Group II revealed a shorter embryo/blastocyst development period between microinjection and 5-cell stage compared to group I. The difference in SOD concentration between groups I and II and between groups II and III was statistically significant. In group II, 34.62% of pregnancies were obtained, whereas in group I this number reached 20% (NS). Myoinositol increased embryo development dynamics and accelerated blastocyst stage reaching time; however, no effect was shown on clinical pregnancy. Furthermore, it restored SOD concentration, lowered in PCOS women, but did not exert any effect on CAT concentration.The aim of this study was to investigate the activity of myoinositol, in a court of 217 PCOS women undergoing intracytoplasmic sperm injection (ICSI), on pregnancy rate, embryo development, estradiol, and progesterone concentration in blood serum, superoxide dismutase (SOD), and catalase (CAT) in follicular fluid. Concerning the court of patient, 112 (groups I and II) out of 217 were PCOS women, whereas group III consisted of healthy subjects (not PCOS). Group I patients were treated with 400  Polycystic ovary syndrome (PCOS) is the most commonly reported ovulation disorder in women –3. It isSuccess of IVF depends on multiple factors  and espeAn antral follicle constitutes a microenvironment, where ovum develops, which simultaneously influences the future quality of embryo. Oxidative stress plays an important role during folliculogenesis and oogenesis, although our knowledge related to its effects remains still insufficient . It is aAs far as PCOS is concerned, it was possible to confirm several degrees of insulin resistance reported in patients, which could also influence fertility treatment success –3, 9, 10The primary outcome of this study was to evaluate the influence of myo-Ins on the pregnancy rate and on the dynamics of embryo development in PCOS patients undergoing ICSI. As secondary outcome, our study aimed at testing the myo-Ins effects on oestradiol and progesterone concentration in blood serum, as well as on SOD and CAT concentration in the follicular fluid of these patients. We considered not only the well-known mechanisms of action, proper to myo-Ins, based on the improvement of insulin and FSH signalling, but also their possible activity as antioxidant molecule.This retrospective study was carried out on the data of patients undergoing ART in 2013 and 2014 at the Ovum Fertility Treatment Centre in Lublin. The study covered 217 women treated for infertility by means of intracytoplasmic sperm microinjection (ICSI) technique. All patients were qualified to undergo ICSI due to a moderate, masculine infertility factor that makes it impossible to conduct classic IVF. A total of 198 from treated pairs had 4 to 6 previous intrauterine inseminations during the last 1-2 years that did not lead to pregnancy, and in the case of 19 women we have reported bilaterally blocked fallopian tube. For the subjects enrolled into the PCOS groups, the inclusion criteria required that such patients had to meet the Rotterdam criteria (2004). Women constituting the studied group were between 27 and 35 years old; they all had FSH < 10 IU/mL and appropriate AMH (Anti-Müllerian hormone) value. The exclusion criteria were the following: presence of severe endometriosis, BMI < 17 and >30, and metabolic diseases as well as lowered ovarian reserve.The study obtained a consent of Bioethical Committee at the Institute of Rural Health in Lublin. All patients signed an informed consent before participating to the study.All patients were treated with the ICSI procedure, taking advantage of fresh oocytes and fresh sperm.2) (pg/mL) and progesterone (ng/mL) levels were detected in the morning. These two parameters were determined when the largest oocyte in the evaluation of ultrasound exceeded 17 mm diameter. The puncture was conducted 36 hours after administering recombinant HCG (r-hCG) (Ovitrelle: Merc-Serono). A total of 52 patients (group II) among the PCOS subjects were administered with 4000 mg of myo-Ins and 400 μg of folic acid (folate)  for 3 months before undergoing ICSI. The other PCOS subjects were treated with 400 μg of folic acid alone.GnRh analogues (Diphereline: Ipsen Pharma) and FSH recombinants  were used in short protocols to stimulate ovulation from the 3rd cycle day (to a maximum of the 17th cycle day). In the day of implementing ovulation induction, the oestradiol , whereas CATs were detected with the Catalase Assay Kit (Sigma-Aldrich) according to the manufacturer instructions. The final level of the SOD activity and CAT activity was reported as a unit of enzymatic activity per protein mg (mIU/mg).μL drops of Cleavage medium  under mineral oil in an automatic 5% CO2 incubator at 37°C until the second day (stage of 2–5 cells). Fifty hours after the ICSI, the culture medium was changed with blastocyst medium .Oocytes were separated from cells in the granular layer, which was followed by the ICSI procedure 3 hours after ovarian puncture. The inseminated cells were grown in 25 t0, tF, and tC times were defined as the hour of the ICSI, the first moment when pronuclei became visible, and the last moment of their visibility, respectively. The moment when a single cell embryo appeared after syngamy was defined as t1, and then the superseding divisions as t2, t3, t4, t5, t6, t7, and t8. The beginning of morula formation was called tM, whereas tB is when the first signs of blastocyst cavity could be seen. Blastocysts were evaluated according to criteria declared by ASRM and ESHRE, and only one of them was transferred in order to avoid multiple pregnancy. During the 7th week of pregnancy, the echo of the embryo and heart rate were evaluated by means of ultrasound examination.Embryo culture was evaluated by means of constant monitoring performed in 10-minute intervals with a camera placed inside the incubator. During all the observation period, the embryos remained in the incubator. The The measurable parameters were shown as mean and standard deviation, whereas the nonmeasurable ones were presented as numerical amount and percentage.2 test was utilized to detect any existing differences. The ANOVA variance analysis was used to test differences among groups, whereas R Pearson's correlation was used to verify dependence between embryo development times and selected parameters. The difference was considered statistically significant at p < 0.05. The database and statistical tests were conducted using Statistica 9.1 programme .For qualitative features, a Chin = 112) and group with healthy patients (n = 105). In the first group, myo-Ins was administered to 52 women (treated group), whereas 60 were treated with 400 μg of folic acid alone (control group). Both treatments were daily and lasted for 3 months before ICSI. Among the healthy subjects , we obtained 33.33% of pregnancies, whereas among the PCOS women this value reached 26.79%. These differences were not statistically significant   (Patients were divided into the following groups: group with PCOS women (= 0.293) . However 0.0810) .tF  between group I and group III and tC  between group I and group II, as well as groups I and III. Of note, statistically significant differences were observed between group I and group II, as well as group I and group III, in the following times: t1 , t2 , t3 , t4 , t5 , and finally tB , as shown in When we compared the embryo development times between the groups of PCOS controls (group I), PCOS patients treated with myo-Ins (group II), and healthy subjects (group III), statistically significant differences were observed: F = 6.558, p = 0.002), whereas in the same day progesterone did not show significant differences. The concentration of SOD in follicular fluid revealed statistically significant differences among the three groups, namely, between group I and group II as well as between group II and group III . On the contrary, no significant differences were observed for CAT in follicular fluid , t4 , t5 , and tB . In the above-mentioned times, the increase in oestradiol level exerted a considerable influence on embryo developmental dynamics; however, no effect was observed in the remaining times. In group II, similar negative correlations concerned E2 and t1 , t3 , t4 , and t5 . In the other times no such statistically significant correlations were detected. Group III showed negative correlations only between E2 and the following times: t4 , t6 , and tB . In group I a positive correlation between progesterone level and t2  was found, whereas in group II negative correlations between progesterone level and tF  and t5  were reported, and in group III a negative correlation between progesterone level and t8  was reported, next to a positive correlation with tB . No other statistically significant correlations concerning progesterone level appeared. In PCOS patients treated with myo-Ins (group II), researchers observed a negative correlation between SOD levels in follicular fluid and the following times: tF , tC , t1 , t2 , t3 , t4 , t5 , t9 , and tB . In the other times, no significant dependencies were observed. Moreover, in these subjects no significant correlations between SOD and embryo developmental times were discovered. In healthy patients a negative correlation was observed for the same parameter only with tM . In group I positive correlations were observed between CAT levels and t5  as well as tB , and nothing else. In group II we can see positive correlations between the activity of CAT and tF  and t1 , next to t5 . In group III positive correlations between CAT concentration and t1 , t2 , t3 , and t4  were noted, without other significant correlations  and Myo-ins with 30 rFSH obtained from 30 patients and reported a lower oestradiol concentration in myo-Ins group, which is not compliant with results obtained by Gerli et al. and the results obtained in our study , 13. TheChiu et al. conducted studies on the group of 53 female patients taking myo-Ins during stimulation before undergoing IVF and they observed a lowered level of myo-Ins in follicular fluid collected from samples with immature oocytes . Their sSeleem et al. compared SOD concentration in follicular fluid of 20 PCOS patients and in a group of 20 healthy patients during the ICSI procedure, and they reported statistically significant higher SOD levels in the group without PCOS, in accordance with our results .2 values on increasing the embryo development times; however, they used a different method of statistical analysis and a diverse study model  to analyse their results. In this way, it was impossible to unambiguously relate their results. Similarly, to our findings, Muñoz et al. reported that progesterone concentration in blood serum exerts an influence on the dynamics of embryo development only to a slight extent.In the studies conducted by Muñoz et al., 774 IVF cycles were analysed by means of the technology based on observing embryos in real time, as we made in our study . AuthorsStudies devoted to fertility treatment in PCOS patients require further tests, using more numerous groups of patients and with more standardised diagnostic techniques to understand in-depth some specific points; however, the studies so far performed have confirmed the validity of using myo-Ins in ART with PCOS patients.The use of myo-Ins before and during IVF stimulation increases the dynamics of embryo development in the first two days of culture and reduces the amount of time required to achieve the blastocyst stage. However, the influence of these results in the number of clinical pregnancy was not confirmed.The concentration of superoxide dismutase in follicular fluid is lower in PCOS women when compared with healthy patients; however, when PCOS women are treated with myo-Ins, this concentration reaches values that are observed among healthy women. The effect of oestradiol concentration in serum, as well as the activity of SOD in follicular fluid, increases the dynamics of embryo development. On the other hand, myo-Ins did not have any influence on CAT activity in follicular fluid. SOD increase may be explained by means of the antioxidant activity exerted by myo-Ins, whereas its inefficacy on the rise of CAT levels should be investigated in-depth. The results we obtained confirm also the necessity to conduct further studies focusing on myo-Ins therapy.


Aim. To compare the differences between medial and intercondylar infragenicular femoropopliteal prosthetic bypasses in terms of their midterm patency and limb salvage rates. Methods. Ninety-three consecutive patients with peripheral arterial disease who underwent a simple distal femoropopliteal bypass using a reinforced polytetrafluorethylene graft were included in this retrospective study. The bypass was constructed in the intercondylar route in 52 of the patients (group A) and in 41 in the medial route (group B). Results. Median observation time of the patients was 12.7 (IQR 4.6–18.5) months. There were 22 and 24 interventional or surgical procedures  performed to restore patency of the reconstruction in groups A and B, respectively (p = 0.14). The 20-month primary, assisted, and secondary patency rates and limb salvage rates were 57%, 57%, 81%, and 80% in group A compared to 21%, 23%, 55%, and 82% in group B . Conclusion. Despite better primary, assisted, and secondary patency rates in patients with a prosthetic infragenicular femoropopliteal bypass embedded in the intercondylar fossa compared to patients with the medial approach, there is no benefit in terms of the limb salvage rate and the number of interventions required to maintain patency of the reconstruction. Femoropopliteal bypass surgery has been, for a long time, the most common infrainguinal reconstruction procedure in vascular surgery. Although the saphenous vein is usually the preferred conduit due to its superior patency rates, in around 20–40% of patients, it is simply not available for bypass in sufficient quality or length , 2. PrevIn the infragenicular femoropopliteal bypass, the distal part of the graft can be either embedded subcutaneously on the medial side of the knee or placed dorsally between the femoral condyles. However, little data exists to compare midterm performance of both approaches, which is influenced by numerous factors, including the outflow bed, type of the graft, anastomosis angle, and compliance mismatch –9.In the present study, we compared the midterm primary, assisted, and secondary patency rates and limb salvage rates of the infragenicular femoropopliteal bypasses between patients with the intercondylar or medial course of the bypass.This retrospective study was approved by the institutional review board  and informed consent was waived. The research was performed in accordance with the Declaration of Helsinki. Between July 2009 and September 2015, 93 consecutive patients with peripheral arterial disease stage IIb to stage IV according to the Fontaine classification who underwent a simple distal femoropopliteal bypass  using a reinforced polytetrafluorethylene (PTFE) prosthesis were included in the study . The bypThe surgical procedures were performed by three consultant vascular surgeons. In all the patients, a reinforced vascular PTFE prosthesis either VascuGraft SOFT  or fusion vascular graft  with a diameter from 6 to 8 mm to match the diameter of the popliteal artery was used .The proximal anastomosis was always connected to the common femoral artery and the distal anastomosis to the popliteal artery, below the knee joint space. If the subcutaneous layer on the medial side of the knee was sufficiently thick , the bypass was routed there (group B), with subsequent diversion towards the popliteal artery below the knee . In the For antimicrobial prophylaxis, four doses of 1.5 g ampicillin/sulbactam  were given intravenously 8 hours apart. Postoperatively, all patients received acetylsalicylic acid  and low-molecular-weight heparin . After mobilization and removal of drains , patients continued with dual antiplatelet therapy ) only, except for those who had received anticoagulation therapy preoperatively and needed reintroduction of the oral anticoagulant treatment.All patients were discharged with statin  and dual antiplatelet therapy or, in case of anticoagulant treatment, with warfarin  and acetylsalicylic acid (100 mg daily).Follow-up examinations were scheduled approximately 1, 3, 6, 12, and 24 months postoperatively, unless clinical problems occurred. Apart from clinical examination, patency of the reconstruction was examined by ultrasound. CT angiography or digital subtraction angiography was indicated if the patient's complaints or clinical examination suggested stenosis or occlusion. In case of restenosis or occlusion, angioplasty, stenting, thrombolysis, thrombectomy, or correction of the anastomosis was performed according to interdisciplinary consent based on the patient's complaints and imaging findings. During follow-up, primary, assisted, and secondary patency limb salvage (preserved foot) and mortality were recorded.t-test, Mann-Whitney U test, or Fischer F-test as appropriate. Life tables were compared using the log rank test. A p value below 0.05 was considered significant.Statistical tests were performed using MedCalc ver. 12 . To test for statistical significance, we used p = 0.38) and 6 days  in group B  min and 6 days (IQR 5–10), respectively, in group A, compared to 130 (IQR 102–160) min . Comparison of primary, assisted, and secondary patency, limb salvage rates, and survival is shown in p = 0.14).Median observation time of the patients was 12.7 (IQR 4.6–18.5) months. The 20-month primary, assisted, and secondary patency rates and limb salvage rates were 57%, 57%, 81%, and 80%, respectively, in group A compared to 21%, 23%, 55%, and 82% in group B (Since the introduction of a prosthetic femoropopliteal bypass, there has been ongoing research into improvement of its patency, which has rarely matched that of reconstructions with autologous grafts , 11. MajIn this study, we retrospectively evaluated midterm results of an infragenicular prosthetic femoropopliteal bypass with regard to its route in the knee region. The patency rates observed in group A were in accordance with a meta-analysis by Takagi et al.  that incIn theory, we assumed that a medial approach in group B would result in a less acute angle, because the graft needed to pass from the medial side of the knee to the dorsal side below the knee to attach to the popliteal artery , 9. ThisAlthough the medial route would be generally preferred, we believe that embedding a bypass medially if the subcutaneous layer is thin may carry an increased risk of bypass infection, compression against bony structures, and wound complications. Conversely, the intercondylar route possesses an increased risk of injury to the structures of the neurovascular bundle and the surrounding venous plexus in particular. In the infragenicular prosthetic femoropopliteal bypasses, we rarely use patch or cuff techniques because their advantage is controversial, with their construction additionally requiring about 15 minutes of operation time . In our The most important limitations of this study include a relatively short follow-up time, small number of patients in each group, and a lack of randomization because the study is retrospective. To overcome this limitation, a randomized trial with exclusion of patients with thin subcutaneous layer on the medial side of their knee would be necessary. Considering the event rate of 15% for limb amputation at 30 months, with hazard ratio of 1.5, such study would require randomization of 550 patients and a multicenter design to achieve 80% power with 5% significance level. To demonstrate the difference in the anastomosis angle, its perioperative measurement or early postoperative imaging would also be required.Despite better primary, assisted, and secondary patency rates in patients with a prosthetic infragenicular femoropopliteal bypass embedded in the intercondylar fossa compared to patients with the medial approach, there is no benefit in terms of the limb salvage rate and the number of interventions required to maintain patency of the reconstruction.


In The Mood website. Mood responses in three samples  were analyzed using agglomerative, hierarchical cluster analysis, which distinguished six distinct and theoretically meaningful profiles. K-means clustering further refined the final parameter solution. Mood profiles identified were termed the iceberg, inverse iceberg, inverse Everest, shark fin, surface, and submerged profiles. Simultaneous multiple discriminant function analysis showed that cluster membership was correctly classified with a high degree of accuracy. Chi-squared tests indicated that the six mood profiles were unequally distributed according to the gender, age, and education of participants. Future research should investigate the antecedents, correlates and consequences of these six mood profile clusters.Mood profiling has been a popular assessment strategy since the 1970s, although little evidence exists of distinct mood profiles beyond the realm of sport and exercise. In the present study, we investigated clusters of mood profiles derived from the six subscales of the Brunel Mood Scale using the  Mood has been described as “a set of feelings, ephemeral in nature, varying in intensity and duration, and usually involving more than one emotion”  spread over three samples. Sample A included 2,364 participants , Sample B included 2,303 participants , and Sample C included 1,865 participants . Participants in each sample were aged from 18 to 65 years or older, and reported a range of educational levels and ethnicities. The sociodemographic composition of each sample is detailed in Table not at all, 1 = a little, 2 = moderately, 3 = quite a bit, and 4 = extremely. The BRUMS has six subscales , with four items each. Total subscale scores range from 0 to 16. The BRUMS measurement model was validated using multi-sample confirmatory factor analysis , adult athletes , young athletes (n = 676), and schoolchildren (n = 596). Subscales have demonstrated adequate internal consistency, with Cronbach alpha coefficients reported as: tension = 0.74, depression = 0.85, anger = 0.82, vigor = 0.85, fatigue = 0.90, and confusion = 0.83 .Adult participants (≥ 18 years) were recruited from the general population via the ite Lim, . Responda priori. Given that hierarchical and partitioning computations will group even random unrelated data , it is typical that the distribution of negative mood scores show large numbers at the lower end of the scale, and smaller numbers at the upper end , H3 (n = 474), H4 (n = 455), H5 (n = 302), and H6 (n = 630). H1 (n = 394) was not as stable as the other five clusters, in that H1 (n = 284) and H7 (n = 110) combined immediately before the sixth step. However, given that the scree plot showed a distinct elbow, and distance coefficients increased from case 2,358, a 6-cluster solution was judged to provide the best fit to the data.Data were analyzed using agglomerative, hierarchical cluster analysis. Ward's method was the chosen clustering algorithm, given theoretical considerations that both the shape and magnitude of the mood profiles would be relevant. Squared euclidean distance was used as the proximity measure to maximize differences between heterogeneous groups. Three checks were conducted to verify the cluster solution  was used to calculate the extent to which cases could be correctly classified into clusters. DFA is a two-step statistical procedure that involves significance testing of discriminant functions followed by calculation of correctly classified cases. The ratio of cases to independent variables was 394 to 1, which far exceeded the requirement of ≥ 20 to 1. The number of cases in the smallest cluster was 64, which exceeded the preferred number of cases  per group. Five discriminant functions collectively accounted for 100% of the variance, and each function predicted significant variance . Additionally, when the discriminant functions were used to predict group membership, the hit ratio was very high. A total of 95.2% of cases were correctly reclassified back into the original categories. This percentage was notably higher than the minimum classification accuracy rate of 46.5% , suggesting that the overlap of the distributions was small, and the function was a good discriminator between groups.DFA showed cluster membership to be classified correctly with a high degree of accuracy for all clusters  were also evident in the other two samples. Descriptive statistics and cluster sizes for Sample B and Sample C are shown in Tables 1B included high levels of depression, confusion, tension, anger, and fatigue. The predictor variables strongly associated with DF2B included a high level of vigor and low fatigue. The predictor variables strongly associated with DF3B included a high level of vigor and fatigue, together with a low level of depression. The predictor variables strongly associated with DF4B included low tension and high depression, while the predictor variables strongly associated with DF5B included low levels of anger and confusion, and a high level of tension. Based on the structure matrix for Sample C, the mood dimensions strongly associated with DF1C included high levels of anger, fatigue, depression, tension, and confusion. The predictor variables strongly associated with DF2C included a high level of vigor and low fatigue. The predictor variables strongly associated with DF3C included high levels of fatigue and vigor. The predictor variables strongly associated with DF4C included low levels of depression and high tension, while the predictor variables strongly associated with DF5C included high tension and low levels of confusion. The structure matrix and unstandardized canonical coefficients for each sample are shown in Table Based on the structure matrix for Sample B, the mood dimensions strongly associated with DF2 + 0.0362 + 0.1502 + 0.2982 + 0.1382 + 0.2542 = 0.211 and 0.0932 + 0.0242 + 0.1482 + 0.2802 + 0.1652 + 0.2902 = 0.221, respectively).The DFA showed that cluster membership was correctly classified with a high degree of accuracy for both Sample B and Sample C. Prior probabilities for Sample B and Sample C respectively were 12.3, 3.6, 15.0, 29.8, 13.8, 25.4% and 9.3, 2.4, 14.8, 28.0, 16.5, 29.0%, for the inverse iceberg profile, inverse Everest profile, surface profile, iceberg profile, shark fin profile, and submerged profile. The proportional by chance accuracy rates were also computed  were analyzed using a two-step clustering procedure. Six mood profiles were identified, including two established profiles  and four novel profiles . Cluster membership was correctly classified with a high degree of accuracy. Results were replicated in Sample B  and Sample C . Chi-squared tests of goodness-of-fit indicated that the distributions of sociodemographic variables  across the six mood profiles were significantly different from expected cell counts in all three samples.Three distinct mood profiles  were previously identified within athletic samples Morgan, . We inveThese findings raise many research questions worthy of future investigation relating to the antecedents, correlates and consequences of the four novel mood profiles, as well as the profiles previously identified in the literature. The iceberg and Everest profiles have long been associated with healthy cognitive functioning and high to superior levels of physical performance Morgan, . DespiteThe inverse iceberg profile has frequently been associated with debilitating conditions among athletes, including overtraining syndrome Budgett, , risk ofThe influence on human functioning of the shark fin profile, the second of the novel mood profiles, is also unknown. Notably, the shark fin profile lacks some markers of negative mood, such as high levels of tension, depression, anger, and confusion. It is reasonable to speculate, however, that a profile with the lowest vigor scores of all the profiles, combined with higher fatigue scores than any profile except the inverse Everest  apart. Hence, the submerged profile is characterized by being relatively devoid of emotion, including vigor, which may well be described as feeling y Morgan , suggestOur findings and those derived from subsequent investigations of the six mood profiles identified in the present study may serve to extend and/or refine existing theoretical frameworks related to the mood construct. For example, Lane and Terry's  conceptuThe finding of sociodemographic differences in the incidence of specific mood profiles clearly warrants further investigation. Males were more likely to report the iceberg profile, whereas females were more likely to report the inverse iceberg and shark fin profiles. Given that the lifetime prevalence of clinical mood disorders in women has been shown to be approximately twice that of men  and over 65 age group (range = 0–0.7%) raises the question of whether they adequately represent the underlying populations of interest. Additionally, small cell sizes made results for some analyses uninterpretable and others questionable due to violation of underlying assumptions. Analyses involving the inverse Everest mood profile were most affected given the modest number of participants reporting that profile. Finally, the ecological validity of the mood profiles is still to be determined. However, further investigation of relationships between various aspects of human functioning and the distinct mood profiles identified in the present study seems likely to yield new insights.This study was carried out in accordance with the protocol approved by the University of Southern Queensland's Office of Research and Higher Degrees, Human Research Ethics Committee . All subjects gave informed consent in accordance with the National Statement on Ethical Conduct in Human Research (2007).RP-S—Conception and design of study, acquisition of data, performed analysis on all samples, interpreted data, drafting the manuscript and acted as corresponding author. Final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. PT—Conception and design of study, acquisition of data, supervised development of work, helped in data interpretation, drafting and revising the manuscript critically for important intellectual content, final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. MM—Conception and design of study, co-supervised development of work, revising the manuscript critically for important intellectual content, final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Statistical models for assessing risk of type 2 diabetes are usually additive with linear terms that use non-nationally representative data. The objective of this study was to use nationally representative data on diabetes risk factors and spline regression models to determine the ability of models with nonlinear and interaction terms to assess the risk of type 2 diabetes.We used 4 waves of data (2005–2006 to 2011–2012) on adults aged 20 or older from the National Health and Nutrition Examination Survey  and multivariate adaptive regression splines (MARS) to build risk models in 2015. MARS allowed for interactions among 17 noninvasively measured risk factors for type 2 diabetes.A key risk factor for type 2 diabetes was increasing age, especially for those older than 69, followed by a family history of diabetes, with diminished risk among individuals younger than 45. Above age 69, other risk factors superseded age, including systolic and diastolic blood pressure. The additive MARS model with nonlinear terms had an area under curve (AUC) receiver operating characteristic of 0.847, whereas the 2-way interaction MARS model had an AUC of 0.851, a slight improvement. Both models had an 87% accuracy in classifying diabetes status.Statistical models of type 2 diabetes risk should allow for nonlinear associations; incorporation of interaction terms into the MARS model improved its performance slightly. Robust statistical manipulation of risk factors commonly measured noninvasively in clinical settings might provide useful estimates of type 2 diabetes risk. Consensus is lacking on whether to screen asymptomatic adults for type 2 diabetes in the United States ,2, despiDiabetes prediction models usually are additive models and use linear terms , and mosData were extracted from NHANES, a cross-sectional and nationally representative survey of the noninstitutionalized US civilian population. Data from 4 waves (2005–2006 to 2011–2012), excluding data for pregnant participants and those aged younger than 20 years, were combined to obtain the sample . The samThe classification of survey participants into 2 groups — those who had type 2 diabetes and those who did not — was based on survey questions and biochemical measures. Survey participants were considered to have diabetes if they 1) answered yes to the question “Other than during pregnancy, have you ever been told by a doctor or health professional that you have diabetes or sugar diabetes?”; 2) answered no to the preceding question but had a measured fasting plasma glucose of 126 mg/dL or greater or a measured HbA1c of 6.5% or more ; or 3) self-reported diagnosed diabetes but had a measured fasting plasma glucose of less than 126 mg/dL and a measured HbA1c of less than 6.5% (which did not conform with the self-reported results) and reported taking insulin or oral hypoglycemic medication. This method for classifying diabetes status was used in previous studies ,12 and iAlthough type 1 diabetes and type 2 diabetes are distinct diseases, NHANES data do not clearly differentiate between them. We followed a method outlined previously  to diffeThe following demographic variables were treated as covariates: age, sex, marital status, and race/ethnicity . The age2), ranged from 13.0 to 57.0 ; waist circumference, ranged from 59.7 cm to 166.4 cm ; and subscapular skinfold, ranged from 4.4 mm to 42.0 mm .We measured overall adiposity, visceral adiposity, and back-of-the-trunk subcutaneous adiposity: body mass index  was computed by adding up the minutes of reported MVPA during transportation, work, and leisure activities from 7 days of recall and dividing the total number of minutes by 7. MVPA ranged from 0 to 1,371 minutes per day .Participants who self-reported smoking at least 100 cigarettes in their lifetime and smoking every day or some days at the time of the interview were classified as current smokers. Participants who reported smoking at least 100 cigarettes in their lifetime and not smoking at all at the time of the interview were classified as ex-smokers. Participants who reported smoking fewer than 100 cigarettes in their lifetime were classified as never-smokers. This smoking classification is widely used . AccordiThe depression score was computed from the Patient Health Questionnaire-9 (PHQ-9 ). The scThe socioeconomic covariates were education level and family income-to-poverty-ratio; the latter was calculated by dividing family income by the federal poverty threshold specific to household size and year. A ratio of 1.0 or greater indicates income above the poverty level. The mean income-to-poverty-ratio was 2.5 and ranged from 0 to 5.0. NHANES assesses household food security through a 10-item questionnaire and classifies adults into 4 categories: 1) full food security (no food security concerns), 2) marginal food security (1 or 2 concerns), 3) low food security (3–5 concerns), and very low food security (6–10 concerns). Accordingly, 69.8% of the study participants had full food security, 10.8% marginal food security, 12.3% had low food security, and 7.1% had very low food security. Education level was categorized as less than a high school diploma (27.8%), high school diploma (24.2%), and some college or higher (47.9%).basisfunctions, or splines. MARS is better than typical regression models because it can handle studies with many predictors and can account for nonlinearity, multicollinearity, and a high degree of interaction among predictors (Multivariate adaptive regression splines (MARS) was used to build a diabetes risk-prediction model. MARS, a type of algorithmic modeling, was selected because it accounts for both linear and nonlinear relationships as well as piecewise interactions between predicting variables. It is a powerful method that is rarely used in public health. This approach enabled us to model the interaction effect of predicting variables because it segments variables into pieces known as edictors .R2 was averaged  from the left-out subset, which is an estimate of model. performance on independent data  and test data (25%). The MARS model was trained and validated using a 5-fold cross-validation technique on model-building data (75% of the data). The prediction error was averaged across all out-of-fold predictions. The out-of-fold ent data . The genFinally, the validated model was tested on test data (25% of the data), and the performance of the model was evaluated by measuring area under curve (AUC) receiver operating characteristic . The larModel building began with 17 variables that are associated with an increased probability of diabetes but do not involve invasive measurement procedures. The initial model included age, sex, race/ethnicity, marital status, education level, family history of diabetes, cigarette smoking, alcohol consumption, household food security, depression score, family income-to-poverty ratio, waist circumference, BMI, subscapular skinfold, systolic and diastolic blood pressure, sleep duration, and MVPA. The baseline model for the performance of MARS models was a logistic regression model.The additive MARS model showed that the forward selection process transformed the variables into 35 basis functions with 13 variables, including the intercept. The variables in the forward selection process were age, waist circumference, family history of diabetes, race/ethnicity, family income-to-poverty ratio, education level, BMI, subscapular skinfold, systolic blood pressure, diastolic blood pressure, MVPA, sleep duration, and depression score. The forward MARS selection process overfitted the prediction model; thus, in a backward selection process, we removed 1 basis function at a time. The final additive MARS model had 21 basis functions but still comprised the same 13 variables in the forward selection process. The second model incorporated 2-way interactions between basis functions. The final selection process produced 28 basis functions, including the constant. The interactions between the 12 pairs of basis functions were incorporated into the final model, which had 20 basis functions. Eight variable basis interactions included in the model are: waist circumference and subscapular skin fold, age and systolic blood pressure, depression score and diastolic blood pressure, race/ethnicity and diastolic blood pressure, race/ethnicity and sleep duration, education and alcohol consumption, race/ethnicity and age, and BMI and systolic blood pressure.J-shaped curve: the risk of diabetes slightly decreased below 125 mm Hg, did not increase between 125 mm Hg to 158 mm Hg, and then started to spike at 158 mm Hg. On the other hand, diastolic blood pressure reflected a U-shaped curve: the risk of diabetes increased below 55 mm Hg and above 83 mm Hg. For MVPA, the risk of diabetes decreased starting at 29 minutes of MVPA per day. For sleep duration, the risk of diabetes increased at less than 7 hours. A depression score of 11 or more increased the risk of diabetes.In the additive MARS model, we observed a high risk of diabetes at ages older than 69 . HoweverThe interaction between a waist circumference greater than 123.4 cm and an age greater than 69 increased the risk of diabetes . The int2 = 5.19, P = .02). The additive MARS model and 2-way interaction MARS model each had 87% accuracy. In addition, net reclassification index and integrated discrimination improvement matrices also showed significant improvement of the MARS models over the logistic model in the classification of diabetes risk status  and for the 2-way interaction MARS model was 0.851  . The difk status .The accuracy and AUC of the MARS models were higher than those for the logistic regression model and for models described in similar studies using noninvasive measurements, such as the model described by Kahn et al , which hIn the 2-way interaction MARS model, 2-way interactions between the basis functions showed a slight improvement over the additive MARS model. The difference in the AUC between the MARS models and the logistic regression model was significant. The performance level of the MARS models is a new achievement in invasive and noninvasive diabetes risk-prediction models in the United States.J-shaped and U-shaped, respectively. However, the cutoff point for blood pressure control in people with diabetes and people without diabetes is being debated. The United Kingdom Prospective Diabetes Study showed that patients with tight blood pressure control (a mean of 144/82 mm Hg) reduced the number of macrovascular and microvascular events (More importantly, the MARS models identified nonlinear and interactive relationships between some of the predicting variables and the risk of diabetes. Among the most important nonlinear associations were the relationships of systolic blood pressure and diastolic blood pressure to the risk of diabetes, which are r events . Recent r events ,29.U-shaped association between sleep and diabetes risk observed in our study, where sleep duration of less than 7 hours or more than 8 hours increases the risk of diabetes, is similar to the association found by Yaggi et al (The gi et al . Howevergi et al ; thus, sThe strengths of this study are that the model used noninvasive measurements. The information used for this model can be obtained through surveys and nonclinical measurements in nonclinical settings. Another strength is that the NHANES data used are representative of the US population. In addition, the MARS models add new, never-used-before information as diabetes prediction markers, such as depression score and sleep duration. The performance of the model decreased when these factors were excluded. This study also has limitations. First, the prediction model was not validated on an independent and representative sample outside NHANES. Second, our model was built on the complete case analysis; data may not be missing completely at random. In addition, NHANES data are research data; trained personnel accurately and repeatedly collect the anthropometric and blood pressure measurements. At the community level, such trained personnel and other resources may not be available, so replicating research standards of accuracy at the community level may be challenging. Finally, the study could be replicated as a more purely predictive model by using data on respondents who were classified as having type 2 diabetes per laboratory testing and who had not previously received a diagnosis.The goal of our study was to improve the performance of diabetes prediction models. Our results suggest that statistical models of type 2 diabetes risk should allow for nonlinear associations and that the incorporation of interaction terms in the models may not improve their predictive ability. The results confirm previous findings that useful estimates of the risk of type 2 diabetes can be derived from risk factors commonly measured noninvasively in clinical settings. The MARS method produced additive and interaction models with a predictive ability that exceeds that of previously described prediction models. The nonlinearity of the model helped identify cutoff points for some risk factors, such as systolic blood pressure and diastolic blood pressure, sleep duration, and waist circumference. In short, the model maximized prediction from measurements that were noninvasive and feasible to measure in nonclinical settings. However, the practical application of this model as a screening tool needs to be replicated and further developed, particularly in community settings.


The porcine cytomegalovirus (PCMV) is a herpesvirus that may pose a risk for xenotransplantation using pig cells, tissues, or organs. Here, three orthotopic pig heart transplantations into baboons were studied. To detect PCMV, a real-time PCR and a Western blot assay based on four PCMV protein sequences, including two tegument proteins, were used. The transmission of PCMV from the donor pig to the recipient baboon was found in two cases, despite PCMV not being detected in the blood of the donor pigs by real-time PCR. Although it was not in the blood, PCMV was detected in different organs of the donor pigs, and in sibling animals. Immunohistochemistry using an antiserum that is specific for PCMV detected virus protein-expressing cells in all of the organs of the recipient baboon, most likely representing disseminated pig cells. Therefore, for the first time, the distribution of PCMV in organs of the donor pigs and the recipient baboons was described. In addition, baboon cytomegalovirus (BaCMV) was found activated in the recipient, and a screening for hepatitis E virus (HEV) and porcine lymphotropic herpesviruses (PLHV) was performed. For the first time, a cross-reactivity between antibodies directed against PCMV and BaCMV was found. Xenotransplantation is at present the most promising solution for overcoming the increasing shortage of human organs for the treatment of organ failure . This shTo detect PCMV in pigs, sensitive PCR-based methods ,18,19,20Recently, we reported on the transmission of PCMV after transplantation of a pig heart into a baboon, even though PCMV was not detected in the blood of the donor pig . Here, tAll of the donor pigs  and their siblings  were triple genetically modified; all were crossbreeds of German Landrace and Large White, with an alpha1,3-galactosyltransferase gene-knockout , expressing human CD46 and human thrombomodulin (hTM) . GalT-KO pigs do not express the Gal epitope, which is the major cell surface determinant toward natural occurring antibodies that induce the so-called hyperacute rejection (HAR). Animal 5014 was a male Landrace pig with GalT-KO and expressing hTM, animal 5016 was a male Landrace pig with GalT-KO, and animal 5017 was a male Landrace pig with GalT-KO expressing CD46, all born 4 May 2016.The first transplant recipient, a four-year-old male baboon (#57), was transplanted and treated as described . The traFor the establishment of a new testing strategy, peripheral blood monoculear cells (PBMCs) from pigs 89030, 91106, 91107, 91111, 91112, 91117, 01118, 91119, and 91122 were used. These pigs were DanAvl basic hybrid sows; they are not genetically modified, and they were derived from a specified pathogen-free (SPF) facility in Fehmarn, Germany. In December 2016, at an age of approximately 11 months, they had been tested positive for hepatitis E virus (HEV) using PCR, and negative for porcine lymphotropic herpesviruses (PLHV)-1, -2, -3 and porcine circovirus 2 (PCV2) using PCR.The baboons were obtained from the German Primate Center, Göttingen, Germany. Housing of the baboons was in accordance with national regulations (see Ethic statement). Enrichment for primates was individually adapted to each animal. In addition, a “clicker training” was performed. Blood samples were taken from the pigs and baboons after the induction of anesthesia before the transplantation. Second blood samples, as well as tissue samples, were taken after euthanisation of the baboons.® 100 mg/mL, Pfizer Deutschland GmbH, Berlin, Germany), azaperone 10 mg/kg , and atropine sulfate . General anesthesia was induced with an intravenous bolus of 20 mg propofol  and 0.05 mg fentanyl . Anesthesia was maintained with the continuous infusion of propofol 7 mg/kg/h; analgesia was achieved by intravenous bolus injections of 2.5 µg/kg fentanyl repeated every 30 min if necessary. Donor pigs were euthanised by applying cardioplegia and subsequently explanting the heart in deep anesthesia.Premedication of the pigs consisted of intramuscular injections of ketamine hydrochloride 10–20 mg/kg  was applied after transplantation to ensure analgesia. Baboons were euthanised by explanting the pig heart in deep anesthesia.Premedication of the baboons consisted of an intramuscular injection of ketamine hydrochloride 10 mg/kg and 0.5 mg/kg midazolam . General anesthesia was induced with an intravenous bolus of 1.5–2.5 mg/kg propofol and 0.05 mg fentanyl. Anesthesia was maintained with the continuous infusion of 5–15 mg/kg propofol, and analgesia was achieved by intravenous bolus injections of 6–8 μg/kg fentanyl repeated every 45 min. A continuous infusion of fentanyl, ketamine hydrochloride, and metamizole , Sachgebiet 54, 80534 München . Applications were reviewed by the ethics committee according to §15 TSchG German Animal Welfare Law. The xenotransplantation experiment was approved by the Government of Upper Bavaria, Munich, Germany . Housing, feeding, environmental enrichment, and steps taken to minimise suffering, including the use of anesthesia and method of sacrifice, was in accordance with the recommendations of the Weatherall report “The use of non-human primates in research”.PCMV testing was performed as described ,21,27,28To test for HEV, RNA was isolated and a real-time PCR was performed as described ,30 usingTo test for baboon cytomegalovirus (BaCMV), a real-time PCR was performed as described  using sp5 to 0.1 copies per reaction to obtain a standard curve by real-time PCR. DNA from different organs, and the blood before and post mortem of baboon 64 and the donor pig, were analysed.To determine the copy number of PCMV, a standard plasmid covering the target DNA of the PCMV PCR was generated. For this, the amplificate was cloned into a TOPO vector using the Zero Blunt TOPO PCR kit . The generated plasmid was diluted from 2 × 10A droplet digital PCR was established to measure the BaCMV copy numbers using the QX200™Droplet Digital™PCR System . PCR reactions were carried out as duplex PCR using baboon CCR5 as a housekeeping gene. Primer and probes were adopted from Mueller et al. . PCR reaPorcine PBMC were isolated by Ficoll gradient centrifugation , using Falcon tubes without porous barriers. The isolated PBMCs were washed with culture medium  and 10% foetal calf serum (FCS) . DNA was isolated before and after incubation. In some experiments, PBMCs were incubated with mitogen phytohemagglutinin (PHA) .E. coli BL21cells . The constructs were grown in 2 L 2YT-Medium at 37 °C until an optical density (OD) of 0.7, followed by induction with 1 M isopropyl β-d-1-thiogalactopyranoside (IPTG). After 3 h of induction, cells were pelleted by 8000 rpm for 15 min, and stored at −20 °C until purification. Cell pellets were resuspended in buffer , sonicated three times for 20 s, and incubated on ice for 20 min. The cell debris was removed by centrifugation , and pellets were extracted with lysis buffer  overnight at room temperature. Solubilised proteins were separated from the remaining insoluble material by centrifugation , diluted to 100 mL lysis buffer, and loaded on HisTrap 5-mL excel columns . The columns were equilibrated with lysis buffer, and loaded with solubilised proteins. After washing with lysis buffer and a second wash buffer  or 20 mM (UB) imidazole, 20 mM disodium phosphate pH 7.5), the proteins were eluted using a 10-column volume gradient with elution buffer .In Western blot analyses for the detection of antibodies against PCMV, two His-tagged recombinant parts of the glycoprotein gB of PCMV, designated R1  and R2 , were cloned, produced, purified, and used as described . In addiIn order to detect antibodies against HEV, the GT3 antigen was used ,30.Western blot analysis was performed using 500 ng/lane of the corresponding antigen. The proteins were dissolved in sample buffer , and denaturated for 5 min at 95 °C prior to electrophoresis. The proteins were analysed using 10% or 14% polyacrylamide gel and the PageRuler prestained protein ladder . Proteins were transferred for 50 min to nitrocellulose membranes by electroblotting (15 V), stained with Ponceau red, cut into strips, and blocked overnight at 4 °C with 5% blotting grade dry milk (Roth) in PBS with 0.05% Tween 20 (blocking buffer). Strips were incubated with sera diluted 1:300 in blocking buffer for 2 h at room temperature. Polyclonal goat anti-pig immunoglobulin G (IgG)–alkaline phosphatase (AP)  was diluted 1:1000 in blocking buffer. For the detection of the gB protein, a 1:1000 dilution of the Penta-His antibody  as the primary antibody, and 1:1000 polyclonal rabbit anti-mouse IgG–horseradish peroxidase (HRP) or IgG–AP  were used as well. Staining was performed with 3,3′-diaminobenzidine (DAB) (Thermo Fisher) or with 5-bromo-4-chloro-3- indolyl-phosphate nitro blue tetrazolium (NBT/BCI) . Testing for PLHV-1, -2, and -3, using PCR assays and Western blot analyses, were performed as described .2O2 in methanol for 30 min at room temperature. The obtained antibody was applied as the primary antibody at a dilution of 1:1024 for 30 min at room temperature. The sections were lightly counterstained with hematoxylin, and assessed using light microscopy. Simultaneously, sections of porcine kidney that were known to be infected with PCMV were immunolabelled as positive controls. Negative controls were prepared by replacing the primary antibody with a commercial Tris-HCl buffer (antibody diluent with background reducing components) .A PCMV-specific rabbit polyclonal antibody was used in this study, which was produced by immunising a rabbit with PCMV J1, which was the first field isolate in Japan. Immunohistochemistry (IHC) was performed as described previously  using thIn order to confirm the immunological cross-reactivity between PCMV and BaCMV at the sequence level, an alignment of the sequences of the glycoproteins of both viruses was performed using BioEdit version 7.2.5, pairwise alignment  .Three orthotopic pig heart transplantations into baboons were performed . The donAll three donor pigs  were found negative when the blood of these animals was analysed by PCR for PCMV infection. Although PCMV was not detected by real-time PCR in the blood of donor pig 3701, the virus was transmitted to the recipient baboon 57 and a high virus titre in the recipient suggested replication in the transplant and/or recipient . WhereasThe recipient of the heart from pig 5154, baboon 64, was found PCMV-positive 40 days after transplantation (see below). In the third transplantation experiment , blood fAlthough the donor pigs were HEV-positive as shown by real-time PCR, baboons 57 and 64 were HEV-negative ,42.When the blood of donor pig 5154 was tested, it was found negative for PCMV on the day of transplantation day 0, . To idenIn addition, tissue samples from pigs 5014 and pig 5016 were analysed by immunohistochemistry (IHC), using antibodies specific for PCMV. No PCMV was detected using this method in the liver, spleen, kidney, heart, lung, and lymph node of both animals . When tiSince pig 5154 was PCMV-positive in a few organs, but negative when the blood was tested using the same real-time PCR, an analysis was performed as to whether testing for PCMV in the blood can be improved by incubating the isolated PBMCs from the animal under study for five to seven days in culture medium. Freshly isolated PBMCs from nine pigs were incubated and tested before and after incubation . These aWhereas in the case of baboon 57, no tissues with the exception of blood and serum were available to test for PCMV by PCR, in the case of baboons 62 and 64, different organs were available, and were tested using real-time PCR . In the In order to confirm the PCR results and obtain additional information about the distribution of PCMV in the baboon, an IHC analysis was performed using PCMV-specific rabbit antibodies A.In the case of baboon 57 (where unfortunately no organs were available to screen for PCMV by PCR), positive cells were found by IHC in the pig donor heart, as well as in all of the analysed organs of the baboon  at the end of the experiment. The highest amount of positive cells was found in the baboon lymph node and testis, while a lower amount was found in the pig heart, and lower amounts were also found in the baboon liver, spleen, kidney, and lung. A semiquantitative presentation of the distribution in different organs as detected by IHC is given in Surprisingly, when a control baboon (number 43) was analysed by IHC using the rabbit serum against PCMV, a slightly positive reaction was observed in some cells in different organs . Since tRecently, a Western blot analysis was developed using two recombinant proteins corresponding to the N-terminal (R1) and the C-terminal (R2) parts of the glycoprotein B (gB) of PCMV, which had been used when screening for antibodies against PCMV in infected pigs . Later, To summarise, using two real-time PCR that specifically detect BaCMV or PCMV, both viruses were detected in baboons 57 and 64. When the sera of baboon 64 before and after the transplantation were analysed using Western blots with PCMV glycoprotein B and tegument proteins, a reactivity of the baboon sera with the PCMV proteins was observed, suggesting a immunological cross-reactivity . AlthougHere, for the first time, the distribution of PCMV in the organs of genetically modified donor pigs used for orthotopic heart xenotransplantation and in organs of the transplanted baboons are described. It is important to note that PCMV was found in several organs of the donor pig—but not in the blood—when tested at an earlier time point, indicating that testing blood is not an efficient way to detect PCMV in young pigs. Based on this result, the testing of organs from sibling animals of the same litter is recommended. In addition, in a recent study, which was not yet finished when we analysed the pigs in these transplantation experiments, we observed that testing non-invasive oral and anal swabs by PCR may be more efficient for PCMV detection compared with testing blood . HoweverAs mentioned above, our results showing an increase in virus load after the in vitro cultivation of isolated pig PBMCs from infected animals  propose The detection of PCMV in cells in all of the organs of baboon recipients 57 and 64, as shown by IHC, is one of the most impressive results of this study. Although it is still unclear whether PCMV is able to infect baboon or human cells, most data indicate that herpes viruses are species-specific . This waBaboons are naturally infected with a cytomegalovirus, BaCMV, which has been isolated and described by several laboratories . Most ofAlthough the severe shortage of human organs for transplantation is the driving force behind xenotransplantation research, non-human primates, particularly baboons, are for several reasons not potential sources of organs and tissues. One of these reasons is that BaCMV is endemic in baboon populations and infects human cells, and is therefore a potential cause of donor-associated disease after xenotransplantation. In addition, it is well known that the human cytomegalovirus (HCMV) is the most common donor-associated infection, and poses a major risk for allotransplantation. Therefore, all efforts should be undertaken to prevent the transmission of PCMV during xenotransplantation using cells, tissues, or organs from pigs.


AbstractEuderus Haliday, Euderussetsp. n., is described and illustrated from the southeastern United States, where it parasitizes the crypt gall wasp, Bassettiapallida Ashmead, 1896, on live oaks in the genus Quercus (subsectionVirentes). This is the 1st species of the genus reported from the southeastern United States to parasitize cynipid gall wasps and the 3rd species of the genus reported to attack cynipids in North America. Modified sections of the identification keys to subgenera and species of Euderus  are included to integrate the new species.A new species of the genus  Euderus Haliday, 1844 is a group of parasitic wasps in the family Eulophidae (Hymenoptera) with approximately 77 described species  and stem boring and other herbivorous Coleoptera . Rarely, Euderus has been reported to attack gall-making Hymenoptera (Cynipidae) or exhibit hyperparasitism on other Hymenopteran parasitoids (Ichneumonidae). Detailed host records from field observation across the genus are summarized in Table 1 of Bassettiapallida Ashmead, 1896  on live oaks , including Quercusvirginiana and Quercusgeminata. We modify the key published by Here, we report the first species of this genus from the southeastern United States to attack cynipid gall wasps, where it is associated with the crypt gall wasp Quercusgeminata, infested with the asexual generation of the crypt gall wasp, Bassettiapallida, were collected July 15, 2014, August 1, 2015, and October 1, 2015, in Inlet Beach, Florida . Additional populations were collected across the U.S. Gulf coast on Quercusgeminata and Quercusvirginiana in 2014, 2015, and 2016 . Emergence of all inPageBreakdividuals was monitored regularly for a year or dissected out of Bassettiapallida crypt galls in the lab. All individuals were preserved in 96% EtOH and frozen in a -80°C ultrafreezer.For the type locality, branches of the sand live oak, Descriptions of the species have been made under a Leica M125 Stereoscope, with lighting achieved through a Leica LED5000 SLI - Spotlight illumination with 2 HiPower LEDs and a Leica TL5000 Transmitted Light Base with Rottermann Contrast TM, brightfield and two sided darkfield. For images, 75-150 stacked photographs were produced by a Canon 7D Mark II , with a Mitutoyo M Plan Apo 10x objective mounted onto the Canon EF Telephoto 70 – 200mm zoom lens, which was mounted on a Stackshot Automated Focus Stacking Macro Rail . The Canon MT–24EX Macro Twin Lite Flash with custom made diffusers was used to minimize hot spots. Images were processed using Zerene Stacker  and plates were finished with Adobe Illustrator CC. Pictures of slide-mounted wings were taken using a Leica ICC50W camera.Euderus in Morphological nomenclature follows American Museum of Natural History (AMNH) . AMNH specimen identification codes: Holotype - AMNH_IZC 00238642; 8 paratypes - AMNH_IZC 00238643 – 00238650.Type material is deposited in the PageBreakmitochondrial cytochrome oxidase (mtCOI) gene using standard PCR protocols  ecologically similar Euderus species attacking cynpid gall wasps, (2) geographically overlapping Euderus species in the coastal southeastern United States, (3) geographically proximate Euderus speices from the Caribbean, and (4) morphologically similar species within the Euderus genus. We also provide the DNA sequence data to complement the morphological taxonomy in Suppl. material Details on the on the type material and type localityare provided, with a thorough description with images, a differential diagnosis of the new species, and a modification to the identification key published by  it from  ecologicTaxon classificationAnimaliaHymenopteraEulophidaeHaliday, 1844Euderus Haliday, 1844. Trans. Ent. Soc. Lond. 3: 298.Entedonamphis Walker.PageBreakTaxon classificationAnimaliaHymenopteraEulophidaeEgan, Weinersmith, & Forbessp. n.http://zoobank.org/44A7E50B-5E80-4A60-9170-DD1B9A7221B7AMNH ID#: AMNH_IZC 00238642Holotype, ♀, Inlet Beach, FL, collected July 15, 2015 by Scott P. Egan, Allotype, same data as holotypeParatypes, 2 ♂, same locality as holotypeSpecimens from each collection locality Table .FEMALE. Length 1.6 – 2.3 mm. Holotype 2.3 mmColor. Head, scrobal depression, pedicel, flagellum, mesoscutum, scutellum, coxae and metasoma metallic, olive green to turquoise to iridescent blue (colors depends on lighting and age of specimen); antennal scape white to yellow; femora and tibiae concolorous with mesoscutum but color lightens apically; tarsi white, except terminal segment dark brown . The god Set is also reported to have trapped his brother Osiris in a crypt to kill him, later retrieving the body and chopping it up into small piece, which also fits with Euderusset, the crypt-keeper wasp, which kills its host in a crypt, and devours the host from the inside out, leaving major sections of exoskeleton  chopped up and distributed in the crypt .Named after the ancient Egyptian god nd chaos  and was PageBreakEuderusset, to be included in the North American key to species of the genus Euderus by Euderus, where characters referring to the male flagellum should be removed. Neoeuderus and Euderus, but at that time only a single male specimen of subgenus Neoeuderus was available for study, and this individual was from the species Euderusviridilineatus for which no females had been found. As the male Euderusset. do not have antennae with pronounced whorls of long hairs, this is not an appropriate diagnostic trait for the subgenus.Two changes are required for the new species, We propose the following revision to the Neoeuderus should be revised as follows:Secondly, Euderusset mtDNA-COI sequences were 98% identical to each another and each was most similar to other previously identified Euderus in the BOLD database. Sequence 1 was 88.4% identical to Euderus sp. D0703 on BOLD and sequence 2 was 89.8% identical to another Euderus sp. on BOLD , where it emerged from a stem crypt gall on Quercusgeminata induced by the crypt gall wasp, Bassettiapallida. We have also collected Bassettiapallida galls from live oaks across the Gulf coast of the southeastern United States, where additional Euderusset have been found, including additional sites in Georgia, Florida, Mississippi, Louisiana, and Texas . We also observed a smaller pulse in September and October, which could have been a natural occurrence, or induced by harvesting galled tissue and bringing it into a controlled environment. Regardless of harvest time (August or October), a similar emergence window was observed in February and March.Emergence of Euderusset is strongly associated with a behavioral phenotype in its host, the crypt gall wasp, Bassettiapallida, where infected gall wasps cut an emergence hole through the gall tissue as an adult, but then die and remain partially in the crypt to plug the emergence hole with its head . When Euderusset emerges, it cuts an emergence hole directly through the head capsule plugging the hole . The host’s behavioral phenotype may benefit Euderusset by making it easier for the adult stage to emerge from the crypt . This putative behavioral manipulation of the host by its parasitoid Euderusset is the first time this has been described by the species-rich and economically important Chalcidoidea and is also the inspiration behind both the scientific name, Euderusset, and the common name, the crypt-keeper wasp.Euderusset, we have also reared eleven additional natural enemy species from Bassettiapallida galls on live oaks , including two inquilines (genera Synergus and Ceroptres) and nine parasitoids including three species from the genus Sycophila, two species from genus Ormyrus, one each from the genera Eurytoma, Acaenacis, and Brasema, as well as a parasitoid from the platygastrid subfamily Platygastrinae that we have not yet been able to key to genus. The natural enemy community requires further description.In addition to


Disease modifying treatments (DMTs) are the main responsible for direct medical costs in multiple sclerosis (MS). The current investigation aims at evaluating possible associations between healthcare costs for treating relapsing remitting MS (RRMS) and disease evolution.The present cohort study retrospectively included 544 newly diagnosed RRMS patients, prospectively followed up for 10.1±3.3 years. Costs for DMT administration and management were calculated for each year of observation. Following clinical endpoints were recorded: time to first relapse, 1-point EDSS progression, reaching of EDSS 4.0, reaching of EDSS 6.0, and conversion to secondary progressive MS (SP). Covariates for statistical analyses were age, gender, disease duration and EDSS at diagnosis.At time varying Cox regression models, 10% increase in annual healthcare costs was associated with 1.1% reduction in 1-point EDSS progression , with 0.7% reduction in reaching EDSS 6.0 , and with 1.0% reduction in SP conversion .Higher healthcare costs for treating MS have been associated with a milder disease evolution after 10 years, with possible reduction of long-term non-medical direct and indirect costs. Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system causing neurodegeneration, axonal injury and demyelination, and is responsible for significant physical disability and social impact. Direct and indirect costs associated with MS are high, due to its chronic management and to early onset in economically productive years ,2.The introduction of Disease Modifying Treatments (DMTs) during the last 20 years has been changing the natural history of MS. Clinical trials support the early initiation of chronic DMTs in order to avoid short term disease activity (i.e. relapses), and to reduce or, ideally, to prevent long term disability accrual –6. HowevSoaring healthcare costs for DMTs have received much attention in health policy contexts, with some studies estimating DMT costs being beyond the health care system tolerance, whereas others below the threshold ,12. In pThe present observational cohort study is a retrospective analysis of prospectively collected data, recorded in the database of the MS Clinical Care and Research Centre of the Federico II University Hospital of Naples, Italy.In compliance with current Italian applicable laws and regulations, considering that all clinical assessments were part of clinical practice in a University setting and that the retrospective analysis included anonymized data, specific ethics approval was not required. All subjects signed the general informed consent form, authorizing the use of personal data for research purposes. The study was performed in accordance with the good clinical practice and the Declaration of Helsinki.Inclusion criteria were: 1) new diagnosis of RRMS from January 2001 to January 2010 ,15; 2) iExclusion criteria were: 1) progressive forms of MS at baseline ; 2) age Due to the observational nature of the study, DMTs were possibly discontinued or changed during the study period in accordance with the local regulatory indications for clinical practice  ,17. In pEconomic resources for the present study included the healthcare costs for the DMTs, for the staff involved in DMT administration , for neurological visits, for other specialist visits related to DMT safety procedures , for MRI procedures, for laboratory exams, for psychological and neuropsychological evaluations, performed in accordance with current guidelines and clinical practice ,17,19,20Healthcare costs were inflated to the most recent values, obtained from the National Drug Formulary for DMT costs , and from the National Tariffs for Healthcare of the Italian National Health System for resource utilization costs , as previously performed in similar studies on Italian MS populations, in order to avoid variations in price per unit of service through different years ,21.Healthcare costs were referred to each year of observation , in order to obtain annual healthcare costs before study endpoints were reached. Overall annual healthcare costs were calculated for each patient . Healthcare costs were included in the statistical models as continuous variables in order to avoid information loss . For staDuring the whole study period, MS subjects attended the MS Centre for visits which were scheduled according to the clinical practice for the follow-up of their disease and treatment, or for the occurrence of a clinical relapse, and were evaluated for 5 different clinical endpoints that have previously been demonstrated as being milestones of MS evolution :Occurrence of clinical relapse: relapses occurring during the study period were recorded; the time occurring from MS diagnosis to the first relapse was calculated (time to first relapse); then, the overall number of relapses was reported on an annual basis ; relapsing patients presented a range of motor/sensory symptoms and met commonly used standards for relapse as determined by clinical neurologists ;1-point Expanded Disability Status Scale (EDSS) progression : the cohort was categorized according to the 1-point EDSS progression or not, and the time from the diagnosis to the 1-point EDSS progression was calculated (time to 1-point EDSS progression);Reaching of EDSS 4.0 (confirmed after 12 months): the cohort was categorized according to the reaching of EDSS 4.0 or not, and the time from the diagnosis to the reaching of EDSS 4.0 was calculated (time to EDSS 4.0);Reaching of EDSS 6.0 (confirmed after 12 months): the cohort was categorized according to the reaching of EDSS 6.0 or not, and the time from the diagnosis to the reaching of EDSS 6.0 was calculated (time to EDSS 6.0);Transition from RR to SP course: the cohort was categorized according to the conversion to SP or not, and the time from the diagnosis to the SP conversion was calculated (time to SP); MS was considered SP when a progressive accumulation of disability occurred after an initial relapsing course, and was associated with a worsening of the same functional system, independently from relapse activity ,24.Endpoints of disability progression  were chosen because clinically important and unlikely to remit once sustained ,23,25. TCovariates included in the models were recorded at the time of diagnosis, and included demographic characteristics , disease duration , and the EDSS , which hMeans and proportions of demographic features, clinical findings and healthcare costs were calculated for the MS population. All healthcare costs underwent logarithmic transformation before being included in the statistical models as continuous variables.Time varying Cox regression models were performed to evaluate the associations between the annual healthcare costs before the specific study endpoint was reached, and the hazard ratios (HR) of relapse occurrence (time to the first relapse), of 1-point EDSS progression, of reaching of EDSS 4.0, of reaching of EDSS 6.0, and of conversion to SP; 95% confidence interval (95%CI) were subsequently calculated. Models were adjusted for age, gender, disease duration and baseline EDSS. In order to improve data presentation, results from the Cox regression models were discussed considering the effect of a 10% increase in the healthcare costs using the following formula: Poisson regression models were performed to evaluate associations between overall annual healthcare costs and ARR; coefficients (Coef) and 95%CI were subsequently calculated. Models were adjusted for age, gender, disease duration, baseline EDSS, and follow-up duration.Covariates were preliminary studied in relation to the overall annual healthcare costs by using t-test for categorical variables, linear regression analysis for continuous variables, and ordinal regression analysis for ordinal variables.For data presentation in Kaplan-Meier curves, healthcare costs have been categorized on the median value (10801.92€ per year). Patients treated with either Fingolimod or Natalizumab during at least a period of the observation time were more likely to be part of the group with healthcare costs higher than the median value (p<0.001).Results have been considered statistically significant if p<0.05. Stata 12.0 has been used for data processing and analysis.Among 685 eligible subjects, the present study included 544 newly diagnosed, drug naïve RRMS patients, who were followed-up for 10.2±3.4 years. Details of subjects assessed for eligibility and excluded or included in the present study, are reported in Demographic features, clinical findings and healthcare costs of the MS cohort are reported in In agreement with the indications of the Italian regulatory agency, the first prescribed DMT was discontinued and possibly changed in 291 patients (53.4%), which underwent 2.2±1.1 different DMTs during the 10-year study period. When considering the entire observation time, subcutaneous Interferon beta-1-a was the most frequently prescribed DMT (32.1%), followed by intramuscular Interferon beta-1-a (19.7%), Interferon beta-1-b (16.6%), Natalizumab (6.4%), Fingolimod (5.4%), Glatiramer acetate (5.4%), Teriflunomide (2.3%), Dimethyl Fumarate , and no DMT (10.0%). Specifically, a limited number of patients, after starting the first prescribed DMT, spent at least part of the study without any DMT.Overall annual healthcare costs were associated with age , and with baseline EDSS , but not with gender (p = 0.595), and with disease duration .After adjusting for different covariates, 10% increase in the annual healthcare costs was associated with 1.1% reduction in the rate of 1-point EDSS progression  .The present study found that higher healthcare costs for the use of more expensive DMTs from the early phases of MS, were associated with a lower risk of reaching milestones of short- , and long-term disease evolution . For instance, the use of treatments for highly active RRMS such as Fingolimod and Natalizumab, which are twice more expensive compared with Interferons , might hThere are different possible reasons underlying the association between healthcare costs for DMT administration and management, and MS progression. Healthcare costs in MS are driven by the use of DMTs, which are prescribed depending on the initial severity of MS and on its subsequent evolution. Indeed, the costs for DMTs are usually determined on the basis of their clinical effects as evaluated in clinical trials, and, so, they represent a surrogate marker of DMT efficacy. However, healthcare costs for DMT administration and management have never been tested in long-term real-life scenarios in relation to MS clinical outcomes. In previous observational studies trying to investigate the long-term efficacy of DMTs, Natalizumab determined more positive disease outcomes, compared with Interferon-beta or Glatiramer acetate, in a large propensity-matched cohort . SimilarAmong additional results, in the present cohort a higher relapse rate was associated with more expensive healthcare choices. Accordingly, previous studies showed that financial consequences of relapses depended not only on the temporary loss of productivity, but also on the subsequent change of treatment –37. IndeLimitations of the present study include the retrospective and non-randomized design, which, although generally considered acceptable for long-term studies in MS , might bIn conclusion, the current landscape with newer and more expensive DMTs is a noticeable challenge for the economic performance of the healthcare systems. Hence, even if requiring short-term resource investments, a higher expenditure for treating MS has been associated with better disease evolution after 10 years. Current findings highlight the need for highly active and, possibly, expensive DMTs, in order to delay disease progression and its long-term burdensome economic consequences.


Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs) and cylindrospermopsin (CYN) as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis, and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis. The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R2 = 0.392−0.740). The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters.Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants.  Lakes and reservoirs are important drinking water sources in Taiwan and many other countries. Due to improper management of nutrients in their watersheds, many reservoirs are facing the risk of eutrophication, increasing the possibility of cyanobacterial blooms in the source waters . CyanobaAnabaena [Anabaenopsis [Aphanocapsa [Microcystis [Nostoc [Oscillatoria [Planktothrix [Microcystis spp. are the most commonly detected toxigenic cyanobacteria in the reservoirs in Taiwan [Anabaena bergii [Anabaena lapponica [Aphanizomenon gracile [Aphanizomenon flos-aquae [Aphanizomenon ovalisporum [Cylindrospermopsis raciborskii [Raphidiopsis curvata [Umezakia natans [C. raciborskii has been considered to be a major problem for water management on a global scale [MCs have been reported to be produced by several planktonic cyanobacterial genera, including Anabaena , Anabaenaenopsis , Aphanocanocapsa , Microcyrocystis , Nostoc  [Nostoc , Oscillallatoria , and Plaktothrix . Howevern Taiwan . CYN hasa bergii , Anabaenapponica , Aphaniz gracile , Aphanizos-aquae , Aphanizlisporum ,31,32, Ciborskii ,26,33,34 curvata , and Umea natans . Howeveral scale .When high concentrations of cyanobacteria are present or expected to be present in source water, such as cyanobacterial blooms, an estimation of the risks associated with cyanotoxins is always very important for water utilities. Conventionally, the procedures to identify such risks includes a collection of samples, delivery of these samples to a central laboratory, and enumeration of cyanobacterial cells that are potentially toxigenic . NormallIn the last two decades, quantitative real-time polymerase chain reaction (qPCR)-based molecular techniques ,45,46,47Microcystis and potentially-toxigenic Microcystis, and the other for Cylindrospermopsis and cylindrospermopsin-producing cyanobacteria. The developed methods were then tested for the influence of DNA concentration on detection, and then applied in the monitoring of the samples collected from 29 drinking water reservoirs throughout Taiwan. Finally, the data measured by qPCR were then compared with cell data and cyanotoxin data, which were determined by microscope method and by ELISA, respectively.The four aims of this study are to develop a qPCR system to detect the cyanobacteria that produce two common cyanotoxins, microcystins and cylindrospermopsin, to test method reliability, to apply the method in field monitoring, and to compare the data with conventional methods for cell numbers and cyanotoxin concentrations. Two duplex qPCR systems were first developed, one for quantifying Microcystis aeruginosa and Cylindrospermopsis raciborskii, are used in this study. Microcystis aeruginosa PCC7820 strain, a producer of microcystins [Microcystis cells and potentially-toxigenic Microcystis cells. Cylindrospermopsis raciborskii CYP026J strain, a producer of cylindrospermopsin isolated by the Australian Water Quality Centre culture collection , was used for the qPCR standard curves for both total Cylindrospermopsis cells and cylindrospermopsin-producing cyanobacteria. Both strains were grown in ASM medium [−2 s−1 of light intensity.Two cyanobacterial strains, ocystins , was purM medium  at 25 °CWater samples were collected from 29 different drinking water reservoirs (DWRs) located throughout the main island of Taiwan and its two off-shore islands, Kinmen Island and Matsu Island. The sampling locations for all the studied reservoirs were all near the water intakes of their associated water treatment plants. The locations of the studied DWRs are shown in Microcystis cells were then counted under a microscope  at ×400 magnification. For the filamentous Cylindrospermopsis, the cross walls between cells were often indistinct, making this measurement imprecise [Cyanobacteria cells were enumerated following the procedures prescribed in the Standard Method 10200F . In briemprecise . Thus, tTo extract DNA from cyanobacterial cells, cyanobacteria-laden water samples (10 mL) were first concentrated using a centrifuge  rotating at 16,060 rcf for 5 min. After centrifugation, the supernatant was removed and the cyanobacterial pellets remaining were re-suspended with 5 mg/mL of Lysozyme , and followed by a freezing-thawing process that was performed three times using liquid nitrogen and a 37 °C water bath for the lysis of cyanobacterial cells . Then, D®II; Cepheid, Sunnyvale, CA, USA). The first system helps differentiate potentially-toxigenic Microcystis cells from that of total Microcystis cells, while another system helps distinguish cylindrospermopsin-producing cyanobacteria from that of total C. raciborskii cells.Each of the developed duplex qPCR systems was performed individually and separately with a qPCR device  region unique to potentially-toxigenic Microcystis cells [® software version 2.0 .For the is cells , while mCylindrospermopsis, primer and probe set m4/k18/pks is efficient for targeting the polyketide synthase (pks) region of cylindrospermopsin-producing cyanobacteria [rpoC1 helps to specifically target the rpoC1 region unique to C. raciborskii [® software.For identification of bacteria ,45,66, wiborskii   were obtained for all the four targeted genes in both uniplex and duplex qPCR systems in this study.Microcystis cells and the mcyB region were amplified from the extracted DNA of M. aeruginosa PCC7820 strain using the primer sets of Micr184F/Micr431R and mcyB#04F/mcyB#04R, respectively. In a similar fashion, the PCR product for the rpoC1 region and the pks region was amplified from the extracted DNA of C. raciborskii CYP026J strain using the primer sets of cyl2/cyl4 and m4/k18, respectively.The PCR product of the 16S rRNA region unique to Sterile deionized water was then added to a PCR mixture, which consisted of 5 µL of the extracted DNA solution, 2 µL of 10× Ex Taq™ buffer, 200 µM of dNTP mix, 0.2 µM of the forward and reverse primers, and 0.1 µL of TaKaRa Ex Taq™ DNA polymerase , such that the volume totalling 50 µL was then placed in a 96-well thermal cycler . The PCR experiment was executed with a pre-incubation at 95 °C (5 min), and a 30-cycle of 3-step amplification of denaturation , annealing , and extension , then a final extension at 72 °C for 10 min.® TA Cloning®, Invitrogen™, Life Technologies, Carlsbad, CA, USA) following the suggested protocol. The plasmid DNAs from cloning were used to develop the standard curve between the Ct values and the copy numbers. Longer sequences of the target region from plasmid DNAs were amplified by using the universal M13 primers to avoid the possibility of mispaired nucleotides at the ends of the target genes. The M13 PCR product was quantified by measuring the fluorescence intensity with PicoGreen® dsDNA Reagent and Kits . The DNA copy number (copies/μL) was then calculated by dividing the DNA concentration (g/μL) by the molecular weight (g/mole) of the targeted gene and then multiplied by 6.02 × 1023 copies/mole [Then, four PCR products were cloned by using a commercial kit (TOPOies/mole .2 to 108 copies/reaction), and the Ct values were plotted against the logarithm of the copy number of the target genes with an amplification efficiency (E) of 100% ± 10%, where E = 10−1/S − 1 (S is the slope of the logarithm regression).Series of 10-fold dilutions of M13 PCR product of the target genes were prepared , was further used to compare the slope coefficients of different regression lines. The slopes are considered to be statistically significantly different when a P value is less than or equal to 0.025 with two-tailed 95% confidence intervals. Another statistical method, the Chow test [The field data obtained in this study were analyzed for the correlation between cell enumeration, toxin concentrations, gene copies and cell equivalents of the samples. Although the gene copies in nditions ,69, in tnditions . The reghow test , was alsMicrocystis and Cylindrospermopsis systems, results clearly demonstrate that the calibration curves obtained from the duplex quantification system are almost identical to those for the uniplex system, suggesting the equal performance of both systems , Kinmen, Taiwan. In the meantime, the samples were also analyzed with the uniplex qPCR systems. The duplex system results were compared with their respective uniplex system for the detection of the targeted cyanobacteria. The data, which are shown in ermopsis . Althougpks gene . The faich lower . This isMicrocystis system when mcyB gene copies were 102 times higher than that of 16S rRNA gene, the system fails to detect the less abundant gene  was transferred to the cell equivalents concentration (cells-mL−1) through the calibration curves constructed for the laboratory cultures [Microcystis cell equivalents, potentially-toxigenic Microcystis cell equivalents, and the total number of Cylindrospermopsis cell equivalents in a sample can be estimated, from the abundance of the 16S rRNA gene, mcyB gene, and rpoC1 gene, respectively. It is noted that the primer and probe set for the pks gene is efficient for targeting the polyketide synthase (pks) region of cylindrospermopsin-producing cyanobacteria, including Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, Raphidiopsis curvata, Anabaena bergii and Oscillatoria sp. [The two duplex systems were further applied to quantify the targeted genes in 338 samples collected between February 2013 and August 2016 from 29 reservoirs throughout Taiwan, consisting of 10 drinking water reservoirs (DWRs) on the main island of Taiwan, 10 DWRs in the Kinmen islands, and 9 DWRs in the Matsu islands . In addicultures , as showoria sp. ,45,66. Aoria sp. ,69, the Microcystis and rpoC1 gene for Cylindrospermopsis, were mostly similar to cell concentrations enumerated with microscopy, with only 5 of the 13 Kinmen samples having a difference of more than one order of magnitude  and copy number of cylindrospermopsin producers (based on the pks gene) follow an increase in the concentration of MCs and CYN, respectively. Note that for each particular toxin, the data obtained for two of the sampling dates did not follow the trends. microcystis system, only mcyb gene and no MCs were detected in the samples collected in February 2013 and May 2014; while in cylindrospermopsis system, only CYN and no pks genes were detected in the 2 samples collected in December 2014 and August 2015. The discrepancy between the two methods, gene detection, and ELISA, may be attributed to a few reasons. First, detection of mcyb and pks genes represents the presence of potential producers, but not necessarily the presence of products (toxins) [Cylindrospermopsis system, the lysed cells or extremely low cell concentrations may lead to no detection of the pks gene in the samples. However, the released CYN was still measurable.The increase of the cell-equivalent concentration of toxigenic (toxins) ,78,79,80(toxins) . Thus, iMicrocystis (detected with 16S rRNA gene) was always higher than that of potentially toxigenic species (mcyB gene). Previous research demonstrated that toxic and non-toxic strains of the same cyanobacterial species often coexist in the environment [Microcystis cells detected were capable of producing MCs, confirming the observations of previous field studies that not all strains of Microcystis found in natural samples are capable of producing toxins [In ironment ,40,41,42g toxins ,78.Microcystis and Cylindrospermopsis data collected from all the reservoirs were separated into three main groups based on differences in geographic location: Taiwan main island ; Kinmen islands ; and Matsu islands . Analyses of both Microcystis and Cylindrospermopsis were further separated. In order to better understand the effectiveness of this study’s new duplex system, two comparisons were made for Microcystis at each geographic location: cell equivalents concentrations (based on the 16S rRNA gene) was compared to the cell abundance (microscopy); potentially toxigenic cell equivalents (mcyB gene) was compared to MC concentration (ELISA). The comparisons for Cylindrospermopsis: cell equivalents (rpoC1 gene) were compared to cell abundance (microscopy); cylindrospermopsin producing the gene copy number (pks gene) was compared to CYN concentration (ELISA).Microcystis concentrations obtained via duplex and microscopy were relatively moderate to strong, ranging from R2 = 0.566−0.769  showed that, regardless of geographic grouping, there were no significant differences for the three geological locations, with p = 0.704, 0.2620, and 0.206 (different if p ≤ 0.025 (two-tailed) for 95% confidence interval) for Taiwan main island and Kinmen islands, Taiwan main island and Matsu islands, and Kinmen islands and Matsu islands, respectively. Similar results were also observed for comparison of all other three cell/cyanotoxin to gene correlations for the three geological locations, with p ≥ 0.025 for the cases of mcyB and MCs concentration, rpoC1 and Cylindrospermopsis abundance, and pks and CYN concentration.During analyses, the methodology developed in this study revealed consistent results, regardless of geographic grouping. In general, results showed that coefficients for linear regression amongst all data were similar, showing that the method in this study is consistent regardless of location. For cell abundance of the total Microcystis and Cylindrospermopsis systems were further tested for regression stability, using Chow test [p < 0.01 in p < 0.05 in p < 0.001 in p < 0.05 in Several parameters may affect the correlation slope of the target compound to the genes, including toxin production rate and cell quota , toxin-producing species, and gene copy of the producing species . Therefohow test . The resIn this study, the slopes of correlations obtained for Taiwan main island, Kinmen islands and Matsu islands fall in a narrow range. Therefore, all the data for the three geographical locations were further correlated. Microcystis and Cylindrospermopsis obtained from this study showed somewhat strong correlation values with the cell number information obtained from microscopy and the cyanotoxin concentration from ELISA, respectively, the qPCR-based method may be a better alternative for the monitoring of cyanobacteria and metabolites in natural water. In addition, since the qPCR system is able to distinguish toxic and nontoxic species, the approach has the potential to quickly provide useful information for water utilities to estimate the potential risks of toxins and toxigenic cells in the source waters.In Microcystis and microcystin-producing Microcystis using 16S rRNA and mcyB, respectively; the second set consisted of Cylindrospermopsis and cylindrospermopsin-producing cyanobacteria using rpoC1 and pks gene, respectively. Field and laboratory tests showed that, in the duplex qPCR systems, presence of a highly abundant targeted gene may inhibit the detection of the other less abundant one. Although mcyB gene was detectable even when the concentration of 16S rRNA gene was four orders of magnitude higher, however, for the Cylindrospermopsis system, when either pks or rpoC1 gene copies were 102–103 times higher than the other one, the system would fail to detect the lower abundant gene. Analysis of the samples collected from 29 reservoirs in Taiwan and her offshore islands show good correlation values between cell  numbers and gene copies, and between toxin concentrations and gene copies for both Microcystis and Cylindrospermopsis systems, with R2 mostly >0.60 for the reservoirs in three geographical locations. Relatively high coefficients of determination were found for the correlations between Microcystis cells and 16S rRNA gene (R2 = 0.740), microcystin and mcyB (R2 = 0.683), Cylindrospermopsis cells and rpoC1 gene (R2 = 0.659), and cylindrospermopsin and pks gene (R2 = 0.392), for all the samples collected in the reservoirs. The results demonstrate that the developed duplex qPCR systems were able to quantify the targeted cyanobacteria and toxin producing genes. In particular, the obtained gene information correlated well with cell numbers enumerated with microscopy and toxin concentrations measured with ELISA. The qPCR approach may be used as a tool for water utilities to diagnose the potential risk associated with toxigenic cyanobacteria and cyanotoxins in drinking water lakes and reservoirs.Two sets of duplex qPCR systems were successfully developed for quantifying two commonly observed harmful cyanobacteria in this study; the first consisted of