (0.4–1.58 mmol/l), hypotriglyceridaemia: < 35 mg/ dl (< 0.4 mmol/l) and hypertriglyceridaemia: > 140 mg/dl (> 1.58 mmol/l). Triglyceride ranges were defined for men as normal: 45–175 mg/dl (0.51–1.98 mmol/l), hypotriglyceridaemia: < 45 mg/dl (> 0.51 mmol/l) and hypertriglyceridaemia: > 175 mg/dl (> 1.98 mmol/l).15All statistical tests were performed using SPSS software . For quantitative variables, data are presented as mean or median ± standard error of means (SEM). The Student’s t-test for independent samples or Wilcoxon– Mann–Whitney test were used to compare the means or medians of two groups, while one-way ANOVA or the Kruskal–Wallis test were used to compare the means or medians of more than two groups. For qualitative variables, data are presented in tables using numbers and percentages, and chi-squared or Fisher’s exact tests were used to compare characteristics between the groups. Spearman’s correlations were used to test possible correlations between two parameters. The results were considered statistically significant for p < 0.05.The study population was distributed equally between men and women in the two regions, north and south. The distribution according to age group showed a significant difference (p < 0.001) between the two regions.Populations from the south consumed more alcohol than those in the north, who drank little or nothing (p < 0.001). The tobacco consumption was zero in the north and in the south very low (p < 0.03).The intense or moderate practice of sport was very low in the north compared to the south (p < 0.001). However, northerners had physical rather than intellectual professions compared to people of the south (p < 0.001).The percentage consumption of polyunsaturated oils was higher among the northerners compared to southerners. The majority of the southern population consumed both crude and refined palm oils, while a small portion of the southern people consumed only crude palm oil. In the north, people consumed mainly soybean oil and refined palm oil .WC and BMI were higher among southerners than northerners . However, the percentage of obese population, determined by measuring either the WC  or BMI , was statistically comparable between the two regions.The average and range of TC, HDL-C, LDL-C and TG levels were not significantly different according to region. By contrast, BG levels, SBP and DBP were higher in the northern populations than the south . The percentages of individuals with diabetes, 10% in the north and 2% in the south, were not statistically different. There was however an over-representation of hypertension (SBP and DBP) in the north compared to the south (p < 0.001) .In men from the two regions, there was no statistically significant difference between WC and BMI. By contrast, women from the south had a WC and BMI statistically higher than northern women . The percentage of obesity was statistically higher among northern than southern men, based on BMI (p = 0.03) but not WC. By contrast, the percentage of obese women was higher in the south compared to the north, based on WC (p < 0.001) but not BMI.BG, TC, HDL-C, LDL-C and TG levels were not significantly different between women and men in the two regions. Both SBP and DBP were higher in men from the north than from the south (p < 0.001), whereas those parameters were comparable in women from the two regions.Northern men had a higher incidence of diabetes (16%) than men from the south (4%) (p = 0.027). Similarly, a greater percentage of men from the north had a SBP and DBP higher than that of men from the south (p < 0.001). The incidence of hypertension was higher among women from the north than from the south (p = 0.01) .The characteristics of the populations showed that there was a significant difference in age between the volunteers from the north and south. Taking this into consideration, anthropometric and biochemical parameters among subjects from the two age groups were statistically compared. The results showed no significant difference between the two age groups for WC, BG, SBP and DBP. However, BMI was higher among people in the 35–51-year age group compared to people in the 51–65-year age group (p = 0.01). The percentage of obese people and people with normal SBP tended to be higher among people in the 35–51-year age group compared to people in the 51–65-year age group , but these were not statistically significant  .In the north, there was a positive correlation between TC and TG levels ; SBP and DBP ; SBP and WC ; SBP and BMI  and DBP and WC . There was a negative correlation between HDL-C and TG levels  . In the To the best of our knowledge, CVD risk factors in northern and southern Cameroonians have not been studied before. Average WC and BMI from the studied population were significantly higher in the south than the north. However, the prevalence of obesity, determined by either BMI (± 21%) or WC (± 65%), was comparable between northern and southern Cameroonians, but based on BMI, higher than that in the entire Cameroonian population (± 10%).17The range limits used to establish each category  could explain the lack of difference in obesity prevalence between southerners and northerners, despite differences in average BMI and WC values. Based on BMI measurements, southern women displayed higher prevalence of obesity compared to northern women (34 vs 20%). Obesity prevalence determined in southern women (34%) was close to the values determined in the women’s labour force from Douala ,19Based on WC measures, in our study, the prevalence of obesity in southern women was 100%, versus 82% for northern women. Our data are higher than that obtained in urban southern women (43%), based on WC.17Based on WC measures, in our study, northern men displayed higher prevalence of obesity compared to southern men (40 vs 30%). These values were higher or lower than the obesity prevalence determined for the men’s labour force from Douala .Strong and positive correlations existed between SBP and WC, SBP and BMI, and DBP and WC among the northerners. In the southern rural zone of Cameroon, obesity in women was not correlated with blood pressure and blood glucose values due to their farming activities, which can be equated with permanent physical exercise.14Higher WC and BMI values observed among southerners could have been related to their food habits and/or lifestyle, despite comparable total energy intakes observed in south and north Cameroon.21The higher prevalence of obesity among southerners could also have been related to the high percentage of individuals consuming alcohol occasionally or regularly (92%), compared to the low percentage (1%) of northerners consuming alcohol, for cultural reasons. Our data corroborate a previous study performed on Cameroonians from urban workplaces, showing that the prevalence of obesity was positively correlated with excessive alcohol consumption.17Among southerners, strong positive correlations existed between lipid parameters and other cardiovascular risk factors studied (hypertension and diabetes). Palm oil, rich in saturated fatty acids and consumed by southerners, is a well-recognised risk factor for CVD and metabolic diseases, as it induces a larger increase in plasma concentrations of TC and LDL-C.8BG, SBP and DBP were significantly higher among northerners than southerners, with northerners being characterised by high carbohydrate consumption compared to southerners.Age has been considered a non-modifiable risk factor for the onset of CVD.3The prevalence of obesity was higher in southern Cameroonians  compared to northern Cameroonians. Lipid parameters  were positively correlated with cardiovascular risk factors such as BG levels, SBP and DBP among southern Cameroonians. The prevalence of diabetes and high blood pressure was higher among northern than southern Cameroonians, despite the lack of correlation between lipid parameters and cardiovascular risk factors assessed among northern Cameroonians.The main objective of this pilot study was to attract the attention of national health authorities and medical communities to the impact of dietary habits on the increasing prevalence of cardiovascular risk factors by comparison of the two main regions of Cameroon. Hopefully, the risk factors uncovered by this study will encourage public health authorities to promote  a widespread multicentre study, allowing healthcare personnel to assess the looming health issues and to eventually provide public health recommendations. Furthermore, additional studies aimed at assessing food consumption and lifestyle factors in these two populations would increase our understanding of cardiovascular risk factors in Cameroon.


Neuroblastoma is the most common extra-cranial solid pediatric cancer and causes approximately 15% of all childhood deaths from cancer. Although lymphatic vasculature is a prerequisite for the maintenance of tissue fluid balance and immunity in the body, little is known about the relationship between lymphatic vascularization and prognosis in neuroblastoma. We used our previously-published custom-designed tool to close open-outline vessels and measure the density, size and shape of all lymphatic vessels and microvascular segments in 332 primary neuroblastoma contained in tissue microarrays. The results were correlated with clinical and biological features of known prognostic value and with risk of progression to establish histological lymphatic vascular patterns associated with unfavorable histology. A high proportion of irregular intermediate lymphatic capillaries and irregular small collector vessels were present in tumors from patients with metastatic stage, undifferentiating neuroblasts and/or classified in the high risk. In addition, a higher lymphatic microvascularization density was found to be predictive of overall survival. Our findings show the crucial role of lymphatic vascularization in metastatic development and maintenance of tumor tissue homeostasis. These patterns may therefore help to indicate more accurate pre-treatment risk stratification and could provide candidate targets for novel therapies. PDPN and LYVE1 may be predictive of immune microenvironments in melanoma. Additionally, the lymphatic system, previously thought to play a passive role in cancer metastasis, is now known to play an integral role in the metastatic spread of disease [The lymphatic system is essential for the maintenance of tissue fluid homeostasis, gastrointestinal lipid absorption, and immune trafficking. Whereas lymphatic regeneration occurs physiologically in wound healing and tissue repair, pathological lymphangiogenesis has been implicated in a number of chronic diseases such as lymphedema, atherosclerosis, and cancer . Althoug disease . Moreove disease . Various disease –10. In t disease , 9, and MYCN oncogene amplification (MNA) and segmental chromosomal aberrations (SCA) [Neuroblastoma is the most common extra-cranial solid pediatric cancer and is responsible for approximately 15% of all childhood cancer deaths , 12. Prons (SCA) . The majns (SCA) –16, few ns (SCA) , 17. Desns (SCA) , this clns (SCA) . Therefons (SCA) , 21.Recently, in addition to vascular density, other parameters such as the size and shape of the microvessels, have been shown to be significantly related to prognosis for blood vascularization in NB  and in o2 (range 1–979.4), 75% corresponding to small capillaries. The distribution according to size of all detected lymphatic vessels together with the statistical descriptors of density and the variables that provided statistically significant results are presented in Table 332 samples included in the tissue microarrays (TMAs) were evaluable . Anti-D2-40 immunoreactivity was observed in 206 of these samples 62.1%) Figure . Figure % Figure p-values for all morphometric variables of all lymphatic vessel microvascularization classes related with the INRG pretreatment risk stratification factors are shown in Table The following features were identified: NB with undifferentiated or poorly differentiated phenotype presented wider small capillaries with more branching than NB with differentiating neuroblasts occupying a large area in the tumor. A high proportion of intermediate capillaries were present in tumors from patients with metastatic stage, >18 months, MNA and/or classified as high-risk, the capillaries were more irregular in NB without differentiating cells. Irregular small collector vessels were associated with metastatic stage, >18 months, undifferentiated NB and high risk. The A binary logistic regression analysis was performed to combine the different lymphatic vessel morphometric variables to predict risk (high risk or non-high risk). The lowest values of the median were taken as a reference category and the coefficients Exp(B) indicated an increase or decrease in high-risk susceptibility Table .The logistic equation can be written as:5–15 μmRel.SA × 0.18215–20 μmWidth × 5.91615–20 μmRel.SA × 0.19320–50 μmWidth × 4.07220–50 μmRoundness × 0.27020–50 μmRel.density × 0.221TotalBranching0.114According to this equation, lymphatic vessel impact on risk was: i) High values of relative %SA, relative density of intermediate capillaries and roundness of large capillaries increased six, three and four times, respectively, the probability of being high risk. Thus, the presence of more abundant intermediate capillaries and irregular large capillaries indicated predisposition to high-risk NB disease. ii) High values of small microvascularization (5–15 μm) relative %SA, 15–20 μm width, 20–50 μm width, 20–50 μm relative density and total branching reduced the probability of being high risk by around 80% for each parameter. Therefore, a wider lymphatic microvascularization (15–50 μm) with more branching is a better predictor of being high risk. Histograms for parameters related to the high risk are shown in Figure versus 75.7 ± 5.9 for lower lymphatic microvascularization)  (5-year EFS% of 65.8 ± 5.5), and tended to be related to lower overall survival (OS), compared to that of patients with samples presenting a lower lymphatic vessel density (5-year EFS% 79.8 ± 4.5) Figure . We also) Figure . Additio) Figure . Examplep-value: 0.033), after metastatic stage (Exp(B): 11.280; Hazard ratio (95% CI): 4.703 (1.906–11.608); p-value: 0.001), MNA (Exp(B): 5.057; Hazard ratio (95% CI): 2.662 (1.134–6.251); p-value: 0.025), and age >18 months (Exp(B): 4.809; Hazard ratio (95% CI): 3.032 (1.125–8.171); p-value: 0.028). 11q status and histopathology were excluded from the equation . OS was only influenced by the INRG variables of stage, MYCN status and age.When considering all INRG variables together with total lymphatic vascularization, a Cox regression analysis demonstrated that a higher microvascularization density was still found to influence EFS (Exp(B): 4.547; Hazard ratio 95% Confidence interval (CI): 2.403 (1.073–5.381); Several studies have revealed the importance of lymphatic vascular density in the prognosis of different malignancies –27. The Tissue vascularization has usually been described following three different quantitative methods , based on the detection of the differential staining of the vessels with more or less specific techniques , undifferentiated NB , NOS (n = 41) and differentiating NB , histopathologically categorized following the International Neuroblastoma Pathology Classification (INPC) [MYCN oncogene  or the 11q chromosome arm , following the International NB Risk Group (INRG) classification [p-values = 0.000 in all cases.Material from 332 primary NB tumors (at least two representative cylinders of 1mm of each tumor) were included TMAs, comprising poorly differentiated NB  . All samfication . The stafication –61 and tHistologic and genetic studies were approved by the Spanish Society of Pediatric Hematology and Oncology (file number: 59C18ABR2002), and the European Committee (file number: 2010-021396-81), as well as by the Ethical Committee of the University of Valencia (file numbers: H1270128195640 and A1420714159483). Participants or their family members/legal guardians provided written informed consent for histological and genetic studies performed in our laboratory. Clinical data were provided by the pediatric oncologists in charge or by the Reference center for NB clinical studies, including outcome information .One 3μm section of each TMA was cut and immunostained with anti-D2-40 monoclonal antibody (1/40)  that stains podoplanin, a transmembrane mucoprotein selectively expressed in lymphatic vessel endothelium Figure .® tool used here measures the internal lumen of the vessels regardless of their wall thickness. Moreover, we had previously analyzed the HE and Masson's trichrome stainings of the studied cohort [®: 5–50 μm for capillaries  and 50–200 μm for collectors   and relative %SA (rel. %SA) and relative density (rel. density); 2. Size  and 3. Shape , as previously reported by our group for blood vascularization [All immunostained slices were digitized with the whole-slide scanner Aperio SlideScan XT (Aperio technologies). Individual images of all TMA cylinders were exported by the Aperio ImageScope in tiff format. Histologically, it has been described that the size of lymphatic capillaries in normal tissue ranges from 10–150 μm and the lymphatic collectors measure between 150 μmand 2 mm . In tumod cohort  and obse) Figure . For eacIn all cases, a significance level of 95% was established and the SPSS statistical analysis software (version 22) was used. Non-evaluable samples  were excluded from the analyses. Samples with no immunoreactivity against D2-40 were also excluded from the statistical analysis except for a subsection of the survival analysis.The numerical continuous variables of density, size and shape from all lymphatic vessel classes derived from the morphometric analysis did not follow a normal distribution, therefore the non-parametric Mann–Whitney test was used to related these variables with the prognostic INRG categories. The continuous morphometric variables were dichotomized using the median, as it is the most accurate statistical descriptor for that purpose. A binary logistic regression was performed, using the back elimination method , combining all lymphatic vessels morphometric variables and relating them with the risk group.Survival analyses were performed using Kaplan–Meier curves and log-rank test. The dichotomized variables related to the density of the different lymphatic vessel classes and the total density of lymphatic microvascularization  were used. Additionally, the strength of the relationship of total lymphatic microvascularization with survival was tested using Cox survival regression using Wald (step back) test, including all INRG prognosis-related variables.VL.xlxs containing all the parameters extracted from the histologies of 332 different patients of primary tumor respect to the lymphatic vessels. Each column contained the data associated with one parameter in a particular range of vessels calibers and the total microvasculature (5–50 μm). Using Rice's Rule for choosing the binning , we computed a histogram for each column and we have normalized it dividing the counts of each bin for the total number of counts. In the specific cases of the parameters relative stained area and relative density, we have discarded the 0 values which indicate no vessels in the histology image.For histogram analysis, we analyzed an excel file E is the expected value, X is the experimental data, μ is the mean value and σ is the standard deviation. In our analysis we used another possible definition: Excess Kurtosis, where Excess Kurtosis = 0 corresponds to the normal distribution,Kurtosis analysis is an estimator of the normality of the distribution and it is defined as the fourth standardized moment,The statistical significance of excess kurtosis G2 is defined by Zg2=G2/SEK where SEK is the standard error of kurtosis (≈ 0.34). This estimator defines a leptokurtic distribution with a significance of 95% whenever Zg2 > 2. The range −2< Zg2 < 2 defines a normal distribution with a significance of 95%.


Reduction mammoplasty (RM) is a proven method of treating macromastia, but the risk factors for postoperative complications have not been clearly identified. Through this meta-analysis, the authors aimed to identify the risk factors of RM complications.An extensive search of the literature describing complications after RM was performed using the PubMed Central, Embase, and Cochrane databases. The following risk factors were extracted: age, body mass index (BMI), tissue resection weight per breast (TRW), smoking and radiation therapy. Odds ratios (OR) were pooled with 95% confidence intervals (CI) to evaluate the relationship between these risk factors and complications after RM.2  and smoking . Infection in those with BMI ≥30 kg/m2 showed a significant difference , as well as wound dehiscence in smokers  and infection in irradiated breasts . However, there was no significant difference in age ≥50 years , combined TRW ≥1000 g .A total of 16 unique studies including 10 593 patients were included in the final analysis. It showed that there was a significant difference in complications in BMI ≥30 kg/m2 and smoking increase the risk of complications. Persons who are obese or irradiated are more likely to develop infections, and smokers experienced a higher incidence of wound dehiscence than did nonsmokers. However, patients aged ≥50 years and TRW ≥1000 g are not associated with complications from RM.BMI ≥30 kg/m Macromastia is a common problem among women, leading to disabling symptoms such as neck, back, and shoulder pain; inframammary maceration; heavy breathing during exercise; and great psychological burdens because of unaesthetic appearance. Reduction mammoplasty (RM) is an approved procedure for women with macromastia, which has effectively relieved existing symptoms with high satisfaction, even though complications have often occurred –4. Risk Postoperative complications after breast reduction include infection, wound healing problems, scars, fat necrosis, seroma, lost nipples and reoperations. The complications occurred after breast reduction would be as high as 40 or 50% in reported studies , 5, 6. MAlmost all the risk factors associated with complications were controversial and no published meta-analysis had investigated it. We therefore performed a meta-analysis of all published prospective and retrospective studies to evaluate the important risk factors in women with macromastia and gigantomastia and provide preliminary guidance for clinical treatment and prognosis.We prospectively defined the study objectives, search paramters, eligibility criteria, and analytical methods.The following electronic databases were searched: The PubMed Central, Embase, and Cochrane Library. They were searched for English language studies using the following headings and keywords: macromastia or gigantomastia. Also used were breast reduction, reduction mammaplasty or reduction mammoplasty. No limitation was put on the date of publication, which covered all previously published studies up to December 2015. In addition, selected study references and review articles were examined for further article sources.The initial selection of studies was performed on the basis of titles and abstracts. Next, two investigators (Min-Xia Zhang and Chun-Ye Chen) independently screened the full text of each selected study using the following inclusion criteria: (1) the study must meet the definition of macromastia or gigantomastia; (2) RM as the only surgical procedure of interest; (3) measured complications of the incidence and risk factors of RM; (4) risk factors were BMI, age, TRW, smoking or radiation therapy, any one of which should be researched in the study; (5) Sufficient data on contrasting groups. Studies were excluded if they contained any one of the following exclusion criteria: (1) case reports, abstracts only, letters, comments or reviews; (2) studies with mixed gender or surgical procedures; (3) virginal, adolescent or pregnant macromastia. To avoid double publication, only the most informative or lastest study was included. This meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement checklist.Data extraction was performed independently by two reviewers (Min-Xia Zhang and Chun-Ye Chen), and any disagreement concerning paper eligibility was resolved by discussion and consensus. The data covered the general characteristics of each study and the outcomes measured. In addition, studies were assigned a level of evidence score.Based on the amount of data available and on clinical relevance, five factors were analyzed including BMI, age, TRW, smoking and radiation therapy.I square test, where p value <0.10 for the Chi squared and I2 ≥50% implied heterogeneity [For each risk factor in our study, odds ratios (OR) and 95% confidence intervals (CI) were calculated for outcomes. A p value <0.05 was judged as statistically significant. Random-effects models were used depended on the heterogeneity of the studies included. Heterogeneity was analyzed with both the Chi squared test ogeneity . The forData was processed in Review Manager version 5.3 from the Cochrane Collaboration.A total of 2532 records were identified by the initial database search, from which 63 full-text articles were retrieved for final review after screening titles and abstracts. Of these, 16 studies that met all predefined inclusion criteria were finally included in our meta-analysis , 17–27. Study characteristics for the 16 included studies are summarized in The study set included altogether 10 593 patients whose demographic features  are displayed in The risk factors with sufficient data of complications available for meta-analysis were BMI, age, TRW, smoking and radiation therapy. The results were expressed as ORs, 95% CIs and p-values.2) and obese (BMI ≥30 kg/m2). BMI ≥30 kg/m2 was the strongest risk factor for overall complications in our meta-analysis based on 6 related studies [2 = 0%). The aggregated results suggest that BMI ≥30 kg/m2 was highly associated with a significant increase in the overall incidence of complications after RM   (2 = 0%).We first divided the patients into two groups: non-obese  . Moreove= 0.004) . The tes2 = 0%). The aggregated results of the 2 studies suggest that age ≥50 years was not associated with a significant increase in the overall incidence of complications after RM   .2 = 50%). Using the random-effect model, the aggregated results suggest that TRW ≥1000 g was not associated with a significant increase in the overall incidence of complications after RM   .2 = 41%). Using the random-effect model, the aggregated results suggest that smoking was highly associated with a significant increase in the overall incidence of complications  (2 = 0%).We combined 6 studies , 20–22 c = 0.06) . In addi = 0.06) . The tes2 = 0%). The aggregated results suggest that breast reduction after radiation lead to a significant increase in the incidence of infection  (2 = 0%).We finally selected 3 studies –27 repor 0.0009) . However 0.0009) . The tesFor observational studies, we applied the Risk of Bias Assessment tool for Nonrandomized Studies (RoBANS), which was compatible with the Cochrane risk-of-bias tool.Even though women undergoing breast reduction were generally young and healthy, postoperative complications were relatively common, with an incidence of 14%-53% in reported studies , 17, 28,2 was classified as “underweight”; a BMI between 18.5 and 24.99 kg/m2 was “normal weight”; a BMI between 25 and 29.99 kg/m2 was “overweight” and a BMI of 30 kg/m2 or more was “obese”  [Increased BMI was the strongest predictor of complications among patients with breast reduction in our meta-analysis. The patients’ weights were categorized as follows: a BMI equal to or less than 18.5 kg/mobesity) . The vasobesity) , 17, altobesity) , 23, 30.obesity) . Our assobesity) . In addiobesity) . Our resOur mate-analysis demonstrated that age at surgery ≥50 years bears no relationship to complications in RM compared with younger patients <50 years. According to a positive study by Shermak MA et al., we diviTRW ≥1000 g was not a significant factor in the overall incidence of complications compared with smaller resections. We had arbitrarily set the crossover point between small and large reductions at 1000 g of tissue per breast . ZubowskAnother strong risk factor for complications after RM in our meta-analysis was smoking. Many other studies supported our results of the effect of smoking status on complication rates, such as Schumacher HH et al., Roje Z 2 = 41%), so we used the random-effect model and sensitivity analysis to evaluate the source of high heterogeneity. The results of sensitivity analysis showed that smoking was also highly associated with complications (p = 0.02) with a low heterogeneity (I = 0%) when excluding one study of Roje Z et al.[Although smoking was significant for complications in our meta-analysis p = 0.06), heterogeneity was also significant  are not mutually exclusive, which might have led to overestimating a complication risk. This study was not designed to provide independent risk factors for complications, for the reporting data was insufficient to do a multivariate analysis. Third, this meta-analysis was an observational study so that a randomized controlled trial was necessary. However, performance of any such study would be difficult. Consequently, there was marked heterogeneity among risk factors. So we attempted to account for heterogeneity using a random-effect model for meta-analysis when the Cochran Q statistic was significant, and find the source of high heterogeneity using sensitivity analysis. Finally, perhaps the biggest limitation of this meta-analysis and an important target for future research was the inability to perform outcome subgroup analyses by operative technique.2 and smoking status are risk factors for complications after RM. Moreover, BMI ≥30 kg/m2 and radiation therapy showed a statistically higher incidence of infection and smokers acquired higher significant rates of wound dehiscence. However, age ≥50 years and TRW ≥1000 g are not associated with post-RM complications. Further large-scale, well-designed studies are urgently needed.This meta-analysis demonstrated that BMI ≥30 kg/mS1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.