The oxidative biomarkers play an important role in the genesis ofcardiometabolic risk-related processes.To investigate the total antioxidant capacity of plasma and its associationwith cardiometabolic risk in non-obese and clinically healthy youngadults.University students of the state of Sergipe, Brazil, aged between 18 and 25years, were recruited for this study from May of 2013 and October of 2014.Anthropometric, clinical and biochemical parameters were measured andanalyzed using protocols which were previously standardized and described inthe literature. The measurement of plasma total antioxidant capacity wasbased on the ability that all the antioxidants present in the sample(plasma) have to inhibit the oxidation of the oxidizable substrate ABTS to ABTS•+ bymetmyoglobin.2 = 0.10; β = 0.17; p = 0.001).Approximately 25% of the sample presented more than one component ofcardiometabolic risk. Low HDL-cholesterol was the most prevalent component.Compared to absence of components, the subjects with at least one componentpresented greater body weight and waist circumference, higher levels ofdiastolic blood pressure and fasting glucose, greater totalcholesterol/HDL-c ratio, and lower levels of HDL-c (p < 0.05). Fastingglycemia was the only parameter which was associated with total antioxidantcapacity (RThe plasma total antioxidant capacity was not able to predict thecardiometabolic risk components due possibly to the establishment ofcompensatory mechanisms that become activated in physiologicalconditions. The hypothesis of this study is that decreasedTAC increases CMR, even in non-obese, healthy young adults. Evaluating changes inCMR components, as well as in their predisposing factors, may be a preventivemeasure to the development of NCDs, since clinical consequences of NCDs, such asclinical complications, increasing prevalence of premature death, and its social andeconomic costs may be prevented by interventions.This is a cross-sectional study on a convenience sample. The volunteers’recruitment occurred through invitations by email, posters, and classroomvisits. The data collection was performed between May of 2013 and October of2014 at two universities, one public and one private, located in the city ofAracaju, Northeast, Brazil.Non-obese, clinically healthy young adults, who were students of schools ofhealth sciences, aged between 18 and 25 years of both sexes, participated in thestudy. Exclusion criteria included the evidence of any disease related to OS,chronic inflammation, gestation, lactation, water-electrolyte imbalance, andself-reported changes in body composition or in absorption and/or metabolism ofnutrients. Exclusion criteria still included recent use of medicaments and/ordietary supplements, follow-up of nutritional treatment that may affect energybalance, food consumption, lipid profile, plasma insulin levels, and metabolismof glucose; regular use of birth control pills in the 2 months before theparticipation in the study; unstable body weight in the past 6 months ; being an elite athlete or planning to changelifestyle during the period of the study; and follow-up of special diets  in the 3 months prior to the participationof the study.11 considering a prevalence of 9.9% for highwaist circumference among university students,12 significance level of 5.0%, sampling error of5.0% and population size of 8,951, considering the number of university studentsenrolled in health majors. A minimum sample size of 135 individuals wasestimated.The sample was calculated according to Miot (2011),13Height was measured to the nearest 1 mm using a stadiometer . Weight was measured to the nearest 100 grams using anelectronic digital balance  withmaximum capacity of 180 kilograms. Body Mass Index (BMI) was calculated bydividing body weight (kg) by height squared (m) and classified according to thecutoff points proposed by the World Health Organization (WHO).13Waist circumference was measured to the nearest 1 mm between the last rib and theiliac crest using a flexible and inelastic tape measure.Lange caliper, CambridgeScientific Industries Inc., Cambridge, Maryland, USA). Truncal fatpercentage was calculated from the ratio of the sum of subscapular andsuprailiac skinfolds and the sum of the four skinfolds.14Triceps, biceps, subscapular, and suprailiac skinfolds thickness were measured tothe nearest 1 mm using a skinfold caliper , from which body fatand fat-free mass were calculated in kilograms. Blood collection was performed by venipuncture after fasting of 12 hours and nointake of alcohol, coffee or tea for 24 hours. Samples of heparin and plasmawere separated by centrifugation at 2,465 g to 5°C for 15 minutes and stored at-80°C.Serum concentration (mg/dL) of glucose, total cholesterol, high-densitylipoprotein (HDL-c), and triglycerides (TG) were analyzed by a colorimetric orturbidimetric assay by an automatic analyzer using specific assay kits.TAC in plasma was determined by colorimetric assay using a specific assay kit. The assay was based on theability that all the antioxidants present in the sample (plasma) have to inhibitthe oxidation of the oxidizable substrate ABTS  to ABTS•+ by metmyoglobin.The amount of oxidized substrate (ABTS•+) was monitored by absorbancereading at 750 nm. The decrease in absorbance at 750 nm was directlyproportional to the concentration of antioxidants in plasma expressed as mMTrolox equivalents, a synthetic water-soluble analogue of vitamin E.15Systolic and diastolic blood pressure levels were measured to the nearest 2 mmHgusing a mercury sphygmomanometer according to Perloff et al.1abdominal obesity (waist circumference >80 cm for women and > 94 cm formen); fasting hyperglycemia (> 100 mg/dL); hypertriglyceridemia (> 150mg/dL); low HDL-C (< 50 mg/dL for women and < 40 mg/dL for men); andhypertension .The CMR components were diagnosed according to the criteria of the InternationalDiabetes Federation (2005):17 using the Estimated Average Requirement (EAR) and theAdequate Intake (AI) values as cutoff points. Inadequate energy intake wasdetermined by the intake < 90% or > 110% from the Estimated EnergyRequirement (EER), calculated by predictive equations proposed by the Instituteof Medicine (2005).15 Somemethodological precautions were adopted around the food consumption assessment,such as the use of visual aids to assist the participants estimate the portionsizes during the FFQ application, training of interviewers, pilot test toclarify questions and inadequacies in the FFQ, and standardization ofrecipes.Usual dietary intake was obtained by the application of a semiquantitative foodfrequency questionnaire (FFQ), developed for this study population. The VirtualNutri software was used to quantify energy and nutrient intake. Inadequateintake was evaluated according to the recommendation proposed by the DietaryReference Intakes (DRI) ,18We collected information on vitamin supplements, smoking, number of cigarettesper day, regular physical activity, and its intensity. To characterize andquantify physical activity, we used the short version of the InternationalPhysical Activity Questionnaire (IPAQ), which is recommended by the World HealthOrganization and has been validated in Brazil by the Center of Studies of thePhysical Fitness Research Laboratory of São Caetano do Sul -CELAFISCS.Continuous variables were presented as mean ± standard deviation whilecategorical variables as absolute (n) or relative frequency (%).Kolmogorov-Smirnov test was used to verify the normality of the distribution.Unpaired Student’s t-test was adopted to compare the categorized groups by thepresence of the CMR components. To track the correlation between TAC and othervariables of interest related to the components of the CMR, we used Pearsontest. Multivariate linear regression was performed with the fasting glucosevalues (mg/dL) as being the dependent variable and TAC value (mM), sex and ageas being the independent variables. A 95% confidence interval was used todescribe the values of the linear regression coefficient (β).Statistical significance was accepted at p < 0.05. All analyses were conductedusing Statistical Package for Social Science, SPSS version 20.0, forWindows.The study was approved by the Human Research Ethics Committee of the FederalUniversity of Sergipe (C.A.A.E.: 0113.0.107.000-11).In accordance with the principles of the declaration of Helsinki, all volunteerswere informed about the study protocol and then signed the consent form. Thevolunteers were informed about the methods and procedures used in the datacollection, the possible benefits and inconveniences, the privacy of results andthe voluntariness of participation.A total of 139 non-obese and clinically healthy young individuals, aged 21.4 ±1.9 years, participated of the study. Women predominated in the distribution ofgender (77%). The anthropometric, clinical and biochemical parameters are describedin Although the individuals of this study were non-obese and clinically healthy, theyalready presented CMR components. About 15% (n = 20) of the sample had low HDL-cconcentrations followed by high diastolic blood pressure levels ,triglyceride , glucose , and abdominal obesity .Almost one-quarter of the sample  had at least one component of theCMR followed by 2  and 3 or more CMR components ,respectively.Among the nutrient intake inadequacies were considerable those relating to theconsumption of saturated fat , fiber , and vitamin D .The individuals were categorized by the presence of the CMR components . Those wGlycemia was the only CMR component which correlated with plasma TAC .2= 0.10; β = 0.17; p =0.001). TAC had a positive predictive effect on the serum glucose levels. Anincrease by 1 unit in the levels of TAC was associated with an increase by 0.17mg/dL in fasting glycemia. It is noteworthy that 10.0% of the increase in glycemiawas due to the effect of TAC , non-smoking, university students from health sciencesarea, mainly nutrition (39%), and with low prevalence of alcohol consumption.Determining the presence of CMR components in young populations is relevant for theearly diagnosis, and establishment of specific interventions and preventivemeasures. In our study population, although the prevalence of alterations in theoutcome measures was low - according to the proposed reference values - almostone-quarter of the sample had at least one component of the CMR. Low HDL-c was themost prevalent component (15%). On the other hand, studies on college students, bothin Brazil23 and international22 studies varied from 30 to 77% among individuals with atleast one risk component, from 12 to 13% for those with two, and from 3 to 16% forthose with three components. Low HDL-c and high blood pressure are the mostprevalent CMR components.The prevalence of CMR components in college populations in national24 The resultof this combination is the greater weight gain in the first year of college whencompared to adulthood.25The early development of these components in young adults has been attributed to pooreating habits, commonly observed in young populations, due to factors related tothis life stage, including independence, inability to make healthy food choices,lack of time, convenience, costs, and influence of both physical and socialenvironments.23 and from othercountries.22In this study, it was observed that the presence of at least one risk component inthe study population was associated with greater anthropometric (body weight) andbiochemical  parameters. These resultsare corroborated by studies conducted with college students from Brazil26 acute myocardial infarction,27 and atherosclerosis.28 Studies have showed that highvalues of TG/HDL-c are correlated with increased CMR in the population.29 In addition, TG/HDL-c ratio has been positively correlatedwith biochemical  and anthropometric parameters and negatively withHDL-c.30TG/HDL-c ratio has been largely discussed as an atherogenic risk indicator forpredicting the development of coronary artery disease,31 hypertriglyceridemia, low HDL-cand hypertension,33 has been associated to thedevelopment of NCDs, especially, type 2 diabetes, cardiovascular diseases, andcancer. In Brazil, a study was conducted to evaluate the association betweencardiovascular risk factors and anthropometric indicators in patients withNCD.34 The authors foundthat 74% of the sample presented glycemia > 100 mg/dL, 56% low HDL-c, 82% highwaist circumference, and 78% overweight.34The presence of the CMR components, such as abdominal obesity,6 Several studies have identified the association between plasmaTAC -an important biomarker of OS for expressing the synergistic action betweenvarious antioxidant compounds5 -and alterations in the anthropometric, clinical and biochemical CMRcomponents.6 It is worthmentioning that all these studies were conducted with individuals with NCDs. Thereis still a dearth of studies with healthy individuals.Excessive ROS production is another factor that has been associated with the presenceof CMR components and, consequently, to the development of NCDs.2= 0.10, β = 0.17, p = 0.001). Thepositive correlation between TAC and fasting glucose found in the study does notcorroborate with the literature.35Hyperglycemia increases the expression of OS by the increase of NADPH concentrationsand ROS production due to the intense mitochondrial metabolism of glucose.36 This results in an increasedproduction of electron donors (FADH2 and NADH) in the Krebs cycle and, hence, in ahigh mitochondrial membrane potential (DmH+) by pumping protons across the innermembrane, inhibiting electron transport at complex III, and increasing the half-lifeof free-radical intermediates of coenzyme Q (ubiquinone) which reduces O2to superoxide. Thus, studies have shown a negative correlation between fastingglycemia and plasma and dietary TAC,37 as well as greater amount of products from oxidative reactions,which reduce the level of the substances that make up the antioxidantsystem.38 However, allthese studies were conducted with individuals with NCD already established.Although the individuals of the present study were non-obese and clinically healthy,it was possible to identify an association between TAC and fasting glycemia afteradjustment by sex and age  is associated to the increase of 0.17 mg/dL in the fasting glycemialevels, 40 in young adults(1.60 mM). This result may be associated with the low prevalence of behavioral riskfactors, such as sedentary life style (36%), low consumption of alcohol, beingnon-smoker, in addition to be university students in health sciences.The lack of associations between TAC and the other anthropometric, clinical andbiochemical variables in the study may be explained by the low prevalence ofalterations on these parameters and by the characteristics of the studiedpopulation: young, predominantly women, non-obese, clinically healthy, physicallyactive, students of health sciences, low alcohol consumption, non-smokers, andmarkers of adiposity - waist circumference (71.1 ± 5.6 cm) and body fatpercentage (23.0 ± 9.7%) - below the risk for triggering metabolicalterations. Nevertheless, it is worth pointing out the high TAC value found in thepresent study  compared to the values found byBarbosa et al.in vitro,without similarity to biological systems. Their results should therefore beinterpreted with caution since they do not measure bioavailability, invivo stability, retention of antioxidants in the tissues, andin situ reactivity.Some limitations in this study must be acknowledged: the sample loss of somevariables due to incomplete information and/or study dropout; the assessment of foodconsumption by instruments available in the literature are subject to error becauseof their large inter- and intra-individual variability, as well as the dependence onthe respondents' memory about past habits, low accuracy in quantifying the intakedue to the use of standardized measures and food lists. Finally, the methods used toevaluate plasma TAC strictly reflect chemical reactions In this study, CMR components were present in some young, clinically healthy,non-obese, and high-TAC adults.The observed positive correlation between plasma TAC and fasting glycemia suggeststhe establishment of an adaptive mechanism. The increase in glycemia in a biologicalsystem, in homeostasis, would determine a compensatory increase of the plasmaTAC.Thus, different from what occurs in populations with NCD already set in, TAC was notassociated with CMR components in this sample of young, non-obese and clinicallyhealthy individuals due possibly to the establishment of compensatory mechanismsthat become activated in physiological conditions.


The high expression of MALAT1 could be considered a biomarker of the early detection of lymph node metastasis and predictor of poor survival in RCC patients, who likely manifested short overall survival . For diagnostic value, the pooled result showed that lncRNA maintained a sensitivity of 0.89 and specificity of 0.91 in RCC diagnosis, The area under the curve of 0.94  for lncRNA in RCC diagnosis also indicated a significant advantage over other biomarkers. Our systematic review and meta-analysis demonstrated that lncRNAs could be considered biomarkers to detect lymph node metastasis and distant metastasis in early stages. LncRNAs could function as potential prognostic markers in RCC. LncRNAs could also display high accuracy for RCC diagnosis.We conducted a systematic review and meta-analysis to investigate the clinical values, including clinicopathology, prognosis, and diagnosis of different long non-coding RNAs (lncRNAs) in renal cell carcinoma (RCC). A total of 14 eligible studies, including 10 on clinicopathological features, 11 on prognosis, and 3 on diagnosis were identified. Results revealed that metastasis-associated lung adenocarcinoma transcript 1(MALAT1) expression was associated with tumor stage (odds ratio [OR], 3.46; 95% confidence interval [CI], 1.63-7.36;  It is estimated that about 66,800 Chinese will suffer from kidney cancer in 2015, and about 23,400 Chinese will die from this cancer , and micLncRNAs are a class of RNA with transcripts longer than 200 nucleotides and lack functional open reading frames . These RHowever, single study may be inaccurate and insufficient because of limitations related to sample size and research programs. As such, studies should be systematically analyzed to determine the potential clinical values of lncRNAs in RCC. Thus far, meta-analysis has yet to be performed, although some reviews have been conducted regarding the evaluation of the clinical values of different lncRNAs in RCC. Likewise, the clinical values of lncRNAs have been rarely analyzed. Therefore, we systematically reviewed studies that have identified the relationship between lncRNA expression and clinical outcomes in RCC. We also included these studies in our meta-analysis if extracted data could be merged. We mainly discussed our findings in terms of the following aspects: clinicopathological features, diagnosis, and prognosis.A total of 464 records were retrieved from PubMed, Embase and Web of science. A total of 173 duplicate reports were excluded. After the titles were reviewed, 181 records were excluded. After the abstracts were screened, 73 records were excluded. Subsequently, the 37 remaining full-text articles were assessed, and 23 studies, including 10 without clinical data, 1 with less than 30 sample numbers, 2 with description on genetic variation, 2 duplicate articles, 2 with discussion on other diseases, 2 with discussion on lncRNA-methylation, and 4 microarray articles, were further excluded on the basis of the exclusion criteria. A total of 14 studies, including 10 on clinicopathological features, 11 on prognosis, and 3 on diagnosis, were eligible for the final analysis. All of the selected studies were nonrandomized. A flow diagram of the study selection process is shown in Figure p value of these two studies by extracting the raw patient number. We then combined these two studies with a total of 6 groups ; therefore, a random effects model was used in this study; for other cases, a fixed effects model was utilized. After combining these two studies, we found that the statistical significance of one group changed. Before combination was performed, Hirata et al. [p values indicated that this correlation was indeed significant. The other comparisons of MALAT1 did not indicate significant changes.A total of 9 lncRNAs were described in the 10 included studies on clinicopathological features. renal cell carcinoma related transcript-1(RCCRT1) , proteins Figure . Moderata et al.  reported2 = 0.0%, p = 0.591). Therefore, the fixed effects model was applied. The model revealed that high MALAT1 expression could predict short OS  was performed to evaluate the lncRNA expression in 1,510 tissue samples. Ten different lncRNAs were associated with the prognosis of patients with RCC. The tumor type of patient in ten studies was ccRCC. The characteristics of these 11 eligible studies are presented in Table 2 = 64.00% and I2 = 89.28%, respectively). Therefore, the random effects model was used in this meta-analysis to calculate the pooled diagnostic parameters of the included studies. The pooled estimates of lncRNAs for the diagnosis of RCC were as follows: sensitivity(SEN), 0.89; specificity(SPE), 0.91; positive likelihood ratio(PLR), 9.4 ; negative likelihood ratio(NLR), 0.12 ; and overall diagnostic odds ratio (DOR), 75 . Figure In the diagnosis category, the main characteristics and Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2)  scores oOver the past decade, there were a lot of researches studying on lncRNAs. Increasing evidence showed that aberrant expression of lncRNAs was associated with clinical outcomes for cancer patients. LncRNAs were also closely related to cancer, they were considered eminent players in cancer biology. The up-regulation or down-regulation of many lncRNAs contributed to oncogenesis by affecting many cellular processes . In ordep-value. However, this conclusion may be insufficiently persuasive because these two studies used different cutoff values to distinguish high and low expression levels. Further studies should be performed because of the limitations of the included studies to verify these conclusions.In the classification of clinicopathological features, dysregulated RCCRT1 , SPRY4-IRegarding the prognostic value, the increased expression of four lncRNAs was associated with poor prognosis, as was the decreased expression of six lncRNAs. Although these lncRNAs were associated with the prognosis of patients with RCC, only MALAT1 was reported by two studies. We then conducted a meta-analysis on the relationship of MALAT1 expression and the OS of patients with RCC. The results revealed that a high MALAT1 expression predicted poor survival among patients with RCC; as such, these patients likely exhibited a short OS. However, this conclusion may be insufficiently persuasive because of the small size of this study. Thus, further research should be conducted to verify this conclusion.et al. [et al. [For diagnostic value, the pooled result showed that lncRNA maintained high sensitivity and specificity in RCC diagnosis. PLR and NLR were applied to judge the clinical applicability of lncRNA for diagnosis, and the PLR > 10 and NLR < 0.1 represent a high diagnostic accuracy . The pooet al.  reportedet al. . Jicheng [et al.  reportedet al. [Our study revealed that MALAT1 was correlated not only with clinicopathological features but also with RCC prognosis; MALAT1 was the most investigated lncRNA in RCC. Thus, the application of MALAT1 is possibly the most promising lncRNA for future studies. MALAT1 is a widely expressed and highly conserved nuclear-abundant lncRNA with a length of approximately 8000 nucleotides . MALAT1 et al.  reportedDespite the scope of this systematic and comprehensive meta-analysis, several limitations should be considered. First, the number of studies included in our meta-analysis was insufficient and the sample size was limited. Most of these studies contained diverse lncRNAs and used different follow-up endpoints, and one lncRNA was identified by two studies. Hence, a premature result may be obtained. Further studies should be conducted when more eligible studies are published. Furthermore, we did not evaluate publication bias in our study because of inadequate data. As literature-based analyses, studies with positive results were more likely to be published. However, the lack of these analyses can amplify the association between lncRNAs and clinical values of RCC, which may partly affect the interpretation and reliability of results. Second, the cutoff value and method for low or high levels of lncRNA varied in different studies, although qRT-PCR was the standard method used to evaluate the expression of lncRNA, which may cause the heterogeneity of results. Therefore, researchers should develop a cutoff value with enhanced consistency and establish a method to classify high or low lncRNA expression. Finally, a remarkable heterogeneity was observed in the analysis of diagnostic value. The heterogeneity of the subjects was composed of different factors, such as patients' baseline characteristics and different cut-off methods. Although we performed the subgroup analysis and found that lncRNA expression level was one of the sources of heterogeneity, this bias could not be accounted for the entire source.In summary, our study is the first meta-analysis to evaluate the expression of lncRNAs and clinical values of patients with RCC. Despite these limitations, our analysis revealed that lncRNAs could be considered biomarkers for lymph node metastasis and distant metastasis in early stages. Furthermore, lncRNAs could be potential prognostic markers for RCC. LncRNAs also exhibited high diagnostic accuracy for RCC diagnosis. What's more, MALAT1 was associated with tumor stage and could be considered a biomarker for lymph node metastasis in early stages, and high MALAT1 expression predicted poor survival among patients with RCC. However, further comprehensive, large-scale, and good quality studies should be conducted to confirm our findings and to verify the clinical values of lncRNAs in RCC.(lncRNA OR lncRNAs OR lincRNA OR lincRNAs OR “long non-coding RNA” OR “long noncoding RNA” OR “long intergenic noncoding RNA” OR “long non protein coding RNA” OR H19) AND  AND  OR “renal cell cancer” OR ). The literature covered was limited to human and English. Additionally, we screened the references from many relevant literatures, including all of the identified studies, reviews, and editorials.We performed a literature search on up-to-date electronic databases, including Pubmed, Embase, and Web of Science for studies that analyzed the relationship between lncRNAs and clinical values  in RCC patients on May 25th, 2016. We mainly searched three key aspects “lncRNA”, “cancer”, and “renal”, following was the detail search strategy in Pubmed: p value, and Kaplan-Meier curves or required data obtained by contacting corresponding authors.The inclusion criteria were as follows: (1) studies that investigated the association between the expression of lncRNAs and clinicopathological features, and the expression level of lncRNAs had to be divided into two levels: high or low; or (2) studies that detected lncRNA concentrations in serum or tissue and presented sufficient data, including sensitivity, specificity, and sample size, to allow us to perform statistical analysis and construction of two-by-two tables; or (3) studies that investigated the association between lncRNA expression and survival outcome and provided a HR or relative risk (RR), 95% CI or The exclusion criteria were as follows: (1) non-English paper; (2) non-human data; (3) studied only in cellular level; (4) letters, case reports, commentaries, conference abstracts or review articles; (5) sample cases fewer than 30; (6) studies focusing on lncRNA genetic alterations, including methylation patterns or polymorphisms; (7) HRs calculated on the basis of multiple lncRNAs; and (8) insufficient data for HR and 95% CI estimation. We included the most recent and informative article when overlapping studies were retrieved.Two investigators  independently assessed the quality of all the included diagnostic studies according to the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) criteria . The QUAp-value for survival analysis, if available, these data were obtained from the original article; otherwise, corresponding authors were contacted to collect these data; if Kaplan-Meier curves were available, data were extracted from graphical survival plots and HRs were estimated [Data were retrieved independently by two investigators , and the information of all the included studies were extracted using a predefined sheet which based on the reporting checklists of Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) . Data restimated .2 values of > 50% indicated that there was a substantial between-study heterogeneity existed. The potential sources of heterogeneity were further identified by subgroup analysis. A fixed effect model was applied for the meta-analysis with moderate heterogeneity (I2 < 50 %); otherwise, a random effect model was used [p < 0.05 was considered to be significant.A test of heterogeneity among studies was conducted using I-squared statistic, Iwas used , 48. A dwas used . (3) Sen


Kumimanu biceae, n. gen. et sp. is larger than all other fossil penguins that have substantial skeletal portions preserved. Several plesiomorphic features place the new species outside a clade including all post-Paleocene giant penguins. It is phylogenetically separated from giant Eocene and Oligocene penguin species by various smaller taxa, which indicates multiple origins of giant size in penguin evolution. That a penguin rivaling the largest previously known species existed in the Paleocene suggests that gigantism in penguins arose shortly after these birds became flightless divers. Our study therefore strengthens previous suggestions that the absence of very large penguins today is likely due to the Oligo-Miocene radiation of marine mammals.One of the notable features of penguin evolution is the occurrence of very large species in the early Cenozoic, whose body size greatly exceeded that of the largest extant penguins. Here we describe a new giant species from the late Paleocene of New Zealand that documents the very early evolution of large body size in penguins.  Kumimanu biceae, an extinct penguin from approximately 55–60 million years ago, which represents an independent origin of giant size soon after the evolutionary transition from flight to diving.The oldest known penguin fossils date to approximately 62 million years ago. Here, Mayr et al. describe  Waimanu manneringi and W. tuatahi, are the oldest and phylogenetically most basal Sphenisciformes reported so far3.New Zealand has yielded several fossils of Paleocene Sphenisciformes, which shed considerable light on the early evolution of penguins. All of the described specimens come from exposures of the Waipara Greensand in the Canterbury region and the two named species, 4. These fossils, an incomplete tarsometatarsus and associated pedal phalanges, belong to an unnamed species that is phylogenetically closer to the crown group (the clade including the extant species) than Waimanu. A definitive taxonomic assignment of the fragmentary fossils is, however, not possible and this is also true for Crossvallia unienwillia, an equally large stem group penguin from the late Paleocene of Antarctica6.Recently, remains of a very large penguin have also been found in the Waipara GreensandAptenodytes forsteri), is much better documented in post-Paleocene penguins, and the Eocene Anthropornis nordenskjoeldi and Pachydyptes ponderosus were for a long time considered to be the largest known penguin species8. Pachydyptes ponderosus, from the late Eocene of New Zealand, is known only from a few wing and pectoral girdle bones7; however, numerous isolated skeletal elements as well as a few partial skeletons have been reported for A. nordenskjoeldi, from the late Eocene and early Oligocene of Antarctica10. Recently, it was hypothesised that the well-preserved Kairuku grebneffi from the late Oligocene of New Zealand may have been taller, although less massive than P. ponderosus; for the largest individual of K. grebneffi, a total body length of about 1.5 m was estimated8. Based on isolated limb bones, lengths of 1.6 and 1.5 m were also calculated for Anthropornis nordenskjoeldi and the very large Palaeeudyptes klekowskii from the Eocene and Oligocene of Antarctica11. While partial skeletons of P. klekowskii indicate a somewhat shorter body length of about 1.4 m13, a recently described humerus fragment and a tarsometatarsus may come from individuals with an estimated length of about 2.0 m14. A large size is reached by other Palaeeudyptes species from the Eocene and Oligocene of Antarctica and New Zealand16, and further, very large Sphenisciformes occurred in the late Eocene of Australia17 and the late Eocene of Peru19.Gigantism, that is, the evolution of a size exceeding that of the extant Emperor Penguin , a large mythological monster, and manu (Maori), bird. The species epithet honors Beatrice (“Bice”) A. Tennyson, the mother of AJDT, who fostered his interest in natural history (pronounced “bee-chee-ae”). Type locality and horizon. Hampden Beach, Otago, New Zealand ; Moeraki Formation, late Paleocene  contained a specimen of the dinoflagellate Palaeocystodinium australinum and an unnamed dinoflagellate taxon that support a Teurian age for this sample; C. Clowes, pers. comm.). Diagnosis. A very large-sized sphenisciform species, which is characterized by proximodistally low and widely spaced condyles of the tibiotarsus. Distinguished from the late Paleocene Crossvallia and all post-Paleocene Sphenisciformes of which humeri are known in the dorsoventrally narrower humerus shaft, with ratio of maximum width of proximal end of humerus to minimum width of shaft being 2.4 . Distinguished from Waimanu tuatahi in having the bicipital crest of humerus not forming a distally directed bulge. Distinguished from Waimanu manneringi (the humerus of which is unknown) in having the tibiotarsus with proximodistally lower and more widely spaced condyles. Dimensions (in mm). Coracoid, maximum length as preserved, 141.8; humerus, maximum length as preserved, 149.6; dorsoventral width of proximal end, 75.0; Tibiotarsus, length from distal margin of condylus lateralis to preserved proximal end, 151.3; width of distal end across condyles as preserved, 51.9; estimated actual width of distal end, ~48. Femur, maximum length along longitudinal axis, 161 mm. Pachydyptes , whose description is pending24, has two such flanges on the caudal and cranial surfaces of the bones, respectively: the olecranon and the dorsal cotylar process .The coracoid lacks the sternal end Fig.  and the ahi Fig. , there iWaimanu tuatahi  is furthermore situated in a very lateral position. The sternal keel appears to have been unusually thick, but is too incompletely preserved to determine its original shape.Of the sternum, the cranial margins of the carina and the corpus are preserved Fig. , but onlW. tuatahi, the lateral surfaces of the corpus do not exhibit well-delimited fossae.A thoracic vertebra Fig.  has a slW. tuatahi, but less stout than the femora of Kairuku and Inkayacu. On the distal end, the fibular trochlea is strongly developed and there is a projecting tubercle for the gastrocnemialis lateralis muscle.The new species has a longer femur than all other fossil penguins, for which this skeletal element is known Table . The bonWaimanu manneringi  and a femur length of 161 mm, we calculate a weight of 101 kg and a body length of 1.77 m for K. biceae. These values exceed the weight and length estimates of all other stem group Sphenisciformes except for the exceptionally large specimens of P. klekowskii, for which a body length of about 2.0 m was estimated14. There exists no overlap between the bones of the holotype of K. biceae and those of the recently described large penguin foot bones from the Waipara Greensand (CM 2016.158.19)4. However, the proximal end of the tarsometatarsus of the latter species has a width of about 40.5 mm, and with a width of about 46 mm for the restored distal end of the tibiotarsus  has a more derived morphology than the tarsometatarsus of aft Fig. .11 that some sphenisciform species achieved a large size very early in the evolution of penguins. What is unexpected, however, is how fast these birds reached even their maximum size in the earliest Paleogene. That a penguin rivaling the largest previously known fossil species existed in the Paleocene may indicate that gigantism in penguins arose shortly after these birds became flightless divers. Gigantism therefore may be an inherent feature of Paleogene penguins, which may have evolved soon after aerodynamic constraints ceased to exist.Irrespective of the exact affinities between the new species and other Paleocene stem group Sphenisciformes, our study corroborates the observation of earlier authorsKumimanu furthermore documents that a very large size occurred in a phylogenetically basal penguin, which in its osteological features closely resembles the Sphenisciformes from the Waipara Greensand. Because the new taxon is phylogenetically separated from the giant Eocene and Oligocene species by various smaller taxa, such as Perudyptes, Delphinornis, Mesetaornis, and Marambiornis  being due to positive, directional selection on population level, which benefits survival and mating success25. In penguins, a size increase may have also constituted a selective advantage in competition for suitable breeding grounds. A correlation with improved diving capabilities has likewise been discussed2.There exists no correlation between palaeotemperatures and body size in fossil Sphenisciformes, with most giant taxa having evolved in a ‘greenhouse world’27, and competition with gregarious pinnipeds for safe breeding was also considered28. The disappearance of giant penguins indeed coincides with the rise of marine mammals, that is, odontocete cetaceans and pinnipeds27, but the exact causes and mechanisms of a competitive replacement remain poorly understood. In any case, the evolution of penguins appears to have been strongly influenced by non-avian vertebrates: whereas the end-Cretaceous extinction of larger marine and terrestrial predators may have been the ecological driver for the loss of flight capabilities in the earliest Sphenisciformes, the radiation of pinnipeds and odontocetes towards the Oligocene27 seems to have terminated the existence of giant species towards the Miocene.To understand size evolution in penguins we therefore must not seek an explanation for the presence of giant species throughout the Paleogene, but one for the absence of equally large species in the Neogene and today. Earlier authors proposed that feeding competition with marine mammals played a role in the extinction of giant penguins and other very large wing-propelled diving birdsThe fossils referred to here are in the collections of Canterbury Museum, Christchurch, New Zealand (CM); Museo de La Plata, La Plata, Argentina (MLP); Museum of New Zealand Te Papa Tongarewa, Wellington, New Zealand (NMNZ).8. Character scoring for the new fossil is as follows: 122:1, 123:0, 129:0, 131:0, 136:1, 137:1, 139:0, 140:2, 141:0, 149:0, 150:0, 151:1, 154:2, 155:0, 156:1, 157:1, 159:0, 160:1, 161:0, 196:1, 199:1, 235:1; all other characters were scored as unknown (?). Based on our study of the published fossils and various new specimens24, character 140  was scored as “2” (absent) for Waimanu tuatahi; the character was scored as “0” (complete) for Anthropornis nordenskjoeldi29. Character 141 (foramen nervi supracoracoidei) was scored as “0” (absent) for W. tuatahi and A. nordenskjoeldi. Based on the referred tibiotarsi in ref. 10, character 199 (position of sulcus extensorius) was scored as “1” (close to midline of tibiotarsus) for Mesetaornis exilis, Marambiornis polaris, Delphinornis larseni, D. arctowskii, D. gracilis, D. wimani, Palaeeudyptes klekowskii, and P. gunnari. The nexus file used in the analysis can be found in the Supplementary Data Phoebetria palpebrata  as an outgroup taxon. To improve the resolution of the resulting phylogeny, we furthermore restricted the coverage of crown group Sphenisciformes to six species and deleted fossil taxa, which are poorly represented or irrelevant for the present study . For this second analysis, two characters were newly added: character 246  was scored as present (0) for P. palpebrata, W. tuatahi and the new taxon, absent (1) for Delphinornis larseni (based on a referred ulna in ref. 10), Anthropornis nordenskjoeldi, Palaeeudyptes klekowskii, Kairuku grebneffi, K. waitaki, Inkayacu paracasensis, Icadyptes salasi, and all extant sphenisciform species, and unknown (?) for all other species included in the analysis; character 247  was scored as 2.4 or more (0) for P. palpebrata, W. tuatahi and the new taxon, unknown (?) for Mesetaornis polaris, Marambiornis exilis, and Delphinornis spp., and < 2.4 (1) for all other species included in the analysis. All analyses were performed with the heuristic search modus of NONA 2.030 through the WINCLADA 1.00.08 interface31, using the commands hold 10,000, mult*1000, hold/10, and max*. Character ordering was as in ref. 8. Bootstrap support values were calculated with 200 replicates, ten searches holding ten trees per replicate, and TBR branch swapping without max*.The phylogenetic analysis is based on a published morphological character matrix http://zoobank.org/urn:lsid:zoobank.org:act:3EF7C725-A115-4A80-8FA8-2461342AEDEE and http://zoobank.org/urn:lsid:zoo-bank.org:act:596D5C8A-052A-48A4-81E2-67F0183BBAAA.This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the International Code of Zoological Nomenclature. The ZooBank Life Science Identifiers for the taxa in this publication are The nexus files used in the phylogenetic analysis are available in the Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Patients were from 14 public clinics of 10 cities from all geographical regions of Brazil. A high prevalence of MS was found, especially among adults (32.3%), and this was related to age, female gender, acid uric levels, and the presence of acanthosis nigricans. ALT above the normal range was associated with triglyceride levels , serum uric acid, age, male gender, HbA1c, and non-Caucasian ethnicity. Patients with T1D, metabolic syndrome, and the aforementioned factors may be at a higher risk of NAFLD and should be referred to ultrasound for NAFLD evaluation. Further studies are necessary to establish the prevalence of NAFLD in individuals with T1D and to determine the disease’s progression in these patients.Obesity is increasing worldwide, affecting even patients with type 1 diabetes (T1D). A higher prevalence of associated comorbidities is expected, such as non-alcoholic fatty liver disease (NAFLD). This paper reports a cross-sectional multicenter study on a population with T1D ( Type 1 diabetes (T1D) is an increasingly recognized disease worldwide, associated with enhanced risk of micro- and macrovascular complications, comorbidities, reduced life expectancy, and higher health care costs ,3,4,5. OThe obesity epidemic also affects the clinical features of T1D, usually diagnosed in a lean patient, with more cases being recognized in individuals with overweight and even obesity . FurtherNAFLD is the most common cause of chronic liver disease, and affects 25 to 30% of the world’s population. It is defined as the presence of ≥5–10% of hepatocytes with fat deposits, ranging from steatosis to high degrees of inflammation. NAFLD is often diagnosed in asymptomatic individuals without excessive alcohol intake (<20 g/day for women and <30 g/day for men), with persistent increase in aminotransferases, particularly alanine aminotransferase (ALT) or those with normal aminotransferases but with metabolic syndrome. These patients are usually referred to abdominal ultrasound. The finding of increased hepatic echogenicity leads to the clinical diagnosis of the disease, although definitive diagnosis depends on liver biopsy, performed only in specific situations, since it is an invasive procedure that is not required for the treatment this condition. NAFLD increases the risk of cardiovascular disease and hinders the achievement of glycemic goals, since it is associated with insulin resistance ,19,20. Due to these changes in the clinical and metabolic profile of T1D, we aimed to evaluate clinical and laboratory parameters that can be used as initial screening tests for fatty liver disease in T1D, as most studies approach only T2D. We also investigated the prevalence of metabolic syndrome in a multicenter population of T1D patients in Brazil and its relationship with elevated aminotransferases.This was a cross-sectional study, based in a multicenter cohort of T1D patients, evaluated between August 2011 and August 2014 at 14 public clinics of secondary and tertiary care, located in 10 cities in all Brazilian geographic regions . The methods have been described previously . Briefly, all patients attended secondary and tertiary clinics from the National Brazilian Health Care System and were seen by an endocrinologist. They had access to free neutral protamine Hagedorn (NPH) and regular insulin, syringes, needles, glucometers, and strips for blood glucose monitoring.The inclusion criteria were: patients were older than or equal to 13 years of age, diagnosed by a physician with T1D through classical clinical findings , and had attended medical follow-up for at least six months in each center.The exclusion criteria included: being pregnant or breastfeeding at the time of inclusion; acute infectious process or history of ketoacidosis in the three months prior to recruitment; or exacerbation of comorbidities, such as congestive heart failure, cardiac arrhythmias, acute respiratory failure, or severe obstructive lung disease at the time of enrollment.Each center provided data for at least 50 T1D patients, which were analyzed by a trained coordinator. Participants underwent a clinical-demographic survey by a standardized questionnaire in which data were collected on gender, current age, self-reported ethnicity, age at diagnosis, as well as diabetes duration, diet (characteristics and adhesion in last month), level of physical activity, smoking (defined as the current use of more than one cigarette per day), alcohol consumption, type of insulin and daily dose, frequency of self-monitoring of blood glucose, self-reported frequency of hypoglycemia in the last month (number of times the patient had blood glucose levels ≤70 mg/dL or needed help to overcome hypoglycemic symptoms), use of other medications, associated diseases, and hospitalization due to any cause in the last year. Economic status was evaluated according to the Brazilian Economic Classification Criteria, which also considers education status. Participants were examined and the following clinical variables were evaluated in medical visits: weight (kilograms), height (centimeters), BMI, blood pressure (BP), heart rate (HR), waist circumference , and the presence of acanthosis nigricans.The following laboratory variables were obtained at the last clinical visit: fasting plasma glucose, glycated hemoglobin A1c , urea, creatinine, total cholesterol (TC), HDL cholesterol (HDL-c), triglycerides (TG) and LDL cholesterol (LDL-c) calculated by Friedewald’s equation , serum aminotransferases (AST and ALT), ultrasensitive C-reactive protein (CRP), complete blood count, and speed erythrocyte sedimentation. Fasting plasma glucose, TG, HDL-c, and TC were measured using enzymatic techniques.Our original sample included 1760 patients. All patients were diagnosed with T1D between 1960 and 2014. Patients between 13 and 18 years of age were classified as adolescents, and patients 19 years of age or older were considered adults, based on the American Diabetes Association (ADA) criteria.Of the 1760 patients, we subsequently excluded those with known liver disease, including viral hepatitis, hemochromatosis, alcoholic fatty liver disease, autoimmune hepatitis, cirrhosis, and hepatocellular carcinoma, as well as those who were using medications that are known to cause NAFLD: corticosteroids, methotrexate, amiodarone, and tamoxifen. Twelve individuals using corticosteroids and five with known liver disease were excluded. Also, we excluded 45 patients without data on aminotransferases and 36 patients with missing data on metabolic syndrome criteria.The study was approved by the Ethics Committee of Pedro Ernesto University Hospital, at State University of Rio de Janeiro (protocol: CEP/HUPE 2769/2010), which was the coordinator center, and by the local ethics committee of each center. All participants or their guardians signed informed consent. Investigations were carried out following the rules of the Declaration of Helsinki.Patients were stratified according to metabolic syndrome criteria by the International Diabetes Federation (IDF) . ConsideAdolescents aged 16 years or older were classified according to the same criteria as adults. Adolescents aged 13 to 15 years old were classified according to the following criteria: (1) abdominal obesity: WC > 90th percentile for age and sex; (2) TG ≥ 150 mg/dL (1.7 mmol/L); (3) HDL-c < 40 mg/dL (1.03 mmol/L); (4) elevated BP ≥ 130 × 85 mmHg . As therFurther analysis was performed with patients stratified into two groups: normal vs. elevated aminotransferases, according to the range of normality recommended by the American College of Gastroenterology (ALT ≤ 25 U/L for women and ≤ 33 U/L for men) . Then, wt-tests or Mann-Whitney U test was used. A linear-by-linear association was performed to evaluate the relationship between quartiles of aminotransferases and the prevalence of metabolic syndrome. A binary logistic regression was performed to access the predisposing factors to metabolic syndrome, considering clinically relevant factors that were statistically significant in the descriptive analysis and that were not involved in the diagnostic criteria. The following factors were included: gender, age, diabetes duration (years), uric acid levels, family history of type 2 diabetes and family history of obesity in first degree relatives, presence of acanthosis nigricans, years of education, and practice of physical exercise. The Nagelkerke R2 value was calculated. Odds ratios with 95% confidence intervals (CI) are expressed as indicated. In another analysis, a stepwise linear regression was performed to determine factors that influence ALT levels. The same factors used on the binary logistic regression were included in addition to the criteria of metabolic syndrome, use of medications , uric acid levels, HbA1c, and self-reported ethnicity. A receiver operating characteristics (ROC) curve was also performed in an attempt to establish the best cut-off for triglycerides that discriminates elevated ALT in this population with T1D. Differences were considered significant at a two-sided p-value less than 0.05. All statistical analyses were performed with Statistical Package for Social Sciences (SPSS) 24.0 .Continuous variables are expressed as means ± standard deviations (SD) or medians [interquartile range], and categorical variables as frequencies and percentages. For the analysis of the differences of categorical variables, the Chi-square or Fisher’s exact test was used, when indicated. To compare the means of the independent continuous variables, Student’ p < 0.01).A total of 1662 individuals were analyzed: 55.2% were female, with a mean age of 30 ± 12 years, 53.9% were Caucasian, from the low (47%) or middle (43.7%) social class. Diabetes duration was 15.5 ± 9.3 years for the whole population study, with a significant difference between adults and teenagers .A linear-by-linear association was observed between quartiles of ALT levels and the prevalence of MS (p < 0.001). A total of 110 patients had ALT above the range of normality recommended by the American College of Gastroenterology (ALT > 25 U/L for women and >33 U/L for men) , 48 of w2 = 280.723, p < 0.001, and explained 22.5% (Nagelkerke R2) of the variance in metabolic syndrome. The odds of having MS was 2.63 for females compared to males . Acanthosis nigricans exhibited the highest odds , and age and acid uric levels were also significant predisposing factors to MS. Data are shown in In the logistic regression analysis, 99.2% of patients were included. The model was statistically significant, χA linear regression established that ALT levels have a direct relationship with triglyceride levels, serum uric acid, self-reported ethnicity as non-Caucasian, age, male gender, and HbA1c. A total of 95.7% of patients were included in this analysis. Data are shown in p < 0.001) and a triglycerides level of 129.5 mg/dL established the highest acceptable sensitivity of 64.5% and specificity of 80.2% to detect high levels of ALT. In the ROC curve analysis , the areThe present study showed that patients with type 1 diabetes in Brazil have a high prevalence of metabolic syndrome. Adults with MS had higher rates of comorbidities such as hypertension and dyslipidemia, as well as parameters linked to insulin resistance such as higher serum acid uric, higher rates of acanthosis nigricans, and a more frequent use of metformin. Adolescents did not show as many alterations, probably due to shorter diabetes duration. Although we found significantly lower doses of daily insulin in the group with MS compared to the group without MS, HbA1c levels were similar in these groups and the small difference does not seem to have a clinical impact. Diet adherence was not related to the presence of MS. However, this might be related to information bias, since it was self-reported. Even so, diet is still recognized as an important treatment tool for metabolic syndrome and should always be reinforced. Predisposing factors to MS were female gender, age, acid uric levels, and the presence of acanthosis nigricans. Both groups, with and without metabolic syndrome, had poor glycemic control, as shown by HbA1c and FPG levels. Good glycemic control is still an important issue in Brazil, related to insulin and diet adherence. Even among patients with good adherence, the majority does not have HbA1c in their target range ,21. We cBecause MS is a known risk factor for NAFLD, most guidelines suggest that these individuals have their levels of aminotransferases evaluated, especially ALT, as it correlates to liver fat content ,20,24. IRegarding the criteria for metabolic syndrome, triglyceride levels were the most relevant parameter in the linear regression. They showed a direct association with ALT levels, even after correction for the use of lipid-lowering drugs. For each 10 mg/dL increase in triglyceride levels, ALT increases 0.5 UI/L. Serum uric acid, age, male gender, and HbA1c also showed a positive relation with ALT increase. Uric acid is a product of fructose metabolism  in the lParticular strengths of our study include the multicenter design with standardized protocol, the large admixed population sample of type 1 diabetic patients, and the study population from across all geographic regions of Brazil. Our study has some limitations. First, there are different classifications for MS. IDF classification was chosen because it establishes different reference values of waist circumference according to ethnicity. As we have analyzed a highly mixed population, this seemed to be the best option. Also, in the classification for MS, the use of angiotensin converter enzyme inhibitor or angiotensin 2 receptor blocker was not considered for the hypertension criterion, as it is frequently used in patients with albuminuria. Mean blood pressure and/or diagnosis of hypertension recorded on medical charts were used. Still, we found a high prevalence of metabolic syndrome, especially in adults. Second, a misclassification of diabetic patients could have occurred because we did not consider autoantibodies or C-peptide levels for the diagnosis of T1D. However, we do not believe that this has great impact in our results, since 93.2% of the studied population was diagnosed before 30 years old, and most likely were correctly classified. Third, liver images and biopsies for the diagnosis of NAFLD were not performed because in this study we did not intended to diagnose NAFLD, but to established the possible predictors for screening. NAFLD is an important cause of liver disease, associated with cardiovascular disease, cirrhosis, liver cancer, and impaired glucose control. Most studies approach only type 2 diabetes, because of its association with hepatic insulin resistance that leads to an inability to suppress hepatic glucose production and in turn, favors fatty acid liver deposits. Although type 1 diabetic patients have low levels of hepatic insulin in comparison to type 2 diabetics, thereby favoring β oxidation, it is still controversial whether this protects  them agaAlthough reference values for ALT have frequently been questioned by specialists  and stanPatients with T1D and metabolic syndrome may be at a high risk of NAFLD. More studies are necessary to establish the prevalence of NAFLD and to define the progression of this disease in type 1 diabetic patients.


Supplemental Digital Content is available in the text The objective of this study is to systematically evaluate the efficacy of renal denervation (RD), adjusted drugs, or combined therapy for resistant hypertension (RH) through a systematic review and meta-analysis of controlled studies.Publications were comprehensively searched. Studies that investigated the effects of RD and/or adjusted drugs in lowering blood pressure (BP) were included. After quality assessment and data extraction, subgroup analyzes were first performed according to blinding method. Meta-regression and inverted funnel plots were also conducted.A total of 13 studies containing 1604 RH patients were included. Compared with control, the meta-analysis showed that RD significantly reduced office-based BP and ambulatory BP in 6 months in the unblinded studies, while no significant difference was found in the blinded studies. Meta-regression demonstrated the significant influence of blinding method on BP reduction, and further analysis revealed a significant BP reduction compared with baseline even in the control arm of blinded studies. RD had similar effects compared with adjusted drugs, and combined therapy seemed to further reduce the level of BP.The efficacy of RD was different between blinded and unblinded studies, and our data revealed a significant BP-lowering effect in the control arm of blinded studies, which was helpful to explain this finding. Furthermore, RD seemed to be equivalent to adjusted drugs, and also we suggested a potential advantage of combined therapy of RD and adjusted drugs compared with monotherapy for RH. However, more studies are warranted to better address the issue. Although RH did not account for the majority of hypertension, more and more attentions were focused on it in clinic. As true RH was always associated with many other risk factors, such as increased age, being overweight, diabetes, smoking, elevated creatinine, sleep apnea, and previous cardiovascular disease,,4 the risk of severe cardiovascular events increased undoubtedly together with long-standing and high level of BP.The reported prevalence of hypertension in the globe was 26.4% of adults with a total number of 972 million, and it was estimated that the prevalence would be 29.2% with 1.56 billion patients in 2025. Due to its high invasion and disability, as well as the development of antihypertensive drugs, the clinical application was gradually abandoned. Recently, a catheter-based renal denervation (RD) system was designed to ablate sympathetic fibers along renal artery, and this option was attempted to treat RH through inhibiting sympathetic outflow to renal by radiofrequency energy. Meta-analysis based on noncontrolled studies had reported the primary clinical outcomes of RD, and found both promising BP-lowering effects and rarely related complications.,7 Meanwhile, Europe and Canada approved the application of RD, and guidelines also mentioned that such a technique may be considered for drug-ineffective patients. A series of controlled studies and randomized controlled studies (RCTs) adopted kinds of outcome measures, including ambulatory BP, office-based BP, and home-based BP, and were further designed to investigate the BP-lowering effect.–22 The efficacy of RD, adjusted drugs, and combined therapy of both RD and adjusted drugs compared with monotherapy of adjusted drugs were all covered.Sympathectomy acted as a radical and surgical procedure for severe hypertensive patients without available drugs previously. Based on the finding, we performed a meta-regression to further clarify the BP-lowering effect according to blinding method, and also reviewed current evidence of adjusted drugs and the combined therapy.However, the results of BP-lowering effect seemed to be varied across the studies. A new published article revealed the significance of blinding method to BP-lowering effect, and concluded that “adding a randomized control arm does not reduce bias unless it is blinded.”22.1 AND (vascular OR nervous OR nerve) AND denervation AND hypertension. References, citations and related articles were also screened to increase the recall ratio. Reviews, animal studies, and case series were excluded.To identify all relevant studies investigating the efficacy and safety of RD, adjusted drugs, and combined therapy for RH, online searches were conducted in MEDLINE, EMBASE, the Cochrane Library, and other supplementary sources, such as clinical register center and Google scholar. Search time was up to March 3, 2016. We used search terms as follows: Inclusion of studies was mainly on the bias of screening and reading the titles, abstracts, and full-texts. First, only prospective controlled studies and RCTs were considered for the analysis. Noncontrolled and cross-over studies that compared the BP changes from the level of baseline to post-RD were excluded. Participants were RH patients, who were diagnosed by investigators and physicians. Patients were divided into RD group or control group. The interventions were previous drugs administration (unblinded studies), or sham procedure + previous drugs (blinded studies) in control group, while RD + previous drugs in RD group. The doses and kinds of drugs were stated to be not allowed to alter in each study, except a pharmacist demonstrated a certain BP increase or decrease. Outcome measures should include BP changes and RD-related complications.2.2After final inclusion, detailed information in each article was extracted to present general characteristic, outcomes data, and methodological quality. General characteristic recorded included first author, publication year, case number, sex, age, intervention, and important factors, such as study design, blinding method, baseline systolic blood pressure (SBP), and diastolic blood pressure (DBP).2.3Primary outcome measures were office-based and ambulatory SBP/DBP changes in 3 and 6 months. Secondary outcome measures were nonresponse rate and operation-related complications. Nonresponse was defined as an office-based SBP reduction ≦10 mm Hg in 6 months.2.4Quality of RCTs was assessed by the Cochrane tool of risk bias in 7 items: random sequence, allocation concealment, blinding of participant, blinding of outcome assessment, incomplete outcome data, selective reporting result, and other bias. According to the reported information in each study, all the items were judged to be with low, high, or unclear risk of bias. Quality of controlled studies was assessed by the Newcastle–Ottawa Scale recommended by the Cochrane Collaboration in 8 items with a total of 9 stars, and a study achieved more than 5 stars was considered high quality.2.5χ2 statistic and presented by using I2 value. When the value of I2 <50%, the heterogeneity was considered to be nonsignificant, and a fixed-effects model was used. Otherwise, the heterogeneity would be significant, and a random-effects model was used. P value <0.05 was judged statistically significant.To pooled analyze the data of outcome measures, RevMan  was used. To pooled analyze the data of outcomes in single RD arm and control arm, MetaXL  was used. Subgroup analyses were first performed according to the blinding method. We presented overall effects by using mean difference (MD) with respective 95% confidence intervals (95% CI) for continuous data. Statistical heterogeneity was tested by 2.6P <0.1), then they were combined analyzed after excluding factors having overlapping effects. Inverted funnel plots were used to assess the risk of publication bias in each outcome measure.To investigate the source of heterogeneity, and the influence of important factors including study design, blinding method, baseline BP, body mass index, and pulse pressure, meta-regression based on individual study was quantitatively compared. MetaAnlyst  was used in random-effects models. Factors were converted to continuous variances, and all the factors were first analyzed by single-factor analysis  guideline, and did not involve any ethical issue.33.1,15–19,21 2 studies compared RD with adjusted drugs,,20 and 1 study compared combined therapy with monotherapy of adjusted drugs. Baseline SBP ranged from 144 to 181 mm Hg and DBP ranged from 81 to 97 mm Hg in RD group. Two kinds of catheters were reported, and 13 studies adopted a Symplicity Cather System except for 1 study adopted a EnglisHTN.A total of 194 publications were initially searched, and finally 15 articles that reported 13 studies containing 1065 patients in RD group and 539 patients in control group were included. The process of study inclusion is shown in Fig. ,20 Only 3 studies had low risk of bias in blinding of participants,,21,22 and 4 studies had low risk of bias in blinding of outcome assessment, as shown in Fig. Quality assessment of RCTs showed that 2 studies had unclear risk of bias in allocation concealment.3.23.2.1Four unblinded studies reported the data of office-based BP reduction in 3 months. Meta-analysis in random-effects model showed that RD reduced SBP by a mean of 22.92  mm Hg and DBP by a mean of 6.87 (3.41–10.33) mm Hg compared with control.Seven studies reported the data of office-based BP reduction in 6 months. Meta-analysis in random-effects model showed that RD reduced SBP by a mean of 23.32 (16.63–30.01) mm Hg in the unblinded subgroup, while by a mean of 3.5 (0.11–6.88) mm Hg in the blinded subgroup Fig. .3.2.2Four studies reported the data of 24 hours ABP in 6 months. Meta-analysis in fixed-effects model showed that RD significantly reduced the SBP  and DBP  in the unblinded subgroup. While no significant difference was found in both SBP  and DBP  in the blinded subgroup, as shown in Supp Figs. 1 and 2.3.3P <0.001, Fig. P = 0.03), and DBP  were significant. Further analysis showed that only blinding method was significant in a combined factors analysis .To explore the influence of other clinical factors, meta-regression was performed based on the data of office-based SBP reduction in 6 months. Single-factor analysis showed that blinding method , as shown in Fig. 3.4.2Compared with baseline, both unblinded subgroup and blinded subgroup showed a reduction of office-based SBP and DBP in 6 months. The results of ABP were similar that both unblinded and blinded subgroups showed a significant reduction.3.4.3In the subgroup of unblinded studies, both office-based SBP and DBP in 6 months were not significantly changed compared with baseline. But, in the subgroup of blinded studies which additionally adopted a sham-procedure compared with unblinded studies, both office-based SBP  and DBP  were significantly reduced, as shown in Supp Figs. 3 and 4. Meta-analysis of ABP also showed similar trends that ABP did not significantly reduce in the subgroup of unblinded studies in control arm, but in the subgroup of blinded studies, as shown in Fig. 3.5 and no significant difference was found in ambulatory SBP , although adjusted drugs showed a superiority of office-based SBP reduction . The other study included 106 cases, and reported nonsignificant differences in SBP reduction of both ambulatory  and office-based  outcomes.Two studies compared the results of RD with adjusted drugs in 6 months, and both of them were RCTs. One study included 19 cases,3.6Only 1 study containing 101 cases compared combined therapy of RD and adjusted drugs with monotherapy of adjusted drugs. The result showed that combined therapy further achieved a significant reduction of ambulatory SBP , while no difference in office-based SBP .4The included studies compared the indexes of renal function, including eGFR, serum creatinine, and cystatin C, and all of them demonstrated no significant difference. It was reported that RD-related complications were minor and mild during periprocedure, such as femoral artery pseudoaneurysm, transient intraprocedural bradycardia, transient blood pressure drop, and pain.5Our study that was an updated meta-analysis included the latest 7 RCTs and 6 controlled studies. With high reliability, we confirmed the BP-lowering effect of RD. We adopted multiple outcomes to clarify the influence of blinding method to the efficacy of RD for RH, and to be the first reported the estimated nonresponse rate of RD, which was as high as 21%. The efficacy of RD with adjusted drugs, and a potential advantage of combined therapy were also addressed.,25 Also, other studies reported similar reductions in 12 and 24 months.,27 However, some differences emerged when the results were limited to only blinded studies. Meta-analysis results showed that RD reduced the office-based BP by a mean of 3.5/1.82 mm Hg in 6 months, which was significantly lower than the level in unblinded studies.To confirm the BP-lowering effect, this study analyzed ambulatory and office-based BP. The combined effect of unblinded studies showed that RD reduced the office-based BP by a mean of 22.92/6.87 mm Hg (SBP/DBP) in 3 months and 23.32/9.22 mm Hg in 6 months, which were similar to previously published studies.There was no doubt that average ABP was the most certain and reliable index to reflect the real effect of RD. We further analyzed the data of ABP, and the results of unblinded studies still showed a significant reduction of ambulatory BP by a mean of 10.97/5.59 mm Hg in 6 months. But subgroup of blinded studies showed absolutely different results that no statistical difference was found compared with control although RD reduced the ambulatory BP by a mean of 2.2/0.9 mm Hg. Therefore, blinding method influenced the BP-lowering effect of RD for RH.,21 which were designed as large sample, blinded, and sham-controlled trials. However, both of them failed to meet the primary efficacy endpoints. Their combined nonresponse rate was 36%, which was obviously higher than the rate of unblinded studies (17%). Analysis of HTN-3 trials supposed that baseline SBP >180 mm Hg and non-African-American might be the predictors of significant BP reduction. However, available data based on individual study in current meta-regression excluded other potential factors including baseline BP, pulse pressure, and body mass index, while demonstrating the significance of blinding method.Current analysis included the latest published papers, among them there were 2 RCTs of Symplicity HTN-3 and FLEX, To design such blinded studies of surgical interventions, an additional sham procedure was decisive, and the blinding to all of participants, doctors, and investigators can be realized only when a sham-procedure was first conducted. Thus, the above results supposed that such a sham-procedure seemed to also have “BP-lowering effect.” It was clear that the difference between unblinded and blinded subgroups was mainly caused by the unexpected significant BP reduction in the control arm of blinded studies. However, it was hard to explain why blinded studies showed a significant BP reduction, as they stated that administrated antihypertensive drugs were comparable without statistical differences, and also no reporting of BP-lowering effect of angiography (the sham-procedure) was achieved in databases.To clarify the influence of blinding method on BP reduction, we first investigated the BP changes compared with baseline in separate RD and control arm. The results confirmed that office-based BP as well as ABP significantly reduced in RD arm regardless of blinding. Besides, the results directly revealed an interesting finding that both ABP and office-based BP significantly reduced also in the control arm of blinded studies. Certainly, the 2 blinded RCTs enabled the reliability of outcomes through enhancing blinding to reduce the risks of performance bias, detection bias (e.g. investigator bias) as well as selection bias.Currently, it was safe that RD was related to minor and mild complications. And it was still recommended as a supplementary treatment on the basis of drugs. As mentioned in the studies, the dose and kinds of antihypertensive agents were mostly not allowed to change, while this intervention seemed actually not to be clinically practical and useful, because BP fluctuation happened commonly in clinic and severe fluctuation must be handled in time. So it was important to evaluate the efficacy of RD compared with adjusted drugs in accordance with clinic, and the results found no significant difference of ABP control in 6 months. And interestingly, what if adjusted drugs combined with RD for RH? However, only 1 study addressed the issue, and concluded that RD further enhanced the effect of antihypertensive drugs.,31 While it seemed that other mechanisms may also be involved when both nerves and vascular were ablated and mechanical dilated. Some studies reported that RD also improved blood glucose and insulin sensitivity,,32 and RD even improved augmentation index (which was independent of BP and muscle sympathetic nerve activity) and had beneficial effects on arterial stiffness. Without clear explanations, we suggested that the combined effects of dilation and ablation on vascular and reduced activities of both renal and systemic sympathetic nerves would have complex influences on the whole body.Although RD was designed to ablate the sympathetic nerves around renal artery, and to reduce the activity of sympathetic nerves in overactive patients through inhibiting renin angiotensin aldosterone system without renal function impairment.,33 and only 1 study used a different one in our analysis. Another study first reported a PVI catheter, and it was applicable and useful in undeveloped and developing countries. However, our study did not include it as its significant shortcomings in the trial design. Information about experience and learning curve of the doctors performing RD was absent, and the determination and judgement of ablation efficacy were also ignored. Renal noradrenaline spillover or vascular muscle sympathetic nerve activity was reported to be validated to determine the inhibition effect of sympathetic activity,,15 while few studies tested them. The study of Mahfoud et al included part of patients in the study of HTN-2,,12 and slight overlapping effects may exist. Results of funnel plots suggested risks of publication bias existed , which may have negative influence on the outcomes. Besides, more and more studies summarized predictors to ensure the efficacy of RD for specific and true RH patients. All of baseline BP, race, and drug agents were potential predictors, which need to be confirmed. And further BP reduction would be beneficial to reach the treatment goal and to reduce the risk of cardiovascular incidence, although the combined therapy seemed promising, more supports were needed, however.Limitations and implications for future study were as follows. Different study design would induce a heterogeneity, and especially blinded RCTs eliminated patient’ subjective bias, although suggestive therapy was sometimes specially adopted in clinic. Some studies concerned that potential conflicting of interests might exist for RD device,6The efficacy of RD was different between blinded and unblinded studies, and our data revealed a significant BP-lowering effect in the control arm of blinded studies with a sham procedure, which was helpful to explain this finding. Furthermore, RD seemed to be equivalent to adjusted, and also we suggested potential advantages of combined therapy of RD and adjusted drugs compared with monotherapy for RH. However, more studies are warranted to better address the issue.


Arabidopsis thaliana. A single green fluorescent protein labeled plant was placed in the center of a grid of different planting densities of neighboring unlabeled plants or empty spaces, into which different treatments were made to the media. The root system of the central plant showed changes in the vertical distribution with increasing neighbor density, becoming more positively kurtotic, and developing an increasing negative skew with time. Horizontal root distribution was initially asymmetric, but became more evenly circular with time, and mean direction was not affected by the presence of adjacent empty spaces as initially hypothesized. To date, this is the first study to analyze the patterns of both vertical and horizontal growth in conspecific root systems. We present a portable imaging platform with simplicity, accessibility, and scalability, to capture the dynamic interactions of plant root systems.Intraspecific competition is an important plant interaction that has been studied extensively aboveground, but less so belowground, due to the difficulties in accessing the root system experimentally. Recent in vivo and in situ automatic imaging advances help understand root system architecture. In this study, a portable imaging platform and a scalable transplant technique were applied to test intraspecific competition in  Plants need water, nutrients, light, and space, and compete for these resources in agricultural systems and natural communities ,2,3,4,5.Arabidopsis thaliana have been focused on aboveground parts  and at the organismal and ecological scale [A. thaliana [Trifolium spp. [A. thaliana plants invest in expression of photosynthesis and auxin biosynthesis genes, and sacrifice expression of stress and defense mechanism genes [In intraspecific competition, aboveground tissues of a plant need to grow faster and taller than those of their neighbors in order to shade their competitors ,15 whileal scale ,19,20. Ial scale . The undthaliana ,23, and ium spp. . Conspecsm genes . ConspecL) is found to increase with nutrient availability, while biomass allocation to belowground roots (MR) decreases with nutrient availability [A = ML + MS, consisting of biomass of leaves and stems) has been modeled to scale equivalently to belowground root biomass (MR) [If representing the fraction of whole plant biomass (M), biomass allocation to aboveground leaves  .Abutilon theophrasti, for example, was shown to integrate information of nutrient distributions and competing neighbors to optimize root behaviors [In agriculture, the three-dimension (3D) structure of root systems  has been taken advantage of in crop improvement . Root syehaviors . Moreoveehaviors . A. thaliana plant not only contributes root debris as sloughed-off dead root cap cells and live border-like cells to the rhizosphere [The roots and rhizosphere of a plant contain a diverse array of associated microbes, plant hormones, and root exudates ,49 that zosphere ,53,54,55zosphere ,57. Recent automation of high-throughput imaging of 3D objects and development of image processing prompts phenomics to become an important research field. Concurrently, non-destructive imaging in vivo and in situ can advance the understanding of root systems from 2D to 3D, and from early seedlings to mature plants ,66,67,68A. thaliana.Here, we present a portable imaging platform with simplicity, accessibility, and scalability. To assess the effects of planting density and two nutrients and root exudate treatments on conspecific root systems, a media transplant technique was applied, and statistical analyses of root circularity and symmetry were incorporated to unravel growth of conspecific root systems over time. Coupled with the transplant technique and the imaging platform, we present new insights into the interaction of root systems in Arabidopsis thaliana, images of the root system of a single  plant was captured using a simple, portable and non-destructive imaging platform   which has been shown in previous studies to have an effect on above ground rosette diameter [https://github.com/jbmatthewgeisler/Roots) and deployed on a lab desktop computer. To assess conspecific root crowding response to planting densities and media treatments in form see . Three pdiameter  was compdiameter  was useddiameter ,79, depoA. thaliana first grew downward, and then reached the bottom of the MagentaR box at 14 days after planting (DAP) (Pisum sativum) roots [The primary roots of ng (DAP) . Next, lng (DAP) . Conspecng (DAP) . This obm) roots ,81,82.R box was 33 mm. The depth of maximum root density (as “location of max variable”) increased with DAP as roots grew towards the bottom of the MagentaR boxes  as a main effect, but there were no significant effects of planting density, treatment, or any interactions . The totaR boxes . By 19 DSkewness of the vertical root density distribution was affected by separate main effects of planting density and DAP . SkewnesKurtosis of vertical root density was affected by two separate interactions, i.e., between planting density and DAP, and between planting density and treatments . From DAp < 0.01 @ n = 80). Taking the magnitude of the Rayleigh’s statistic as a measure of root asymmetry, there was a significant interaction between planting density and DAP , but no effect of treatment, either as a main effect or in an interaction  . Plants in the D4 boxes initially (at 12 DAP) had the highest asymmetry in the distribution of roots around the central plant, which decreased thereafter. By contrast, central plants in the D3 boxes showed no change in asymmetry with time, except at DAP 26, when, in common with plants in the D4 boxes, asymmetry was lowest  matrices. Root distributions around the stem of the central plants  were asymmetric, exhibiting a departure from a uniform circular distribution at all DAPs, in both planting densities and under all treatments  matrices were also employed to exhibit growth direction(s) of root systems in windroses . Roots fR box were at the Growth Stage S1.12  according to Boyes and associates [At 26 DAP (the last time point to image conspecific root systems in our study), the plants in the Magentasociates ,84. At 23−/NO2− and NH4+) was tested, to confirm that over a course of 30 days, gel nutrients of the high concentration (as 1/2X MS) and low concentration (as 1/4X MS) could reach an equilibrium, at least in their outermost rings of 3 mm thickness  density distribution of roots in response to planting density (number of competitors) and time . Root systems grew with a negative kurtosis, meaning an evenly vertically distributed root system at early time points in all plants. At later time points, the lower planting density (D3) remained negatively kurtotic in contrast to a shift to positive kurtosis (where more roots occurred at a specific depth) in higher planting densities (D4) c. A negathaliana . Primarythaliana  and endothaliana ,94. Laskthaliana . This peR boxes) can be utilized to scale up the experiments. Square plastic boxes were chosen to avoid the optical distortion of a curved container, and only four images (one from each face) were used in this study. The root systems of A. thaliana, when grown in agar media at the laboratory, were reported to be regulated by the uptake of externally provided sucrose (in the media) in their aerial leaves [A. thaliana are also controlled by loci and environments [Hordeum vulgare cv. Proctor), seminal  root systems grew only in the nitrogen-rich segment of soil matrix, but not in the nitrogen-deficient segment [An important product of this study was the proof of the method itself. A simple, low cost system is presented to non-destructively explore and quantify the belowground root system over time. 3D heatmaps  and 2D wl leaves . The rooronments ,98. In t segment . The sam segment . This phIn addition to nutrients, root systems can be affected by associated environmental factors; available water shapes hydropatterning , while sA. thaliana are under mutual signaling according to its available resources. Puig and associates provided a first and direct link of the NITRATE TRANSPORTER 1.1 (NRT1.1) transporting auxin [Physiological coordination has been observed between shoot and root development in recent studies. Shoot branching is found to be controlled by strigolactone synthesized in the roots . Shoot dng auxin . That ling auxin ,30,31,32Arabidopsis thaliana ecotype Columbia wild type  and a transgenic GFP marker line (herein referred to as ER::GFP), also in Col-0 background with a green fluorescent fusion protein that was localized in vivo to the endoplasmic reticulum (ER) [w/v) gellan gum  for support in a MagentaR box . Half strength of MS medium is non-limiting for the growth of A. thaliana. MagentaR boxes were chosen due to their semi-transparency and square growth area. Gellan gum is approximately 80% optical transparent, and suitable for high resolution digital imaging [lum (ER) , were us imaging . Col and® Write-On Transparency Film, Staples, Carbondale, IL, USA) for exact positioning according to −1 m−2. The clear MagentaR boxes sat in black pots to prevent root exposure to light and production of reactive oxygen species [Seeds were sown in five designated positions, one central (position 5) and four adjacent to each corner (position 1–4) using a plastic sheet  with either Col plants or media  in the other four positions. Positions were numbered 1–4, starting at the left on a marked side (A) and counting clockwise around the box see . The medR box , such that two sides  corresponded to the X-axis as horizontal and Y-axis as vertical, and another two  corresponded to the Z-axis as horizontal and Y-axis as vertical. Images of roots were taken from four sides of each Magentahttps://github.com/jbmatthewgeisler/Roots). A total of 20 plant boxes were examined at 5 time points (2 densities * 4 treatments * 2 replicates) for a total of 320 images (16 boxes * 5 time points * 4 sides of each MagentaR box = 320). The entire procedure, from a cropped image of the root to quantified root density matrices, are detailed in the Root images were processed in Adobe Photoshop to help isolate the roots of the (green) ER:GFP plants growing in position 5 from the gel media, conspecific (non-fluorescent) competitors, and other artifacts . Images were also taken with white light for comparison. The processed images were cropped to a standard size without losing any roots , and wer3−) and nitrite (NO2−) levels. The nitrate test followed USEPA method 353.2 [Inorganic nitrogen (nitrate/nitrite and ammonia) analysis of the replacement gel blocks (for H treatments) was made at 30 DAP, when the preparatory setups were established. The same analysis of the gel media for the position 5 plants was made at 28 DAP, after the experimental study was completed. The extraction method for gel media followed that of Keeney and Nelson . A plug od 353.2 , in whicR boxes, where they remained for a course of 30 days in the aforementioned growth chamber. Outer and inner 3 mm thick rings of gel were scrapped/removed from the half strength gel plugs and quarter strength surrounding media, respectively, for inorganic nitrogen contents [Diffusion of inorganic nitrogen in gel media over 30 days was assessed in the following experiment. Three plugs (drilled by a #7 hole puncher) of half strength MS nutrient media  were inserted into quarter strength nutrient media  in Magentacontents ,113.p < 0.05.An XZ root matrix was generated by taking average pixel counts from the XY and ZY images of each plant, reducing each column (collapsing the Y-axis) to an estimated number of root pixels at that XZ coordinate. The matrix was transformed to a table of polar coordinates in R  by choosOur proof-of-concept study exhibited that our easy-to-operate and portable imaging platform could capture both phenotypic characteristics of root systems in 3D—combination of 2D of X and Z axes and vertical Y axis. This study also highlighted the potential and advantages of the simple transplant technique, and paved a scalable framework with precise manipulation of added ingredients for future studies in ecological questions  and possible environmental problems .


Objectives: The nutraceutical approach to the management of metabolic syndrome (MetS) might be a promising strategy in the prevention of cardio-metabolic risk. Low-dose bergamot-derived polyphenolic fraction (BPF) has been proven effective in patients with MetS, as demonstrated by a concomitant improvement in lipemic and glycemic profiles. The present study was aimed to further explore, in a sample of subjects receiving second generation antipsychotics (SGAs), the effects on body weight and metabolic parameters of a low dose of BPF (500 mg/day) administered for 60 days.Methods: Twenty-eight outpatients treated with SGAs assumed BPF at single daily dose of 500 mg/day for 60 days. Body weight, BMI, fasting levels of glucose, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides were determined; moreover, Brief Psychiatric Rating Scale (BPRS) was administered.Results: Low-dose BPF administration did not change clinical and metabolic parameters, as well as clinical symptoms in the study sample. At the end of the trial, among completers (n = 24) only nine patients (37.5%) reached an LDL reduction >0 but <50%.Conclusions: Our results demonstrate that patients treated with SGAs may need higher BPF doses for obtaining the positive effects on body weight and metabolic parameters previously found in the general population at lower doses. Metabolic syndrome (MetS), a cluster of risk factors including obesity, dyslipidemia, glucose intolerance, insulin resistance (or hyperinsulinemia) and hypertension, is highly predictive of type 2 diabetes and cardiovascular disease  is an endemic plant of the Southern Italy, which differs from other citrus fruits for flavonoids and flavonoid glycosides composition , and for their high amount  and in a trend for body mass index (BMI) decrease (P = 0.005); however, no effects on metabolic parameters were observed  has beneficial effects in patients with MetS, as demonstrated by a concomitant improvement in lipemic and glycemic profiles, and by the improvement of altered endothelium-mediated vasodilation  administered for 60 days.This was a 60-day, open-label, preliminary study aimed to evaluate the efficacy and safety of adjunctive BPF to atypical antipsychotics therapy. BPF , Italy) was administered in capsules at the oral daily dose of 500 mg/day and was maintained unchanged until the end of the trial at day 60. The concentration of 5 main flavonoids per capsule was: neoheriocitrin , naringin , neohesperidin , melitidin  and brutieridin ; excipients of any type were not present. During the study, no additional medications, including aspirin or non-steroidal anti-inflammatory drugs, were allowed. The study was carried out at the Psychiatry Unit of the University Hospital of Messina, Italy. The protocol has been approved by the Ethics Committee of the University of Messina, Italy.Twenty-eight outpatients, 15 men and 13 women, aged 21–65 years, in treatment with SGAs , were included in this study. All patients had been on monotherapy for at least 3 months; the dose had been stable for at least 1 month before the study and was left unchanged throughout the study. The patients did not receive any antidepressant or anticonvulsant drugs for a period of 2 months before the study. Furthermore at enrollment, all subjects received standardized dietary advice in order to reduce the variability of their baseline metabolic values. Patients with any significant concurrent medical illnesses, organic brain disorder, mental retardation, pregnant or lactating women, or a current diagnosis of alcohol/drug dependence were excluded. All the patients provided written informed consent after a full explanation of the protocol design, and the study was conducted according to the Declaration of Helsinki.Patients attended three visits: initial screening (week −1), inclusion (day 0), and final visit (day 60).A physical examination was performed to measure vital signs .Standard laboratory methods were used to determine fasting levels of glucose, total cholesterol, HDL, and triglycerides. Low density lipoprotein (LDL) cholesterol was determined by the Friedewald et al. calculation   .Adverse effects, either observed or spontaneously reported, were recorded at each visit and classified in terms of onset, duration, severity, action taken, and outcome. In order to monitor the adherence to the study protocol, weekly telephone calls during the study period and a pill count on the last day (day 60) were carried out.Steady-state serum concentrations of clozapine, olanzapine, risperidone, 9-hydroxyrisperidone  and quetiapine were measured by HPLC at day 0 and day 60  with last-observation-carried forward (LOCF) was performed. Continuous data were expressed as mean ± SD and the within group differences in efficacy ratings between baseline and final test were analyzed by the Wilcoxon rank sum test. To measure the magnitude of a treatment effect, effect size was provided by using Cohen's d statistic and was considered small when lower than 0.50, moderate when ranging from 0.50 to 0.79, and large when equal to or greater than 0.80. The statistical analysis was performed with SPSS 16.0 software .Baseline characteristics, duration of SGAs prior treatment, SGAs type and daily dose in enrolled/completers subjects are detailed in Table Table LDL reduction was chosen for treatment efficacy evaluation because it is the most commonly used surrogate endpoint for cardiovascular disease reduction, and it is the primary target for statin therapy in cholesterol-lowering guidelines , 9 individuals (37.5%) experienced an LDL reduction >0 but <50%, whereas 15 subjects (63.5%) experienced no reduction or an increase in LDL compared with baseline. None of the patients experienced an LDL reduction ≥50%.Among completers  and quetiapine were not affected significantly by administration of BPF. Concerning the two antipsychotics more frequently administered to patients participating to the study, namely olanzapine (7 patients) and clozapine (6 patients), mean steady-state serum concentrations of olanzapine were 29.1 ± 12.4 ng/mL at day 0 and 25.0 ± 9.7 ng/mL at day 60, while serum concentrations of clozapine were 551 ± 139 ng/mL at day 0 and 572 ± 124 ng/mL at day 60.The metabolic effects of atypical antipsychotics represent a serious medical problem to be addressed with different therapeutic approaches in order to improve the life expectancy of subjects with mental disorders. Cross-sectional studies have reported MetS prevalence rates of 11–69% in pharmacologically treated psychiatric patients, and of 4–26% in drug-naïve patients; longitudinal studies have showed baseline prevalence rates between 0 and 14% in drug-naïve patients, increased up to a maximum of 52% after 3 months of antipsychotics treatment  for 30 days significantly reduced body weight in SGAs-treated patients, suggesting that BPF could be an effective and safe agent to prevent weight gain associated with atypical antipsychotic use  reached a minimal LDL reduction according to the current statin guidelines  among ethnic groups, it seems difficult to generalize these results to other populations.Our current and earlier results tend to suggest that patients treated with SGAs may need higher BPF doses  for obtaining the positive effects on body weight and metabolic parameters previously found in the general population at lower doses.Further clinical trials with adequately-powered and well-designed methodology are needed to better evaluate the effectiveness of the BFP on SGA-induced metabolic side effects, and to identify potential nutraceutical strategies for the prevention of MetS in subjects with mental disorders.AB, RZ, and MM designed the study, wrote the protocol, and supervised the Method procedures, the various drafts and the final version of the manuscript. GP and ES managed the literature searches and wrote the first drafts of the manuscript. MCr, MCa, and VS organized recruitment and collected data, contributed to the literature search and writing of the manuscript. All Authors contributed to and have approved the final manuscript.ES has previously received honoraria for speaking and consultation from AstraZeneca, Boheringer-Ingelheim, Eli Lilly, Janssen, Lundbeck, and Pfizer. The remaining authors declare no conflict of interest.