This study was conducted to describe the changes in repeat multiparametric MRI (mpMRI) occurring in prostate cancer (PCa) patients during active surveillance (AS), and to study possible associations between mpMRI-related parameters in predicting prostate biopsy (Bx) Gleason score (GS) upgrading >3+3 and protocol-based treatment change (TC).The study cohort consisted of 76 AS patients with GS 3+3 PCa and at least two consecutive mpMRIs of the prostate performed between 2006–2015. Patients were followed according to the Prostate Cancer Research International Active Surveillance (PRIAS) protocol and an additional mpMRI. The primary end points were GS upgrading (GU) (>3+3) in protocol-based Bxs and protocol-based TC.Out of 76 patients, 53 (69%) had progression , while 18 (24%) had radiologically stable disease, and 5 (7%) had regression  in repeat mpMRIs during AS. PIRADS scores of 4–5 in the initial mpMRI were associated with GU (p = 0.008) and protocol-based TC (p = 0.009). Tumour progression on repeat mpMRIs was associated with TC (p = 0.045) but not with GU (p = 1.00). PIRADS scores of 4–5 predict GU ; 0.51–0.95, specificity 0.62 ) with PPV and NPV values of 0.34  and 0.93 , respectively.mpMRI is a useful tool not only to select but also to monitor PCa patients on AS. Prostate cancer (PCa) is the most common male cancer . The morMultiparametric MRI (mpMRI) and MRI-targeted fusion biopsies (FBx) have the potential to select patients for AS and to assist in clinical decision making when indicated . HoweverThe specific purposes of this study were 1) to characterise changes occurring in repeat mpMRI during AS, and 2) to study possible associations between mpMRI-related parameters, and changes thereof, in predicting Bx GS upgrading >3+3 (GU) and protocol-based treatment change (TC).The study approval by the Institutional Ethics Committee of the Helsinki University Hospital (diary number 386/13/03/02/2014) waived the need for consent based on the retrospective nature of the study and on the research conducted using only the local registry data and images. The study’s population selection is presented in http://dx.doi.org/10.17504/protocols.io.j66crhe. The majority of the patients had their first mpMRI within the first initial diagnostic year on AS, i.e. during the first year of diagnosis before the confirmatory Bx, which was scheduled according to the PRIAS protocol. Repeat mpMRIs were not systematically performed but repeated on most patients within the first two years on AS and thereafter whenever deemed clinically necessary (often when PSA DT <10 years). The mpMRI parameters included T2WI, DWI with ADC mapping and DCE. Imaging was performed between April 2007 and May 2015 using a 3.0 T Philips Achieva MRI scanner with 4 mm  or 3 mm (T2WI from 2013 on) slices. The MRI protocol evolved over the study period according to then current knowledge. Following their publication in May 2012, the European Society of Urogenital Radiology (ESUR) guidelines for mpMRI were followed [3 in tumour volume. mpMRI regression was defined as a decrease in PIRADS score or the disappearance of lesion(s) or a decrease of ≥0.1cm3 in tumour volume in subsequent mpMRIs. Pearson’s χ2 test of independence and Cramer’s V were performed to examine the association between categorical variables. All analyses were performed using SPSS® Statistics software (IBM® Version 21). A two-tailed p-value of less than 0.05 was considered statistically significant.The mpMRI protocol can be found here followed . For insThe study’s population demographics and mpMRI data are detailed in Tables 2 = 0.12; φ = 0.039; p = 1.0) whereas the moderate association between mpMRI progression and TC was statistically significant . PIRADS scores of 4–5 in the first mpMRI strongly associated with TC  and GU . PIRADS scores of 3–5 in the first mpMRI did not associate with TC  or GU . PIRADS scores of 4–5 predicted GU with a sensitivity of 0.80 ; 0.51–0.95), specificity of 0.62 , and PPV and NPV values of 0.34  and 0.93 , respectively. GU ≥7 in protocol-based random Bxs occurred in 15 out of 76 patients (19.7%). Over two-thirds (69%) of the patients with initially very low-risk PCa showed progression on serial mpMRI during AS. Tumour progression on serial mpMRI was also associated with treatment change from AS to definitive treatment. Furthermore, AS patients with a negative mpMRI were very unlikely to show histological upgrading in follow-up Bx. These results suggest that mpMRI is a valuable tool for PCa risk stratification not only at diagnosis but also during AS. In addition, the PIRADS scoring system seems to be effective in assessing prostate mpMRI changes.The major limitations of the study are its retrospective nature and lack of a standardised Bx protocol based on mpMRI findings. Cognitively targeted Bxs were taken in addition to the systematic Bxs within the sextants that contained tumour as according to the mpMRI. The FBx technique was not available when the first patients, included in this study, were diagnosed, and, therefore, additional targeted cores were occasionally taken. However, due to the retrospective nature of the study, location and accompanying data for these cores cannot be accurately assessed. In 2015, our Bx protocol changed and MRI-ultrasound-FBxs  from patThe strengths of the study include the use of the categorised, structured PIRADS scoring system for suspicious lesions in mpMRI, the use of a well-defined protocol for AS (PRIAS) and relatively long intervals between MRIs. Our early experience with the use of prostate MRI scans in an AS cohort prior to the PIRADS era was recently published and it suggested a poor correlation between MRI and GU . The datOur PCa patient population, with two mpMRIs, was highly preselected towards a very low-risk patient cohort. Patients with large and high-grade tumours are more likely to be detected earlier during surveillance, and to subsequently receive active treatment. Accordingly, patients available for the second mpMRI have a low risk for large GS >6 disease. However, 69% of the 76 patients showed progression on mpMRI during surveillance, even though the remaining AS cohort likely represents a cohort of men with only “negligible” risk PCa that remains after implementing PRIAS follow-up protocol. This suggests that current PRIAS protocol, mainly relying on PSA-DT and random Bx, is not sufficient to identify patients at risk of harbouring clinically significant disease. A similar discontinuation rate was recently observed in a large AS Movember Global Action Plan (GAP3) cohort consisting of more than 14 000 patients. This highlights the importance of proper follow-up during AS even if very low-risk patients are selected for AS. It also reflects our inability to predict PCa behaviour accurately at diagnosis using traditional clinical tools. The selection bias may also explain the lack of association between mpMRI change and GU, as more sophisticated targeting methods may be needed to detect the remaining smaller GS >6 cancer foci. We have previously shown that adherence to repeat Bxs in the PRIAS cohort is greatly attenuated by Bx-related complications such as infections ,17. The All the currently published cohorts demonstrate that treatment-free survival curves are far from optimum, which is mainly due to diagnostic inaccuracy, i.e. routine diagnostics fail to detect a substantial proportion of GS >3+3 cancers as do the follow-up protocols. In this respect, our data further support the use of mpMRI not only at diagnosis , but also as a tool for monitoring patients during AS. Therefore, an appropriate aim would be to more accurately select patients for early change in their plan of treatment and perhaps to simplify and personalise subsequent follow-up. Data on serial mpMRI among AS patients are scarce and suffer from a lack of standardisation for definition; this is particularly problematic in progressing prostate mpMRI. Recently, Diaz et al. and Felker et al. presented similar cohorts of PCa patients with serial mpMRI data ,20. Whilhttp://clinicaltrials.gov, NCT02914873).Our finding that low PIRADS scores were less useful for detecting high GS PCa is consistent with findings reported by others ,15. ThisProstate mpMRI is a useful tool not only for guiding patient selection for AS at diagnosis, but also for monitoring during surveillance.S1 FileThe data behind the statistics.(TXT)Click here for additional data file.


Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but their genetic causes remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we estimated the heritability of whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) activities measured in platelets. Overall, heritability was higher for NAS (0.72 ± 0.091) and ASMT (0.59 ± 0.097) compared with serotonin (0.31 ± 0.078), AANAT (0.34 ± 0.077) and melatonin (0.22 ± 0.071). Bivariate analyses showed high phenotypic and genetic correlations between traits of the second step of the metabolic pathway  indicating the contribution of shared genetic factors. A better knowledge of the heritability of the melatonin synthesis variability constitutes an important step to identify the factors that perturb this pathway in individuals with ASD. Large-scale genetic studies revealed a complex genetic architecture, involving combined effects of multiple low risk common variants, rare and de novo deleterious mutations4. Endophenotypes are measurable markers with potentially reduced genetic heterogeneity compared to the disease itself and they can be used to overcome the genetic and phenotypic heterogeneity. They facilitate the stratification of patients into more homogeneous subgroups5, increase the power of quantitative genetic analyses to detect variants, genes or pathways associated with the disease of interest and they reveal underlying biological mechanisms.Autism Spectrum Disorders (ASD) are complex neurodevelopmental disorders characterized by deficits in social communication/interaction as well as restricted interests and repetitive patterns of behaviors. Although ASD are highly heritable disorders10. A recent meta-analysis including 739 patients and 868 controls estimated an elevated whole-blood serotonin in 28.3% of the patients with ASD compared to 5% of controls11. Low melatonin levels in urine, plasma and pineal gland have also been described in ASD individuals15. Melatonin derives from serotonin, which is successively converted into N-acetylserotonin (NAS) and melatonin by the enzymes arylalkylamine N-acetyltransferase  and acetylserotonin O-methyltransferase . Melatonin biosynthesis essentially occurs in the pineal gland, following a marked circadian rhythm with a maximal secretion at night. A global disruption of this pathway was observed in patients compared to relatives and controls, including hyperserotonemia, deficits in AANAT and ASMT platelet activity, increased platelet NAS and melatonin deficit15. These alterations were also observed to a lesser extent, in the relatives of patients with ASD, compared to controls15, but familial correlations and heritability have never been assessed for both steps of the melatonin synthesis pathway in families with ASD.Hyperserotonemia is one of the most replicated endophenotypes in ASD17. NAS is an agonist of melatonin receptors and activates TrkB in mice, a receptor of the brain-derived neurotrophic factor (BDNF), in a selective and circadian manner19. Melatonin is a pleiotropic neuroendocrine molecule essential for synchronizing circadian and seasonal rhythms, as well as sleep/wake cycles, but also displays antioxidant, neuroprotective, or immunomodulatory effects22. Biochemical alterations of this pathway could thus be related to the core symptoms of autism or the comorbidities such as cognitive problems, epilepsy, sleep and gastrointestinal disorders observed in ASD25.Serotonin is involved in a wide range of central processes, such as brain development, emotion, learning, memory or cognitive functions2), defined as the ratio of total genetic variance  to phenotypic variance, or high narrow sense heritability (h2), reflecting only the additive part  of the genetic contribution to the phenotypic variance (Supplementary Note). Several studies in humans and in animal models have demonstrated relatively high heritability for serotonin28 and melatonin32. In humans, serotonin narrow sense heritability estimates range from 0.2 to 0.5133 and urinary melatonin narrow sense heritability estimate was 0.53 in families with acute intermittent porphyria32. To our knowledge, NAS, AANAT and ASMT heritability have never been investigated. In this study, we estimated the narrow sense heritability of all five quantitative traits  in 717 individuals including 213 children with ASD, 364 parents, 128 unaffected siblings and 12 other relatives   for each biochemical parameter: hyperserotonemia, increased platelet NAS, and deficit in platelet ASMT, platelet AANAT and plasma melatonin  with a mean age of 31.3 years and 59.3% males (425/717). As frequently observed in ASDRegarding the distribution of the biochemical traits within pedigrees, different categories of families could be determined Fig. . We obseWe first calculated the correlation coefficients for parent-offspring (P-O), sibling-sibling (S-S) and father-mother (F-M) pairs Table . When alWe then estimated the heritability of each trait. When all individuals were taken into account, the narrow sense estimates of heritability (reflecting additive effects) were all significant , as previously reported15. When only children with ASD were included, these phenotypic correlations were significantly different from 0  . Regarding genetic correlations, two negative genetic correlations were significantly different from 0 in children with ASD  and in unaffected children, only one genetic correlation was significant (NAS-ASMT: ρG = −0.90)  and for ASMT-melatonin (ρE = 0.48) in children with ASD  Fig. . For una90) Fig. . These gASD Fig. . In concASD Fig. .Figure 336. These processes are closely related to light-dark cycle39 and their regulation might depend on environmental factors, such as sleep-wake cycle and artificial light exposure. Thus, synchronization could be observed between parents that share daily habits and life rhythms.Very few heritability estimates for serotonin and melatonin are available for humans in the literature Table  and thiset al.27 in an inbred founder population of Hutterite families. They found respectively 0.51 and 1.0 for narrow and broad sense heritability, suggesting a very strong genetic influence with both additive and dominance components. Our estimation of serotonin narrow sense heritability was lower (0.31) than the one reported by Abney et al., but very similar to the one obtained in a large cohort of 866 monozygotic and 878 dizygotic unaffected twin pairs  ranged between two previous estimates32. Wetterberg et al. (1983) estimated melatonin heritability to 0.53 in 107 families with acute intermittent porphyria32. In contrast, a twin study measuring late night plasma melatonin in nine monozygotic and 11 dizygotic twin pairs, reported a high monozygotic correlation (rMZ = 0.928) and a high dizygotic correlation (rDZ = 0.867)31. Using these correlations and the Falconer’s formula (H2 = 2(rMZ - rDZ)), the heritability for melatonin would be 2(0.928-0.867) = 0.12.We then estimated the heritability of the five traits. In the literature, the first estimate of blood serotonin heritability was obtained by Abney  lower 0.1 than thAll the five traits studied here are not expected to be independent since they belong to the same metabolic pathway. Using a bivariate approach, we estimated pairwise phenotypic, genetic and environmental correlations in samples including only children with ASD or only unaffected children. In both groups, phenotypic correlations appeared to be particularly high for the last step of melatonin biosynthesis pathway  Fig. . Therefo40. Another limitation is the absence of analyses in the pineal gland. Finally, our estimations for unaffected children might not reflect the heritability the melatonin synthesis variability in the general population. Nevertheless, for serotonin and melatonin, our heritability estimates are very similar from those obtained in previous studies  at the Institut Pasteur in Paris (France) approved the study. Methods were performed in accordance with the relevant guidelines and regulations. Written informed consents were obtained from all participants. For the patients who were unable to consent for themselves, a parent or legal guardian consented to the study on their behalf.10. All the subjects were recruited into the Paris Autism Research International Sib-pair (PARIS) study. The ASD diagnosis was based on clinical expert assessment including the Autism Diagnostic Interview – Revised (ADI-R)45 and the Autism Diagnostic Observation Schedule (ADOS)46. Intellectual Quotient (IQ) was measured using an age-appropriate Wechsler scale. For the most severe and/or non-verbal patients, the Raven’s Standard Progressive Matrices and the Peabody Picture Vocabulary test were used.Clinical evaluations of patients with ASD, their relatives and control subjects have been detailed previously14. Subjects were asked to avoid food with high content of tryptophan and/or serotonin two days before blood sampling and individuals receiving exogenous melatonin were not included in this study. Melatonin was measured in plasma using a radio-immunoassay  according to the manufacturer’s instructions. Whole-blood serotonin  was measured by high-performance liquid chromatography47. NAS, as well as enzyme activities of AANAT and ASMT were determined in platelets by radio-enzymology48. Patients who were receiving melatonin for treatment of sleep disorders were not included in the analyses. Only pedigrees with both parents and at least one child were included in the following analyses. The overlap between the biochemical data for serotonin, AANAT, NAS, ASMT and melatonin is illustrated in Supplementary Fig. 15.Blood samples were collected in the morning, between 8:30 and 10:30 as described previously49. Because some of the studied traits were not normally distributed, nonparametric statistical tests (Wilcoxon rank-sum test) were preferred to compare groups of individuals. For familial correlation and heritability analyses, serotonin, ASMT and melatonin values were logarithmically transformed in order to conform more closely to a normal distribution  7.2.5 55, when their effect as covariates  were significant at the threshold of P < 0.1. Individuals at more than three standard deviations (SD) from the mean were considered as outliers and were removed. Depending on the biochemical trait and the subgroup considered, the number of outliers was always very low, ranging from 0 to 7  and R software56. For each relative pair types available in the sample pedigrees, FCOR calculates sibling-sibling (S-S), parent-offspring (P-O) and father-mother (F-M) familial correlations with their asymptotic standard errors. S.A.G.E then tests the difference of the correlation coefficients from 0 using Fisher’s z-transformation.Parent-offspring (P-O), sibling-sibling (S-S) and father-mother (F-M) correlations were measured for the five biochemical traits using the Family Correlations (FCOR) module of the Statistical Analysis for Genetic Epidemiology (S.A.G.E.) 6.3 software package Narrow sense heritability was estimated using the variance component-based program SOLAR 7.2.5 with a maximum likelihood estimation method and polygenic models. When the effect of the covariates  was significant (P < 0.1), relative proportion of variance explained by known covariates was estimated and they were included in the model.57. The trait pairs included all pairwise combinations and the same covariates (age and sex) significant at the threshold of P < 0.1 were used. Three parameters were estimated for each pair of traits: the additive genetic correlation (ρG), the shared environmental correlation (ρE) and the total phenotypic correlation (ρP) (Supplementary Note).SOLAR can extend the variance component model to a bivariate analysis that maximizes the model for two dependent traitsFamilial correlations and univariate heritability analyses were first performed on all individuals, including parents and their affected children as well as their unaffected children (Supplementary Fig. Supplementary Information


Quercus acuta Thunb. (Fagaceae) (QA) is cultivated as a dietary and ornamental plant in China, Japan, South Korea, and Taiwan. It has been widely used as the main ingredient of acorn tofu, a traditional food in China and South Korea. The aim of this study was to determine in vitro and in vivo xanthine oxidase (XO) inhibitory and antihyperuricemic activities of an ethyl acetate extract of QA leaf (QALE) and identify its active phytochemicals using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) systems. The QALE was found to possess potent in vitro antioxidant and XO inhibitory activities. In vivo study using hyperuricemic mice induced with potassium oxonate demonstrated that the QALE could inhibit hepatic XO activity at a relatively low oral dose (50 mg/kg) and significantly alleviate hyperuricemia to a similar extent as allopurinol. Several active compounds including vitamin E known to possess XO inhibitory activity were identified from the QALE. To the best of our knowledge, this is the first study that reports the active constituents and antihyperuricemic effect of QA, suggesting that it is feasible to use QALE as a food therapy or alternative medicine for alleviating hyperuricemia and gout. Hyperuricemia is a condition with an abnormally high concentration of uric acid in the blood. It can be caused by regular intake of purine-rich food and invariably accompanied by gout, chronic kidney disease, and/or other metabolic syndrome . Xanthin Carthamus tinctorius, Prunus persica, and Angelica gigas) were reported to be effective for the prevention and anesis of gout . Therefore, we preliminarily screened in vitro xanthine oxidase (XO) inhibitory activity of extracts from 229 dietary and/or medicinal plants including the three plants mentioned above. However, unfortunately, the XO inhibitory activity of the three plant extracts was not so high, while, unexpectedly, an extract from the leaf of Quercus acuta Thunb. (Fagaceae) (QA) showed profoundly higher activity compared to the other plant extracts (data not shown). Based on these preliminary results, we designed the present study on QA, which could contribute to developing a novel complementary and alternative medicine for treating hyperuricemia or gout.One of the therapeutic approaches to treat and prevent hyperuricemia is to use XO inhibitors that can block the final step of XO reaction to synthesize uric acid, thus reducing the levels of uric acid. Allopurinol is the only XO inhibitor approved for clinical use. However, it can cause several side effects such as hypersensitivity reactions, nephropathy, fetal liver necrosis, and enhancement of 6-mercaptopurine toxicity , 9. TherQA is cultivated as a dietary and ornamental plant in China, Japan, South Korea, and Taiwan . The fruTherefore, the objective of this study was to determine antioxidant, XO inhibitory activity, and antihyperuricemic activities of the QALE and identify its major active phytochemicals. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography analyses were performed for chemical profiling. In vitro DPPH free radical assay was used to determine antioxidant activity. XO inhibitory and antihyperuricemic activities were assessed in potassium oxonate-induced hyperuricemic mice.QA leaves were collected from the Wando Arboretum  and identified by Dr. Deuk-Sil Oh affiliated to the Wando Arboretum. A voucher specimen (MNUCSS-QA-01) was deposited at Mokpo National University . Air-dried and powdered QA leaves (10 g) were extracted twice with ethyl acetate (100 mL) at room temperature for 3 days. The resultant ethyl acetate solution was evaporated, dried, and stored at −50°C. It was used for further in vitro and in vivo experiments.Male ICR mice (4-weeks old) were purchased from Orient Bio, Co. . They were retained in a clean room at a temperature of 20–23°C with 12 h light (07:00–19:00) and dark (19:00–07:00) cycles at relative humidity of 50 ± 5%. Mice were housed in ventilated mice cages  with filtered and pathogen-free air. Food  and water were provided ad libitum. All animal experiments were carried out in accordance with the Guidelines of the Animal Investigation Committee of Jeonnam Bioindustry Foundation  with approval number of JINR1517. l. × 0.25 mm i.d., 0.25-μm film thickness). GC-MS was tuned using perfluorotributylamine (PFTBA) with mass fragments of 69.0, 219.0, and 502.0 m/z under electron ionization (EI) conditions. GC oven was heated using the following program: isothermal at 65°C for 10 min and 10 min−1 to 300 with helium (He) as carrier gas. Transfer line was heated at 300°C. The mass spectrometer was operated in scan mode of 50–550 amu. All mass spectra were compared to the data system library (NIST 2008). The operation parameters for the GC were shown in GC-MS analysis was conducted using a previously reported method with slight modifications . Brieflyμm, 150 mm l. × 5 mm i.d.) analytical column was used with a mobile phase consisting of solvent A (acetonitrile) and solvent B (water containing 0.2% phosphoric acid). Gradient elution  was performed at flow rate of 1.0 mL/min  equipped with a photodiode array detector. Agilent Zorbax extended C18 (5 0 mL/min . Column 50 (μg/mL) values  were identified by GC-MS analysis. Additionally, three compounds  were identified by HPLC analysis.GC-MS and HPLC analyses were performed to identify active constituents from QALE with antioxidant and XO inhibitory activities. Typical GC-MS and HPLC chromatograms of phytochemical contents and their retention times are shown in Figures This study aimed to determine the antioxidant, XO inhibitory, and antihyperuricemic activities of QALE and identify its active constituents. In our preliminary study, air-dried powder of QA leaf was extracted with various solvents  and the ethyl acetate extract was found to possess higher XO inhibitory, DPPH free radical scavenging activity, and reducing power than other extracts (data not shown). The ethyl acetate extraction condition was then optimized with respect to its antioxidant and XO inhibitory activity (data not shown). Based on these results, the ethyl acetate extract was selected for further in vitro and in vivo characterization.Our present results clearly indicate that the QALE is enriched with phenols and flavonoids . PolypheAs shown in Figures 2α-amyrin, linolenic acid, friedelin, and all-trans-squalene by GC-MS analysis. A previous study using vitamin E-deficient rabbits has reported that deficiency of vitamin E could be responsible for increased accumulation of hepatic XO [α-Amyrin has been shown to exert anti-inflammatory effect by reducing the levels of prostaglandin E2 and COX-2 expression in mice with 12-O-tetradecanoylphorbol-acetate- (TPA-) induced skin inflammation [2O2 generation both by neutrophils and in the XO system [As shown in patic XO . Moreovepatic XO . Stigmaspatic XO . α-Amyriammation . Squalenammation . Friedelammation , 29. Friammation . SimilarO system . LinolenO system . AdditioO system , 32.Taken together, the present study shows that QALE possesses antioxidant, XO inhibitory, and antihyperuricemic activities. In addition, several phytochemicals identified from the QALE might be responsible for its biological activities. This study provides a good basis for future development of QA-based dietary or medicinal preparations as an alternative to allopurinol.The present study investigated the antioxidant and antihyperuricemic effects of QALE and identified major active phytochemicals therein. The ethyl acetate extract of QA leaf was shown to possess potent in vitro antioxidant and XO inhibitory activities. Our in vivo mouse study demonstrated that QALE at a relatively low oral dose (50 mg/kg) could inhibit hepatic XO activity and significantly alleviate hyperuricemia to the extent comparable to allopurinol. Several active compounds including vitamin E known to possess XO inhibitory activity were identified from QALE by GS-MS and HPLC analyses. To the best of our knowledge, this is the first study that reports the active constituents and antihyperuricemic effects of QA. Our results suggest that it might be feasible to use QALE as a food therapy or alternative medicine to alleviate hyperuricemia and gout.


There are few reports about resection of portal vein (PV)/superior mesenteric vein (SMV) and reconstruction by using allogeneic vein. This case-control study was designed to explore the feasibility and safety of this operation type in patients with T3 stage pancreatic head cancer.A total of 42 patients (Group A) underwent PV/SMV resection and reconstruction by using allogeneic vein were 1:1 matched to 42 controls (Group B) with other types of resection and reconstruction. The two groups were well matched.P = 0.067) and increased intraoperative blood loss  was found between the two groups. R1 rate of PV/SMV was slightly reduced in group A compared to group B , although no significant difference was found. The incidences of main postoperative complications between the two groups were similar. A slightly increased 1-year and 2-year overall survival rate (OS)  and disease-free survival rate (DFS)  could be found in group A compared to group B, although the differences were not significant.There was no significantly prolonged total operation time (Group A vs. Group B [490.0 min vs. 470 min], The operation types of PV/SMV resection and reconstruction by using allogeneic vein is safety and feasible, it might have a potential benefit for patients. With increasing incidence and mortality, cancer is the one of leading causes of death worldwide and it has become a major public problem. Among the malignant solid tumors, pancreatic cancer is characterized by low curative rate and high mortality –5. CanceRadical pancreatic surgery accompanied with PV/SMV resection, has commonly been undertaken for pancreatic cancer with infiltration of PV/SMV since the first report of pancreatoduodenectomy (PD) with resection and reanastomosis of vein in 1951 , 9. TillIn this study, we conducted a case-control study to explore the feasibility and safety of PV/SMV resection and reconstruction by using allogeneic vein in patients with pancreatic head adenocarcinoma.From January 2012 to November 2016, patients with pancreatic head cancer who underwent surgical treatment at our institution were reviewed from our prospectively collected clinical database. Clinicopathological data of patients with duct adenocarcinoma of pancreatic head with PV/SMV infiltration by postoperative pathology were used for analysis. Patients with advanced pancreatic cancer, or with T4 stage pancreatic cancer, or without PV/SMV infiltration were excluded from this study, neither patients with cancer history nor patients with neoadjuvant therapy were enrolled in this study.Patients underwent segmental PV/SMV resection with reconstruction using allogeneic vein were 1:1 matched with patients underwent PV/SMV resection with reconstruction by other methods . Matching criteria included age, gender, body mass index (BMI), American Society of Anesthesiology (ASA) score, concomitant disease, blood test, surgical procedures (extended PD with extended Lymphadenectomy), postoperative pathology, and adjuvant therapy  or reconstruction using allogeneic vein (length of invasion > 2 cm), whereas for patients with type three or type four, only segmental PV/SMV resection with reconstruction using allogeneic vein can be performed  was used for justifying the condition of PV/SMV 1 month after operation, and then 6 months postoperatively; meanwhile Doppler was also used for evaluation of PV/SMV blood flow.Demographic and clinicopathologic parameters, operative details, perioperative complications and prognosis recorded to our database were used for analyzed. Drain amylase of > 3 times serum amylase after the third postoperative day, as defined by ISGPS , was defPhysical examination, laboratory tests, and image examinations were used for follow-up, the follow-up interval was 2 months. SPSS 16.0  was used for data analysis. Quantitative variables were expressed as median and range, and comparisons were analyzed with the Student t-test according to data distribution. Categorical variables were presented as number and percentage and were analyzed by chi-square test or Fisher exact test, as appropriate. Kaplan-Meier method was used to analyze the survival of patients, and the curve of survival between groups was analyzed by log-rank test.As shown in Fig. Patients with reconstruction using allogeneic vein (group A) were 1:1 matched to patients with reconstruction by other methods including segmental excision with direct end-to-end anastomosis, partial venous excision with direct closure, partial venous excision using a patch and segmental excision with artificial vascular graft (group B). In group B, type 1 PV/SMV invasion was confirmed in 17 patients and type two in 25 patients according to our classification of venous invasion. Of the 42 patients, 13 cases received partial venous excision with direct closure, four cases received partial venous excision using a patch, 22 cases received segmental excision with direct end-to-end anastomosis, and three cases received segmental excision with artificial vascular graft. Demographics and clinical characteristics are shown in Table P = 0.067), although the time to vein reconstruction in group A was longer compared with that in group B . The volume of intraoperative blood loss was 650 mL and 450 mL in group A and group B, respectively (P = 0.108). The length of PV/SMV resected was measured in this study, and the median length was 3.0 cm in group A which was obviously longer than that in group B. Although no significant difference for the R1 rate of PV/SMV, the R1 rate decreased almost 10% in group A compared with group B (4.8% vs. 14.3%). The incidences of pancreatic fistula were 14.3 and 19.0%, no significant difference was found (P = 0.558), the same result could be found for incidence of bleed (4.8% vs. 4.8%). The stump bleeding of gastroduodenal artery was confirmed for all four patients. Emergency laparotomy was carried out for two patients, interventional embolization was used for another two patients, and none died from bleed. No portal vein thrombosis was found by doppler on the third and seventh day after operation in group A, but one patient had portal vein thrombosis conformed by doppler B-ultrasound. There was no patient with infectious complication related to allogeneic vein.As shown in Table P = 0.501). The 1- and 2-year DFS were 43.9 and 10.5% in group A, 36.6 and 7.4% in group B, respectively (P = 0.502), which were shown in Fig. As shown in Fig. Radical resection is still the only potential method for the cure of pancreatic cancer although the development of non-surgical fields –18. TherFour types of venous resection are recommended by ISGPS : partialCompared with routine venous resection and reconstruction, the coexistence of advantages and disadvantages can be observed for allogeneic vein reconstruction. We conclude the main advantages of this technique: Firstly, extending surgical indications of pancreatic cancer. Without excessive limitation by infiltration scope of PV/SMV, patients have more chance to receive surgical resection. In our study, we could see that the length of PV/SMV resected in group A was obviously longer than group B. Secondly, improving en-bloc resection rate and R0 rate, more patients can benefit from radical resection. In this study, R1 rate of PV/SMV in group A decreased by nearly 10% compared with that in group B although no significant difference. Thirdly, avoiding incidences of vascular tension after anastomosis, vascular stenosis, anastomotic stricture and diameter mismatch. Recovering normal anatomic structure of vascular for special location such as superior mesenteric-portal-splenic vein confluence to the most extent. Fourthly, avoiding rejection induced by artificial vascular and long-time anticoagulation treatment.The main disadvantages of this technique are easily observed. Obviously, the procedure of allogeneic vein reconstruction is more complex compared with routine types, thus more operation time is needed. In this study, we could see that the time to vein reconstruction was significantly longer in group A than that in group B, whereas the total operation time and intraoperative blood loss between the two groups were comparable. Preservation of blood vessel is another issue of concern, there has been no unified standard till now.The complications did not increase in patients underwent allogeneic vein reconstruction, and no increased vein-related complications were found; meanwhile, the prognosis in these patients had slight improvement although no significant difference.PV/SMV resection and reconstruction by using allogeneic vein is safety and feasible, it may extend surgical indications and increase R0 resection rate compared with routine types.


Bacterial community characteristics including abundance, diversity and composition were revealed by quantitative PCR and high-throughput sequencing. The bacterial 16S rRNA gene copies significantly correlated with soil carbon and nitrogen contents, suggesting the control of nutrients accessibility on bacterial abundance. The bacterial diversity was strongly related to soil pH, with higher diversity in neutral samples and lower in acidic samples. Soil pH was also identified by an ordination analysis as important factor shaping bacterial community composition. The relative abundances of some dominant phyla varied along the pH gradient, and the enrichment of a few phylotypes suggested their adaptation to low pH condition. In contrast, at the current pollution level, PAH showed marginal effects on soil bacterial community. Overall, these findings suggest pH was the primary determinant of bacterial community in these arable soils, indicative of a more substantial influence of acidification than PAH pollution on bacteria driven ecological processes.Acidification and pollution are two major threats to agricultural ecosystems; however, microbial community responses to co-existed soil acidification and pollution remain less explored. In this study, arable soils of broad pH (4.26–8.43) and polycyclic aromatic hydrocarbon (PAH) gradients (0.18–20.68 mg kg Microorganisms play vital roles in soil processes such as substance turnover, biogeochemical cycling and plant growth, contributing essentially to the soil ecosystem services. Soil contains a myriad of bacteria of which the diversity and community structure are crucial to soil functional stabilityBoth natural and anthropogenic processes contribute to soil acidification, and the resulting pH variation is often an important factor affecting soil bacterial diversity and community composition at different geographic scales45610Soil pollution has shown extensive effects on below-ground microbes. Among the pollutants studied, polycyclic aromatic hydrocarbons (PAHs) has been found to be widespread in terrestrial environments15http://www.mlr.gov.cn/xwdt/jrxw/201404/t20140417_1312998.htm) in China. The community responses to overlapped acidification and PAH pollution are therefore crucial to assess the function changes in impacted ecosystems. In our previous study, we observed co-existed pH (4.26–8.05) and PAH gradients (0.18–20.68 mg kg−1) within soil samples collected from a farmland adjacent to a smelting plant. As revealed by fingerprinting technique, the community of Thaumarchaeota, one of major archaeal phyla in these arable soils was tightly related to soil pH rather than to PAH, suggesting variation in archaeal nitrification in this farmlandBoth soil acidification and pollution might co-exist in arable soils, as suggested by wide occurrence of acidification10 g−1, and were significantly (p < 0.01) related to total carbon (TC), total nitrogen (TN) and nitrate  copies are often used as a surrogate of degrading bacterial abundancewith PAH . Howeverr ≥ 0.7, p < 0.01, p < 0.01, The Illumina MiSeq sequencing generated 180 876 reads from 29 libraries after quality control, with an average of 6 237 reads for each sample and median length of 408-bp. The reads were clustered into OTUs based on a 97% threshold of sequence similarity. Observed species, Chao1 estimator and phylogenetic diversity were calculated, and were compared at the same level of subsampling (2 000 reads). All measures of bacterial diversity were tightly related to pH , and partly addresses the link between ammonium and bacterial diversity , PAH showed negligible influence on bacterial diversity (29Despite a two orders of magnitude variation in soil concentration (0.18–20.68 mg kgiversity  and commp < 0.05), hence were included in CCA analysis. The first two axes of the CCA biplot accounted for 9.5% and 4.6% of the total community variation, respectively, with pH the most correlated factor with the first axis  were highly enriched in soils of low pH, which may accounted for >30% of the library of low-pH sample , which is consistent to our previous studyThe influence of pH on other dominant phylotypes could be depicted with a heatmap , in whicH sample . RhodanoRhodococcus was directly related to PAH , and to copies of GP PAH-RHDα gene . Degradation of PAH by Rhodococcus sp. has been widely recognizedSimilar correlation between PAH and individual bacterial phylotypes were found. Among others, the relative abundance of Gram positive Taken together, this study examined bacterial abundance and structure in arable soils affected by both acidification and PAH pollution. The results reveal that pH was a primary determinant of bacterial community diversity and composition, suggesting acidification has potentially significant impacts on soil ecological processes driven by bacteria. By contrast, the influence of PAH on soil bacteria appears to be negligible within the measured pollution range, which may be in relation to the interactions among pollutants, microbes and soil matrix. Large-scale soil survey and simulated exposure to PAH will facilitate a deep insight into the ecological effect of PAH on soil microbes.In total 29 soil samples were collected from three separated agricultural plots in southwest Nanjing, Jiangsu Province, China. Upon the sampling, the three plots were used for cultivation of varied vegetables. Each soils was obtained by mixing five 50-g subsamples from a 10 × 10 m grid. A 50-g subsample of each soil was stored at −20 °C for molecular analysis, and the rest was air-dried, homogenized, and stored at 4 °C for physicochemical analysis.−1  PAH-ring hydroxylating dioxygenase (PAH-RHDα) genes were determined with primer sets GP-F/GP-R, respectively1–107 copies μl−1) was used per assay. The control was always run with water as the template instead of DNA extract. The qPCR amplification efficiency was 85.9% with r2-value of 0.995 for bacterial 16S rRNA gene, 71.0% with r2-value of 0.995 for GP PAH-RHDα gene.QPCR was performed on a CFX96 instrument (Bio-Rad) and SYBR green-based reactions were performed in triplicate for each sample. The components of qPCR assays were described previouslyBacterial community analysis was performed by sequencing bacterial 16S rRNA gene as previously describedhttp://greengenes.lbl.gov/). In this study, 2 000 sequences per sample were randomly subsampled to calculate the Faith’s phylogenetic diversity, the observed species and the Chao 1 estimator. The correlations between microbial diversity and soil variables were calculated using SPSS 13.0. Canonical correspondence analysis (CCA) was performed with OTU table and all available environmental parameters using the vegan package of R. Heatmap was generated with centered dominant phylotypes data using the R package pheatmap, with phylotypes and samples aggregated into clusters using the complete neighbor method.Raw paired Illumina MiSeq data were assembled and analyzed using QIIMEThe sequences obtained in this study have been deposited in the European Molecular Biology Laboratory (EMBL) database.How to cite this article: Wu, Y. et al. pH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution. Sci. Rep.7, 40093; doi: 10.1038/srep40093 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Guidelines have recommended significant reductions in dietary sodium intake to improve cardiovascular health. However, these dietary sodium intake recommendations have been questioned as emerging evidence has shown that there is a higher risk of cardiovascular disease with a low sodium diet, including in individuals with type 2 diabetes. This may be related to the other pleotropic effects of dietary sodium intake. Therefore, despite recent review of dietary sodium intake guidelines by multiple organizations, including the dietary guidelines for Americans, American Diabetes Association, and American Heart Association, concerns about the impact of the degree of sodium restriction on cardiovascular health continue to be raised. This literature review examines the effects of dietary sodium intake on factors contributing to cardiovascular health, including left ventricular hypertrophy, heart rate, albuminuria, rennin–angiotensin–aldosterone system activation, serum lipids, insulin sensitivity, sympathetic nervous system activation, endothelial function, and immune function. In the last part of this review, the association between dietary sodium intake and cardiovascular outcomes, especially in individuals with diabetes, is explored. Given the increased risk of cardiovascular disease in individuals with diabetes and the increasing incidence of diabetes worldwide, this review is important in summarizing the recent evidence regarding the effects of dietary sodium intake on cardiovascular health, especially in this population. High dietary sodium intake has been related to high blood pressure for more than 4,000 years . The conBased on this body of evidence, multiple dietary guidelines for sodium intake have been published Table . The 201However, low dietary sodium intake has pleotropic effects, which could contribute to cardiovascular health. Therefore, rationalizing that low sodium intake reduces adverse cardiovascular outcomes based on its blood pressure lowering effects alone may not be appropriate. In 2013, the Institute of Medicine (IOM) in the U.S. examined the evidence on the effect of dietary sodium intake on health outcomes in the U.S. general population and higher risk subgroups . It was In Australia, the National Health and Medical Research Council (NHMRC) recommended sodium intake to be ideally less than 1,600 mg/day (70 mmol/24 h) and at a maximum of 2,300 mg/day (100 mmol/24 h) for adults in 2013 . This reA literature search in MEDLINE (1946–July 2016) was performed using a combination of the following search terms: salt, salt intake, dietary salt intake, dietary sodium intake, dietary sodium, dietary sodium chloride , hypertension, heart rate, immune system, cardiovascular, cardiovascular disease, cardiovascular mortality, mortality, diabetes, type 2 diabetes, and type 1 diabetes. Combinations of limitations including English language, core clinical journals, and journal article were placed on the search terms. References from the relevant papers were also sourced.Recommendations to reduce sodium intake have been based on the prevailing view that high sodium intake is detrimental to blood pressure, which is a surrogate endpoint for cardiovascular morbidity and mortality . There iOver the recent years, dietary sodium has been shown to have other effects such as impacting on rennin–angiotensin–aldosterone system, left ventricular hypertrophy, heart rate, albuminuria , insulin sensitivity, lipids, immune function, endothelial dysfunction, and sympathetic nervous system activity.However, studies demonstrating the effects of dietary sodium on these factors have demonstrated inconsistent results. The discrepancy in results may be attributed to methodological differences among studies. This includes differences in the methods of measurement and ranges of dietary sodium intake, study populations, study outcomes, and failure to explore non-linear associations . Many ofHowever, many studies estimate dietary sodium intake through dietary surveys or overnight and spot urine collections due to the lower burden on participants . DietaryOn the other hand, overnight and spot urine collections are weak surrogates for 24-h urine collections because they have not been sufficiently validated and could underestimate or overestimate 24-h urinary sodium excretion , 28. AltIn addition to the method used to estimate dietary sodium intake, the inconsistency in results could also be attributed to limitations of study design. Since many of the studies, which showed the effect of sodium intake on cardiovascular health are observational studies, they are susceptible to confounders and reverse causation. Potential confounders can be reduced in observational studies through restricting or matching participants and performing stratified or multivariate analysis . HoweverIn addition, there is a lack of consistency in definitions of “low,” “moderate,” and “high” dietary sodium intake and “severe” and “moderate” sodium restriction. For example, many studies used the term “moderate sodium restriction” when the mean or median reduction in dietary sodium intake is less than 120 mmol/24 h. However, Grassi et al. used this term despite having a 140 mmol/24 h reduction in sodium intake (mean sodium reduction 129–136 mmol/24 h) . ConversHigher sodium intake was proposed to be associated with left ventricular hypertrophy –35, whicIncreased heart rate was demonstrated to be independently associated with increased cardiovascular and all-cause mortality , 38. ThiJ-shaped relationship between sodium intake and albuminuria, where both lower (sodium intake of <170 mmol/24 h) and higher (sodium intake of >203 mmol/24 h) dietary sodium intake were associated with higher urinary albumin excretion , which is considered the gold standard method for assessing sympathetic outflow in humans , which indicated that the inhibition of basal release of endothelium-derived nitric oxide was reduced (4) bioavailability and reduced oxidative stress during sodium restriction  cells, which predominantly have a Th1 phenotype -induced M1 macrophages are pro-inflammatory and produce pro-inflammatory cytokines, which promote endothelial dysfunction, destabilization of atherosclerotic plaques in advanced atherosclerosis and thrombus formation in acute coronary syndromes , and dirvia a renal-independent mechanism, resulting in an approximate 40mM (40 mmol/L) increase in interstitial sodium compared to plasma in rodents (in vitro (increased by 40 mmol/L) have been demonstrated to promote activation of Th17 cells and M1 (pro-inflammatory) macrophages and blunt activation of M2 (non-inflammatory) macrophages and Treg cells (in vitro (increase by 40 mmol/L) (+ and CD8+ T cell proliferation were blunted (++CD16+) monocytes and increased production of intracellular reactive oxygen species  , 121. On blunted . However blunted . In addi species . Moreove species , which p species . Convers species . This su species  addresse species . Convers species .in vitro and during increased sodium intake in vivo (in vitro (40 mmol/L sodium chloride) and in vivo (mouse models) (in vitro promoted the stable induction of human and murine Th17 cells through the activation of the p38/MAPK pathway involving nuclear factor of activated T cells 5 (NFAT5) and SGK1 (in vitro (increased by 40 mmol/L in human and murine Treg cells) and in vivo (8 and 1% sodium chloride in immune-deficient NOD-scid IL2Rgnull mouse models) significantly impaired the suppressive function of Treg cells and promoted a pro-inflammatory Th1-type effector phenotype associated with a SGK1-dependent increase in IFNγ secretion in Treg cells. This illustrated that T cell populations can exhibit plasticity depending on the microenvironment and highlights the importance of environmental influences on T helper cell polarization.Serum-and-glucocorticoid-regulated kinase 1 (SGK1) acts as a mediator of sodium homeostasis by regulating sodium reabsorption through activation of epithelium sodium channels (ENaC) in the kidneys . Express in vivo , 108. Inand SGK1 . Moreoveand SGK1 . In addiand SGK1  demonstrin vitro or in experimental models where genetic modification is possible   . This fi2) , 133. In2) . On the 2) . Despite2) , there a2) , 130. Th2) , 130. Th2) . Additio2)  also repJ-shaped  in the general population aged 35–70 years old in the Prospective Urban Rural Epidemiology (PURE) study. Another study in individuals at high risk of cardiovascular disease supported this by showing that the risk of cardiovascular events was higher with estimated sodium intake less than 3 g/day (130 mmol/24 h) and greater than 7 g/day (304 mmol/24 h)  and higher sodium intake of at least 7 g/day (304 mmol/24 h) were associated with a higher risk of cardiovascular events and death than a sodium intake of 4–5 g/day (174–217 mmol/24 h). Conversely, in normotensive individuals, only lower sodium intake was associated with increased risk of cardiovascular events and death   showed a failure . Mente e failure  supportend death . This suCardiovascular disease accounts for up to 80% of deaths in individuals with diabetes . Since dIn addition, central sympathetic hyperactivity was reported in individuals with type 2 diabetes, with the greatest sympathetic hyperactivity seen in those with concurrent essential hypertension , 151. Tyvia sodium-glucose cotransporters (SGLT) in the luminal side of proximal tubule epithelial cells (Diabetic nephropathy is the leading cause of end-stage renal disease worldwide , 154. Deal cells . This isal cells . The tubal cells . In contal cells , individal cells . In addial cells , which mal cells . They haal cells . Therefoal cells . Moreoveal cells .Given these differences between people with diabetes and the general population Figure , cardiovIn individuals with type 1 diabetes, both high and low dietary sodium intakes were shown to be associated with increased all-cause mortality . MoreoveDietary sodium intake recommendations support sodium restriction based on previous evidence suggesting a reduction in blood pressure. It was proposed that this would be associated with a subsequent reduction in cardiovascular morbidity and mortality. However, increasingly, it is now being understood that sodium intake has other pleiotropic effects that affect cardiovascular health, highlighting that the association between sodium intake and cardiovascular outcomes cannot be based on blood pressure alone. Therefore, current dietary sodium intake guidelines have been revised since the IOM reported that there was no clear benefit or harm of sodium restriction to less than 100 mmol/24 h in 2013. However, recently, the current dietary sodium guidelines have been challenged because there is emerging evidence to suggest an associated increase in morbidity and mortality with lower dietary sodium intake in high-risk groups, including those with diabetes. These studies suggest that the current dietary guidelines may be too strict; therefore, they may not be suitable and may need further revision. However, there is a lack of data from randomized controlled trials to determine the optimal level of dietary sodium intake for specific populations. In addition, there is a paucity of data with a lack of randomized controlled trial data in humans to explain the possible mechanisms contributing to the adverse outcomes associated with lower dietary sodium intake in high-risk populations.YK undertook the literature review and wrote the first draft of the manuscript. EE, SB, and GJ planned the study, reviewed, and edited the manuscript.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.


This study aimed to compare the mid-term adverse cardiovascular outcomes associated with Coronary Artery Bypass Surgery (CABG) and Percutaneous Coronary Intervention (PCI) with Everolimus Eluting Stents (EES). Electronic databases were searched for studies comparing the mid-term (>1 year) adverse cardiovascular outcomes between CABG and PCI with EES. Odd Ratios (OR) with 95% Confidence Intervals (CIs) were calculated and the pooled analyses were performed with RevMan 5.3 software. A total number of 5207 patients were involved in this analysis. No significant difference was observed in mortality between CABG and EES with OR: 0.90, 95% CI: 0.73–1.10; P = 0.30. Moreover, CABG was associated with a high stroke rate, with OR: 0.73, 95% CI: 0.45–1.17; P = 0.19, without any statistical significant. CABG was associated with significantly lower Major Adverse Cardiac Events and Myocardial Infarction with OR: 1.46, 95% CI: 1.05–2.04; P = 0.03 and OR: 1.46, 95% CI: 1.01–2.12; P = 0.05 respectively whereas PCI was associated with a significantly higher repeated revascularization with OR: 2.21; 95% CI: 1.76–2.77; P = 0.00001. In conclusion, significant differences were noted in several subgroups analyzing the mid-term cardiovascular outcomes between CABG and EES. Even if Percutaneous Coronary Intervention (PCI) is a short procedure resulting in rapid discharge from the hospital when compared to Coronary Artery Bypass Surgery (CABG) which is time-consuming, more expensive and associated with late hospital discharge following surgery, the latter is associated with significantly better long-term adverse clinical outcomes, when compared with first generation Drug Eluting Stents (DES)1245PubMed, Medline, EMBASE and the Cochrane library were searched for studies comparing CABG and PCI with EES using the following words or phrase ‘coronary artery bypass surgery and everolimus eluting stents’. To further enhance this search, the words ‘percutaneous coronary intervention, and second generation drug eluting stents’ as well as abbreviations such as ‘CABG, PCI, and EES’ were used. References of suitable articles were also checked for relevant studies. In addition, only English language publications were considered relevant to this search strategy.They were Randomized Controlled Trials (RCTs) or observational studies.They compared CABG and PCI with EES.They reported adverse cardiovascular outcomes among their clinical endpoints.They had a mid-term follow up period above one year (more than one year but less than 5 years).Studies were included if:They were meta-analyses, case studies or letter to editors.They did not compare CABG and PCI with EES.They did not report adverse cardiovascular outcomes as their clinical endpoints.They had a follow up period of less than one year.They were associated with the same trial .They were duplicated studies (same study obtained from different databases).Studies were excluded if:Mortality StrokeMyocardial Infarction (MI)Repeated revascularization (including target vessel revascularization and target lesion revascularization)Major Adverse Cardiac Events (MACEs) consisting of death, MI and revascularization. Because only one study reported Major Adverse Cardiovascular and Cerebrovascular Events (MACCEs) consisting of death, MI, revascularization and cerebrovascular events, MACCEs were included in the subgroup analyzing MACEs.The cardiovascular outcomes analyzed included:A mid-term follow-up was defined as a follow up period longer than one year but not exceeding 5 years.The reported outcomes with respective follow up periods have been summarized in Five authors  independently assessed the eligibility of the studies considered relevant to this analysis. Information and data related to the total number of patients involved in the CABG and PCI groups respectively, the types of study involved, the patients’ enrollment period, data concerning the baseline features of the patients included, the reported cardiovascular outcomes and follow up periods were systematically extracted by the same five authors. If any of the authors disagreed about including certain studies or data, disagreements were discussed and solved among themselves. However, if a consensus could not be reached, disagreements were finally resolved and a final decision was made by the sixth author (WQH). The bias risk observed among the trials was assessed using recommendations obtained from the Cochrane CollaborationThe Cochrane Q-statistic test whereby a P value less or equal to 0.05 was considered statistically significant. Any P value greater than 0.05 was considered insignificant.2 statistic test whereby a low value of I2 represented a low heterogeneity, whereas an increasing I2 value represented an increasing heterogeneity among the subgroups assessed.The IThe PRISMA reporting guideline was followed for this systematic review and meta-analysis2 was greater than 50%, a random effect model was used and if I2 was less than 50%, a fixed effect model was used appropriately. Publication bias was visually estimated by assessing funnel plots which were obtained from RevMan. Odd Ratios (OR) with 95% Confidence Intervals (CIs) were calculated for the subgroup analysis of the cardiovascular outcomes. The pooled analyses were performed/conducted with RevMan 5.3 software.In this analysis, if IEthical approval was not required for this type of study. All the six authors had full access to the data and approved the manuscript as written.Two hundred and forty-two articles were obtained during the search process. After a careful assessment of the titles and abstracts, 228 articles were eliminated since they were either not related to the title of this research, or they were duplicates. Fourteen full-text articles were assessed for eligibility. A further 10 articles were eliminated since 6 articles were letter to editors whereas 4 articles were associated with the same trial. Finally, only 4 articles  which satisfied all the inclusion and exclusion criteria of this study were selected and included in this meta-analysis. The flow diagram showing the study selection has been represented in A total number of 5207 patients were involved in this analysis (2605 patients were treated by PCI with EES and 2602 patients were treated with CABG). The general features of the studies included in this meta-analysis have been summarized in For study bangalore2015, only patients who were >80 years were included. Reasons were due to the fact that all the other studies consisted of less patients and therefore, in order to match their patient number, and in order for the result of this analysis not to be influenced by the result obtained in the study Bangalore2015, only patients >80 years were considered.The baseline features of the patients included in this study have been listed in Results of this analysis have been summarized in 2 = 0%. Moreover, CABG was associated with a higher rate of stroke, with OR: 0.73, 95% CI: 0.45–1.17; P = 0.19, I2 = 0%, however, the result was not statistically significant. CABG was also associated with significantly lower MACEs and MI with OR: 1.46, 95% CI: 1.05–2.04; P = 0.03, I2 = 0% and OR: 1.46, 95% CI: 1.01–2.12; P = 0.05, I2 = 0% respectively whereas PCI was associated with a significantly higher repeated revascularization with OR: 2.21; 95% CI: 1.76–2.77; P = 0.00001, I2 = 0%. Results showing the adverse cardiovascular outcomes between CABG and PCI with EES have been represented in This analysis showed that during a mid-term follow up period of more than one year, no significant difference was observed in mortality between CABG and EES with OR: 0.90, 95% CI: 0.73–1.10; P = 0.30, I2 = 0%. This result has been represented in Since our data included patients with multi-vessel diseases and patients with left main coronary diseases, further analyses were conducted separating these two categories of patients and comparing CABG and PCI with EES. In patients with left main coronary artery disease, mortality was also not significantly different between CABG and PCI with EES, with OR: 0.78, 95% CI: 0.58–1.05; P = 0.10, I2 = 0%. MI and repeated revascularization significantly favored CABG with OR: 1.53, 95% CI: 1.04–2.25; P = 0.03, I2 = 0% and OR: 2.25, 95% CI: 1.77–2.87; P < 0.00001, I2 = 0% respectively. However, even if stroke was higher in these patients with multi-vessel diseases revascularized by CABG, with OR: 0.70, 95% CI: 0.43–1.14; P = 0.15, I2 = 0%, the result was not statistically significant. Results showing adverse cardiovascular outcomes between CABG and PCI with EES in patients with multi-vessel coronary diseases have been represented in In patients with multi-vessel coronary artery diseases, a similar mortality rate was observed between CABG and PCI with EES, with OR: 1.02, 95% CI: 0.77–1.36; P = 0.88, IFor the above analyses comparing CABG and PCI with EES, sensitivity analyses yielded consistent results. Based on a visual inspection of the funnel plot obtained, there has been no evidence of publication bias for the included studies that assessed all the mid-term cardiovascular outcomes. The funnel plot representing sensitivity analysis is shown in This study aimed to compare the adverse cardiovascular outcomes associated with CABG and PCI with EES during a follow up period of more than one year (less than 5 years). The current analysis showed no significant difference in mortality between CABG and PCI with EES. Even if a higher rate of stroke was observed with CABG, the result was not statistically significant. However, PCI with EES was associated with significantly higher MACEs, MI and repeated revascularization when compared to CABG. A similar result was obtained when patients with left main coronary artery disease and multi-vessel coronary diseases were separately analyzed.et al., including diabetic patients with multi vessel coronary diseases showed a similar mortality rate associated with EES and CABG during the long termThe study published by Bangalore The Coronary Artery Bypass Surgery and Everolimus Eluting Stent Implantation in the Treatment of Patients with Multivessel Coronary Artery Disease (BEST) trial which involved patients with multivessel diseases also showed a significantly higher rate of MACEs associated with PCI with EES when compared to CABG, at 2 years follow up 11. In addition, 11 cases of stroke were reported with PCI, whereas 13 cases were reported with CABG. The trial which involved 880 patients, was a prospective, open-labeled randomized trial conducted in several sites including Korea, China, Malaysia and Thailand. However, this current study could not analyze MACEs specifically in patients with multi-vessel diseases due to a lack of data.In contrast, the Premier of Randomized Comparison of Bypass Surgery versus Angioplasty Using Sirolimus-Eluting Stent in Patients with Left Main Coronary Artery Disease (PRECOMBAT 2)At last, this current analysis satisfied all the criteria for a meta-analysis in terms of low level of heterogeneity, low risk of bias, and robust results. Therefore, it might be used by physicians and interventionists in order to select treatment strategies in patients with CAD, and to predict mid-term prognosis. However, further studies with long term follow up period should be recommended to further debate on this very interesting issue particularly relevant to clinical medicine.This research is new in the field of interventional cardiology. Only a few trials or cohort studies have compared CABG and PCI with EES. However, this is currently the first meta-analysis combining data obtained from the few published studies to conduct a new analysis showing whether similar or different outcomes were reported between CABG and PCI with EES, representing a new idea in clinical medicine. In addition, a very low level of heterogeneity obtained among all the subgroups analyzed, might represent another new feature of this study. Moreover, physicians and interventionists might be able to predict prognosis or select revascularization strategies in patients with CAD, taking into consideration the cost as well as other benefits or disadvantages of these respective procedures.Limitations in this analysis were as follows: A limited number of patients were analyzed. Lack of data might have had an effect on the result of this analysis. The study Bangalore2015 consisted of more than 15,000 patients. However, only patients above 80 years old were extracted from that study to be included in this current analysis. Reasons for extracting only patients above 80 years old were to match the number of patients obtained from the other studies in order to obtain a smooth result which would not be based only on the dominant study Bangalore2015. Moreover, similar endpoints were not reported in all the studies included in this analysis restricting the comparison of several outcomes, showing another limitation of this analysis. In addition, the fact that patients with diabetes mellitus, multi-vessel coronary diseases and left main coronary artery diseases were combined and analyzed could represent another limitation of this study since this combination of patients with different co-morbidities could also have had a major impact on the results.Significant differences were noted in several subgroups analyzing the mid-term cardiovascular outcomes associated with CABG and PCI with EES. Mortality and stroke were not significantly different between these two revascularization procedures. However, PCI with EES was associated with significantly higher MACEs, MI and repeated revascularization when compared to CABG. Unfortunately, due to limited data, which could have affected the results of this analysis, further studies with larger population size and longer follow up periods are recommended to completely solve this issue.How to cite this article: Bundhun, P. K. et al. Adverse Cardiovascular Outcomes associated with Coronary Artery Bypass Surgery and Percutaneous Coronary Intervention with Everolimus Eluting Stents: A Meta-Analysis. Sci. Rep.6, 35869; doi: 10.1038/srep35869 (2016).


Nosema ceranae/fipronil combination on the life traits and physiology of mature drones were examined following exposure under semi-field conditions. The results showed that the microsporidia alone induced moderate and localized effects in the midgut, whereas fipronil alone induced moderate and generalized effects. The parasite/insecticide combination drastically affected both physiology and survival, exhibiting an important and significant generalized action that could jeopardize mating success. In terms of fertility, semen was strongly impacted regardless of stressor, suggesting that drone reproductive functions are very sensitive to stress factors. These findings suggest that drone health and fertility impairment might contribute to poorly mated queens, leading to the storage of poor quality semen and poor spermathecae diversity. Thus, the queens failures observed in recent years might result from the continuous exposure of drones to multiple environmental stressors.The honey bee is threatened by biological agents and pesticides that can act in combination to induce synergistic effects on its physiology and lifespan. The synergistic effects of a parasite/pesticide combination have been demonstrated on workers and queens, but no studies have been performed on drones despite their essential contribution to colony sustainability by providing semen diversity and quality. The effects of the  Similar impairments have been observed more broadly in wildlife, including vertebrates and invertebrates9. In insects, reproductive disorders induced by environmental stressors can result in fertility decline21, a change in reproductive behavior, such as choice of sexual partner(s) and mating success23, and diverse effects on offspring production and physiology29.Through reproduction, sexual species have the ability to respond to environmental stressors in their habitats by producing adapted offspring. However, pollutants and biological agents might threaten sexual reproduction by impairing fertility. For instance, in humans, infectious agents such as fungi, bacteria and viruses31. For instance, potential drivers of pollinator loss  could result in weakening of pollination services in ecosystems, resulting in significant negative ecological (ex.: wild plant diversity) and economic (ex.: crop production) impacts33. Among various stressors, the combined effects of pollutants and simultaneous infection by parasites on host health are of primary concern31. Depending on the stressor combination, the joint effects can be antagonistic, additive, synergistic or potentiating. A synergistic interaction is defined as a combination of stressors that results in a greater effect than the cumulative expected effect from independent exposure34. The synergistic interaction of chemicals combined with natural stressors, such as pathogens, has been studied in aquatic organisms, such as Daphnia and Artemia35, and, more recently, in the honey bee43. Co-exposure might induce additive or synergistic effects, as shown in honey bee workers41 and queens42 following exposure to Nosema Ceranae microsporidia and several insecticides. However, the drone caste has not been examined despite evidence that reproductive disorders can lead to species weakening. These disorders have rarely been suggested to explain the pollinator decline that has been observed worldwide, particularly in the decline of bee populations exposed to stressors44. The honey bee, Apis mellifera (Hymenoptera: Apidae), is a valuable host model to study the ecological and evolutionary dynamics of host/parasite/pesticide interactions in eusocial insects because of its economic value, genetic diversity, available data on its biology and parasites, and pioneer studies on honey bee/parasite/pesticide interactions.The effects of numerous stressors on the health of organisms have gained increasing interest because of possible impacts not only on animal health but also on ecosystem services46, has a singular mode of reproduction that uses a haplodiploid sex-determination system and polyandry. Thus, at an early stage of its life, a young diploid queen mates with up to 20 haploid drones in specific sites called drone congregation areas, where fierce competition occurs due to a highly male-biased sex ratio48. Following mating, a small proportion of the spermatozoa received from each drone migrates into spermatheca to be used by the queen to fertilize eggs throughout her life48. The diversity of sperm stored, a result of the polyandry, enables high intra-colonial genetic diversity that enhances survival, fitness, productivity and resilience to environmental disturbances51. Moreover, sperm diversity plays an important role, but sperm quality is also extremely important because drone semen quality determines spermathecal content, which is closely linked to queen reproductive potential20. Thus, a drone is a key individual at the origin of a cascade of events required for colony growth and sustainability. However, although drones are key to colony health, the effects of drone exposure to environmental stressors have been poorly investigated. Some studies, mostly focused on the effects of Varroa mites and mite treatments, have shown that drone survival54, fertility55 and mating behavior12 are impacted. More recently, it has been shown that other environmental stressors, such as the microsporidium Nosema apis18, and systemic insecticides, such as neonicotinoids56 and fipronil20, might also affect drone survival or fertility.The honey bee, a species reported to be declining in the Northern hemisphereNosema ceranae, which is suspected to be a major cause of honey bee decline in Southern Europe57, or the insecticide fipronil, which is known to impair drone fertility58,  contamination with the 50,000 spores of the parasite Nosema ceranae could induce synergistic effects in male honey bees, as previously reported in workers38. In our study, the effects on drones were assessed with an original approach examining drone fertility, life traits and primary physiological functions  using a battery of physiological markers in different biological compartments  revealed a parasite load of approximately 30 × 106 spores per individual for the N. ceranae and N. ceranae/fipronil stressors , in contrast to the sperm contamination observed in drones of a similar age exposed to N. apis18.The survival and sexual maturity rates of drones, the semen volume per drone, the spermatozoa concentration in semen and the level of ect Fig. . Moreoveect Fig. , the speely Fig. , correspVarious physiological markers were tested in three biological compartments . These markers are linked to one or more key physiological functions related to the immune system (glucose oxidase (GOX), phenoloxidase (POX) and alkaline phosphatase (ALP)), the xenobiotic detoxification system (glutathione-S-transferase (GST) and carboxylesterases 1, 2 & 3 (CaEs)), antioxidant defense , catalase (CAT), glutathione peroxidase (GP), glutathione reductase (GR)), neural activity (acetylcholinesterase (AChE)) or metabolism , lactate dehydrogenase (LDH), adenosine triphosphate (ATP), CaE-1, CaE-2, CaE-3, GST)  and CAT (P = 0.025) (Table P = 0.017), whereas LDH, GP, POX and CaE-3 increased . Finally, the changes observed from each stressor alone were also observed for the N. ceranae/fipronil combination except for ALP. In the midgut, compared with each stressor alone, the changes in CAT and CaE-3 were enhanced by the combination of stressors, which appeared to generate specific effects in comparison with the controls . Moreover, it appears that the combination of the stressors exerted specific effects that were significantly different from what was observed in the controls (GA3PDH (P = 0.038), ATP (P = 0.017), GST in head (P = 0.045), GST in midgut (P < 0.0001), CAT in head (P = 0.049), and GR (P = 0.01)). However, with the exception of GST in the midgut, for which the combination induced a synergistic effect, for the other parameters, the combination of the stressors did not elicit effects different from those induced in the head by either N. ceranae  or fipronil . Interestingly, GST was increased in the midgut and decreased in the head, indicating tissue-dependent regulation of the synergetic effect.In drones infected with P P = 0.08 and CATN. ceranae/fipronil). Principal component analysis (PCA) provides a visual representation of the physiological states of drones exposed to different stressors , although a trend toward positive CAT modulation was observed in the spermatozoa  in comparison with the control. Finally, for the N. ceranae/fipronil interaction, a significant positive change was observed only for CAT in spermatozoa  in comparison with the control. However, positive tendencies for Red Pot  for antioxidant ability and LDH, ATP and reducing potential (Red Pot) for metabolism)  were grouped. Interestingly, N. ceranae samples maintained heterogeneous distribution in the total cluster, which has been previously observed  to respond to biotic and abiotic stress factors or combinations of these factors. Thus, effects of the N. ceranae/fipronil combination on the drones were investigated from the physiological to the phenotypic level following the exposure of drones, in a semi-controlled environment, from emergence to sexual maturity.In ecosystems of the biosphere, each environmental stressor has the ability to disturb organisms from the molecular to population level. The effects can range from small cellular changes to the death of individuals and might include behavioral and reproductive disorders. However, in ecosystems, organisms are seldom exposed to only one abiotic or biotic stressor and are often exposed simultaneously to several environmental stressors. In combination, these stressors can elicit different types of effects, including additive, synergistic and antagonistic effectsN. ceranae and very important and widespread for the N. ceranae/fipronil  previous studies showing the ability of fipronil to alter the cognitive functions of workers80, (ii) AChE changed in drones  (Table iii) the weakening of individuals, revealed by the generalized physiological disturbances (Table N. ceranae/fipronil), the effects induced by N. ceranae and fipronil alone were still observed and tended to be increased in some cases. In addition, 6 new markers were affected, confirming the specific effects of the interaction in comparison with the controls, which could lead to a significant synergetic effect, as observed for midgut GST  suggests that reproductive functions are more sensitive to stressors than other studied functions. Regardless of stressor, fertility impairment is observed at the phenotypic level, with a decline in sperm concentration of approximately 20%, whereas the maturity and semen volume of the drones are not affected  on the males of a eusocial insect species. Similar effects have been shown on the survival and physiology of drones but not on their fertility. Indeed, the results have shown effects on sperm quality regardless of stress factor applied . Consequently, the high sensitivity of fertility parameters suggests that reproduction could be one of the first functions of drones affected in stressful conditions. As a result, stresses exerted alone might not affect the vitality and survival of individuals but could have many effects on reproductive functions. All of the changes observed in the drones or their fertility could have serious consequences for the life cycle of an A. mellifera colony. Even if the physical integrity of the drone was preserved, allowing him normal behavior and unchanged mating success, damage would result by transmitting poor quality semen, affecting not only queen performance but also the offspring. If the physiology of drones is strongly impacted to jeopardize their survival, mating performance might be compromised and lead to a shortage of healthy males in congregation areas. This might lead to poorly mated queens related to decreased selection pressure or a potential loss of genetic diversity, which would be largely detrimental to the species. This study highlights the need to study the effects of stress factor combinations on fertility problems in the honey bee as one of the mechanistic explanations for queen failure. This study also highlights the need for a multi stressor approaches when studying reproductive disorders affecting many species in the biosphere.In conclusion, this study is the first in the available literature to show specific effects of an insecticide/pathogen combination (N. ceranae under semi-field conditions in Avignon (South France) between early June and late July of 2012. To perform this experiment and control drone age, 15 queens were previously encaged within 15 hives on drone combs for laying. Twenty-four days after laying, newly emerging drones from these 15 rearing hives were homogenously mixed before being introduced to the experimental hives. Thus, the control and treated hives contained drones of same age and genetic diversity. The drones were then reared from emergence to sexual maturity for 20 days in sixteen queenless colonies, which are known to take better care of drones83. These queenless colonies were placed in compartmented tunnels (two hives/compartment), covered with an insect-proof net to control the foraging of food. Homogeneous batches of 300 drones were cloistered in these queenless colonies containing 5000 workers, 1 brood comb and 5 empty combs for food storage, according to a previously described protocol84. Drones, worker bees and brood combs needed for the study were obtained from honey bee colonies that were monitored for their sanitary status and specially checked for the absence Nosema spp. infection.Drones were exposed to the insecticide fipronil and/or the pathogen N. ceranae/fipronil interaction on drones, 4 experimental groups of 4 queenless colonies each were established: the control group (Ctrl), a group infected with N. ceranae (Nos), a group exposed to fipronil (Fip) and a group both infected with N. ceranae and exposed to fipronil (Nos + Fip). Before introduction to queenless colonies, emerging drones were individually fed 2 µL sugar syrup  with or without 50,000 spores of N. ceranae, which is a realistic infectious load85  was provided daily from 8:30 a.m. to 11:30 a.m. Crushed pollen and water were available the rest of the time20. Thus, drones were chronically exposed to the insecticide in the hive via food gathered by foragers mimicking natural exposure conditions. Twenty days after introduction, the surviving drones were caught  the protocol required the individual feeding of 4800 newborn drones to expose them to the microsporidium, (ii) fertility and physiological parameters required a large volume of semen, and a mature drone produces less than 1 µL, and (iii) some parameters, such as survival rate, can be measured only at the colony level.In this study, it was difficult to perform a large number of measurements because  in Kiev solution 20 and counted in triplicate. Considering that spermatogenesis has been initiated at time of drone emergence and that the number of sperm is fixed before the exposure period considered in this study, the spermatozoa concentration is assessed as a parameter for evaluating the cytotoxicity of stressors in sperm58. For each of these previous parameters, one measure per colony was obtained and four colonies were investigated . To determine the level N. ceranae infection in 20 day-old drones, 3 samples of 5 drones per hive were analyzed . Briefly, abdomens were crushed in distilled water, and the resulting suspension was filtered. The spore concentration of the suspension was determined using a Malassez counting cell36.After 20 days of exposure in the hive, drone life traits such as survival, maturity, infection with ® F500 plate reader from TECAN   Fig. .−1 antipain, leupeptin and pepstatin A, 25 units.mL−1 aprotinin and 0.1 mg.mL−1 soybean trypsin inhibitor), 40 mM sodium phosphate, pH 7.4] to obtain a 10% (w/v) extract88. Tissues were homogenized with a Tissue-Lyser II (Qiagen®) homogenizer for 5 periods of 10 s at 30 Hz separated by an interval of 30 s. After 10 min, the homogenization procedure was repeated a second time. The homogenate was centrifuged for 20 min at 15000 g, and the supernatant was used to analyze enzymes activities and non-enzymatic compounds in the biological compartments. For enzymatic assays in semen, the semen was diluted in an equal volume of Kiev solution and centrifuged for 20 min at 16000 g to separate spermatozoa and seminal fluid89. The supernatant, corresponding to seminal fluid, was recovered to measure LDH and SOD activities. The pellet, corresponding to spermatozoa, was rinsed twice by suspension in Kiev solution, centrifuged and recovered. Then, the pellet was diluted 10% (v/v) in lysis buffer to lyse the spermatozoa. The cellular debris were removed by centrifugation for 15 min at 15000 g before measuring CAT activity. All of the procedures were performed at 4 °C. Unlike enzymatic activity measurements, the ATP content and the reducing potential were measured in non-separated semen at room temperature.For assays in the head, midgut and abdomen, the tissue from 6 drones was homogenized in lysis buffer [10 mM NaCl, 1% (w/v) Triton X-100, protease inhibitors (2 µg.mL3PO4) pH 7.0 according to the method of Ellman et al.90 as modified by Belzunces et al.88.AChE is a neural enzyme involved the controls nerve transmission by hydrolyzing the neurotransmitter acetylcholine in the synapse. Head AChE activity was determined, spectrophotometrically at 412 nm in reaction medium containing 0.3 mM acetylthiocholine iodide (AcSCh.I), 1.5 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and 100 mM sodium phosphate (Na2O2), which has antimicrobial properties. Excreted in honey by salivary glands, this enzyme is involved in the social immunity of honey bees. Head GOX was assayed by assessing the oxidation of o-dianisidine by H2O2 at 430 nm. The reaction medium contained 100 mM glucose, 2.5 Units/200 µL peroxidase, 0.3 mM o-dianisidine and 125 mM potassium phosphate pH 7.036.GOX catalyzes the oxidation of D-glucose into D-gluconic acid and hydrogen peroxide (H−1 L-DOPA and 10 mM sodium phosphate (NaH2PO4) pH 7.236.POX plays a role in the individual immunity of insects through the melanization process involved in tissue regeneration and encapsulation of foreign bodies, such as pathogens. Abdomen POX activity was determined by following the conversion of 3,4-dihydroxy-L-dihydroxyphenylalanine (L-DOPA) into melanin at 490 nm. The reaction medium contained 20 mM NaCl, 0.4 mg.mL2), 2 mM p-NPP and 100 mM Tris-HCl pH 8.591.ALP plays an important role maintaining the homeostasis of gut tissues due to its involvement in many metabolic processes linked to immune responses. The midgut PAL dephosphorylating activity was monitored by the conversion of p-nitrophenyl phosphate p-NPP) into p-nitrophenol at 410 nm. The reaction medium contained 20 µM magnesium chloride , β-naphthyl acetate (β-NA) or p-nitrophenyl acetate (p-NPA), respectively. The reaction medium contained 0.01 mM acetylcholinesterase inhibitor BW284C51, 0.1 mM α-NA, β-NA or p-NPA and 100 mM sodium phosphate (NaH2PO4) pH 7.0. For CaE-1 and CaE-2, catalysis proceeded for 1 min and was stopped by adding 0.2 reaction volume of a solution containing 10% sodium dodecyl sulfate (SDS) and 4 mg.mL293.ATP, the energy fuel of the cell, is also a coenzyme involved in numerous metabolic reactions. ATP content in the head, midgut and semen was determined using ATPlite kit (PerkinElmer) based on luminescence measurements produced by the oxidation of D-luciferin by luciferase that involves 1 molecule of ATP and O84.The Red Pot corresponds to the ability of cells to reduce compounds linked to metabolic activity. The Red Pot in semen was assessed using a Prestoblue kit (Invitrogen). The assay was based on the reduction of the cell permeable compound resazurin to a red fluorescent resorufin. The absorbance of resorufin was measured at 570 nm+) into 1,3-biphosphoglyceric acid  and the reduced form of nicotinamide adenine dinucleotide (NADH) during glycolysis and plays an important role in energetic metabolism. GA3PDH can also catalyze the reverse reaction when gluconeogenesis overtakes glycolysis. GA3PDH activity was determined in the head with the reverse reaction using 3-phosphoglyceric acid (3-PGA), which is converted by phosphoglycerate kinase (PGK) into 1,3-BPG. 1,3-BPG is converted to GA3P in the presence of NADH whose oxidation was followed at 340 nm. The reaction medium contained 7 mM 3-PGA, 4 mM L-cysteine-HCL neutralized with sodium bicarbonate (NaHCO3), 2 mM magnesium sulfate (MgSO4), 120 µM NADH, 1.2 mM ATP, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 units.mL−1 3-phosphoglycerate kinase (3-PGK), 80 mM triethanolamine buffer pH 7.043.GA3PDH reversibly catalyzes the conversion of glyceraldehyde-3-phosphate (GA3P) in the presence of nicotinamide adenine dinucleotide (NAD+) into 6-phosphogluconolactone and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). Head G6PDH activity was determined by continuously following the formation of NADPH at 340 nm. The reaction medium contained 100 mM Tris-HCl buffer at pH 7.4, 10 mM MgCl2, 1 mM G-6-P, 0.5 mM NADP+ and 100 mM Tris-HCl pH 7.443.G6PDH is a key enzyme of the pentose phosphate pathway that generates NADPH, which contributes to the regeneration of reduced glutathione (GSH) involved in the defenses against oxidative stress. This metabolic pathway also contributes to the biosynthesis of nucleotides, amino acids and some fatty acids involved in cell metabolism. G6PDH catalyzes the conversion of glucose-6-phosphate (G-6-P) in the presence of the oxidized form of nicotinamide adenine dinucleotide phosphate . Thus, GST plays a role in xenobiotic detoxification, antioxidant defense and metabolic regulation. GST activity in the head and midgut was determined by measuring the conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm. The reaction medium contained 1 mM EDTA, 2.5 mM GSH, 1 mM CDNB and 100 mM Na/K phosphate pH 7.42.−) into hydrogen peroxide (H2O2) to limit oxidative stress. SOD activity was indirectly measured in seminal fluid using the xanthine/xanthine oxidase system to generate O2.− and nitro blue tetrazolium (NBT). SOD competes with xanthine oxidase and limits the generation of reduced NBT, which was followed at 560 nm. The reaction medium contained 0.1 mM EDTA, 0.1 mM xanthine, 0.025 mM NBT, 0.00833 U/mL xanthine oxidase, and 50 mM phosphate/carbonate pH 7.884.SOD converts the superoxide anion . Thus, GP contributes to the regulation of reactive oxygen species (ROS) involved in oxidative stress. Head GP was assayed using tert-butyl hydroperoxide (TBHP) as the substrate. The generated GSSG was reduced by glutathione reductase (GR) in the presence of NADPH to generate GSH and NADP. The conversion of NADPH in NADP+ was followed at 340 nm. The reaction medium contained 1 mM EDTA, 0.2 mM TBHP, 0.85 mM GSSG, 0.16 mM NADPH, 0.25 U/mL GR and 50 mM Na/K phosphate pH 7.468.The GP catalyzes the destruction of peroxides, such as H+. The reaction medium contained 1 mM EDTA, 0.85 mM GSSG, 0.16 mM NADPH and 50 mM Na/K phosphate pH 7.468.As described above, GR enables the regeneration of GSH involved in the regulation of oxidative stress. Head GR was followed at 340 nm by the conversion of NADPH into NADP2O2 into oxygen (O2) and water (H2O) to protect cells against oxidative stress. The decomposition of H2O2 by CAT was followed in head, midgut and spermatozoa at 240 nm. The reaction medium contained 30 mM H2O2 and 100 mM sodium phosphate pH 7.096.The CAT catalyzes the decomposition of Hi.e., drone survival rate, sexual maturity rate, semen volume per drone, spermatozoa concentration in semen and physiological markers in semen; n = 4 per modality of treatment) used a post-hoc Wilcoxon test because the small number of data points required a non-parametric test. Parameters for which several measures per hive were performed  used a generalized linear mixed model with random effect on the hive from which drones came. These statistical analyses were performed using the package “lme4” in R software97. Bars with the same letters indicate no significant difference between the groups. Bars with different letters indicate a significant difference at the 0.05 level between groups.Parameters for which one measure per hive were performed  for analyzing and visualizing data99. The distance measure used was Euclidian distance, with UPGM as the linkage rule for clusters. For the latter approach, data normalization was required to convert each measure to the rate of variation compared with the average of controls. These analyses were performed considering physiological marker responses, first in drone and then in drones plus semen.The effects of different treatments on drone physiology were described with 2 complementary approaches. A principal component analysis (PCA) was performed using the package “ade4” in the R software