Primary closure following laparoscopic common bile duct exploration (LCBDE) has been widely adopted because of the efficacy and safety in treatment of common bile duct (CBD) stones. However, the risk factors for bile leakage, the most common complication after primary closure, has not been clarified yet.A retrospective cohort study of patients who underwent LCBDE with primary closure after choledochotomy between Feb. 2012 and Jun. 2016 was performed. Risk factors for bile leakage were identified by logistic regression inculding demographic factors, preoperative condition and surgical details.vs ≥1 cm, 31.6% vs 7.0%, p = 0.04) and those managed by inexperienced surgeons . After multivariable regression, the diameter of CBD  and experience of surgeons  were significantly related to bile leakage.Between Feb. 2012 and Jun. 2016, a total of 265 LCBDE procedures were applied in our hospital and 141 patients with primary closure were included in this study. Bile leakage occurred in 11.3% (16/141) of these patients, and happened more frequently in patients with slender CBD (<1 Slender CBD and inexperienced surgeons were the high risk factors for bile leakage after primary closure following LCBDE. Common bile duct (CBD) stone is the strong indication for surgical therapy, especially in patients with obstructive jaundice. With the development of laparoscopic equipment and technology, laparoscopic common bile duct exploration (LCBDE) has been widely used in clinical practice –4. Many Primary duct closure after open CBD exploration was first described by Halstead as early as 1917. Since then, the debate between primary closure and T-tube drainage continued even in the era of laparoscopic surgery. In the past decade, numerous studies comparing primary with T-tube were published and revealed the feasibility and safety of primary closure –10. MetaPatients with CBD stones who were admitted to our center between Feb. 2012 and Jun. 2016 were screened and those who received primary closure after LCBDE were included in this study. In our institution, the indications for primary closure following LCBDE are as follows: (1) CBD stones are confirmed by preoperative MRCP or CT with no intrahepatic bile duct stone; (2) The diameter of CBD is more than 0.8 cm; (3) No obvious inflammatory changes of CBD are detected intraoperatively; (4) No. of stones is less than 5; (5) Function of Oddi sphincter is normal without residual stone which is confirmed by intraoperative flexible choledochoscope. Patients with previous biliary surgery history or converted to open surgery were excluded from this study. This study was approved by the Ethics Committee of Xuanwu hospital.2 in abdominal cavity was maintained at 12 to 14 mmHg. Four trocars were placed in umbilical, medial epigastric, right anterior axillary line and right midclavicular area, respectively. The 10 mm trocar in right midclavicular area was placed for choledochoscope examination. After dissection of the gallbladder, the cystic duct was clipped with Hem-o-lock. Then the dissection continued to expose the cystic duct-CBD junction and the anterior wall of the CBD. The 1.0–1.5 cm longitudinal choledochotomy was made in the anterior surface of the CBD. After the choledochotomy, small stones were washed out by saline irrigation. Then, a flexible choledochoscope was routinely used to examine distal and proximal bile duct. The residual stones were extracted by using a stone basket. For big stones, electrohydraulic lithotripsy was used through the working channel of the choledochoscope. After complete remove the stones, the flexible choledochoscope was preformed to confirm the clearance of the intrahepatic/extrahepatic bile duct and the normal function of Oddi sphincter. Then, primary closure was performed with continuous or interrupted suture using Vicryl 3–0 or 4–0 suture line. Intraoperative cholangiography (IOC) was not performed  for patients with drainage tube, bile was detected in the drainage for more than 3 days or the volume of drainage containing bile was more than 100 ml/day; (2) for patients without drainage tube, local or general peritonitis was detected and bile was found in ascites by puncture or reoperation.Possible risk factors for bile leakage after primary closure following LCBDE are divided into three groups: demographic factors, preoperative condition and surgical details.Demographic factors: (1) age; (2) sex; (3) ASA score.Preoperative condition: (1) serum leukocytes; (2) total bilirubin; (3) diameter of CBD; (4) No. of stones; (5) gallstone.Surgical details: (1) operative time; (2) total blood loss; (3) method of suture; (4) length of choledochotomy; (5) surgeon’s experience.p < 0.1 were considered for the multivariable model. Results were presented as odds ratios (ORs) with 95% confidence intervals (CI). A P value <0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 18.0 .The data is expressed as means ± SD or median (range). For pairwise comparisons of groups, we used the chi-square test or Fisher exact test for categorical variables and Student t test for quantitative variables. The logistic regression analysis was used to identify variables associated with risks for bile leakage after primary closure following LCBDE. All variables with univariable Between Feb. 2012 and Jun. 2016, 3109 patients with gallstones and 589 with CBD stones were treated in our hospital. Of the patients with choledocholithiasis, 376 patients received surgical therapy with 111 open and 265 laparoscopic procedures, respectively. In laparoscopic operation group, 8 patients converted to open surgery. The data about the patients who successfully received LCBDE was shown in Table No perioperative death occurred during the study period. The overall complication rate was 21.3 (30/141), and biliary-specific complication rate was 12.7% (18/141). According to Dindo-Clavien classification, most of the complications were as grade I, while one patient with uncontrolled bile peritonitis (grade IIIb) received reoperation (Table The median follow-up time was 34 (range 1–76) months, stone recurrence was detected in 2 (1.4%) patients. Repeated LCBDE followed by T tube drainage was performed in these two cases. No bile duct stricture was found during the follow-up period.vs ≥1 cm, 31.6% vs 7.0%, p = 0.037) and managed by inexperienced surgeons  (Table Bile leakage occurred in 11.3%(16/141) of the patients with primary closure following LBCDE. Then, the patients were divided into two groups, bile leakage group and no bile leakage group. Most possible risk factors, such as age, ASA score, total bilirubin, operative time, total blood loss and et al were comparable between two groups. However, bile leakage occurred more frequently in patients with slender CBD , p = 0.04] and inexperienced surgeons  were found to affect bile leakage.The influences of age, sex, ASA score, serum leukocytes, total bilirubin, diameter of CBD, No. of stones, gallstone, operative time, total blood loss, method of suture, length of choledochotomy and surgeon’s experience on bile leakage after primary closure following LCBDE were summarized in Table .In the era of mini-invasive surgery, laparoscopic cholecystectomy (LC) has been the standard therapy for symptomatic gallstones. However, debate continues regarding the best treatment for managing cholecystocholedocholithiasis, and a consensus has not been reached , 13. In The role of ERCP in diagnosis of CBD stones has been replaced by MRCP, however, it is widely used to remove CBD stones in one-stage or staged procedures. EST is associated with serious short-term complications, including bleeding, post-ERCP pancreatitis (PEP), and perforation of the digestive tract. In addition, EST may increase the incidence of long-term complications such as biliary infection due to the dysfunction of the Oddi’s sphincter after the procedure –16. In op = 0.17], postoperative morbidity , mortality , and length of hospital stay  . In experienced hands, the incidence of bile leakage decreased significantly form 15.7% to 7.0% (p = 0.04). The data form LC, LCBDE and LCTBDE also revealed the importance of surgeon’s experience for decreasing the complication rate [vs ≥1 cm, 31.6% vs 7.0%, p = 0.04). Possible reasons for this tendency were as follows: (1) the wall was thin in patients with slender CBD, and the bile could leak from the needle pinprick; (2) when suturing, surgeons might stitch too little tissue because of the fear of CBD stricture; (3) after suturing, transient stenosis of CBD might occur due to the tissue edema, and pressure increased within the biliary tree, then bile leakage occurred. So, in these cases, transcystic biliary drainage should be considered to reduce the pressure within the biliary tree.Bile leakage negatively influences the postoperative recovery and patient might need additional imaging study and even reoperation. However, the risk factors for bile leakage after primary closure following LCBDE has not been fully discussed. In our study, we found that the surgeon’s experience was the most important risk factor for bile leakage [OR 95% CI, 4.228 1.330–13.438), ion rate , 26, 43.8, ion raion rate . In patiIn our cases series, one patient received reoperation due to the uncontrolled bile peritonitis. We found intraoperatively that the first suture was too far away from the superior edge of bile duct incision. So in the following cases, we usually preform the first suture above the superior edge of incision to avoid bile leakage resulting from excessive traction in following sutures. Absorbable sutures were used in our hospital to avoid CBD stricture and stone recurrence. In the follow-up period , no CBD stricture was observed and 2 patients experienced stone recurrence.Despite the limitations of retrospective study design and limited case volume, it was the initial try to detect the risk factors for bile leakage after primary closure following LCBDE. We found that slender CBD and inexperienced surgeons were associated with high bile leakage risk independently in primary closure after LCBDE. However, we believed that primary closure after LCBDE was safe in selected patients preformed by experienced hands.Slender CBD and inexperienced surgeons were the high risk factors for bile leakage after primary closure following LCBDE.


Results showed that oral microbiota from caries-active twin possessed higher metabolic activity and produced more lactic production. 16S rRNA sequencing analysis showed that more than 80% of family taxa could be transferred into gnotobiotic-mice. Key caries-associated genera were significantly different between twins and the same difference in genus level could be found in mice as well (p < 0.05). This study suggested that oral microbiota of twins could be distinguished from each other despite the similarities in genetic make-up, living environment, and lifestyle. The difference in microbiota was applied to develop a mice model which may facilitate the investigation of core microbiota of dental caries.Oral microbiome is potentially correlated with many diseases, such as dental caries, periodontitis, oral cancer and some systemic diseases. Twin model, as an effective method for studying human microbiota, is widely used in research of relationship between oral microbiota and dental caries. However, there were few researches focusing on caries discordant twins. In this study,  The etiology and related risk factors of dental caries have been a hot topic for a long period of time. The microbial factors are known as indispensable causes of the onset and progress of dental caries. Recently, core microbiota was considered to play important roles in the development of diseases, such as obesity and dental peri-implantitis3. Previous studies claimed that core microbiota was one of the most important elements in dental caries development6. More efforts should be put on exploring how core microbiota determines the progress of dental caries via in vitro and in vivo experiments. Therefore, an effective and accurate model is highly in need. The existing in vitro mono- or multi- species biofilm model8 or saliva biofilm model9 were utilized to simulate cariogenesis environment. Streptococcus mutans biofilm is one of the examples which contributed to the development of anti-caries material10. In our previous studies, we found that the growth media, not the substrates, have significant effects on the microbial community of saliva-derived biofilm in vitro11. However, in vitro model was unable to completely restore and delegate inner oral environment and microbiota due to the pH, temperature, enzymes and so forth. It can only reflect the condition of biofilm in exact situation.Dental caries, a multi-factors related disease, is a major factor of irreversible tooth decay and will in further lead to pulpitis, severe toothache, and chronic infections13, dietary habits, physical status, genetic factors and living conditions16. The common in vitro model can hardly take these factors into account. Thus, in vivo animal model is likely to be a better model than in vitro to imitate a relatively similar oral environment of human being. In our previous study, human oral microbiota-associated mouse model had developed via transplanting human saliva to mice. It successfully reconstructed human oral microbiota in gnotobiotic mice (results under review) and it can technically be used.Furthermore, there are other factors that can contribute to onset of dental caries, for instance, speed of salivary flow, saliva composition, fluoride exposure19. These brand-new technologies refresh the previous views on microbes in human body and relevant researches emerge to the public in an endless stream. A suitable model can possibly enhance the efficiency along with these technologies. For this purpose, a more effective research model is supposed to be established. Combining the utilization of biotechnology and bioinformatics, the highly precise results in human microbiome researches could be possessed.Biotechnology and bioinformatics methods develop rapidly in recent years, which stimulate the establishment of a better understanding of both cultured and un-cultured bacteria and the microbiota-related diseases. At the same time, those methods enable the determination of the composition and possible functions of microbiota in specific niches22. According to these comparisons, researchers can estimate the importance of genetic or environmental factors that influence the variation of human physical characteristics24. For example, Townsend et al. emphasized the value of twin studies in clarifying the effects of genetic factors on common dental problems, such as dental caries, periodontal diseases and malocclusion25. Because of the one hundred percent identical genes and comparable living conditions and lifestyles, monozygotic twins are supposed to own the same body features. Nonetheless, the twins discordant for obesity have been recruited in a research of gut microbiota26 implying the influence of the gut microbiota on the development of obesity using the germ-free mice model.Twins research involves comparisons of similarity or discrepancy between monozygotic twins or between monozygotic and dizygotic twinset al. reported that twins have similar experience of dental caries27. Nevertheless, it was observed that identical twins with the same diet, lifestyle and living environment can keep different DMFT. Therefore, twins with discordant caries experience might be a useful model for investigating the core microbiota of dental caries. Besides, it is important to mimic the different oral microbiota derived from the monozygotic twins with different dental caries experience. So far, there is no study investigating the difference of caries experience between those monozygotic twins. Therefore, based on the in vitro biofilm model and oral microbiota associated mice model, the present research is aimed to compare the oral microbiota between monozygotic twins with discordant caries.Papapostolou In this study, one pair of 23-year-old female monozygotic twins were recruited. The physical conditions were recorded and showed in Table 28 on hydroxyapatite (HA) disks. Then, the biofilms were subjected to 3--2,5-diphenyltetrazolium bromide assays (MTT) and lactic acid measurements.Saliva and plaque samples were used to form biofilm in SHI mediump < 0.05). To the summary, the oral microbiota of the younger twin, who got higher DMFT, metabolized more absorbable nutrition and produced more lactic acid, which was a risk factor of caries. The higher metabolic viability and more lactic acid production may contribute to the formation of dental decay on host.The MTT assay results  illustrated that saliva biofilm of the younger twin on hydroxyapatite(HA) specimen had almost 3 folds greater absorbance than the elder twin (p < 0.05) Fig. . ResultsThe microbial communities in saliva and dental plaque of the discordant caries experienced-twins were characterized through 16S rRNA sequencing analysis and the differences of richness, diversity and relative abundances of each taxon were determined. In total, 16 phyla, 27 classes, 63 orders, 116 families, 294 genera, 484 species and 688 OTUs were detected from samples of the two participants. As for diversity, the Shannon index showed that microbial diversities were higher in both plaque and saliva samples of the elder than the younger twin Fig. .Figure 2In 688 OTUs, 117 of which were detected in all the 4 samples, and 255 and 172 OTUs were shared by twins in saliva and plaque respectively . At genus level, Streptococcus and Neisseria were the majority in saliva and plaque  of the monozygotic twins were gavaged into recipient mice  respectively. After 5 weeks, 14 saliva samples were collected from each mice group and 16S rRNA gene amplicons were sequenced.Firmicutes at phylum level, Proteobacteria and Bacteroidetes also showed high abundance. At genus level, Streptococcus, Leuconostoc, Geobacillus and Enterococcus were very common genera in mice oral cavity  than inactive group (the abundance was 37.77% vs. 26.9%), and the difference was significant (p < 0.05). As for caries-inactive group , there were higher percentage of Leuconostoc (p < 0.05), Fusobacterium (p < 0.05) and Enterococcus (p < 0.05). In addition, Geobacillus and Acinetobacter were also more abundant in caries-inactive group abundances were not significantly different (p > 0.05)  Fig. .Figure 3p > 0.05). Similarly, Simpson, ACE and Chao1 indices were not significantly varied between two groups (p > 0.05)  Fig. .As for the beta diversity, the PCoA was conducted and the result showed that two groups of mice possessed similar saliva oral microbiota Fig. .30. The transplantation rates in the present study reached 88.4% and 81.6% at family level in caries-active group and caries-inactive group respectively, which was consistent to our previous study. Besides, 90% and 80.9% of bacterial orders were transplanted into mice and at the class level, the transplantation rates were 90% and 86.4% in caries-active group and caries-inactive group. The mice model that recruited twins was successfully constructed, and it was able to receive the majority of microbiota from the donors.The saliva microbiota of mice and hosts were compared. In general, we successfully transplanted human oral microbiota into mice oral cavity. Previous researches had developed humanized mice model and transplanted 88% of genus-level taxa and 11 of 12 bacterial classes of donor gut microbiota into mice gutStreptococcus, Haemophilus, Acinetobacter, Rhodococcus and Neisseria and so on were included. In comparison to the previous study proved that Streptococcus, Prevotella, Leptotrichia, Veillonella and Fusobacterium were correlated with onset and progress of caries18, and we found significant difference between the abundance of Streptococcus, Prevotella, Leptotrichia and Fusobacterium of the twins in the present study. 3 of these taxa also showed obvious difference in the mice oral cavity including Streptococcus, Prevotella and Fusobacterium. 75% of key taxa variations in host were successfully transferred into mice and they could reflect the difference between oral microbiota of the monozygotic twins. In addition, Veillonella showed no difference in both twins‘ saliva and mice saliva. The successful transplantation of difference in caries-correlated taxa indicates the possibility of investigation of caries-associated microbiota via our mice model utilized caries discordant twin pairs.Meanwhile, we found that recipient mice could not only presented the oral microbiota of the donors but also displayed the difference of oral microbiota between the donors. In this study, the genera, occupying higher than 1% of total microbiota, whose quantitative difference between two donors was higher than 10% were selected. Meanwhile, the genera occupying over 0.1% but less than 1% and the quantitative differences being more than 0.3% between two individuals were selected  . The written informed consent was obtained from each participant upon admission to oral examination, photos and samples.The samples consisted of 1 pair of twins who were 23-year-old females, and informed consents for inclusion were obtained from participants. Assessment of oral health showed that the elder one had zero DMFT, while the younger sister has 4 DMFT. None of them had systemic diseases and antibiotic usage in last 3 months. They lived together since they were born and had same oral hygiene behaviors.36. Supragingival dental plaque from the buccal surface was collected with a sterile hand probe from 11, 16, 26, 31, 36, 46 teeth of each participant. Samples were deposited into coded sterile plastic tubes with sterile Tris-EDTA buffer solution, transported in ice to the microbiology laboratory and processed within 3 h of sampling. After that, the samples were diluted in sterile glycerol and stored at −80 °C37.Saliva was collected from the adult twin donors, and participants did not use antibiotics within the past three months. The donors were demanded not to brush teeth for 24 h before collection and abstained from food/drink intake for at least 2 hours prior to donating saliva. Stimulated saliva was collected during parafilm chewing and kept in ice. Saliva samples were centrifuged at 2600 g for 10 min to remove large debris and eukaryotic cells, then each sample was diluted in sterile glycerol to a saliva concentration of 70% and stored at −80 °C before experiment28. Plates were incubated at 37 °C under anaerobic conditions to allow biofilm formation.2 mL of each saliva sample was mixed with an equal volume of phosphate-buffered saline (PBS) and mixture was centrifuged at 14,000 g for 3 min. Then, 200 µl supernatant was added to each well that contains one piece of normalized hydroxyapatite(HA) tablet of the 24-well plate to pre-coat the HA tablets and plate were incubated at 37 °C with lid opened for 1 h to dry the saliva coating. Plates were then sterilized under ultraviolet (UV) light for 1 h before 150 µL of each saliva samples were inoculated into pre-coated well containing 850 µL SHI medium. The composition of SHI medium were refer to the previous assay−1 MTT in PBS) was added to each well and incubated at 37 °C under 5% CO2 for 1 h. During the incubation, metabolically active bacteria metabolized the MTT to formazan inside the living cells. Disks were then transferred to new 24-well plates, and 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The plates were incubated for 20 min with gentle mixing at room temperature. Two hundred microliters of the DMSO solution from each well was collected, and the absorbance was measured at 540 nm using microplate reader . The higher absorbance, the higher formazan concentration in wells, indicating higher metabolic activity in the biofilm on the composite surface36.A 3-−2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the metabolic activity of the biofilms. MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to purple formazan. Disks with 2-day biofilms (n = 6) were transferred to a new 24-well plate, and 1 mL of MTT dye (0.5 mg/mL36.Disks with 2-day biofilms (n = 6) were rinsed with cysteine peptone water (CPW) to remove loose bacteria, and then transferred to 24-well plates containing 1.5 mL of buffered-peptone water (BPW) plus 0.2% sucrose. The specimens were incubated for 3 h to allow the biofilms to produce acid. Then, the BPW solutions were collected for lactate analysis using an enzymatic method. Absorbance of BPW was determined at 340-nm with a microplate reader . Standard curves were prepared using a standard lactic acid  as described in previous studies40. In brief, microbial DNA was extracted from the saliva-derived biofilms by using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek) according to manufacturer’s protocols. DNA concentration was assessed by Nanodrop2000  and DNA quality was determined by 1% agarose gel electrophoresis. The universal target V4–V5 regions of the 16S rRNA gene were PCR-amplified using barcoded primers 515 F (5′-GTGCCAGCMGCCGCGG-3′) and 907 R (5′-CCGTCAATTCMTTTRAGTTT-3′). PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The amplicons were then extracted from 2% agarose gels and further purified by using the AxyPrep DNA Gel Extraction Kit  and quantified by QuantiFluor -ST  according to the protocols. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to the instruction. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP092150).The saliva-derived biofilms samples were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd  where the total DNA was extracted, amplified and sequenced according to their standard procedures41. Operational Taxonomic Units (OTUs) were clustered with 98.5% similarity cutoff using UPARSE (version 7.1). The taxonomy of each 16S rRNA gene sequence was analyzed by Ribosomal Database Project Classifier42 (http://rdp.cme.msu.edu/) against the Human Oral Microbiome Database with confidence threshold of 70%43. Alpha diversity indices 44 and richness estimators (ACE index and Chao 1 index)45 calculations were performed using Mothur v.1.30.2. Phylogenetic beta diversity measures such as the unweighted UniFrac distance metrics analysis was determined using the represent sequences of OTUs for each sample, and principal component analysis (PCA) was conducted according to the distance matrices. In this study, a P value threshold of 0.05 (Wilcoxon rank-sum test) and an effect size threshold of 2 were used for all biomarkers discussed. OTUs are picked using a closed-reference OTU picking protocol (QIIME 1.9.1) against the Greengenes database pre-clustered at 70% identify. The obtained OTU table was normalized by 16S rRNA copy number. The other data subjected to non-parametric Mann–Whitney analysis or student t test. Differences were considered significant when P < 0.05 and extremely significant when P < 0.01. Software SPSS19.0  was applied for statistical analysis.Raw reads were demultiplexed and quality-filtered by QIIME (version 1.9.1)in vivo study was approved by the ethics committee of West China School of Stomatology, Sichuan University (WCHSIRB-D-2016-070), and all experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The 30.Germ-free adult male C57BL/6 J mice were maintained in plastic gnotobiotic isolators under a strict 12-hour light cycle, donators ‘saliva were gavage into germ-free recipients (n = 7). Mice were subsequently maintained in separate cages in a gnotobiotic isolator and fed normal diet which was in line with national standard GB-T14924.3-2001. At the 35th day after humanization, mice were killed and saliva samples were collected and stored at −80 °C as soon as possibleThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable requests.Supplementary informationSupplementary Table S1


Current European Guideline for resuscitation recommends a centrally positioned umbilical venous catheter as the best option for administering necessary drugs. Especially in preterm infants, a frequently used alternative is the peripheral venous catheter.2) and heart rate (HR) were measured using pulse oximetry at the right hand/wrist. In each neonate, either NIRO 200NX  or INVOS 5100C  were used to measure cerebral tissue oxygenation index (cTOI) and cerebral regional oxygen saturation (crSO2), respectively. SpO2, HR, and cTOI/crSO2 during and 1 min before and after successful venous punctures were analyzed.Two randomized controlled studies were conducted at the Division of Neonatology, Medical University of Graz. During neonatal resuscitation, a standardized protocol was filled out by an uninvolved observer including time points after birth of all attempts of venous puncture, time point of successful venous puncture, and total number of needed attempts. Arterial oxygen saturation  1–2] to establish a peripheral venous catheter. In median, intravenous (IV) catheterization was successfully established 5 (IQR 4–9) min after birth. SpOPeripheral IV catheterization during postnatal stabilization of preterm infants is feasible and successful in most of the cases at first attempt. Gaining intravenous (IV) access is often needed within minutes after birth, especially in preterm neonates, for administering medications, fluids, and nutrients during postnatal stabilization . There aThe primary aim of the present study was to investigate how many attempts were needed to establish a peripheral IV catheter during postnatal stabilization. Secondary aims were (a) to determine the time needed to establish the peripheral venous catheter and (b) to investigate any potential side effects of the venous puncture on the vital parameters in preterm neonates.The present study is a retrospective analysis of exploratory parameters of two randomized controlled trials , 5 that via IV catheter  within the first 10 min after birth.Peripheral venous catheterization was the primary vascular access during the study periods. Preterm infants with gestational age <34 weeks were included into the analysis, if there was an intention to treat with medication 2) and heart rate (HR) were measured with the IntelliVue MP30 Monitor  using pulse oximetry at preductal level on the right hand/wrist. In each neonate, either NIRO 200NX  or INVOS 5100C  were used to measure cerebral tissue oxygenation index (cTOI) and cerebral regional oxygen saturation (crSO2), respectively. The sensor was positioned on the forehead in each infant. The sensor on the forehead was secured with cohesive conforming bandage . All variables were stored continuously every second in the multichannel system alpha-trace digital MM  for subsequent analysis.Arterial oxygen saturation , as appropriate. Categorical variables are presented as absolute and relative frequencies. Changes over time were evaluated using analysis of variance for repeated measurements. In cases when assumption of sphericity was violated as estimated by Mauchly’s test of sphericity, Greenhouse–Geisser correction was used.SPSS 23.0.0.2 (IBM Corp. 2015) was used for data analysis. A value of 2 was measured in 48/23 neonates). All 61 preterm neonates received respiratory support via face mask, and 10 neonates needed intubation subsequently during postnatal stabilization.Data of 61 of 70 preterm neonates were analyzed—9 neonates were excluded, because there was no intention to treat. They did not get IV catheterization during neonatal stabilization. Mean gestational age was 31.5 ± 2.2 weeks, and the mean birth weight was 1,527 ± 541 g. In median, it needed one attempt (IQR 1–2) to establish a peripheral venous catheter. In median, IV catheterization was successfully established 5 (IQR 4–9) min after birth, whereby in 38 (62.3%) preterm neonates, the peripheral venous catheter was established with the first attempt. All infants had monitoring of cerebral oxygenation , cTOI (p = 0.001), and crSO2 (p = 0.001) rose significantly. HR increased too, but the rise did not reach significance (p = 0.199).Arterial oxygen saturation, HR, and cTOI/crSOThe present study demonstrates that peripheral venous catheterization during postnatal stabilization of preterm neonates is feasible, and in the majority of patients (62%), only one attempt was necessary. The number of first-time success rate in our study was even higher than the previously reported by Legemaat et al. in NICU population .2 and cerebral regional oxygenation saturation, both rose, but changes were along the percentiles, which represent the physiological increase during the first 10 min after birth (The recent European Guideline  suggestser birth –10. FurtPeripheral IV catheterization during postnatal stabilization of preterm infants is feasible and successful in most of the cases at first attempt. In addition, the IV puncture during postnatal stabilization did not affect the physiological neonatal transition concerning arterial and cerebral oxygenation and HR.This study was carried out in accordance with the recommendations of Regional Committee on Biomedical Research Ethics with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Regional Committee on Biomedical Research Ethics.Conception and design: BU, NB-S, and GP. Collection and assembly of data: NB-S, BS, LM, GP, and BU. Analysis and interpretation of the data; critical revision of the article for important intellectual content; final approval of the article: NB-S, BS, LM, GP, BU, and AA.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.


Common bean is a legume of social and nutritional importance as a food crop, cultivated worldwide especially in developing countries, accounting for an important source of income for small farmers. The availability of the complete sequences of the two common bean genomes has dramatically accelerated and has enabled new experimental strategies to be applied for genetic research. DArTseq has been widely used as a method of SNP genotyping allowing comprehensive genome coverage with genetic applications in common bean breeding programs.HE of 0.442 for the whole collection, 0.102 for Andean and 0.168 for Mesoamerican gene pools (FST = 0.747 between gene pools), 0.440 for the group of cultivars and lines, and 0.448 for the group of landrace accessions (FST = 0.002 between cultivar/line and landrace groups). The SNP effects were predicted with predominance of impact on non-coding regions (77.8%). SNPs under selection were identified within gene pools comparing landrace and cultivar/line germplasm groups  and between the gene pools (59 SNPs), predominantly on chromosomes 1 and 9. The LD extension estimate corrected for population structure and relatedness (r2SV) was ~ 88 kbp, while for the Andean gene pool was ~ 395 kbp, and for the Mesoamerican was ~ 130 kbp.Using this technology, 6286 SNPs (1 SNP/86.5 Kbp) were genotyped in genic (43.3%) and non-genic regions (56.7%). Genetic subdivision associated to the common bean gene pools (K = 2) and related to grain types (K = 3 and K = 5) were reported. A total of 83% and 91% of all SNPs were polymorphic within the Andean and Mesoamerican gene pools, respectively, and 26% were able to differentiate the gene pools. Genetic diversity analysis revealed an average For common bean, DArTseq provides an efficient and cost-effective strategy of generating SNPs for large-scale genome-wide studies. The DArTseq resulted in an operational panel of 560 polymorphic SNPs in linkage equilibrium, providing high genome coverage. This SNP set could be used in genotyping platforms with many applications, such as population genetics, phylogeny relation between common bean varieties and support to molecular breeding approaches.The online version of this article (doi:10.1186/s12864-017-3805-4) contains supplementary material, which is available to authorized users. Phaseolus vulgaris L. [Phaseolus vulgaris varieties [It is estimated that approximately 150 plant species are grown directly for human consumption or animal feed worldwide, and 30 of them contribute to 95% of the calories and protein in the human diet . Among lgaris L. , being carieties . In BrazThere is consensus regarding the predominant genetic structure of common bean in the Andean and Mesoamerican gene pools , 8, due Molecular markers have been very helpful in efforts to detect gaps and redundancies in germplasm collections , to elucHigh-density genotyping, combining genome complexity reduction with next-generation sequencing (NGS), allows the identification of an almost unlimited number of SNPs for any species at low cost. The strategies of restriction site-associated DNA sequencing (RADseq)  and genoIn this study, DArTseq derived SNPs were used for the genetic analysis of a common bean germplasm collection of Andean and Mesoamerican origin, being each origin further stratified into cultivar/line and landrace groups. This study also made advances in the detection and characterization of genomic regions with signals of selection imposed by the domestication and breeding of common bean in Brazil. In addition, a set of SNPs with high discriminatory value between gene pools, as well as between groups (landraces and cultivars/lines) within gene pools, was proposed for routine use for the characterization of gene bank accessions and in breeding programs.A total of 188 common bean accessions, including 91 landraces and 97 Brazilian and international cultivars/lines belonging to the Andean and Mesoamerican gene pools, were used . The DArTseq quality markers were determined by the parameters “reproducibility”  and “call-rate” (percentage of samples for which a given marker was scored”) [DArTseq™ represents a combination of DArT complexity reduction methods, based on methyl filtration, and next-generation sequencing platforms . The teca et al. . The ampscored”) .P. vulgaris v 1.0 [Genomic regions flanking SNPs were aligned against the reference genome of is v 1.0  using BLis v 1.0 . Annotatis v 1.0  based onis v 1.0 . The SNPSNPs with putative effects predicted to be moderate or high were functionally annotated using the Blast2GO tool v 3.2  and char2 < 0.5) identified using Golden Helix SNP & Variation Suite v 8  through the LD Prunning command. A population number (K) ranging from 1 to 20, with 20 interactions each, was assumed. The admixture model was applied using a 500,000 burn-in periods followed by 1,000,000 Markov Chain Monte Carlo (MCMC) replications. The most likely K was determined, as proposed by [The genetic structure, based on the Bayesian clustering approach, was implemented by Structure v 2.3.4 . This anposed by  using Stposed by , followeposed by . The orgposed by . Discrimposed by  was perfposed by , 59 to pposed by , based oposed by . The Anaposed by  using SNFST for each window of the genome [π, average pairwise differences among individuals chosen randomly from the sample population) [W, estimation of population mutation rate calculated on the basis of the number of segregating sites) [http://ggplot2.org/) was used to create the graphs for patterns of variation [The e genome , Tajima’e genome , diversiulation) , nucleotulation) , 67 and g sites) , were esg sites) , 69. Theariation .FST joint distribution versus heterozygosity. Those loci that fall outside the 95% confidence interval were considered outliers.The outlier SNPs were detected using two methods: 1) Method proposed by Foll and Gaggiotti  implemen2) and corrected for bias due to population structure (K = 2) and relatedness (r2SV) using the LDcorSV package for R [LD was estimated using SNPs with MAF ≥ 0.05 and the pairwise LD measures were calculated by the usual method  was performed applying an individual-centered approach as described by [HE estimates were obtained using a hierarchical procedure, with a 150 km neighborhood grid used to avoid spatial autocorrelation between groups. In cases in which only one accession was represented in a given region, HE represents diversity only for this accession. This analysis was performed using the “sHe” function of the R package “biotools” [Spatial analysis of genetic diversity . Data from rainfall and climate/temperature were obtained from the Institute of Forest Research and Studies (IPEF). The software ArcGIS, based on Geographic Information System (SIG), was used to define areas on the maps. Landraces were geographically placed on the maps using the associated coordinate information.The 188 beans analyzed by DArTseq comprised a mini core group derived from the Brazilian common bean core collection (600 accessions) and are representative of the most genetically diverse accessions identified by microsatellite markers analysis (data not shown). For the SNP markers generated in DArTseq  showed alignment in the genome, of which 5311 (89.09%) aligned to a single region and 650 (10.90%) presented multiple alignments (ranging from two to 88). The sequences aligned to the 11 chromosomes and 12 scaffolds. The average number of SNPs per chromosome was 541, ranging from 389 on chromosome 4 to 792 on chromosome 2 , low impact (14.22%), moderate impact (7.92%), and high impact (0.05%). Most SNPs with predicted effects were observed in genic regions (6950), of which 20.82% and 17.30% were observed within exons and introns, respectively, with the remaining in non-translated regions. In genic flanking sequences (5 kb window) 5,267 effects were identified, of which 58.21% and 41.79% occurred in downstream and upstream regions, respectively. SNP effects categorized as moderate and high, were identified in 901 transcripts, of which 810 were mapped and 777 were fully annotated  and Mesoamerican (111) gene pools and six genotypes (3.87%) as admixture Fig. . Five ofThe tools implemented in DAPC revealed a more complex population structure in the Mesoamerican by landraces and lines/cultivars Fig. . The denFST value between groups (0.747). The combined set of markers generated an overall exclusion power of 100%, whereas 28 SNPs distinguished all genotypes  and 0.012 (p >0.002) for Mesoamerican and Andean, respectively. Within the Andean gene pool, cultivars/lines presented 1,217 private alleles, while in landraces it was 533 (Table High numbers of polymorphic SNPs were identified for Mesoamerican (87.51%) and Andean 88.39%) cultivars/lines compared to the landraces (Mesoamerican = 90.78%. Andean = 73.49%). The 9% cultivFST was high between the gene pools for the majority of the chromosomes, with the highest level at chromosomes 1 and 9  compared to the Mesoamerican  and was greatly reduced for the Andean  and Mesoamerican  groups  for the cultivars/lines and 0.0043 for the landraces of Mesoamerican origin, with a similar distribution of SNPs into MAF classes. Reduced values were observed for the cultivars/lines (0.0022) and landraces (0.0013) of Andean origin, probably due to the additional set of SNPs with MAF > 0.1 and ≤ 0.2  for all individuals was 0.00071 (±0.00001), with a lower value estimated for the 33 Andean landraces (0.00058). The θW, Tajima’s D, and FST estimates were highly variable across the P. vulgaris genome  and reduced θW and Tajima’s D  and Arlequin (p < 0.05), respectively, of which 16 loci were common to both analyses. From the 59 SNPs, 54 aligned over the 11 chromosomes (with the highest abundance on chromosomes 1 and 9), with an average of one SNP every 8.6 million bases. Across the genome, ~41% of SNPs were identified within genes , while the remaining (~59%) were in intergenic regions. The analysis of SNP effect on the outliers revealed a total of 110 effects predicted for 54 outliers, of which 11% were low-impact, 10% moderate and 79.09% modifier type. We identified 91 transcripts affected by 54 SNPs under selection, of which 82 presented homology to the non-redundant (nr) protein database and 71 were annotated , 15 outlier SNPs common to both analyses were identified distributed around chromosomes 2, 7, 8, and 9. 131 transcripts were affected by these SNPs, and 116 of these have been annotated  at a distance of ~2055 Kb and ~395 Kb for r2 and r2SV, respectively, while for the Mesoamerican, half decay was observed at distances of ~312 Kb and ~130 Kb. For accessions overall without correction (r2), no decay was observed within the 3000 Kb window  LD decreased to half at 88 Kb  to 62 (chromosome 8) were identified. A total of 4354 SNPs (82.57%) were distributed in these blocks, with an average of ~10 SNPs per block. Chromosomes 9 (90.12%) and 4 (71.77%) had the highest and lowest percentage of SNPs within blocks, respectively. The average block size was 842.2 Kb, and the largest block was in chromosome 3, with 8634 Kb and 21 SNPs. The maximum and minimum frequency of haplotypes was 0.850 and 0.010, respectively, with the most common haplotype located on chromosome 7. On average, 71.66% of the genome was covered by the blocks , FIS = 0.826 (±0.001), and FIT = 0.897 (±0.001). DAPC revealed a structure similar to those obtained for the whole set of SNPs (5531)  was 0.411. For the regions with low precipitation (≤700 to 1000 mm), the HE of the seven accessions  was 0.419.The highest estimates of sis Fig. . Within P. vulgaris genome, supporting the analysis of population structure, LD, and identification of genomic regions under selection that have an impact on crop improvement research. The average call-rate was 92%, close to the value of 91.3% previously reported for watermelon [Pstl-Msel) resulted in the more frequent appearance of SNPs, as reported by Schröder et al. [The SNP markers derived using DArTseq demonstrated that this technology is an efficient method of genotyping with broad genome coverage and can be useful for analyses of genetic diversity in a common bean germplasm pool composed of landraces and cultivars/lines. The sequencing of two important varieties of common bean representative of the Andean and Mesoamerican groups , 18 has termelon , and thetermelon . A high termelon , increasr et al. . Within r et al. . The conn = 181, HE = 0.442), compared to the SNPs identified by RAD , Valdisser et al. [n = 88, HE = 0.390), Müller et al. [HE = 0.168; n = 111) was close to values obtained by Rodriguez et al. [HE = 0.157; n = 100); however, lower compared to the studies of Cichy et al. [HE = 0.233; n = 21), who characterized a more diverse set of accessions that are from different geographic origin, breeding program, grain type, and growth habit. In our Mesoamerican germplasm, the only structure observed was by grain type (K = 4) [π = 0.001781), compared to the Mesoamerican (π = 0.004541) germplasm. The lowest diversity in the Andean is expected  due to the prevalence of Mesoamerican introduction and domestication in Brazil [DArTseq also allowed the detection of SNPs with high diversity  , 31, witn Brazil , in addin Brazil . The intn Brazil , whereasn Brazil , 88.HE and π were reported for the Andean, compared to the Mesoamerican. Moreover, the diversity of the Andean Brazilian landraces  reduces only in the order of 1.3 x compared to the diversity estimated by Cichy et al. [HE = 0.128). Therefore, there is a strong indication that Brazil may be a center of secondary domestication with high diversity that deserves a further characterization of the remaining accessions and integrates into the Brazilian core collection. For the Andean cultivars/lines (n = 31), even represented by accessions of 22 countries, the πC/L (0.002171) was almost half the value for the Mesoamerican  used predominantly in breeding program for improving agronomic traits [In the present study, all landraces originated from Brazil and only the breeding germplasm included introductions. From the 87 Brazilian landraces, lower estimates of Regarding the spatial distribution of the genetic diversity, no significant relationship between genetic and physical distances was identified (data not shown). By overlapping the thematic and diversity maps, important sites to collect landraces, considering both genetic diversity and adaptability under hydric restriction and high temperature were identified. For breeding purposes, this is extremely valuable in terms of understanding adaptive responses and identification of target accessions genetically diverse with the potential to be integrated into bean pre-breeding programs, as well as germplasm resource management. Genetically diverse accessions originated from geographic areas with high temperature and reduced rainfall could be of potential use in common bean breeding programs in attempt to increase the frequency of favorable alleles, and consequently, increasing the potential to obtain inbred lines more tolerant to drought stress, which can be obtained, for example, through recurrent selection.As expected, the SNP effects categorized as modifiers was more abundant and SNP with impact on protein efficiency and loss-of-function, that have a direct impact on gene function with adaptive interference during the course of selection, were reported in a smaller proportion . ConsideFST = 0.86 and chromosome 9, FST = 0.87), suggesting that distinct and broad genetic mechanisms were involved in the process of common bean domestication. Similar finds were reported by Schmutz et al. [The process of domestication and artificial selection imposed by agriculture resulted in changes to allelic frequencies and allowed the identification of genomic regions under adaptive evolution using high-density SNP genotyping , 91, 92.z et al. . Among tz et al. , 22 and,z et al. .Outlier SNPs associated with the same GO terms were reported in both gene pools. Among these, we highlighted integral components of membranes that could respond to plant demand to be more efficient in the process of water and nutrient transport, as well as the location of photoassimilates. Furthermore, several common transcripts related to the development of morpho-anatomical structures were reported, corroborating previous studies of QTLs involved in the domestication and diversification processes . SelectiOur data showed that, several genes under selection in both pools were related to pathways of primary metabolism, such as sucrose, amino acids, lipids and starch metabolism. These pathways are source of energy and carbon that broadly affect a range of cellular mechanisms. Interestingly, 11 distinct putative genes under selection were identified in both pools, six Mesoamerican and five Andean genes, related to the same Purine and Thiamine metabolic pathway revealing different signatures of selection associated with the same processes. Products derived from the Thiamine biosynthesis play role as a cofactor in important metabolic pathways, such as glycolysis, Krebs cycle, nitrogen assimilation, and have been shown to have functions in response to abiotic and biotic stress in plants . The Pur2, and after the correction, an increase in decay was observed, showing that the evolutionary and breeding history [c/L = 4040 Kbp; Meso LDc/L = 428 Kbp) presented slower LD decay compared to the landraces , consistent with previous studies [A high proportion of alleles at low frequencies were observed within the gene pools, whereas for the whole set of accessions, most SNPs were present at high frequencies (≥0.3), reflecting the presence of fixed loci for alternative alleles between the gene pools. Without correcting for relatedness and structure, the LD presented elevates estimates of r history , 85 stro history  and lobl history . This wa studies . The pat studies , and thi studies . The mor studies . The red studies .Furthermore, the variation in size of the haplotype blocks across the common bean genome Table  revealedDArTseq analysis over a diverse group of common bean germplasm allowed the identification of a panel composed of 560 SNPs, selected from the whole set of 6286, with nearly 90% genome coverage. For breeding purposes, this panel of SNP, which allows identification of genetic intervals at low to moderate resolution, would be readily incorporated to routine genetic analysis of breeding programs. The benefits of marker-assisted breeding using this panel over a large set of SNPs are due to the increase in the efficiency of genome sampling at a lower cost. This panel certainly will be of great utility for germplasm characterization, linkage mapping, and assisted backcrossing, meeting the research demands with impacts on bean crop systems. For studies that demand improved genome resolution, such as association and genome selection, whole genome sequencing of multiple samples is the method we should use to allow the detection of the majority allelic variants for relevant traits.The present study has shown that the DArTseq approach generated a large set of useful SNPs for common bean with a comprehensive genome coverage, representative of coding and non-coding regions that allowed an accurate assessment of structuration and quantification of genetic diversity in the Brazilian core collection composed of landrace and improved germplasm. We also were able to identify genomic regions under selection in domesticated germplasm associated with molecular functions that could be used as target in further studies to determine the nature and relevance of these loci in the process of adaptation. In addition we observed that the extent of LD was variable throughout the genome and in different strata of germplasms, which was helpful for determination of a reduced set of SNPs useful for genetic analysis. Through this study, we are adding value to the common bean Genebank at Embrapa publicly available for worldwide signatory institutions of the International Treaty on Plant Genetic Resources. This information, in combination with phenotypic evaluation, hold much promise for breakthroughs in the elucidation of genetic control of complex traits.Additional file 1:Identification of the common bean accessions used in the SNP analysis, the gene pool origin, type of germplasm, institution of origin, and the commercial type of grain. (XLSX 18 kb)Additional file 2:The genotyping data and SNP quality parameters for the whole set of accessions. (XLSX 5705 kb)Additional file 3:Functional annotation of transcripts affected by SNPs with high (*) or moderate predicted impact. (XLSX 75 kb)Additional file 4:Enzymes associated with SNP sequences with high and moderate impact predicted. (PDF 22 kb)Additional file 5:The KEGG pathway maps for SNPs with high or moderate predicted impact. (XLSX 25 kb)Additional file 6:Gene models representing all SNP with high impact effects predicted by SnpEff. A) High impact effects classified as “splice_acceptor_variant”, defined as two bases before exon start, except for the first exon. B) High impact effects classified as “stop_gained”, a sequence variant whereby at least one base of a codon is changed, resulting in a premature stop codon. The nucleotide described corresponds to the disruptive high impact allele. (PNG 3026 kb)Additional file 7:FST), Watterson’s θ (θW), and Tajima’s D for all 11 chromosomes in the P. vulgaris genome for each group. FST is given as an average across all pairwise comparisons between Andean cultivars/lines and landraces (green), and between Mesoamerican cultivars/lines and landraces (red). The results of Tajima’s D and θW are given for each group separately, Andean cultivars/lines (dark green) and landraces (green), and Mesoamerican cultivars/lines (red) and landraces (yellow). FST, Tajima’s D and θW related summary statistics were calculated for each 100 kb non-overlapping sliding window. (PDF 240 kb)Genome-wide loess curves for genetic differentiation  classes. (A) Distribution of the number of SNPs into MAF classes for the whole population (grey), Andean (green), and Mesoamerican (red) genotypes. (B) Distribution of the number of SNPs into MAF classes for each group: Andean cultivars/lines (dark green) and landraces (green) and Mesoamerican cultivars/lines (red) and landraces (yellow). (PDF 23 kb)Additional file 9:Functional annotation of the transcripts affected by outlier SNPs. (XLSX 21 kb)Additional file 10:Functional annotation showing the most relevant GO terms for the outlier SNPs. The terms were filtered according to the node score. The numbers represent the amount of transcripts related to each term. (PDF 122 kb)Additional file 11:The KEGG pathway associated with outlier SNPs. (XLSX 15 kb)Additional file 12:Functional annotation of transcripts affected by SNP outliers in the Mesoamerican gene pool. (XLSX 24 kb)Additional file 13:Functional annotation of transcripts affected by outlier SNPs in the Andean accessions. (XLSX 15 kb)Additional file 14:Functional annotation showing the most relevant GO terms for the outlier SNPs within each gene pool. (A) Mesoamerican; (B) Andean. The terms were filtered according to the node score. The numbers represent the amount of transcripts related to each term. (PDF 233 kb)Additional file 15:Information of the selected 560 SNPs to compose a panel for genetic analysis of common bean. (XLSX 56 kb)Additional file 16:DAPC analysis based on the 560 SNP panel showing the division between Mesoamerican cultivars/lines (red) and landraces (yellow). M_CL: Mesoamerican cultivars/lines; M_L: Mesoamerican landraces; A_CL (dark green): Andean cultivars/lines; A_L (light green): Andean landraces. (PDF 38 kb)


IL16 polymorphisms and plasma IL16 level with OS risk in a Chinese population. We genotyped IL16 rs4778889, rs11556218, and rs4072111 in 358 patients with OS and 402 controls using a polymerase chain reaction-restriction fragment length polymorphism assay. Plasma IL16 level was measured by enzyme-linked immunosorbent assay. Rs11556218 was associated with an increased risk of OS in heterozygote comparison , dominant model , and allele comparison . Moreover, rs11556218 TG/GG genotypes were associated with higher levels of IL16 as compared to TT genotype (P = 0.03). However, no significant association of rs4778889 and rs4072111 and OS was found. These findings suggest that rs11556218 TG/GG genotypes may be associated with increased susceptibility to OS, probably by increasing the production of IL16 level.Interleukin (IL) 16 plays a key role in inflammatory diseases as well as in tumorigenesis of osteosarcoma (OS). The aim of this study was to investigate the association of  IL6, and IL12 121314Osteosarcoma (OS), derived from primitive transformed cells of mesenchymal origin, is an aggressive malignant neoplasm in bone and often occurs in children and young adults123356810IL16 is a pleiotropic cytokine and functions as a modulator not only in inflammatory processes but also in tumorigenesis. By binding to the CD4 molecule, IL16 can activate monocytes and stimulate the secretion of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL1β, IL6, and IL15. These cytokines were related to the development of OS1617181920212223IL16, located on chromosome 15q26.3 in the human genome, encodes IL16 cytokineIL16 with risk of a series of cancer types, including colorectal cancer262628293135IL16 and OS risk. In this study, we evaluated the association of the three SNPs in IL16 with susceptibility to OS in a Chinese population. Moreover, the effect of IL16 polymorphisms on plasma level of IL16 was also assessed.The study protocol was approved by the Review Boards of Affiliated Hospital of Youjiang Medical College for Nationalities. The study was carried out in accordance with the relevant guidelines. Informed consent was signed by each adult participant or guardians on the behalf of the children participants.This study population included 358 OS patients and 402 healthy controls from the Affiliated Hospital of Youjiang Medical College for Nationalities between January 2008 and June 2015. Diagnosis of OS was confirmed by histological examination. Clinical information was extracted from medical records, including age of diagnosis, gender, family history of cancer, tumor location, and metastasis. We excluded those patients with a history of familial cancer from this study. The control subjects were recruited from individuals who underwent routine health examination at the same hospital during the same period. The selection criteria for control subjects were as follows: healthy volunteers without OS, hypertension, and diabetes mellitus; no history of any cancer, cardiovascular diseases, and other inflammatory diseases; no family history of any cancer. The controls were frequency matched to cases in terms of age, gender, and residence area. All subjects were unrelated Han Chinese.IL16, which have been identified to be functional previously3839We selected three SNPs in IL16 rs4778889, rs11556218, and rs4072111. PCR primer sequences, annealing temperature, restriction enzymes used, and length of PCR products were prepared as described previouslyGenomic DNA was extracted from peripheral blood using a commercial kit according to the manufacturer’s manuals . The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay was carried out to genotype Blood samples were obtained from patients with OS and healthy controls. After centrifugation at 1000 g for 10 min, the plasma was stored at −80 °C until analysis. Plasma IL16 concentration was measured by enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions. The minimum detectable dose of the human IL16 ELISA kit was 5 pg/mL. No cross-reactivity with other cytokines was tested. All the samples were analyzed in duplicate with intra-assay coefficients of variation less than 10%.t-test, whereas differences of gender were evaluated using Pearson χ2 test. Hardy-Weinberg equilibrium (HWE) of the three polymorphisms in IL16 was tested using a goodness-of-fit χ2 test. Genotype and allele frequencies between cases and controls were compared using the chi-square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed using unconditional logistic regression based on age and gender adjustment. Differences of IL16 plasma level among cases and controls were compared using Mann-Whitney U test. Statistical significance was set at P < 0.05.All statistical analyses were done using SPSS statistical software package version 13.0 . Differences of age between the study groups were assessed using the Student’s P = 0.23) and gender (P = 0.91). Most of the tumors (72.1%) located on long tubular bones. Only 29.3% patients had tumor metastasis.The characteristics of patients with OS and controls are summarized in IL16 rs4778889, rs11556218, and rs4072111 in OS patients and healthy controls are shown in P > 0.05). The frequencies of rs11556218 TG and TG/GG genotypes were significantly increased in OS patients compared to controls . Similarly, the frequency of rs11556218 G allele was significantly higher in OS cases compared to controls . However, no significant association between rs4778889 and rs4072111 and OS risk was observed (P > 0.05). When stratification analysis was done according to tumor location and metastasis, no significant difference of IL16 genotype and allele frequencies was detected among any subgroup of OS patients (data not shown). No evidence of linkage disequilibrium was observed for the three SNPs .The distributions of P < 0.001). However, there was no significant difference of IL16 level among metastatic patients and non-metastatic patients (P = 0.65). We further compared the correlation between plasma IL16 level and IL16 polymorphisms. We found that patients carrying rs11556218 TG/GG genotypes had higher levels of IL16 than those carrying TT genotype .As shown in IL16 and risk of OS in a Chinese population. The results revealed that rs11556218 was associated with an increased risk of OS in heterozygote comparison, dominant model, and allele comparison. Moreover, rs11556218 TG/GG genotypes corresponded to higher levels of IL16. These findings indicate that rs11556218 may be responsible for the susceptibility to OS.In this hospital-based case-control study, we investigated the association between three common SNPs in IL16, can result in an asparagine to lysine substitution. In 2009, Gao et al. firstly reported that rs11556218 TG genotype was a risk factor for the development of colorectal cancer and gastric cancer31IL16 rs11556218 increasing cancer risk41The rs11556218, located on exon 6 of IL16, is related to gene expression and transcriptional activity3839IL16 rs4778889 CC genotype was associated with an elevated risk of non-cardia gastric cancerIL16 rs4778889 CC genotype was associated with a decreased risk of renal cell carcinomaIL16 rs4778889 was not associated with the risk of colorectal cancer31IL16 rs4778889 polymorphism and OS risk, which was verified by three independent meta-analyses4142The rs4778889, located at position −295 bp in the promoter region of With regard to rs4072111 and cancer risk, contradictory results were also observed26VEGF) gene4445IL6 can reduce VEGF expression and abolish conditional medium-mediated angiogenesis in human OS cellsIL16, produced by CD8+T cells, can stimulate the secretion of tumor-related cytokines, such as TNF-α, IL1β, IL6, and IL1522IL16 polymorphisms in the etiology of OS. Only OS patients were investigated in this study. Therefore, the results cannot be expanded directly to other pediatric cancers until confirmation was made in different cancer types.Some limitations in this study should be discussed. Both cases and controls were enrolled from a single hospital, which we cannot rule out the possibility of selection bias. However, no deviation from HWE was tested in each SNP, suggesting the possibility is minimal. As is known, OS is composed of complex interactions between genetic and environmental factors. In this study, gene-environment interaction cannot be assessed due to lack of available data. Further gene-environment interaction analysis may provide strong evidence of IL16 rs11556218 TG genotype had an elevated risk of OS in the Chinese population. Further well-designed investigations involving in various populations are warranted to verify these results. In the future, further functional analysis of rs11556218 will help us to clarify the potential biological mechanism of OS.In conclusion, we reported for the first time that How to cite this article: Tang, Y.-J. et al. Association of interleukin 16 gene polymorphisms and plasma IL16 level with osteosarcoma risk. Sci. Rep. 6, 34607; doi: 10.1038/srep34607 (2016).