The impact of MMP-1 , MMP-3 Lys45Glu (A/G), MMP-7 -181A/G, and MMP-12 -82A/G variants and plasma MMP levels on obesity and microvascular reactivity in Tunisians.  Our population included 202 nonobese and 168 obese subjects. Anthropometric, biochemical, and microvascular parameters were determined according to standard protocols. PCR-RFLP and ELISA were used to determine the genetic variants and levels of MMPs, respectively. 2): 30 ± 0.51 versus 27.33 ± 0.8, P = 0.004; MMP-3 levels: 7.45 (4.77–11.91) versus 5.21 (3.60–10.21) ng/ml, P = 0.006). The MMP-12 -82G allele was also associated with higher BMI values when compared to subjects carrying the AA genotype . Individuals carrying the MMP-3 45G or MMP-12 -82G variants were also associated with a higher risk for severe forms of obesity . Similarly, the MMP-7 -181G allele was associated with a higher MMP-7 level and an increased risk for morbid obesity when compared to AA genotype carriers (0.32 (0.31–0.60) versus 0.18 (0.17–0.24) ng/ml, P = 0.01; OR = 1.67, P = 0.02, resp.).  The MMP-3 45Glu (G) allele associates with higher anthropometric values and MMP-3 levels compared to AA genotype carriers (BMI (kg/m MMP-3, MMP-7, and MMP-12 polymorphisms associate with obesity risk and its severity. Obesity is a pathological condition that is closely related to genes with environmental modulators, including sedentary life and positive energy balance . It is cUntil now, SNPs in MMP genes have been described to alter their expression . As an eUp to now, only a few studies have determined the impact of MMP variants and plasma MMP levels on obesity-related phenotypes and microvascular reactivity. The goals of the current report were to explore the impact of MMP-1 , MMP-3 Lys45Glu (A/G), MMP-7 -181A/G, and MMP-12 -82A/G SNPs on the development of obesity and its severity among Tunisians. We also determined whether these SNPs affect biochemical and microvascular reactivity, as well as plasma levels of the corresponding proteins.2) while 168 subjects were obese (BMI ≥ 30 kg/m2). Obese individuals were categorized according to the WHO classification as class I with 30 ≤ BMI < 35 kg/m2, class II with 35 ≤ BMI < 40 kg/m2, and class III (morbidly obese) with 40 kg/m2 ≤ BMI [2 were excluded from the study. Diabetic individuals and subjects with a history of cardiovascular disease, smoking habits, malignancy, and renal, thyroid, and liver diseases were also excluded from the study. Other exclusion criteria were subjects using medications that might alter the endothelial or smooth muscle-dependent responses. All participants signed a written informed consent, and the study was approved by the Farhat Hached Hospital Ethics Committee.370 randomly selected Tunisian subjects (181 men and 189 women) with a mean age of 38 years were selected for the current study. 202 individuals were classified as nonobese subjects (18.5 ≤ BMI < 25 kg/mm2 ≤ BMI . Overwei2), waist circumference (WC) (cm), and hip circumference (HC) (cm) were determined according to standard protocols. Waist-to-height ratio (WHtR) and waist-to-hip ratio (WHR) were also measured by WC/height ratio and WC/HC ratio, respectively. The fasting glucose (FG), total cholesterol (TC), and triglycerides (TG) were determined by the corresponding oxidase methods . The immune-inhibition method  was used to measure high-density lipoprotein cholesterol concentration (HDL-C) while LDL-C was determined with the Friedwald formula [For all participants, weight (kg), height (m), BMI (kg/m formula . The senmax) represented the endothelium-dependent response, registered after three cumulative doses of acetylcholine (ACh) (arbitrary unit). The forearm flow response to heating the skin (FSBFRHS) represented the endothelium-independent response registered after the increase in skin temperature (44°C) without ACh infusion (arbitrary unit). Data were also presented as cutaneous vascular conductance (CVC) that represents the cutaneous blood flow (CBF)/mean arterial pressure (MAP) ratio. The MAP was determined as 1/3 SBP + 2/3 DBP. The difference between the peak CVC upon ACh stimulation (after three doses of ACh) and the baseline CVC was considered as the endothelium-dependent response (ΔACh-CVC). The difference between the peak CVC following RHS-induced vasodilation and the baseline was considered as the endothelium-independent response (ΔRHS-CVC) [Endothelial function was determined in all subjects by assessing the forearm microvascular cutaneous vasoreactivity using laser Doppler flowmetry coupled with iontophoresis  . The maxRHS-CVC) .μl) contained 100 ng of genomic DNA, 25 μM of each deoxynucleotide triphosphate , 50 pmol of each primer, 2.5 mM of MgCl2, and 0.5 units of Taq polymerase . Details of primers and PCR-RFLP conditions are reported in DNA was isolated as previously described . AllelicMMP-1 and MMP-3 levels were determined by enzyme-linked immunosorbent assays (ELISA) using commercially available kits . MMP-7 and MMP-12 plasma levels were evaluated by Magnetic Luminex .t-test or the Mann–Whitney U test as appropriate. Comparison of categorical variables including calculation for deviation from the Hardy–Weinberg equilibrium (HWE) was performed using the chi-square (χ2) test. The evaluation of odds ratios (ORs) and 95% confidence intervals (CIs) was carried out using an unconditional logistic regression model adjusted for age and sex. Haplotypes and linkage disequilibrium (LD) were analyzed with SNPstats (http://bioinfo.iconcologia.net/SNPstats). Results were considered statistically significant if the P value <0.05. The Bonferroni correction (Pc) was applied to adjust for multiple comparisons.SPSS® 17.0 software  and GraphPad Prism (version 6.04) were used for statistical analyses. Comparisons of quantitative variables were carried out using a 2 ± 0.44. In this study, we used a BMI cutoff point of 30 kg/m2 for obesity status. As expected, higher BMI and abdominal obesity values  were associated with obesity status. SBP, DBP, MAP, and hs-CRP were increased in obese subjects. As indicated in max as well as the FSBF dose-response to ACh expressed as area under the curve (AUC) was decreased in the obese group (P < 0.001). Basal CVC, as well as peak ACh-CVC and ΔACh-CVC values, was also decreased in obese subjects (P < 0.001). BMI and central adiposity (WC and WHR) were inversely correlated with the FSBFmax response to ACh . Additionally, basal CVC and peak ACh-CVC were inversely associated with BMI  and WC values . The abdominal obesity marker (WHtR) was inversely associated with peak ACh-CVC .Anthropometric, biochemical, and microvascular values are indicated in 2 ± 0.61 versus 28.5 kg/m2 ± 0.63, For the MMP-1 (-519A/G) polymorphism, no significant variation in BMI values was observed in individuals carrying the dominant model (AG+GG) genotype in comparison to AA genotype carriers ; WC ; WHtR , 2 ± 0.49 versus 27.19 kg/m2 ± 0.49, P = 0.04) ; WC ; WHtR , Regarding the MMP-3 Lys45Glu (A/G) SNP, individuals carrying the (AG/GG) genotype showed increased BMI, WC, and WHtR values when compared to AA genotype carriers (BMI (30 kg/m = 0.04) ). HigherP = 0.06). No significant correlation was observed between MMP-1, MMP-3, MMP-7, and MMP-12 polymorphisms, FG, and hs-CRP levels. No significant association was also found between MMP-1 and MMP-7 polymorphisms and TG, TC, LDL-C, and HDL-C levels. Regarding the MMP-3 polymorphism, a higher amount of LDL-C was found in subjects carrying the AG or GG genotypes compared to AA genotype carriers . A significant increase in LDL-C was also found among obese subjects carrying the AG or GG genotypes when compared to AA genotype carriers . Investigation of the MMP-12 (-82A/G) SNP also revealed that individuals carrying the AG or GG genotypes present higher LDL-C levels when compared to AA genotype carriers . Analysis of endothelial function in the sampled population revealed, however, no significant relationships between the MMP-1, MMP-7, MMP-3, and MMP-12 variants and microvascular reactivity parameters (data not shown).The SBP, DBP, and MAP revealed no significant association with MMP-1, MMP-3, and MMP-7 polymorphisms. Regarding the MMP-12 (-82A/G) SNP, there was an increase in DBP among subjects carrying the AG or GG genotypes compared to AA genotype carriers . When subjects were categorized according to severity of obesity, a higher level of MMP-7 was observed in morbidly obese subjects carrying the -181G variant (AG+GG: 0.32 (0.31–0.6), AA: 0.18 (0.17–0.24), P = 0.01, Pc = 0.04).We also determined whether the MMP polymorphisms affect plasma levels of the corresponding proteins. For the MMP-1  SNPs, there was no significant impact on MMP-1 levels in the sampled population  ng/ml, AG+GG: 2 (0.75–4.16) ng/ml; –1607 1G/2G), 1G1G: 2.53 (0.75–4.17) ng/ml, 1G2G+2G2G: 1.66 (0.66–3.81) ng/ml). For the MMP-3 Lys45Glu (A/G) polymorphism, subjects carrying the AG or GG genotypes present higher MMP-3 levels compared to individuals having the AA genotype (7.45 (4.77–11.91) ng/ml versus 5.21 (3.6–10.21) ng/ml, P = 0.03, Pc = 0.24; OR = 1.61, P = 0.02, Pc = 0.08, resp., P = 0.01; OR = 2.4, P = 0.006; OR = 2.04, P = 0.004, resp., P = 0.004, Pc = 0.016). When subjects were categorized according to severity of obesity, the frequency of the Lys45Glu (G) allele was more frequent in nonmorbidly obese (48.58%) compared to nonobese subjects  . For the MMP-12 (-82A/G) SNP, the frequencies of AA, AG, and GG genotypes were 76.4%, 23%, and 0.6% in obese participants and 88.6%, 11.4%, and 0% in nonobese participants. We found a significant association between the AG genotype, the combined genotypes AG+GG, and the development of obesity . The frequencies of A and G alleles were 94.28% and 5.72% in nonobese subjects and 87.88% and 12.12% in obese individuals. The G allele was associated with enhanced obesity when compared to A allele carriers . The G allele was also associated with nonmorbid obesity when compared to the A allele . Similarly, the AG genotype alone or in combination with the GG variant was associated with a higher risk for severe forms of obesity when compared to AA genotype carriers . The G allele was also significantly associated with morbid obesity when compared to the A allele .Analyses were carried out to study the distribution of the different SNPs according to obesity status and severity based on BMI Tables  and 4. F, resp.) . BesidesD′ = 0.42, r = 0.13). Haplotypes follow this order: MMP-1 (-1607 1G/2G), MMP-1 (-519A/G), MMP-7 (-181A/G), MMP-3 Lys45Glu (A/G), MMP-12 (-82A/G). Comparison of the haplotype frequencies between obese and nonobese groups revealed an increased risk for obesity for the haplotype s1G−1607:G−519:G−181:GLys45Glu:A−82 (Supplementary File 1 available online at https://doi.org/10.1155/2017/6198526).We further determined the haplotype frequencies of the different polymorphisms in obese and nonobese subjects. Linkage disequilibrium analysis revealed that MMP-3 and MMP-12 SNPs were in mild linkage disequilibrium . Several studies reported an enhanced expression of MMP-1 in preadipocytes from obese subjects suggesting a role in ECM remodeling in obesity . MMP-1 hMMP-3 (stromelysin I) is a proteoglycanase and a key player in ECM degradation and remodeling . MMP-3 wMMP-7 (matrilysin-1) is another metalloproteinase with an inflammatory regulatory role that displays a proteolytic activity against various kinds of ECM components . Previouin vitro adipogenesis decreases adipocyte expansion by increasing endostatin levels and inhibiting adipose tissue vascularity. Up to now, several SNPs were reported in the MMP-12 locus. Among these, the -82A/G SNP was found to affect both AP-1 binding affinity and MMP-12 promoter activity [MMP-12 is a macrophage metalloelastase that has a role in ECM remodeling processes . A previactivity . A reporactivity  revealedactivity  revealedactivity , 54. WitIn the current report, when individuals were stratified by BMI, the MMP-3 45Glu (G) and MMP7 (-181G) variants were more frequent in individuals with severe forms of obesity. Although further studies are required, these results may highlight that the contribution of MMP-3 and MMP-7 SNPs in the development of the morbid phenotype can be substantially higher than in lower classes of BMI and could, therefore, be valuable markers to predict the risk for the development of extreme classes of obesity. Consistently, previous studies provided evidence of a weak population-related risk for common obesity for several genetic variants. As an example, Villalobos-Comparán et al.  found thThe limitations of this study should be considered. A first limitation is the small sample size of the screened population. Our findings need, therefore, to be replicated in a larger population or an independent severe obesity cohort. A second limitation is the lack of measures of MMP activities in serum, as well as in adipose tissue.MMP-3 Lys45Glu (A/G), MMP-7 (-181A/G), and MMP-12 (-82A/G) variants were associated with obesity and its anthropometric indicators. The current findings also highlight a significant correlation between the MMP-3Lys45Glu (A/G) and MMP-7 (-181A/G) variants and severe forms of obesity among Tunisians. Our results also revealed significant alterations in microvascular reactivity in obese subjects. MMP variants may not, however, be determinant factors of endothelial dysfunction in the studied population.Supplementary file 1. Estimated haplotype frequencies of MMP-1, MMP-3, MMP-7 and MMP-12 polymorphisms in obese and non-obese subjects.


Scientific Reports7:1110; doi:10.1038/s41598-017-01016-8; Article published online 24 April 2017The original version of this Article contained errors. In the Abstract,“The use of SC79, an Akt1/2 inhibitor”now reads:“The use of SC79, an Akt1/2 activator”In the Introduction,“wildlife and animal exposure including fetal growth”now reads:“wildlife and animal exposure including reduced fetal growth”In addition,“inducing Sertoli cell injury and Leydig cell steroidogenic function”now reads:“inducing Sertoli cell injury and disrupting Leydig cell steroidogenic function”Finally,“This mechanistic study thus provides additional insight on the molecular mechanism by which PFOS mediates reproductive”now reads:“This mechanistic study thus provides additional insight on the molecular mechanism by which PFOS causes reproductive”The original version of this Article omitted Xiang Xiao and Wing-yee Lui as corresponding authors. Correspondence and request for materials should also be addressed to X.X. (xxiao@zjams.com) or W.Y.L. (wylui@hku.hk)In the Results section, under the sub-heading ‘PFOS perturbs Sertoli cell TJ-barrier function through changes in the spatial expression of actin binding and regulatory proteins’,“Palladin also no longer stretched across the Sertoli cell cytosol to organize actin filaments into bundles, instead, paladin retracted from cell cytosol and found closer to the cell nuclei .”now reads:“Palladin also no longer stretched across the Sertoli cell cytosol to organize actin filaments into bundles as seen in controls, instead, palladin retracted from cell cytosol and found closer to the cell nuclei following PFOS treatment ”The Results section contains an error in a sub-heading, where:“Rescue of PFOS-induced Sertoli cell TJ-barrier disruption by SC79, an activator of Akt through changes in the re-distribution of BTB-associated proteins.”now reads:“Rescue of PFOS-induced Sertoli cell TJ-barrier disruption by SC79, an activator of Akts through changes in the re-distribution of BTB-associated proteins.”There were also errors in the Figure legends. In Figure 2 legend,“Instead, palladin was retraced from the cell peripheries but concentrated to cell nuclei after PFOS treatment.”now reads:“Instead, palladin was retracted from the cell peripheries but concentrated to cell nuclei after PFOS treatment.”In Figure 3(c) legend6 cells/cm2 for 3 days were pre-treated with 2 μg/ml SC79 for 30 min, and then cells were rinsed and treated with 20 μM PFOS for 24 hr. Thereafter, cells were then fixed and examined by immunofluorescence microscopy. SC79 treatment was found to block PFOSinduced mis-localization of TJ proteins CAR and ZO-1, as well as basal ES proteins N-cadherin and β-catenin, apparently through enhanced endocytosis. BTB-associated proteins at the Sertoli cell-cell interface in control and treated cells were annotated by corresponding white and yellow brackets, respectively”“(C) Sertoli cells cultured at 0.04 × 10now reads:6 cells/cm2 for 3 days were pre-treated with 2 μg/ml SC79 for 30 min, and then cells were rinsed and treated with 20 μM PFOS for 24 hr. Thereafter, cells were then fixed and examined by immunofluorescence microscopy. SC79 treatment was found to block PFOS-induced mis-localization of TJ proteins CAR and ZO-1, as well as basal ES proteins N-cadherin and β-catenin. BTB-associated proteins at the Sertoli cell-cell interface in control and PFOS-treated cells were annotated by corresponding white and yellow brackets, respectively”“(C) Sertoli cells cultured at 0.04 × 10In Figure 4 legend“yellow rectangles in FOS-treated cells”now reads:“yellow rectangles in PFOS-treated cells”In Figure 5 legend, Figure 1A now reads Figure 1BIn Figure 6 legend,“Thereafter, cells were fixed with ice-cold methanol and visualized byimmunofluorescence microscopy.”now reads:“Thereafter, cells were fixed with ice-cold methanol and visualized by immunofluorescence microscopy using corresponding antibodies (Table 1)”In addition, the Acknowledgments section now reads:We wish to thank Dr. Ben Margolis and Dr. Shuling Fan who kindly provided us with some anti-CRB3 antibody to validate some experiments in this report. This work was supported by grants from the National Institutes of Health ; National Natural Science Foundation of China (NSFC) (31371176 to X.X.), Qianjiang Talents Program (QJD1502029 to X.X.), Zhejiang Province Funding ; Hong Kong Research Grants Council (RGC) General Research Fund (GRF) , NSFC/RGC Joint Research Scheme (N_HKU 717/12 to W.M.L.), and University of Hong Kong CRCG Seed Funding (to W.M.L.)The Author Contributions statement now reads:C.Y.C., Y.G. and H.C. designed research; Y.G., H.C. and C.Y.C. performed research; X.X., W.Y.L., W.M.L., D.D.M. and C.Y.C. contributed new reagents/analytic tools; Y.G., H.C. and C.Y.C. analyzed data; Y.G. and C.Y.C. wrote the paper; Y.G., H.C. and C.Y.C. prepared all figures. All authors reviewed the manuscript.There were also errors in References 43 and 76, which now read:in vitro a useful model to study molecular mechanisms in spermatogenesis? Semin Cell Dev Biol59, 141–156, doi:10.1016/j.semcdb.2016.01.003 (2016).43. Li, N., Mruk, D. D., Lee, W. M., Wong, C. K. C. & Cheng, C. Y. Is toxicant-induced Sertoli cell injury et al. SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling. Oncotarget7, 60123–60132, doi:10.18632/oncotarget.11164 (2016).76. Gong, Y. These errors have now been corrected in the HTML and PDF versions of this Article.


Bariatric surgery has emerged as a competitive strategy for obese patients. However, its comparative efficacy against non-surgical treatments remains ill-defined, especially among nonseverely obese crowds. Therefore, we implemented a systematic review and meta-analysis in order for an academic addition to current literatures.Literatures were retrieved from databases of PubMed, Web of Science, EMBASE and Cochrane Library. Randomized trials comparing surgical with non-surgical therapies for obesity were included. A Revised Jadad's Scale and Risk of Bias Summary were employed for methodological assessment. Subgroups analysis, sensitivity analysis and publication bias assessment were respectively performed in order to find out the source of heterogeneity, detect the outcome stability and potential publication bias.P > 0.05). The pooled results of primary endpoints (weight loss and diabetic remission) revealed a significant advantage among surgical patients rather than those receiving non-surgical treatments (P < 0.05). Furthermore, except for certain cardiovascular indicators, bariatric surgery was superior to conventional arms in terms of metabolic secondary parameters (P < 0.05). Additionally, the pooled outcomes were confirmed to be stable by sensitivity analysis. Although Egger's test (P < 0.01) and Begg's test (P<0.05) had reported the presence of publication bias among included studies, “Trim-and-Fill” method verified that the pooled outcomes remained stable.25 randomized trials were eligibly included, totally comprising of 1194 participants. Both groups displayed well comparability concerning baseline parameters (Bariatric surgery is a better therapeutic option for weight loss, irrespective of follow-up duration, surgical techniques and obesity levels. Emerging as a costly burden of global healthcare system, obesity has currently attracted worldwide attentions due to its uncontrollably rising incidence, especially in industrialized countries . EconomiAccording to SAGES  and NICEBariatric surgery, initially reported in 1995 , has beeversus non-surgical interventions is rarely described, especially lacking of a well summarized evidence. Additionally, the clinical value of bariatric surgery for nonseverely obese patients requires further analysis. Hence we performed this systematic review and meta-analysis in order to comprehensively make comparisons between both strategies, aiming to provide novel evidences for future guidelines.Nevertheless, long-term (3-year or more) efficacy of surgical The preliminary 1076 entries were rigorously screened to generate 25 eligible studies for pooling analysis, with a total amount of 1194 participants and individually ranging from 16 to 150 Figure . Merely According to Revised Jadad's Scale, 13 trials were appraised as high-quality in methodology, while DSS, Heindorff 1997 and Mingrone 2002 were identified as low-quality investigations  , 2-year (P < 0.00001) or long-term (3 years or more) follow-up duration (P < 0.00001) , Roux-en-Y gastric bypass (P < 0.00001), laparoscopic adjustable gastric banding (P < 0.00001) and biliopancreatic diversion (P < 0.00001)  and severe obesity , metabolic surgery was a more effective tool for losing weight against non-surgical treatments   .P < 0.0001), 2-year (P = 0.004) and long-term follow-up (P < 0.0001)  .P < 0.00001), Roux-en-Y gastric bypass (P < 0.00001), laparoscopic adjustable gastric banding (P = 0.0008) and biliopancreatic diversion (P = 0.001)  .P = 0.0004) and severely obese (P < 0.0001) sufferers significantly benefited from surgical remedies, compared with non-surgical patients   .P < 0.00001), 2-year (P < 0.00001) or long-range follow-up (P = 0.006), bariatric surgery was far more efficient to eliminate excessive weight than non-surgical interventions (Regardless of 1-year (ventions .P < 0.00001), Roux-en-Y gastric bypass (P < 0.00001), laparoscopic adjustable gastric banding (P = 0.0008) and biliopancreatic diversion (P < 0.00001) was performed  and severe obesity (P < 0.00001)  .P < 0.00001)  .P = 0.0005), 2-year (P = 0.0003) or long-term follow-up (P = 0.02), surgical management was significantly effective in decreasing fasting glucose compared to non-surgical interventions (Regardless of 1-year (ventions .P = 0.0005), Roux-en-Y gastric bypass (P < 0.00001) and laparoscopic adjustable gastric banding (P < 0.0001) fell behind in fasting glucose reduction against conserved therapeutics, except for biliopancreatic diversion, which was statistically equivalent to contrastive group (P = 0.05) (None of sleeve gastrectomy ( = 0.05) .P = 0.001) and severely obese (P < 0.00001) patients obtained greater downregulation on fasting glucose following the surgical interventions   .P < 0.00001), 2-year (P = 0.02) and long-term follow-up (P < 0.00001), rather than those with conservative interventions , Roux-en-Y gastric bypass (P < 0.00001), laparoscopic adjustable gastric banding (P = 0.002) and biliopancreatic diversion (P = 0.03), bariatric surgery was significantly superior to non-surgical approaches in terms of reducing elevated glycated hemoglobin (Irrespective of sleeve gastrectomy (moglobin .P < 0.00001) and severely obese patients (P < 0.00001)  .P < 0.00001)  .P < 0.00001), 2-year (P < 0.00001) and long-term follow-up (P < 0.00001), patients that were surgically treated obtained larger decline on waist circumference than those were conventionally cured (Among the three subgroups of 1-year (ly cured .P < 0.0001), Roux-en-Y gastric bypass (P < 0.00001), laparoscopic adjustable gastric banding (P < 0.00001) and biliopancreatic diversion (P < 0.00001)  .P < 0.00001) and severely obese sufferers (P < 0.00001), more reduction on waist circumference was observed following surgical management in contrast to traditional treatments (Based on the pooled outcomes featuring nonseverely (eatments .P < 0.00001)  .P = 0.30), while bariatric surgery was a superior option among sufferers with 1-year (P = 0.001) and long-range follow-up (P = 0.02)  .P = 0.36), laparoscopic adjustable gastric banding (P = 0.36) and biliopancreatic diversion (P = 0.90) was statistically comparable with non-surgical strategies in terms of reduction on systolic pressure, except for the dominant efficacy of Roux-en-Y gastric bypass (P = 0.007) (Bariatric surgery featuring sleeve gastrectomy (= 0.007) .P < 0.00001) and severely obese patients (P = 0.41) respectively, in terms of reduction on systolic pressure   .P = 0.26) and 2-year (P = 0.55). However, surgical interventions were inferior to non-invasive remedies amid patients with long-term follow-up (P = 0.03)  .P = 0.03) were in a significantly superior status, the remaining techniques were statistically comparable to conservative regimens concerning the decrease on diastolic pressure, inclusive of sleeve gastrectomy (P = 0.67), laparoscopic adjustable gastric banding (P = 0.57) and biliopancreatic diversion (P = 0.52) (Although Roux-en-Y gastric bypass ( = 0.52) .P = 0.23) and severe obesity (P = 0.73) (Patients undergoing both interventions exhibited comparable efficacy on diastolic pressure reduction regardless of nonsevere obesity ( = 0.73) .P < 0.00001)  .P < 0.0001) and 2-year (P < 0.0001) follow-up had a greater decline on triglycerides level than those being conventionally treated, except for the comparable efficacy among patients with long-term follow-up (P = 0.06)  .P = 0.0005) and laparoscopic adjustable gastric banding (P = 0.004) exerted a more evident influence among enrolled patients, while sleeve gastrectomy (P = 0.08) and biliopancreatic diversion (P = 0.14) was therapeutically comparable with conventional remedies  and severe obesity (P < 0.0001)  .P = 0.13)  .P = 0.18) and 2-year (P = 0.07), there was no therapeutic difference between both interventions. However, patients with long-term follow-up achieved more reduction on total cholesterol following non-surgical managements, in contrast to bariatric surgery (P = 0.03) (Among obese sufferers being followed up for 1-year ( = 0.03) .P = 0.004), patients receiving bariatric surgery gained comparable efficacy of total cholesterol reduction against non-surgical enrollees, including sleeve gastrectomy (P = 0.67), Roux-en-Y gastric bypass (P = 0.78) and laparoscopic adjustable gastric banding (P = 0.62) (Besides the preponderant role of biliopancreatic diversion ( = 0.62) .P = 0.01), while nonseverely obese counterparts reported no significant difference between surgical and non-surgical interventions (P = 0.76)  .P < 0.00001)  .P < 0.00001), 2-year (P = 0.0005) and long-term follow-up (P = 0.001), metabolic surgery led to more increase on high density lipoprotein among included patients, compared to non-surgical recipients (Despite of 1-year (cipients .P = 0.003) and Roux-en-Y gastric bypass (P < 0.00001) displayed a higher increase on high density lipoprotein. However, the increase on high density lipoprotein was identically obtained between non-surgical treatments and bariatric surgery of laparoscopic adjustable gastric banding (P = 0.19) and biliopancreatic diversion (P = 0.27) (The recipients of sleeve gastrectomy ( = 0.27) .P < 0.00001) or severe obesity (P < 0.00001), bariatric surgery was a more capable pattern of elevating high density lipoprotein than traditional modes   .P = 0.24) and 2-year follow-up (P = 0.14), recipients of conventional strategies exceled those with long-term postoperative follow-up duration, with regard to the reduction on low density lipoprotein (P < 0.00001)  .P = 0.34), Roux-en-Y gastric bypass (P = 0.71) and laparoscopic adjustable gastric banding (P = 0.46), it was mathematically comparable between surgical and non-surgical patients regarding the decrease on low density lipoprotein. Nevertheless, patients undergoing biliopancreatic diversion had an advantage over those were conventionally cured (P < 0.00001) (Based on subgroup statistics of sleeve gastrectomy (0.00001) .P = 0.76) and severely obese patients (P = 0.09)  .Firstly, by interchanging random-effects and fixed-effects models, the overall as well as subgroup outcomes of primary endpoints (weight loss and diabetic remission) were confirmed to be statistically stable Table .Secondly, by eliminating four low-quality trials of DSS, Heindorff 1997, Mingrone 2002 along with O'Brien 2013, the stability of primary endpoints were numerically verified, irrespective of overall or subgroup analysis  and 6 added for diabetic remission   and diabetic remission , Egger's test and Begg's test consistently confirmed the presence of publication bias among the included studies . However 0.0001) .Although Golomb et al had retrospectively doubted the dominant role of bariatric surgery at 5-year follow-up , its conIt is empirically regarded that compared to severe obesity, patients suffering from nonsevere obesity manifest with better general conditions which makes it more economical to be conventionally cured. However, deriving from our pooled evidence, nonseverely obese patients should rather receive bariatric surgery than conservative managements, which is similar to those featuring morbid obesity and is a vital addition to current literatures.Adolescent obesity and overweight crowds have been targeted as further research highlights of bariatric surgery. Uncontrollable weight increase has severer impacts on pubescents than adults, simultaneously damaging their sexual development and intelligence growth . O'BrienNotwithstanding the statistical outcomes are of great strength in both methodology and statistics, there are still some limitations within. First of all, the internal heterogeneity is still in a considerable level despite the subgroup analyses have been additionally performed. Difference from chemical examination methods, surgical manipulation techniques and diversified non-surgical interventions (life-style and medications) may contribute to inerasably internal heterogeneity. More accurate grouping on different confounding factors may benefit the source seeking of heterogeneity. Secondly, raw data of life-quality and survival prognostication are inadequate for meta-analyzing, making it less valuable in terms of comparisons on life expectancy. Moreover, mutual comparison of financial burdens is still lacking, blockading a more comprehensive appraisal of both strategies.Taken together, despite of the shortcomings of surgical therapeutics  , bariatrIn line with the PRISMA Checklist  and CochIn order to guarantee the integrity of literature retrieval, databases of PubMed, Web of Science, EMBASE and Cochrane Library were electronically searched with a search term “bariatric randomized OR bariatric randomised OR obesity surgery”. Both abstracts and full-texts were elaborately examined for fear of omission or ineligible inclusion. Additionally, citation lists of formerly published meta-analysis were screened as well.versus non-surgical interventions among obese patients (body mass index > 30); 3. Adequate raw data of interested endpoints including metabolic and cardiovascular indicators;Studies were eligibly included because of the following criteria: 1. English-written and officially published trials until December 2015; 2. Randomized controlled trials evaluating the comparative efficacy of surgical Studies were eventually eliminated due to the following reasons: 1. Duplicated publications; 2. Inadequate sample-size (< 10); 3. Lack of follow-up materials;With regard to baseline, primary  and secondary parameters , a well-prepared electronic form was designed to facilitate date extraction from tables, figures, text contents and supplementary information within the eligible trials. Overlapped data deriving from a single registered trial was mathematically combined to prevent repetitive counting. All continuous variables were rounded to one decimal place.Firstly, a Revised Jadad's Scale  was emplSecondly, as an addition to Revised Jadad's Scale, Review Manager 5.3 assisted us to summarize the risk of bias amid the qualified literatures. The symbol of green, yellow and red represented low risk of bias, unclear risk of bias and high risk of bias respectively, in terms of seven constituted categories . The higher proportion that green occupies, the lower risk of bias there is .2) [2 < 25%. Otherwise a random-effects model was preferred under the remaining circumstances [via eliminating low-quality trials, exclusion of controversial studies and comparing the outcome variation between fixed-effects and random-effects models. Moreover, the incorporated outcomes were additionally classified into various subgroups for purpose of more specific and instructive discoveries. With aid of STATA 12.0, publication bias was numerically examined by Begg's test and Egger's test [P < 0.05.Review Manager 5.3 was employed as a statistical platform for our quantitative analysis. The effect-sizes of dichotomous and continuous variable were calculated by models of odds ratio and weighted mean difference respectively, along with 95% confidence interval. If the source data on endpoints were not offered explicitly, median was statistically regarded as mean while standard deviation was derived from range, interquartile range or 95% confidence interval as appropriate , 49. A d2) . Revealimstances , in order's test . A “Trim


SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants.Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative  Cytosine DNA methylation, a dominating epigenetic modification mechanism, plays vital roles in gene expression regulation, transposon silencing, gene imprinting, and X chromosome inactivation . It is wde novo through distinct mechanisms (De novo methylation is mainly established by RNA-directed DNA methylation (RdDM), in which domains-rearranged methyltransferases (DRMs) are guided to target locus to direct methylation of all three sequence contexts via 24-nucleotide short interfering RNAs , another CMT subfamily member . Arabidopsis AtC5-MTase proteins were blast-analyzed against the S. miltiorrhiza 99-3 whole genome sequence using tBLASTn algorithm  using the BLASTx and BLASTn program, respectively (http://www.ncbi.nlm.nih.gov/blast/). Obtained raw genomic sequences, open reading frame (ORF) sequences and deduced protein sequences were listed in Sequences of 11 lgorithm . S. milthttp://gsds.cbi.pku.edu.cn/index.php) using the coding sequences and the corresponding genomic sequences as inputs. Protein amino acid number, molecular weight (Mw) and theoretical isoelectric point (pI) were analyzed using the EXPASY PROTOPARAM tool (http://www.expasy.org/tools/protparam.html). Multiple sequence alignment was carried out using ClustalW  plants grown in a field nursery at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, were collected in August and stored in liquid nitrogen until use. Total RNA was extracted from three biological replicates for each tissue using the Quick RNA isolation kit . Each biological replicate represents independent single plant. RNA integrity and quantity were analyzed using agarose gel and NanoDrop 2000C Spectrophotometer , respectively. Total RNA was reverse-transcribed using the PrimeScript™  RT reagent kit . qRT-PCR primers listed in http://frodo.wi.mit.edu/primer3/). qRT-PCR analysis was conducted in triplicate on the CFX96™ real-time PCR detection system  for each biological replicate using SYBR premix Ex Taq™ kit  as described  (S. miltiorrhiza hairy roots treated with yeast extract (YE) and methyl jasmonate (MeJA) were downloaded from SRA database under the accession number SRP111399  were downloaded from SRA database under the accession number SRX1423774  . RNA-seqRP111399 . RNA-seqX1423774 . DiffereX1423774 . Heat maX1423774 .Arabidopsis cytosine DNA methyltransferase proteins against the whole S. miltiorrhiza 99-3 genome sequence resulted in the identification of eight SmC5-MTase genes. The number of SmC5-MTase genes in S. miltiorrhiza is comparable with that in other plants, such as Arabidopsis  varies from 4.89 (SmCMT3) to 8.03 (SmCMT2a) and the molecular weight (Mw) varies from 43.7 kDa (SmDNMT2) to 189.4 kDa (SmMET1)  . These sterparts . Gene stse genes . The undS. miltiorrhiza and Arabidopsis. Four highly conserved motifs, including I, IV, VI and X  and methyl jasmonate (MeJA) are effective elicitors for tanshinone and phenolic acid production in iorrhiza . The resry roots . The trary roots . CompareSmC5-MTases in plant defense, RNA-seq data from SA-treated S. miltiorrhiza cell cultures was analyzed -dependent immunity . In ordeanalyzed . The trareatment , suggestS. miltiorrhiza. Understanding the regulatory mechanism of their biosynthesis and metabolism is important for S. miltiorrhiza quality improvement. Most of the studies about S. miltiorrhiza gene function concentrate on key enzyme genes and transcription factors associated with secondary metabolism  through regulating SA-dependent defense genes in trans or cis (S. miltiorrhiza.Recent studies suggest that DNA methylation is involved in regulation of secondary metabolism . Consistnd xylem . Althougreatment . Dynamiced by SA . Analysis or cis . SimilarSmC5-MTase genes were identified using whole genome sequence and RNA-seq data from S. miltiorrhiza. Based on phylogenetic tree and conserved domain distribution, SmC5-MTase genes were divided into four subfamilies, including SmMET, SmCMT, SmDRM and SmDNMT2. Comparative analysis of SmC5-MTases and AtC5-MTases revealed the conservation and divergence of plant C5-MTases. The transcript abundance analysis of SmC5-MTase genes suggests functional importance of SmC5-MTases in secondary metabolism and stress response in S. miltiorrhiza. The results provide useful information for understanding the role of DNA methylation in medicinal plant development and bioactive compound biosynthesis.Eight 10.7717/peerj.4461/supp-1Table S1Click here for additional data file.10.7717/peerj.4461/supp-2Table S2Click here for additional data file.10.7717/peerj.4461/supp-3Table S3Click here for additional data file.10.7717/peerj.4461/supp-4Figure S1Click here for additional data file.10.7717/peerj.4461/supp-5Figure S2Click here for additional data file.10.7717/peerj.4461/supp-6Figure S3Click here for additional data file.10.7717/peerj.4461/supp-7Figure S4Click here for additional data file.10.7717/peerj.4461/supp-8Figure S5Click here for additional data file.10.7717/peerj.4461/supp-9Data S1Raw data.Click here for additional data file.


Protective effects of boswellic acid (BA) against acetaminophen (APAP)-induced hepatotoxicity in Balb/ cA mice were examined. BA, at 0.05 or 0.1%, was supplied for 4 weeks. Acute liver injury was induced by APAP treatment. Results showed that BA intake increased hepatic BA bioavailability. APAP treatment decreased glutathione (GSH) level, increased reactive oxygen species (ROS) and oxidized glutathione (GSSG) production; and lowered activity and protein expression of glutathione reductase (GR) and heme oxygenase (HO)-1 in liver. BA intake at both doses alleviated subsequent APAP-induced oxidative stress by retaining GSH content, decreasing ROS and GSSG formations, reserving activity and expression of GR and HO-1 in liver, and lowering hepatic cytochrome P450 2E1 activity and expression. APAP treatment enhanced hepatic levels of interleukin-6, tumor necrosis factor-alpha and monocyte chemoattractant protein-1. BA pre-intake diminished APAP-induced release of those inflammatory cytokines and chemokines. APAP up-regulated hepatic protein expression of toll-like receptor (TLR)-3, TLR-4, MyD88, nuclear factor kappa B (NF-κB) p50, NF-κB p65 and JNK. BA pre-intake at both doses suppressed the expression of NF-κB p65 and p-JNK, and only at 0.1% down-regulated hepatic TLR-3, TLR-4 and MyD88 expression. APAP led to obvious foci of inflammatory cell infiltration in liver, determined by H&E stain. BA intake at both doses attenuated hepatic inflammatory infiltration. These findings support that boswellic acid is a potent hepato-protective agent. It is metabolized by hepatic cytochrome P450 system, especially CYP2E1, which leads to the overproduction of reactive free radicals and n-acetyl-p-benzoqui- noneimine (NAPQI) , 4. In aet al. [et al. [Toll-like receptors (TLRs) are pattern recognition receptors mainly responsible for immunomodulation. The activation of TLR-3 and TLR-4 has been implicated in APAP-induced hepatic damage because both TLRs could stimulate the production of adaptor proteins including MYD88 and nuclear factor kappa B (NF-κB), which in turn promote the generation of oxidants and inflammatory cytokines and chemokines , 8. Furtet al.  reportedBoswellia species including Boswellia carteri, Boswellia serrata and Boswellia sacra [via its anti-oxidative and anti-inflammatory activities [et al. [Boswellic acid BA, , a pentaia sacra . It is rtivities . The stu [et al.  revealed [et al. . Thus, iκB, TLR-3 and TLR-4 was evaluated. Furthermore, histological analysis was processed in order to provide more solid evidence to support the benefit of using BA as a hepatic protective agent.The purpose of this animal study was to examine the protective effects and action modes of BA at various doses upon liver of acetaminophen treated mice. The influence of this compound upon GSH, ROS and cytokines variation, CYP2E1 activity and protein expression of associated factors such as NF-2.2.1.Boswellic acid  was synthesized and provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China. APAP (98%) was purchased from Sigma Chemical Co. .2.2.Five-to six-week-old male Balb/cA mice were obtained from National Laboratory Animal Center . Mice were housed on a 12-h light-12-h dark schedule, and fed with water and mouse standard diet . Use of the mice was reviewed and approved by the China Medical University animal care committee (104-305).2.3.BA at 0.05 or 0.1 g was mixed with 99.95 or 99.9 g powder diet to prepare 0.05 and 0.1% BA diets. Mice were divided into five groups: normal group ; BA group (0.1% BA diet), APAP group ; BA-low-APAP group (0.05% BA diet plus APAP treatment); BA-high-APAP group (0.1% BA diet plus APAP treatment). After 4 weeks supplement, normal and BA groups were sacrificed, and the other three groups were treated by a single intraperitoneal injection of APAP (400 mg/kg body weight). APAP was dissolved in phosphate-buffered saline (PBS). Mice were killed with carbon dioxide after 24 h. The mortality of mice due to APAP injection was zero. After sacrificed, liver from each mouse was collected and weighted. There was no mice die before sacrificed. Blood was also collected, and serum was separated from erythrocyte immediately. Liver tissue, 0.2 g, was homogenized on ice in 2 ml phosphate buffer (pH 7.2), and the filtrate was collected. The protein concentration of serum and liver filtrate was determined by a commercial assay kit  with bovine serum albumin as standard.2.4.et al. [g for 5 min at 4°C. Agilent 1100 series HPLC system  equipped with a C18 reversed-phase column  was used. BA quantification was performed using the external standard method. The limit of detection was 0.1 mg/g tissue. The relative standard deviations of precision and accuracy were less than 5.2%.The HPLC method of Lozano-Mena et al.  was used2.5.Serum activities of ALT and AST were determined by using commercial assay kits . CRP level (mg/l) was measured by an ELISA kit .2.6.Liver tissue was homogenized with cold PBS containing 0.05% Tween 20 and 1 mM EDTA. After centrifuging, supernatants were used for measurements. GSH and GSSG concentrations (nmol/mg protein) in liver were determined by commercial colorimetric GSH and GSSG assay kits . ROS level was quantified by using 2’, 7’-dichlorofluores- cein diacetate. Fluorescence value was measured by using a fluorescence microplate reader at excitation and emission wavelengths of 485 and 530 nm, respectively. Relative fluorescence unit (RFU) was the difference in fluorescence values obtained at time 0 and 5 min. Results are expressed as RFU/mg protein. The activity (U/mg protein) of GPX and GR was assayed by using commercial kits purchased from EMD Biosciences Co. .2.7.Hepatic levels of IL-6, TNF-alpha and MCP-1 were measured by using cytoscreen immunoassay kits . The sensitivity of assay with the detection limit was 5 pg/ml for IL-6, and 10 pg/ml for TNF-alpha and MCP-1.2.8.The activity of CYP2E1 in freshly prepared liver microsome was estimated by colorimetrically measuring the formation of 4-nitrocatechol, a product from p-nitrophenol hydroxylation catalyzed specifically by CYP2E1. The protein concentration of CYP2E1 was measured by ELISA, and a rabbit anti-CYP2E1 antibody  was used for detection. The formed 4-nitrocatechol was expressed as nmol/mg protein.2.9.Hepatic tissue, 40 mg, was homogenized in buffer containing protease-inhibitor cocktail purchased from Sigma-Aldrich Chemical Co.  and 0.5% Triton X-100. Homogenate was then mixed with buffer , and followed by boiling for 5 min. Protein sample at 40 was electrophoresed on 10% SDS-polyacrylamide gel, and further transferred onto nitrocellulose membranes  for 1 h. After blocking with a protein solution containing 5% skim milk for 1 h, membranes were treated with monoclonal antibody  against heme oxygenase (HO)-1, CYP2E1 (1:1000), NF-κB p50, NF-κB p65, JNK (1:500), TLR-3, TLR-4, MyD88 (1:2000) at 4°C overnight, and followed by incubating with horseradish peroxidase-conjugated antibody at room temperature for 3.5 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and the bands were quantified by an ATTO image analyzer .2.10.Partial liver tissue from each mouse was fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Paraffin section at 5 mm thickness was cut and processed with hematoxylin-eosin (H&E) stain, and followed by examining under a light microscope for histological analysis. The severity of hepatic inflammatory injury was assayed according to the Ishak scoring system . The inj2.11.P < 0.05.The effect of each treatment was analyzed from 10 mice (n = 10) in each group. Data were reported as means ± standard deviation (SD), and subjected to analysis of variance. Differences among means were determined by the Least Significance Difference Test with significance defined at 3.3.1.. The intake of BA at both doses alleviated subsequent APAP-induced elevation of ALT, AST and CRP levels in serum (P < 0.05).BA intake at 0.1% increased hepatic BA content, and did not affect body weight, feed intake, water intake and liver weight 3.2.P < 0.05); but BA pre-intake attenuated APAP-induced GSH depletion and decreased hepatic GSSG and ROS production (P < 0.05). APAP raised GPX activity and reduced GR activity in liver (P < 0.05). BA intake failed to change hepatic GPX activity (P > 0.05); but significantly maintained hepatic GR activity (P < 0.05). As shown in P < 0.05). BA intake did not affect GPX protein expression (P > 0.05); but significantly retained protein expression of GR and HO-1 in liver (P < 0.05). APAP treatment enhanced CYP2E1 activity and expression (P < 0.05); however, BA intake significantly suppressed subsequent APAP-induced elevation of CYP2E1 activity and protein expression (P < 0.05). APAP treatment also significantly increased hepatic release of TNF-alpha, IL-6 and MCP-1 (P < 0.05). BA intake lowered APAP-induced hepatic production of these cytokines (P < 0.05).As shown in 3.3.P < 0.05). BA pre-intake at both doses down-regulated the expression of NF-κB p65 and p-JNK (P < 0.05), and only at high dose suppressed hepatic TLR-3, TLR-4 and MyD88 expression (P < 0.05).APAP up-regulated hepatic protein expression of TLR-3, TLR-4, MyD88, NF-κB p50, NF-κB p615, JNK and p-JNK (κB p50 expression (P > 0.05).BA did not alter NF-3.4.P < 0.05). BA intake at 0.05 and 0.1% decreased hepatic infiltration by inflammatory cells and lowered Ishak inflammation scores (P < 0.05), in which 0.1% BA treatment exhibited greater anti-inflammatory effects than 0.05% BA (P < 0.05).Histological data revealed that BA intake at 0.1% only (without APAP) did not cause inflammatory stress and showed similar Ishak inflammation score as normal groups . APAP tr4.Boswellia species, increased its deposit in liver and protected liver against subsequent APAP induced oxidative and inflammatory injury. Besides increasing GSH retention, we found that BA effectively decreased CYP2E1 activity and expression, lowered ROS, TNF-alpha and MCP-1 production, as well as suppressed HO-1, TLR-3, TLR-4, NF-κB p65 and p-JNK expression. In addition, our histological results indicated that BA improved diffuse ballooning degeneration and lobular inflammation caused by APAP These findings support that BA is a potent hepatic protective agent.Our present study revealed that the pre-intake of BA, an active compound of APAP at high dose led to GSH depletion and GSSG accumulation . Our datvia its anti-oxidative activities, which definitely led to less stimulation for hepatic NF-κB activation and JNK phosphorylation. Our western blot data regarding hepatic protein expression of NF-κB p65 and p-JNK agreed that BA intake at both doses limited the activation of these two signal pathways. Consequently, it is reasonable to observe lower hepatic levels of IL-6, TNF-alpha and MCP-1 in BA treated mice. The decrease in serum levels of ALT, AST and CRP in BA treated mice also agreed that BA intake declined APAP caused liver inflammatory stress. In addition, our histological data further supported that BA pre-intake effectively improved hepatic inflammatory injury.APAP overdose activated NF-κB and JNK pathways through stimulating ROS generation , 23. Theet al. [et al. [in vivo anti-inflammatory data of BA, and extended BA’s mediating activity to TLRs and MyD88.TLRs play crucial roles in the pathological progression of inflammatory liver diseases because TLRs recognize endogenous damage-associated molecular patterns during inflammatory reactions . TLR-3 iet al.  reported [et al.  reportedBA is naturally present in many edible plants including vegetables and herbs , 30. The5.via maintaining hepatic GSH content, retaining activity and expression of GR and HO-1, decreasing inflammatory cytokines and suppressing protein expression of CYP2E1, NF-κB p65, JNK, TLR-3 and TLR-4. These findings support that boswellic acid was a potent hepatic protective agent.Boswellic acid pre-intake protected mice liver against subsequent acetaminophen-induced oxidative and inflammatory injury All authors claimed that no Conflict of Interest.


Pochazia shantungensis adults and nymphs, newly recorded pests, were evaluated. The LC50 values of Thymus vulgaris, Ruta graveolens, Citrus aurantium, Leptospermum petersonii and Achillea millefolium oils were recorded as 57.48, 84.44, 92.58, 113.26 and 125.78 mg/L, respectively, against P. shantungensis nymphs using the leaf dipping bioassay, and 75.80, 109.86, 113.26, 145.06 and 153.74 mg/L, respectively, against P. shantungensis adults using the spray bioassay method. Regarding volatile components identified in T. vulgaris oil, the LC50 values of carvacrol and thymol using the leaf dipping bioassay against P. shantungensis nymphs were 56.74 and 28.52 mg/L, respectively. The insecticidal action of T. vulgaris oil against P. shantungensis could be attributed to carvacrol and thymol. Based on the structure-toxicity relationship between thymol analogs and insecticidal toxicities against P. shantungensis nymphs similar to the LC50 values against P. shantungensis adults, the LC50 values of thymol, carvacrol, citral, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were 28.52, 56.74 and 89.12, 71.41, 82.49, and 111.28 mg/L, respectively. These results indicate that the insecticidal mode of action of thymol analogs may be largely attributed to the methyl functional group. Thymol analogues have promising potential as first-choice insecticides against P. shantungensis adults and nymphs.The insecticidal toxicities of five essential oils against  Pochazia shantungensis Chou & Lu. is a hemipterous insect that belongs to the Ricaniidae insect family, which includes about 400 described species in over 40 generaP. shantungensis is a newly recorded pest that is economically devastating for various trees including apple, blueberry, peach and persimmon, mainly in Wanjugun, KoreaP. shantungensis was first recorded in Chungcheongnamdo in 2010P. shantungensis has subsequently occurred sporadically in several other provinces across Korea2P. shantungensis adults and nymphs, despite the urgent demandP. shantungensis.Cynanchum paniculatum oilMentha piperita oilMentha spicata oil3789Plant oil is a very complex mixture that can contain approximately 30–65 constituents at various concentrations35Thymus vulgaris oil-derived constituents against P. shantungensis. Therefore, the aims of the present study were first to investigate the insecticidal properties of T. vulgaris oil-derived components against P. shantungensis adults and nymphs, and then to determine the structure-activity relationship between thymol analogs and insecticidal toxicities.So for no report has been received about the insecticidal toxicities of P. shantungensis adults and nymphs. Essential oils of Achillea millefolium flowers, Citrus aurantium fruits, Leptospermum petersonii leaves, Ruta graveolens leaves and T. vulgaris leaves were analyzed  of P. shantungensis adults and nymphs. Differences in the insecticidal toxicities of plant-derived oils may be explained on the basis of species-specific responses to plant species, phytochemicals, and the weight and size of P. shantungensis adults and nymphsThis study was undertaken within the framework of a more general study involving the natural products for insecticidal toxicities against analyzed . The yieexposure . From thP. shantungensis adults and nymphs, the components of A. millefolium, C. aurantium, L. petersonii, R. graveolens, and T. vulgaris oils were investigated by GC-MS analysis. The identified components, together with the percentages present in the essential oils are displayed in A. millefolium oil; limonene (87.75%), citral (3.21%), limonene oxide (2.29%), and (−)-carveol (1.75%) in C. aurantium oil; citral (48.12%), β-citronellal (19.50%), isopulegol (8.14%), geraniol (4.64%), and linalool (3.33%) in L. petersonii oil, and thymol (23.34%), undecyl trichloroacetate (18.52%), methyltridecyl pentanoate (16.18%), and 2,2-dimethylpropanoic acid (7.03%), 2-acetoxytetradecane (6.98%), palmitic acid (5.07%), and methyl linolenate (4.78%) in R. graveolens oil. The major components in T. vulgaris oil were thymol (40.04%), ρ-cymene (29.97%), γ-terpinene (8.17%), linalool (4.99%), terpinolene (3.26%), α-pinene (2.84%), β-caryophyllene (2.50%), carvacrol (2.45%), limonene (1.25%), α-phellandrene (1.20%), myrcene (0.91%), camphene (0.89%), and caryophyllene oxide (0.21%). Together, thymol, ρ-cymene and γ-terpinene made up 78.18% of T. vulgaris oil. The volatile components consisted of 8 monoterpene hydrocarbons , 1 monoterpene alcohol , 2 monoterpene phenols (carvacrol and thymol) and 2 sesquiterpene hydrocarbons (βT. vulgaris oil were borneol (0.98%), camphene (0.60%), carvacrol (2.81%), β-caryophyllene (2.39%), 1,8-cineol (0.96%), p-cymene (25.2%), myrcene (1.93%), linalool (2.86%), α-pinene (1.16%), 1-octen-3-ol (1.19%), α-terpinene (1.02%), γ-terpinene (6.37%), α-thujene (1.50%) and thymol (42.27%). The fact that the essential oil of T. vulgaris leaves dried more at 45 °C than at 30 °C can be explained mainly by the increase of thymol by 8% and carvacrol by 12%, while the quantity of γ-terpinene was decreased by 4.9%T. vulgaris were affected by the environmental conditions, including harvest time, genotype, storage period, handling method and intraspecific variability, and the experimental conditions, which included the extraction method, extracted plant parts, and plant tissue drying temperatureTo further explore the insecticidal toxicities of the five essential oils against oxide 2.2%, and  derived from the five essential oils were evaluated using spray and leaf dipping bioassays against P. shantungensis adults and nymphs -carveol, geraniol, and bornyl acetate) did not exhibit any insecticidal toxicity against P. shantungensis adults and nymphs (data not shown). The insecticidal toxicities of the essential oils appear to be connected to their chemical composition. The insecticidal toxicities of T. vulgaris and R. graveolens oils could be due to the existence of thymol and carvacrol, which exhibited the greatest insecticidal toxicities. The essential oils of C. aurantium and L. petersonii contain citral, which showed insecticidal toxicities against P. shantungensis adults and nymphs, however its toxicity was weaker than thymol and carvacrol. Furthermore, P. shantungensis nymphs were more susceptible to T. vulgaris oil, carvacrol and thymol, when compared to P. shantungensis adults -carveol, (+)-carvone, β-caryophyllene, caryophyllene oxide, β-citronellal, citral, 1,8-cineole, d nymphs . Based os adults . In a prP. shantungensis adults and nymphs, thymol, carvacrol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were selected as thymol analogs for testing , decyl chloroformate (97%), dodecanoic acid (98%), β-farnesene (90%), geranyl acetate (97%), geraniol (98%), isopulegol (98%), linalool (97%), limonene (97%), limonene oxide (97%), methyl linolenate (99%), myrcene (90%), myristic acid (99%), palmitic acid (99%), α-phellandrene (85%), α-pinene (98%), pivalic acid (99%), sabinene (75%), α-terpineol (90%), γ-terpinene (97%), 4-terpineol (95%), terpinolene (90%), and tymol (99%) were obtained from Sigma-Aldrich . Achillea millefolium L. flowers, Citrus aurantium L. fruits, Leptospermum petersonii F. M. Bailey leaves, Ruta graveolens L. leaves, and Thymus vulgaris L. leaves were collected from a local store in Chonju, Korea. Sample specimens were authenticated by Jeongmoon Kim at Chonbuk National University, Korea. Essential oils of the five plants were obtained by steam distillation extraction, and finally dried over Na2SO4 to extract the pure essential oils (Bornyl acetate (99%), camphene (95%), camphor (96%), carvarol (98%), (−)-carveol (95%), (+)-carvone (96%), β-caryophyllene (98.5%), caryophyllene oxide (95%), β-citronellal (95%), citral (95%), 1,8-cineole (99%), ial oils .P. shantungensis adults and nymphs were collected from persimmon trees in Wanjugun, Korea and classified the fourth instar stages of P. shantungensis nymphs and adults as detailed elsewhere2P. shantungensis adults and nymphs were assessed  and were separated with HP-Innowax capillary column and DB-5 column . The conditions of the column were as follows: Helium at 0.75 mL/min; column temperature (51 to 201 °C) at 2 °C/min; injector temperature (211 °C); split ration (48:1); ion source temperature (231 °C); ionization potential (70e V); and mass spectra range (50–800 amu). The components of T. vulgaris oils were evaluated according to retention times, retention indices, and mass spectra and were identified by comparison with a spectrum library (T. vulgaris oil constituent (%) was measured by comparison with internal standards.The components of the essential oil extracted from  library . The rel50) value and the slope of the regression lines were calculated using the statistical package SPSS, version 12.0 for Windows.Data obtained for each dose response bioassay were subjected to probit analysis. The median lethal concentration .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


MiR-486 was found to be associated with cancer’s diagnosis and prognosis. This meta-analysis aimed to investigate the potential effect of miR-486 on cancer detection and prognosis.We searched PubMed, Cochrane library, Embase, Chinese National Knowledge Infrastructure (CNKI) and Wanfang databases to find all correlated articles. The STATA 11.0 was applied to estimate the pooled effects, heterogeneity and publication bias.The pooled sensitivity (SEN), specificity (SPE) and Area under the curve (AUC) were 82% (95% CI: 78–85%), 88% (95% CI: 83–92%) and 0.91 (95% CI: 0.88–0.93). Subgroup analysis indicated miR-486 from circulating samples exhibited higher diagnostic accuracy with the AUC was 0.90 (95% CI: 0.87–0.92) than miR-486 from other specimen with the AUC of 0.78 (95% CI: 0.75–0.82) and miR-486 obtained a better diagnostic value in the Asian population with the AUC of 0.94 (95% CI: 0.91–0.95) than the Caucasian and Caucasian/African population with the AUC of 0.80 (95% CI: 0.76–0.83) and 0.89 (95% CI: 0.86–0.91) respectively. MiR-486 obtained high value for the diagnosis of non-small cell lung cancer with SEN, SPE and AUC were 0.82 (95% CI: 0.0.77–0.87), 0.90 (95% CI: 0.84–0.94) as well as 0.92 (95% CI: 0.89–0.94) respectively. For the 7 prognostic tests, the pooled hazard ratio (HR) was 0.48 (95% CI: –0.13–1.08) for low versus high miR-486 expression.This meta-analysis indicated that miR-486 can be used as ideal biomarkers in the cancer’s diagnosis. However, Low miR-486 expression did not increase the risk of poor outcome. MicroRNA is a group of 19–22 nucleotide, small, single-stranded and conserved non-coding RNA that acts as a regulator of gene expression at both the post-transcriptional and the translational levels through acting on the 3’-untranslated region (UTR) of messenger RNA (mRNA) , SPE [TN/ (FP+TN)], the positive likelihood ratio (PLR) [(SEN/ (1-SEN)], the negative likelihood ratio (NLR) [(1-SPE)/SPE)], the diagnostic odds ratio (DOR) [PLR/ NLR] and the pooled HR with its 95% Confidence Interval (95% CI) respectively. We also constructed the SROC curve and calculated the AUC value. Simultaneously, we assessed the heterogeneity among the selected studies through the I2 value . The P vnificant . As for This was the first meta-analysis to confirm the potential value of miR-486 on cancer diagnosis and prognosis. The expression of miR-486 might be an effective biomarker for detection of human cancer.


A new recombinant norovirus GII.P16-GII.2 outnumbered pandemic GII.4 as the predominant GII genotype in the winter of 2016–2017 in Hong Kong, China. Half of hospitalized case-patients were older children and adults, including 13 young adults. This emergent norovirus targets a wider age population compared with circulating pandemic GII.4 strains. Noroviruses are leading causes of acute gastroenteritis (http://www.rivm.nl/mpf/norovirus/typingtool).Since August 2012, we have conducted an ongoing molecular surveillance of norovirus genotype distribution in all hospitalized gastroenteritis patients in our teaching hospital in Hong Kong . The female-to-male ratio was 1.04:1. We successfully genotyped 357 (90.8%) samples. The top 3 circulating VP1 genotypes during the study period were GII.4 (n = 214 [54.5%]), GII.2 (n = 86 [21.9%]), and GII.3 (n = 16 [4.1%]). Before this season, GII.2 had been a rare genotype, accounting for <1% of total strains circulating locally , panel Ahttp://www.megasoftware.net)  from the samples of 20 case-patients  as previously described (are.net) , panel AThe GII.2 strains we identified clustered most closely with the recombinant GII.P16-GII.2 strains from Germany during the same period . SubsequU-test)  , panel CU-test) , panel CU-test)  Figure. We noted a gradual narrowing in the age distribution of GII.4 infections to young children <5 years of age in the past 5 seasons , presumaWe report the emergence of a recombinant norovirus GII.P16-GII.2 variant that surpassed the previously predominant genotype GII.4 in hospitalized acute gastroenteritis case-patients in the winter of 2016–2017 in Hong Kong. However, unlike the recently emerged epidemic GII.17 Kawasaki variant that predominated only in part of Asia (China and Japan) during 2014–2016  apart from causing outbreaks. Collectively, these findings might have important implications for norovirus vaccine formulation and vaccination strategy. Close monitoring of the global spread of GII.P16-GII.2 is warranted.Incidence of hospitalization in case-patients with GII.4 and GII.2 infections, Hong Kong, China, July 2016–February 2017.


Mycobacterium porcinum is a rapidly growing environmental mycobacterium responsible for opportunistic infections. The 7,025,616-bp draft genome of M. porcinum strain CSURP1564 exhibits a 66.71% G+C content, 6,687 protein-coding genes, and 65 predicted RNA genes. In silico DNA-DNA hybridization confirms its assignment to the Mycobacterium fortuitum complex. Mycobacterium porcinum, a member of the Mycobacterium fortuitum third biovariant complex, is a nontuberculous mycobacterium primarily recovered from swine with lymphadenitis  oleic acid-albumin-dextrose-catalase  under a 5% CO2 atmosphere. Colonies were confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (M. porcinum CSURP1564 genome was incorporated into in silico DNA-DNA hybridization (DDH) (M. porcinum IP141460001 (GenBank accession number MVIG00000000), 49.5% with M. boenickei CIP 107829 (FUWC00000000), 34.5% with M. conceptionense D16 (CTEF00000000), 34.4% with M. farcinogenes DSM 43637 (CCAY000000000) and M. neworleansense ATCC 49404 (CWKH00000000), 31.40% with M. fortuitum CT6 (NCBI reference sequence number NZ_CP011269), and 20.5% with M. avium 104 (NCBI reference sequence NC_008595).trometry . Reads itrometry , and contrometry , GapFilltrometry , and mantrometry  yielded on (DDH)  using GGon (DDH) , with reM. porcinum strain CSURP1564 differs from M. conceptionense in the M. fortuitum complex. This is the first report of MinION technology applied to the genome sequencing of a nontuberculous Mycobacterium species , European Nucleotide Archive (ENA), under the accession number OLMG00000000 (OLMG01000001 to OLMG01000005).The draft genome sequence of


Lactobacillus plantarum Oregon-R-modENCODE strain BDGP2 was isolated from the gut of Drosophila melanogaster for functional host microbial interaction studies. The complete genome comprised a single circular genome of 3,407,160 bp, with a G+C content of 44%, and four plasmids. Lactobacillus plantarum increases amino acid metabolism and promotes larval growth in Drosophila melanogaster under nutrient-scarce conditions (L. plantarum strains include one derived from a laboratory fly strain (KP) and one from wild-caught flies (DF)  . Here, wL. plantarum Oregon-R-modENCODE strain BDGP2 was isolated from a fecal swab. Bacteria were streaked onto Difco de Man, Rogosa, and Sharpe (MRS) agar  agar  agar (BDRS) agar  and sequRS) agar . DNA wasRS) agar , and whoRS) agar . A singlsion 2.0 , 9. The sion 2.0  and the sion 2.0 .L. plantarum strains, ours contains integrated likely prophages . There are seven copies, six ranging in size from 36,225 to 46,235 bp and in sequence similarity from 45 to 70% and a partial copy of only 13,544 kb. Together they constitute 7.5% of the genome. Two cornerstone proteins highly conserved in prophages are the large terminase subunit and the portal protein (L. plantarum strain DF plasmid unnamed1 (GenBank accession number CP013754) and L. plantarum strain TMW 1.277  plasmid pL1277-4 (accession number CP017367), respectively.The chromosomal genome annotation predicts 3,148 protein-coding genes, 131 pseudogenes, 5 rRNA operons, a single 5S rRNA, and 85 tRNAs . Of the 3,148 protein-coding genes, 328 are contained within candidate prophages. Like other  protein . These cL. plantarum Oregon-R-modENCODE strain BDGP2 have been deposited in GenBank under the accession numbers CP023174 (chromosome) and CP023175 (pLtBDGP2A), CP023176 (pLtBDGP2B), CP023177 (pLtBDGP2C), and CP023178 (pLtBDGP2D) (plasmids).The complete genome sequences of the chromosome and four plasmids of


Both spontaneous hepatitis C virus (HCV) clearance and the achievement of sustained virological response (SVR) by anti-viral therapy greatly reduce the incidence of hepatocellular carcinoma (HCC). The current study aimed to compare the risk of HCC between the two patient groupsA total of 313 subjects with spontaneous HCV clearance (SC) and 564 age- and sex-matched patients in the treatment-induced SVR group were enrolled for analysis.Nineteen (2.2%) of the 877 patients developed HCC during 6,963 person-years of follow-up. Fourteen (2.5%) SVR patients and 5 (1.6%) SC patients developed HCC (P=0.004). Cox regression analysis of factors predictive of HCC included SVR , diabetes (HR/CI:3.41/1.21-9.58), and age (HR/CI: 1.07/1.01-1.14). Of the 564 SVR patients, eleven (5.9%) of the 187 patients with fibrosis stage 2-4 (F2-4) and 2 (0.9%) of the 226 patients with F01 developed HCC (P=0.01). Compared to SC subjects, only SVR patients with F2-4 (P<0.001) but not F0-1(P=0.60) had a higher risk of HCC development. Cox-regression analysis using liver fibrosis as a variable demonstrated that factors associated with HCC included SVR with F2-4 (versus SC: HR/CI: 10.06/2.20-45.98), diabetes (HR/CI:3.23/1.14-9.19), and age (HR/CI: 1.08 1.02-1.15).Compared to subjects with spontaneous viral clearance, subjects with antiviral treatment-induced HCV viral clearance remain at high risk for HCC development, especially if they have significant hepatic fibrosis. These results may provide important information for decision-making regarding the prioritization of current direct antiviral agents in resource-limited countries. Spontaneous hepatitis C virus (HCV) seroclearance occurs in 20-30% of patients after acute infection –3. SubjeA total of 313 subjects in the SC group (participants who had anti-HCV seropositivity and undetectable HCV RNA at baseline (n = 291) and those who had anti-HCV seropositivity and detectable HCV RNA levels at baseline, but HCV RNA became undetectable after more than one-year follow-up (n = 22)) and 564 age- and sex-matched patients in the SVR group were enrolled for analysis with a follow-up period of 4,224and 2,739 person-years, respectively. The baseline characteristics are shown in Table Nineteen 2.2%) of the 877 patients developed HCC over 6,963 person-years of follow-up, including fourteen (2.5%) SVR patients and 5 1.6%) SC patients with an incidence of 0.51% and 0.12% per person-year, respectively (P=0.004)  of the 564 SVR patients had liver histology available within six months of initiating the antiviral therapy. Of them, eleven (5.9%) of the 187 patients with F2-4 and two (0.9%) of the 226 patients with F0-1 developed HCC (Log-Rank test P=0.01). Compared to SC subjects, only SVR patients with F2-4 (Log-Rank test P<0.001), but not F0-1(Log-Rank test P=0.60), had significantly higher risk of HCC development  HCV cohort. The R.E.V.E.A.L-HCV study is a community-based cohort and enrolled participants from seven townships in Taiwan during 1991-1992. The details of the study participants, interview, blood collection and laboratory examinations have been described previously in detail. , 23 Sex-Interleukin 28B (IL-28B) rs8099917 was selected as the single-nucleotide polymorphism (SNP) to be tested for its association with HCC. The genetic testing was determined using methods that have been described previously , 25–27.The baseline characteristics of the clinical cohort and R.E.V.E.A.L.-HCV cohort were compared by chi-squared tests. The cumulative risk of the incidence of HCC among patients who occurred spontaneous HCV RNA clearance (R.E.V.A.E.L.-HCV cohort) and treatment-induced RNA clearance was estimated by Kaplan-Meier method, and the statistical significance of the difference was examined by log-rank tests. Cox proportional hazards models were used to estimate multivariate-adjusted hazard ratios (HR) with 95 percent confidence intervals (95% CI) for HCC risk for various groups with RNA clearance after adjustment for other risk factors. Statistical significance levels were determined by a 2-sided P-value of 0.05. The proportionality assumption (non-changing HRs over time) of Cox models was examined, and the assumption was not violated. All analyses were performed using the SAS statistical software package .


ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here the features of 10.1601/nm.1335 Mlalz-1 are described, together with high-quality permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to 10.1601/nm.133510.1601/strainfinder?urlappend=%3Fid%3DIAM+12611T, 10.1601/nm.1334 A 321T and 10.1601/nm.1783110.1601/strainfinder?urlappend=%3Fid%3DORS+1407T, based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as 10.1601/nm.1335. Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata-nodulating 10.1601/nm.1328 strains, but ≤93% with nodC of 10.1601/nm.1328 strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced 10.1601/nm.1335 strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In 10.1601/nm.1334 strain 10.1601/strainfinder?urlappend=%3Fid%3DWSM+419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of 10.1601/nm.1334 strains, which suggests genetic recombination between strain Mlalz-1 and 10.1601/nm.1334 and the horizontal gene transfer of lpiA-acvB.10.1601/nm.1335 Mlalz-1 (INSDC = The online version of this article (10.1186/s40793-017-0270-2) contains supplementary material, which is available to authorized users. Medicago spp.) are some of the most important and extensively grown pasture legumes and their specific symbiosis with strains of rhizobia belonging to either 10.1601/nm.1328 (synonym 10.1601/nm.1339) meliloti or the closely related species 10.1601/nm.1334 [Symbiotic nitrogen fixation by pasture legumes and their associated root nodule bacteria provides a critical contribution to sustainable animal and plant production, and the maintenance of soil fertility in agricultural systems –3. As su/nm.1334 , 6 has b/nm.1334 .Medicago laciniata (L.) Miller (cut leaf medic), an annual native of southern and eastern Mediterranean and Saharo-Sindian countries, is of importance because of its ability to grow in comparatively arid habitats and marginal cropping areas [Medicago sativa L.  or Medicago truncatula Gaertn. [nod genes, in particular a specific nodC allele [M. truncatula and M. laciniata shared chromosomal identity, but differed in their nodC alleles [M. laciniata and bv. meliloti for the classical 10.1601/nm.1335 group that efficiently nodulates M. sativa. However, in subsequent studies the diversity observed within bv. medicaginis strains indicate that this group is certainly heterogeneous [ng areas –11. It i Gaertn. , 13. ThiC allele . For exa alleles . Based oM. laciniata is native to the Canary Islands and is present on all of the islands of this archipelago, growing in environments that range from arid to subhumid. 10.1601/nm.1335 strain Mlalz-1 was isolated from a N2-fixing nodule of M. laciniata grown in alkaline soil (pH 9.0) collected in Guatiza, in the arid Northeast of Lanzarote Island, in 2007. This strain was one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 GEBA-RNB project proposal [proposal , 18. HerLeft and Center). It is fast growing, forming colonies after 3–5 days when grown on ½LA, TY, or a modified yeast-mannitol agar [Right). Minimum Information about the Genome Sequence (MIGS) for strain Mlalz-1 is provided in Table 10.1601/nm.1335 Mlalz-1 is a motile, non-sporulating, non-encapsulated, Gram-negative strain in the class 10.1601/nm.809. The rod shaped form has dimensions of approximately 0.5 μm in width and 1.0–2.0 μm in length  [T (= 10.1601/strainfinder?urlappend=%3Fid%3DLMG+19920T) [T [T, 99.7% to 10.1601/nm.1334 A 321T and 99.5% to 10.1601/nm.1783110.1601/strainfinder?urlappend=%3Fid%3DORS+1407T. In contrast, 10.1601/nm.133510.1601/strainfinder?urlappend=%3Fid%3DIAM+12611T and 10.1601/nm.133710.1601/strainfinder?urlappend=%3Fid%3DLMG+7834T [Previous studies using multilocus sequence typing showed that al group . Phyloge+6133T) , 10.160120T) [T . The avaG+7834T  were onlM. laciniata, which is suited for cultivation in arid environments [10.1601/nm.1335 Mlalz-1 was selected for sequencing at the U.S. Department of Energy funded Joint Genome Institute as part of the GEBA-RNB project , 18. Theronments . The 10.ronments  and a hironments . Sequenc600nm of 0.6 was reached. DNA was isolated from 50 ml of cells by Peter van Berkum according to the method described by van Berkum [−1.10.1601/nm.1335 Mlalz-1 (= 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+1984) was cultured on MAG solid media  for thren Berkum . The finThe draft genome of 10.1601/nm.1335 Mlalz-1 was generated at the DOE Joint genome Institute (JGI) using Illumina technology . An IlluUniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [Walnut Creek, CA, USA.Genes were identified using Prodigal , as partnSE tool  was usednSE tool . Other nnSE tool . AdditionSE tool  developeThe genome is 6,664,116 bp with 62.16% GC content Table  and compT (= 10.1601/strainfinder?urlappend=%3Fid%3DLMG+6133T), 10.1601/nm.1334 A 321T (= 10.1601/strainfinder?urlappend=%3Fid%3DLMG+19920T) and 10.1601/nm.1783110.1601/strainfinder?urlappend=%3Fid%3DORS+1407T. As the genomes of these type strains have not been sequenced or are not publically available, gANI values [repC, which is present as a single copy on 10.1601/nm.1335 pSymA and pSymB. The 10.1601/nm.1335 Mlalz-1 genome carried 2 repC loci (A3CADRAFT_00120 and A3CADRAFT_01676) with highest encoded protein identity to RepC proteins of 10.1601/nm.1335 strains. Mlalz-1 A3CADRAFT_00120 RepC1 had highest identity (98.10%) to the RepC1 protein encoded by SMb20044 on pSymB of 10.1601/nm.1335 1021. 10.1601/nm.1335 Mlalz-1 A3CADRAFT_01676 RepC2 had highest identity (99.00%) to the RepC2 protein encoded by SMa2391 on pSymA of 10.1601/nm.1335 1021. This indicated the presence of two megaplasmids in strain Mlalz-1, and revealed that strain Mlalz-1 has a similar genome architecture to that of 10.1601/nm.1335 1021.10.1601/nm.1335 Mlalz-1 is one of seven strains of 10.1601/nm.1335 that have been sequenced from the GEBA-RNB genome sequencing projects . On the I values  had to bI values . The totgspDOGLMCKEFHIJ necessary for a functional T2SS, but lacks the gspS gene found only in certain genera [.All 29 10.1601/nm.1335 strains within the gANI clique share a core set of 4948 orthologous genes, using cut off values of 1e-5 and 30% minimum protein identity. 10.1601/nm.1335 Mlalz-1 contains 176 unique genes, 96 (54.5%) of which encode hypothetical proteins. The unique genes include those encoding the components of a T2SS, located on scaffold A3CADRAFT_scaffold_5.6 Fig. , as welln genera  , actP, actR, actS, phrR, exoR, exoH, lpiA, acvB, degP1, mdh3, fbaB, groS, kdpB, kdpC, fixN2 and fixO2 [lpiA-acvB operon. One operon (A3CADRAFT_01189-A3CADRAFT_01190) is found on scaffold A3CADRAFT_scaffold_3.4, in a gene region that is conserved in other 10.1601/nm.1335 (sequence similarity >98%) and is located on the chromosome of the fully sequenced 10.1601/nm.1335 1021. The second version of the lpiA-acvB operon (A3CADRAFT_05694-A3CADRAFT_05695) is located on A3CADRAFT_scaffold_47.48, in a gene region that is conserved in 10.1601/nm.1334 genomes (sequence similarity >96%) and is located on the pSMED02 symbiotic plasmid of the fully sequenced 10.1601/nm.133410.1601/strainfinder?urlappend=%3Fid%3DWSM+419. The regulatory gene fsrR, required for the acid activated expression of lpiA in 10.1601/nm.133410.1601/strainfinder?urlappend=%3Fid%3DWSM+419 [lpiA-acvB gene regions of all other 10.1601/nm.1335 sequenced genomes. These findings suggest that 10.1601/nm.1335 Mlalz-1 acquired the plasmid-borne lpiA-acvB operon and associated fsrR regulatory gene by lateral transfer from an 10.1601/nm.1334 strain.The Mlaz-1 genome also contains acid-tolerance or acid-responsive genes that are orthologous to the genes identified in the comparatively acid tolerant strain 10.1601/nm.133410.1601/strainfinder?urlappend=%3Fid%3DWSM+419. Acid-tolerance or acid-responsive genes identified in Mlaz-1 include nd fixO2 –52  genes identified in the 10.1601/nm.1335 Mlalz-1 genome  with nodC of other M. laciniata-nodulating 10.1601/nm.1328 strains in the NCBI database, whereas there is a lower sequence identity (≤ 93%) with nodC of 10.1601/nm.1328 strains that nodulate other Medicago species. Nodulation of Medicago hosts requires Nod factors that are sulfated at the reducing terminus and acylated at the non-reducing terminus, with a polyunsaturated fatty acyl tail [Medicago species since the nodEF, nodL and nodHPQ genes that are required for these specific decorations of the Nod factor are present in the genome. 10.1601/nm.1335 Mlalz-1 also possesses the three nodD genes that mediate host-specific activation of nodABC in the symbiotic interactions of 10.1601/nm.1335 with Medicago [Essential symbiotic  with that of sequenced 10.1601/nm.1334 strains, which infers horizontal gene transfer of this region from 10.1601/nm.1334.10.1601/nm.1335 Mlalz-1 is a rhizobial strain that is able to nodulate and fix nitrogen with the highly specific host  strains . HoweverAdditional file 1: Table S1.Ensifer meliloti Mlalz-1. (DOCX 52 kb)Associated MIGS record for Additional file 2: Table S2-S4.Table S2. Acid responsive gene orthologs present in Ensifer strains. Table S3. The nodulation genes of Ensifer meliloti Mlalz-1. Table S4. The nitrogen fixation genes of Ensifer meliloti Mlalz-1. (DOCX 65 kb)


In the Local score development  subsection of the Results section, there is an error in the model equation following the second paragraph. The correct equation is:Log odds of in-hospital mortality = -4.67 + (moderate hypoxemia x 0.43) + (severe hypoxemia x 1.62) +  +  +  + (wheeze x -0.35) + (unconsciousness x 1.74)