To characterize early amplitude-integrated electroencephalogram (aEEG) and single-channel EEG (aEEG/EEG) in very preterm (VPT) infants for prediction of long-term outcome.Forty-nine infants with median (range) gestational age of 25 (22–30) weeks.Amplitude-integrated electroencephalogram/EEG recorded during the first 72 h and analysed over 0–12, 12–24, 24–48 and 48–72 h, for background pattern, sleep–wake cycling, seizures, interburst intervals (IBI) and interburst percentage (IB%). In total, 2614 h of single-channel EEG examined for seizures. Survivors were assessed at 2 years corrected age with a neurological examination and Bayley Scales of Infant Development-II. Poor outcome was defined as death or survival with neurodevelopmental impairment. Good outcome was defined as survival without impairment.Thirty infants had good outcome. Poor outcome (n = 19) was associated with depressed aEEG/EEG already during the first 12 h (p = 0.023), and with prolonged IBI and higher IB% at 24 h. Seizures were present in 43% of the infants and associated with intraventricular haemorrhages but not with outcome. Best predictors of poor outcome were burst-suppression pattern , IBI > 6 sec  and IB% > 55% at 24 h age . In 35 infants with normal cerebral ultrasound during the first 3 days, outcome was correctly predicted in 82% by IB% .Long-term outcome can be predicted by aEEG/EEG with 75–80% accuracy already at 24 postnatal hours in VPT infants, also in infants with no early indication of brain injury. The high prevalence of neurodevelopmental impairment (NDI) after very preterm (VPT) birth is still a concern . The aetDevelopment of methods that enable early identification of acute brain injury in preterm infants is urgent, not only to direct overall care and inform parents about their infant's condition, but also to obtain predictive information as a basis for neuroprotective intervention. The electrocortical background pattern (BG) in the amplitude-integrated electroencephalogram (aEEG)/EEG has proved to be one of the best early predictors of outcome in term asphyxiated infants . HoweverThe normal EEG background in VPT infants is mainly discontinuous (DC), characterized by high-voltage activity bursts and interburst intervals (IBI) of low voltage. We and others have shown that the highest burst rate per hour (i.e. best background) in aEEG recorded at 24–48 postnatal hours was predictive of outcome in preterm infants with large IVH (grade 3–4) . A neuroIn very preterm infants, the early aEEG/EEG background, as recorded at 24–48 postnatal hours, is predictive of outcome with around 80% accuracy, indicating the impact of early brain damage on long-term outcome.Brief subclinical seizures are common during the first postnatal days and are associated with IVH, but not long-term outcome.With the hypothesis that higher proportions of discontinuous electro-cortical background (i.e. suppressed aEEG/EEG) reflect an unfavourable prognosis, the objectives of this study were to characterize early aEEG/EEG development in VPT infants in relation to outcome at 2 years of age; to assess the predictive value of aEEG/EEG on long-term outcome; and to assess the value of a one-hour high-quality recording as compared to the long-term aEEG/EEG trend.Fifty-four infants were prospectively included in the study between July 2005 and May 2007 after antenatal written parental consent; 45 had a gestational age (GA) <28 weeks , and nine were born at 28–30 weeks GA (VPT). GA was determined by prenatal ultrasound at 17–18 weeks. All infants were treated at the tertiary level neonatal intensive care unit (NICU) at Lund University Hospital, Sweden. The final study cohort consisted of 49 infants; 41 had follow up at 2 years of corrected age and eight infants who died. Five surviving infants were excluded because two children moved prior to follow-up, and three families declined further participation. The Regional Ethical Review Board at Lund University approved the protocol.The infants underwent standard NICU care and had an additional recording of a single-channel EEG during the first 72 postnatal hours. Bolus doses of morphine (most often 0.05–0.15 mg/kg), or continuous infusion of morphine, were administered on clinical indications to mechanically ventilated infants. Thirty infants received in median (range) 3 (1–7) bolus doses of morphine during the study period. In six of these infants, morphine was at some time administered as infusion. Repeated cerebral ultrasound investigations (cUS) were performed  on days 1, 3 and 7 and at 3 and 6 weeks, and at term. Germinal matrix and IVH were classified according to Papile . The preA single-channel EEG recording with the aEEG trend  was initiated as soon as possible after initial stabilization (median 8 h). After gentle skin preparation , hydrogel electrodes  were applied at P3 and P4, according to the International 10–20 System, as well as a frontal reference.Two investigators , blinded for the patient's identity and clinical data, assessed visually the aEEG trend, displayed at 6 cm/h, in 4-h epochs. For each epoch, the dominating (>50%) aEEG BG was scored in six categories using a published scoring system : 0 = flaInterburst intervals were quantified by using a validated automated segmentation algorithm based on a non-linear energy operator (NLEO), reflecting both amplitude and frequency content of the EEG ,12. BefoMedian values of consecutive 4-h epochs of each infant's BG score, SWC score, IBI and IB%, respectively, were calculated over the first 0–12, 12–24, 0–24, 24–48 and 48–72 postnatal hours.For a comparison of continuous and intermittent aEEG/EEG, IBI and IB% were quantified in a 1-h high-quality single-channel EEG selected from each recording as close as possible after 24-h age while meeting the following criteria: No artefacts identified in the original EEG; no handling of the infant; and no morphine  administElectrographic seizures were identified in the single-channel EEG by an experienced neurophysiologist (IR) who examined all recordings  at 10 mm/sec and 70 μV/cm sensitivity. A seizure was defined as an abnormal electrical event, lasting for 10 sec or more, with evolving, repetitive waveforms with a gradual build-up and reaching a maximum before declining in frequency, morphology or amplitude  with the two subscales Mental Developmental Index (MDI) and Psychomotor Developmental Index (PDI) . NDI wasNon-parametric statistics were used for group comparisons and analysis of changes over time in aEEG/EEG measures. Cerebral ultrasound findings during the first three postnatal days and the following aEEG/EEG measures were evaluated as predictors of outcome: background scores, SWC scores, IBI and IB%. Sensitivity, specificity, PPV, NPV and percentage of correctly predicted infants were calculated. Receiver operating characteristics (ROC) curves, with area under the curve (AUC) estimations, were produced for identification of optimal cut-offs. Odds ratios for poor outcome were calculated in logistic regression models, adjusting for GA.The median (range) duration of the aEEG/EEG recordings was 56 (14–71) h, with 32 (7–64) h per infant having sufficient technical quality for analysis of IBI and IB% . Follow-up at 2 years was conducted in 41 infants; eight VPT and 22 EPT infants survived with good outcome, while 11 EPT infants survived with NDI. Eight EPT infants died in the neonatal period, that is, in total, 19 EPT infants had a poor outcome. Clinical data are presented in The aEEG/EEG background scores were significantly lower in infants with poor outcome at 0–12, 12–24, 24–48 and 48–72 h as compared to those with good outcome . Median IBI and IB% were higher in infants with poor outcome at 24–48 and 48–72 h . Background scores, IBI and IB%, differed significantly at 24–48 and 48–72 h between EPT infants with poor and good outcome .Continuous activity (BG score 5) was present in 53% of infants with good outcome (p = 0.002 vs poor outcome) and was identified in 88% of the VPT infants, in 41% of the EPT infants with good outcome but only in 11% of EPT infants with poor outcome (p = 0.038 for EPT comparison). BS dominated at least one 4-h epoch in 84% of infants with poor outcome, but only in 33% of infants with good outcome (p = 0.001). BS was present in 25% of VPT infants, in 84% of EPT infants with poor outcome and in 36% of EPT infants with good outcome (p = 0.004 for EPT comparison). SWC was identified during the first 72 h in 64% of the infants, more often in infants with good outcome (p = 0.029), In infants with good outcome, the median BG and SWC scores increased between 0–24 and 24–48 h , and median IBI and IB% decreased between 0–24 and 24–48 h , but there were no further changes at 48–72 h. These changes were not seen in infants with poor outcome.Thirty-five infants (13 with poor and 22 with good outcome) had normal cUS during the first 3 days. In this subgroup, infants with poor outcome had lower BG scores at 24–48 h  and 48–72 h , longer IBI at 24–48 h , and higher IB% at 24–48  and 48–72 h  as compared to infants with good outcome. Studying survivors only, the 11 infants with NDI had lower BG scores at 24–48 h  and 48–72 h , longer IBI at 24–48 h  and 48–72 h  and higher IB% at 24–48  and 48–72 h  as compared to the 30 infants without NDI.Electrographic seizures were present in 21 infants (43%). Altogether 89 seizures with duration from 12 sec to 6 h 10 min (median 60 sec) were identified . EPT infants with poor outcome had longer IBI and higher IB% . Among iThe prognostic value of aEEG/EEG features and cUS are summarized in Our study demonstrates that the electrocortical background activity, as recorded with single-channel aEEG/EEG, contains predictive information on long-term outcome in VPT infants already at 24 h of age, irrespective of brain damage according to cUS, and GA at birth. Overall, the outcome in around 80% of the infants could be correctly predicted by aEEG/EEG at 24 postnatal hours.Neural dysfunction, reflected by aberrations in aEEG/EEG, may be a sign of developing brain damage. Early identification of such electrocortical dysfunction may thus open a time window for possible neuroprotective interventions. As compared to term asphyxiated infants , the proThe EEG measures IBI and IB% are directly related to the developmental neurobiology because they are based on identification of ‘bursts’, or ‘spontaneous activity transients’, which are considered important for brain wiring during development . In the We assessed the quality of the IBI and visually distinguished the isoelectric IBI during BS from tracé discontinu, the normal DC background activity of VPT EEG. It has been postulated that BS represents a functional thalamo-cortical disconnection in the mature brain . HoweverArtefacts are common in long-term aEEG/EEG monitoring in preterm infants, and therefore, we assessed the predictive value of quantitative data from a 1-h high-quality single-channel EEG obtained at 24 postnatal hours, also with at least a 4-h interval between the recording and administrated morphine. The predictive value of IB% was similar to the predictive value of visual and quantitative assessments in the long-term monitoring, which emphasizes the importance of high recording quality. Still, combined quantitative and visual assessment by an expert reader may be even better, as shown by West et al. . Also, tSeizures in preterm infants are markers of abnormal brain function and carry an increased risk for abnormal outcomes ,19,20. IElectroencephalogram background activity increases during the first postnatal days in preterm infants ,22,24,25In conclusion, our data show that cerebral depression as assessed by early aEEG/EEG is associated with long-term outcome in VPT and EPT infants. The relatively small study population is a limitation, and the findings need to be reproduced in larger cohorts. We included infants up to 30 gestational weeks but focused on EPT infants below 28 gestational weeks, because these infants have the highest risks for subsequent brain damage. Both visual pattern recognition and quantitative analysis of the aEEG/EEG background provide long-term predictive information in VPT and EPT infants. Brief subclinical seizures are common during the first days and are associated with IVH, but not to long-term outcome.


Our work in a rodent model of urinary calcium suggests genetic and gender effects on increased residual variability in urine chemistries. Based on these findings, we hypothesized that sex would similarly be associated with residual variation in human urine solutes. Sex-related effects on residuals might affect the establishment of physiological baselines and error in medical assays.CV) for urinary solutes in paired sequential 24-h urines  in 6,758 females and 9,024 males aged 16–80 submitted to a clinical laboratory.We tested the effects of sex on residual variation in urine chemistry by estimating coefficients of variation . There was no effect of sex on CV for calcium (P>0.3). Males had higher CVs for citrate (P<0.01) from ages 16–45 and females higher CVs for citrate (P<0.01) from ages 56–80, suggesting effects of an extant oestral cycle on residual variance.Females had higher CV by sex might reflect dietary lability, differences in the fidelity of reporting or genetic differentiation in renal solute consistency. Such an effect could complicate medical analysis by the addition of random error to phenotypic assays. Renal analysis might require explicit incorporation of heterogeneity among factorial effects, and for sex in particular.Our findings indicate the effects of sex on residual variance of the excretion of urinary solutes including phosphorus and citrate; differences in  A priori predictions of direction  were not possible since sex differences in residual variation of urinary calcium in our rat model indicated sex-by-strain interaction Given sex and genetic effects on residual variance, we hypothesized that 4; mmoles), sulfate , uric acid  and urea nitrogen . Cr was collected for the purposes of measuring completeness of collection, and Na and UN for the purposes of controlling for dietary inputs of salt and protein. Where a measurement was missing for any urinary solute, that individual was removed from all analysis for that urinary trait. Weight (kg), age (yrs) and sex were collected for each individual but no additional clinical or background information was collected by Litholink Inc. Individuals known to be receiving treatment for stones at the time of collection were excluded from the study, although not all patients had a fully-known treatment history. Use of this data set was approved by the Western Institutional Review Board . Records were collected over the history of Litholink's operation. No experimentation was performed.Paired 24-hour urines with a maximum interval of 72 hours were obtained as part of routine clinical testing for 7,372 female and 9,780 male kidney stone patients and submitted to a single reference laboratory (Litholink Corp) for urine chemistry measurement. Urines were collected at room temperature using an antimicrobial preservative. Individual 24-h urines were measured for all subjects for calcium , citrate , chloride , creatinine , potassium , sodium , magnesium , oxalate , phosphorus , ammonium (NHCV) for paired 24-h measurements of urinary excretion rates were estimated for all individuals as CVs for each trait were ranked in ascending order x1…xn for independent analysis Ranked CVs were used to avoid complications from non-normal distributions in this nonparametric statistic while still permitting multivariate models to account for the effects of significant covariates. Global effects on CV for each urinary solute were estimated simultaneously with covariate selection using multivariate backwards regression in SAS P <0.1, beginning with the starting model (Model 1):y, yCV, yCV, CV-weight interactions on yCV, yCV and yCV relationships to account for changes in CV within or across life history categories, CV for differences in urinary volume between the two samples, yCV, yCV, th percentile were excluded prior to analysis as possibly irreparably biased due to errors of collection. Ranked CV for each trait by sex was estimated using a least-squares (LS) model based on terms retained from backward stepwise regression, above, with sex coded as a fixed effect and all other terms as partial regressions Coefficients of variation  P-values (Pnom) for sex effects on CV were ranked in ascending order from 1 to m . Where the ranked Pi for hypothesis Hi for each trait i satisfied the relation Pi≤αi/m, where α was the nominal a priori significance threshold ( = 0.05), we rejected the null hypothesis of no significant difference in CV between males and females for that test and all tests with lower PiThe significance of sex effects on vs. post-menopausal female physiology on CVs for urinary traits, we divided the population into two classes: 1) a rough ‘menarche-menopause’ (MM) category of 16–45 years and 2) a rough ‘post-menopausal’ (PM) group of 56–80 years. These age categories were selected to coincide with the approximate mean age of inception of menopause in Western women CVs was then tested using backwards regression (Pthr<0.1) in Model 1 as above, within each of categories 1) and 2). Significance thresholds were estimated separately within each group at the FDR as above.In order to test the effects of post-menarche y, i on the CV for trait y, j, i and age group j on yCV and all other terms were as per Model 1 (above). As above, from three to five terms were normally included in the final analysis via backwards regression gression . The sigth percentile of CVs were highest overall for Na and Cl, and lowest for Cr although the range of individual CVs was highest for Ca  at the FDR  a priori thresholds, which were not significant after FDR correction .Females had significantly higher  the FDR . Femalesrrection . Female CVs in in the subdivided Model 1 for the examination of sex effects on variation at ages 16–45 and 56–80. Males had significantly higher P<0.01) and females significantly higher P<0.01) (P<0.01) at the nominal significance threshold but not after multiple correction.A total of 4,128 males and 4,219 females from ages 16–45 and 3,578 male and 1,772 females were available. We detected marked changes in the pattern of male and female (P<0.01) . From 16PFDR>0.05); P<0.1)  .P<0.001) between the two age groups (16–45 years and 56–80 years). There were no significant sex×age group effect for any other urinary metabolite after multiple correction (PFDR>0.1)  .CV for citrate from 16–45 years of age. We did not detect male-female differences for CVs in urinary calcium despite predictions from our rodent model Females had higher coefficients of variation for urinary phosphorus over the entire age distribution and for citrate from 56–80 years, while males had higher Since our study did not employ dietary controls or journaling, our findings might result from day-to-day male-female dietary variability, male∶female genetic or endocrine differences, or differences in the fidelity of urine collections; sex-by-strain and sex-by-genotype effects in our rodent model were detected on controlled, constant diets σ) or more. Our rodent work suggests that IIV can bias estimation of ‘classical’ genetic effects on means: a region of chromosome 1 associated with CV in urinary calcium in our rodent model  obscured the detection of a nearby locus with effects on mean urinary calcium  CV were only detected for two urinary solutes. Notably, females did have significantly higher CVs for urinary phosphorus. Phosphorus and calcium are physiologically related Whether resulting from dietary variability or endogenous endocrine or genetic effects, IIV in urinary solutes may have significant diagnostic consequences: Parks et al CVs for urinary citrate were higher than in females in the menarche-menopause group, and females higher at post-menopausal ages. Both female humans and rats excrete more citrate than males overall Male Exogenous and endogenous sex hormones have major main effects on renal physiology Table S1Final models for partial regression terms in stepwise backward regression (Pacceptance≤0.1) of ranked coefficients of variation (CV3), calcium (Ca), chloride (Cl), citrate (Cit), potassium (K), magnesium (Mg), sodium (Na), oxalate (Ox), phosphorus (P), sulfate (SO4), uric acid (UA) and urine urea nitrogen (UN) in 9,024 male and 6,758 female stone-formers aged 16–80, and in age categories 16–45 years and 56–80 years.) for 24-h urinary ammonium >0.1.(DOC)Click here for additional data file.


Objective: We aimed to investigate if increased F-18 Fluoro Deoxyglucose (F-18 FDG) uptake observed in vocal cords (VC) of the patients on Positron Emission Tomography/Computarize Tomography (PET/CT) scans is connected to speaking of the patients or not. If so, we expected to detect an increased metabolic activity in Broca's area. In this study, we have retrospectively searched for a correlation between the activity in the Broca's area and vocal cords of patients who had undergone FDG PET for different indications. Material and Methods: FDG PET/CT scans of 30 patients with (VC [+]) and 30 patients without (VC [-]) bilateral F-18 FDG uptake on their vocal cords were retrospectively evaluated. Brain quantification was carried out on NeuroQ software with 20 iterations using patients' transaxial brain cross sections. On the 20th-23rd-26th-29th cross sections, area/whole brain ratios of the right (R) and left (L) for Broca’s area were calculated. VC (+) and VC (-) patients' R and L Broca's areas were compared using Student's t-test.Results: There was no significant difference between the Broca's areas of VC (+) and VC (-) patients. L Broca's areas of both VC (+) and VC (-) patients were more active than R Broca's areas (p<0.05). There was a negative correlation between VC (+) patients' SUVmax values in the vocal cords and the activity in their R Broca's region. Conclusion: In our study, we did not find a significant difference between Broca's areas of VC (+) patients and VC (-) patients, so the activity in their vocal cords does not seem to be related to increased metabolic activity in Broca's areas. We have concluded that the vocal cord activity is not related to speaking of the patients. The activity in the vocal cord might be due to inflammation or, as in the eye muscles, may be associated with high metabolism in laryngeal muscles. Conflict of interest:None declared. F-18 Fluorodeoxyglucose (FDG) Positron Emission Tomography/Computed Tomography (PET/CT) is used in a wide range of oncological patients. It helps the clinician in diagnosis, staging, biopsy guidance, response to treatment and re-staging processes .FDG is a glucose analogue and it enters into the brain cells via the neuronal membrane glucose transporter. Deoxyglucose is converted to deoxyglucose-6-phosphate in neurons and captured in the cell.Glucose metabolism varies in different brain regions even in healthy subjects ,3. SpeakFDG uptake is also observed in vocal cords (VC) which may be secondary to malignancy but it may as well be physiological ,8,9,10.In our study, we tested the hypothesis that patients with symmetrical FDG uptake in VCs in PET/CT might have spoken during the imaging process and if they had, Broca's areas in the brain might have been activated secondary to speaking. We have investigated if patients with FDG uptake in their VCs have increased metabolic activity in Broca's areas.Patients referred to our department between 2010 July and 2011 February for F-18 FDG PET/CT due to oncologic diseases were retrospectively evaluated. A total of 60 patients were selected as 30 patients with (VC [+]) and 30 patients without (VC [-]) bilateral F-18 FDG uptake on their vocal cords. Patients with a history of previous head and neck cancer, head and neck surgery, vocal cord paralysis, cerebrovascular disease (CVD), head and neck radiotherapy (RT), brain metastases and neurological disease were excluded. None of the patients had hoarseness. All the patients' fasting blood glucose (FBG) were measured after 6-8 hours of fasting. Patients’ FBG was ≤200 mg/dL. Patients rested in a quiet room after an intravenous (IV) catheter was placed. All patients were scanned about 60 minutes after the injection of 6.8 to 12.4 mCi F-18 FDG intravenously. Imaging was carried out using Phillips Gemini TOF PET/CT . First a low dose CT scan with 120 kV, 50-60 mA, 5 mm cross section was performed between vertex and 1/3 proximal femur. CT images were used for PET attenuation correction and anatomical localization purposes. Then, PET scan was performed for 1.5 minutes per bed position, from 1/3 proximal femur to vertex. Transaxial, coronal, sagittal and 3D fusion images of patients with and without attenuation correction were reviewed and bilateral VC (+) and VC (-) patients were identified. "Standard Uptake Value Maximum" (SUVmax) values were used to characterize the metabolic activity in vocal cords of VC (+) patients. Cross-sections were set parallel to orbitomeatal axis. th, 23rd, 26th and 29th cross-sections were automatically calculated. Area/whole brain ratios of Broca's areas were obtained as left (L) and right (R) Broca 20, Broca 23, Broca 26 and Broca 29. Ratios to whole brain in each ROI were compared to the normal values in the database. L Broca/R Broca ratios were also calculated.After the cross-sections were created, they were processed with 20 iterations using NeuroQ  software.After processing, average pixel values in standardized regions of interest (ROI) in Broca's areas in 20The data were evaluated with the SPSS 16.0 statistical software. The correlation between SUVmax values and area/whole brain ratios of Broca's areas in VC (+) patients were evaluated with Pearson correlation test. The difference between area/whole brain ratios of Broca's areas in VC (+) and VC (-) patients and the difference between the patients' area/whole brain ratios of right and left Broca's areas were evaluated with Student's t-test. p<0.05 was accepted as statistically significant.Fourteen of VC (+) patients were female, 16 were male and average age was 53.3±16.8. Average FBG of VC (+) patients was 104.2±18.9 mg/dL. Average dose of injected F-18 FDG was 9.7±0.7 mCi. F-18 FDG PET/CT was performed in 7 patients for diagnosis , 3 patients for biopsy guidance (3 lung masses), 4 patients for staging , 9 patients for evaluation of response to treatment  and 7 patients for re-staging . Average SUVmax value in vocal cords of VC (+) patients was 4.8±2.2 (2.6-12.3). Laryngeal examination of the patients with SUVmax values greater than 9.9 did not reveal any pathology. Laryngeal examination was not carried out for other patients.Twelve of VC (-) patients were female, 18 were male and average of age was 58.5±12.1. Average FBG of VC (-) patients was 104.3±28.7 mg/dL. Average dose of injected F-18 FDG was 9.9±0.7 mCi. F-18 FDG PET/CT was performed in 5 patients for diagnosis , 3 patients for biopsy guidance (5 lung mass), 4 patients for staging , 9 patients for evaluation of response to treatment  and 8 patients for re-staging . Average whole brain ratios of L and R Broca 20-23-26-29 areas in VC (+) and VC (-) patients were given in Comparison of L Broca 20-23-26-29 and R Broca 20-23-26-29 areas in VC (+) and VC (-) patients are given in L Broca/R Broca ratios of VC (+) and VC (-) patients are given in We could not find a strong correlation between SUVmax values of VC (+) patients and Broca's areas. We have found a very weak correlation with L Broca 20-23-26, R Broca 20-26-29 and a negative correlation with L Broca 29, R Broca 23. The correlation data between Broca areas and SUVmax values are given in PET and Functional Magnetic Resonance (fMRI) are two methods which provide clear appearances of active cortical locations during mental tasks. Both methods operate on the principle that an increase in blood supply is required for supplying oxygen in order to preserve increase in neural activity while a particular region of brain performs a particular task. In general, this condition occurs several seconds after neural activity starts and therefore, those techniques most likely offers indirect appearance of timing and sequence of cortical events . Studying meta-analysis of neuroimaging studies, Indefrey and Levelt plotted several regions related with word generation. They suggested that word selection is related with left middle temporal gyrus and phonological awareness is related with right supplementary motor cortex (SMC), left anterior insula and left posterior superior and middle temporal gyrus (Wernicke’s area). It was suggested that Broca’s area  is significantly related with process of syllabication during word generation. It is not known whether sign generation and voice generation are equally related with those neural regions .Neurosurgeon Wilder Penfield was the first scientist to experimentally prove the relationship between Broca’s area and speech generation. He reported in dozens of cases that in recovered patients who underwent neurosurgical operation due to treatment-resistant epilepsy, inferior frontal gyrus stimulations – there was slight inter-subject variations- via electrical stimulation of frontal lobe had the influence to stop ongoing speech. The coincidence between focus of Penfield’s effect and location of Broca’s area was a very convincing argument favoring motor role of this region .In a study conducted on dead subjects and healthy (with regards to hearing ability) subjects, he revealed that left inferior gyrus was equally used by deaf and healthy subjects during voice and sign generation .As it is estimated, both voice generation and sign generation are related with left inferior frontal gyrus and particularly with BA 45, the anterior section of Broca’s area. Equal activation of Broca’s area for both sign and word indicates that there is a modality-independent role of this region in language generation, consistent with findings of previous investigations. Accordingly, function of Broca’s area has a strong relationship with verbal – acoustic phonological features of verbal language . In another study, presence of activation in left inferior frontal gyrus during motion of talking was demonstrated using fMRI .In anothAll of those studies demonstrate that Broca’s area is activated during speech. In the current study, we examined increased metabolic activity in Broca’s area in order to prove whether FDG uptake or in other words increased metabolic activity in vocal cords is related with the speaking of the patients during examination. However, we could not find a significant correlation between FDG uptake in vocal cords and metabolic activity in Broca’s areas. Moreover, no significant difference was found between Broca’s areas of VC (+) patients and VC (-) patients.However, there was a significant difference between left Broca’s areas and right Broca’s areas of VC (+) patients (inter-group comparison) and also between left Broca’s areas and right Broca’s areas of VC (-) patients (inter-group comparison), left Broca’s areas having higher values than right Broca’s areas in all patients (p<0.03). No comparison was made with other publications since there is no similar study in the literature. It is concluded that activity in vocal cords is not related with increased metabolic activity in Broca’s areas and thus, it does not result from speaking of the patient, since no significant difference was found between Broca’s areas of VC (+) and VC (-) patients. It was considered that the metabolic activity in vocal cords may be secondary to inflammatory processes or it may be related with continuous high metabolism, similar to that of eye muscles. In current study, patients had PET/CT studies for oncologic purposes. Cranial PET/CT was not performed. Functional Brain PET studies need special patient preparation and acquisition techniques/parameters. On the other hand, it is also very well known that standard oncological FDG PET/CT imaging could not meet most of these requirement. Therefore, sensitivity of the study may be low considering the possibility of data loss.We think that the activity in vocal cords is not related to the vocal activity of the patient during the scan. The increased metabolic activity in vocal cords might be due to inflammation or related to a high metabolism in the laryngeal muscles.


Purpose. To assess glutamine/glutamate (Glx) and lactate (Lac) metabolism using proton magnetic resonance spectroscopic imaging (1H-MRS) in order to differentiate between adrenal gland nodules and masses . Materials and Methods. Institutional review board approval and informed consent were obtained. A total of 130 patients (47 men) with 132 adrenal nodules/masses were prospectively assessed (54 ± 14.8 years). A multivoxel system was used with a two-dimensional point-resolved spectroscopy/chemical-shift imaging sequence. Spectroscopic data were interpreted by visual inspection and peak amplitudes of lipids (Lip), choline (Cho), creatine (Cr), Lac, and Glx. Lac/Cr and Glx/Cr were calculated. Glx/Cr was assessed in relation to lesion size. Results. Statistically significant differences were observed in Glx/Cr results between adenomas and pheochromocytomas (P < 0.05), however, with a low positive predictive value (PPV). Glx levels were directly proportional to lesion size in carcinomas. A cutoff point of 1.44 was established for the differentiation between carcinomas larger versus smaller than 4 cm, with 75% sensitivity, 100% specificity, 100% PPV, and 80% accuracy. Lac/Cr results showed no differences across lesions. A cutoff point of −6.5 for Lac/Cr was established for carcinoma diagnosis. Conclusion. Glx levels are directly proportional to lesion size in carcinomas. A cutoff point of −6.5 Lac/Cr differentiates carcinomas from noncarcinomas. Since t data as .Glucose and glycogen levels are low in tumoral cells. Therefore, glycolysis regulation in these structure occurs via alternative carbohydrates, especially glutamine. The degradation of to lactate has been referred to as glutaminolysis .There is no consensus in the literature regarding the association between high or low concentrations of glutamine and tumor growth potential –7. NeverCompared with normal cells, tumors use almost all their ability to degrade glucose, independent of the amount of oxygen present. However, this consumption under anaerobic conditions can be up to 20 times higher than in aerobic conditions . In addi1H MRSI in vivo is not able to distinguish between glutamin and glutamate. As a result, total Glx data are usually assessed, considering a peak between 2.1 and 2.5 ppm [ 2.5 ppm –17. Notw1H MRS in order to differentiate between adrenal nodules and masses .Therefore, the objective of this study was to assess Glx and Lac metabolism using The present study was approved by the appropriate Research Ethics Committee. Informed consent forms were signed by all patients included or their guardians.1H MRSI at the Department of Diagnostic Imaging of our institution. Data collection took place between January 2004 and May 2011.A total of 130 patients  presenting with adrenal nodules or masses were prospectively assessed. All patients had been previously examined with a dedicated adrenal CT and/or a MR protocol. Of the 130 patients, 128 presented nodules or masses either on the right or left adrenal gland only, and two presented bilateral masses or nodules, totaling 132 nodules. All patients underwent 1H MRSI sequence, were not included in the sample.The following inclusion criteria were taken into consideration: (1) presenting with adrenal nodule/mass with a diameter >1.0 cm and previous dedicated adrenal CT or MRI exam; (2) histopathological confirmation by biopsy or surgery in cases of pheochromocytomas, carcinomas, functional adenomas, or nonspecific lesions; (3) lesion stability for over 12 months based on CT or MRI findings in patients with adenomas. Patients on chemotherapy protocols or with previous adrenal biopsy or surgery, as well as those in which it was not possible to schedule the Exclusion criteria were presence of lesion(s) with a diameter >1.0 cm but no voxel eligible for analysis , and presence of adrenal nodule or mass with a diameter around 1.0 cm but presenting surface contaminants.Five patients were excluded as a result of poor band positioning, a low signal-to-noise ratio, and absence of voxels eligible for analysis. As a result, a total of 125 patients  met = all study criteria and were therefore included in the final analysis. Mean age in this group was 54 ± 14.8 years (range: 13–87), and all presented with an adrenal lesion larger than 1.0 cm . A total of 127 masses were diagnosed and classified as adenomas (84), carcinomas (10), pheochromocytomas (28), and metastases (5).Patients were examined after a four-hour fast and 10 minutes after the intravenous administration of an antispasmodic drug (Buscopan). At this occasion, patients were also asked to answer a questionnaire on MRI contraindications.Patients were positioned feet first into the MRI system, with their arms resting alongside their bodies, and lying down on their backs on the spine coil. Once the patient was adequately positioned, the phased-array spine coil was used. n = 123), or a 3.0 T Magnetom Verio scanner  (n = 4). MRI examinations were conducted using either a 1.5 T Magnetom Sonata scanner , a 1.5 T Magnetom Espree scanner   sequences and T1-weighted chemical shift imaging (CSI). 1H MRSI protocol, as described by Faria et al. [HASTE sequences were performed in three orthogonal planes to allow three-dimensional (3D) mass location and thus planning of the shimming and a et al. .Subsequently, spectroscopic imaging data were obtained using a multivoxel system in the selection of the volume of interest and a hybrid two-dimensional point resolved spectroscopy (PRESS)/CSI sequence, commercially available from Siemens Medical Systems. During examination we reinforce for the patient to breathe smoothly. This protocol was adopted so as to minimize any possible artifacts from periadrenal structures.1H MRSI data were obtained using T2-weighted HASTE sequences, in two stages: (1) sagittal images only, at maximum inspiration, maximum expiration, and free breathing, carefully positioning the multivoxel volume of interest grid in the center of the lesion, based on all three sagittal sequences  [Adrenal chnique) .All spectroscopic findings were analyzed by two investigators (Homero J. F. Melo and Suzan M. Goldman) until consensus was reached. Images were processed using a Leonardo workstation . A 1000 Hz Gaussian line-broadening filter was used, and priority was given to the transformation of Fourier data into two spatial directions, using a Hamming filter.1H MRSI matrix was adjusted to the three orthogonal planes obtained during image acquisition, and the most appropriate voxels were selected for analysis. Voxels located in the adjacent fat tissue (not including lesion tissue) were disconsidered. Spectroscopic analysis was conducted in left-right, craniocaudal direction. Following voxel selection, the abscissa was enlarged so that an adequate ppm amplitude (0.5–8.5) could be used to phase correct water peak in relation to other metabolites. Subsequently, the ppm range was reduced (0.5–4.7) again in order to increase spectral resolution. The All spectroscopic data were interpreted by means of visual inspection and by measuring peak amplitudes of the metabolites of interest, namely, creatine (Cr), lactate (Lac), glutamine, and glutamate (Glx) (2.1–2.5 ppm).In addition, Glx and Lac were measured in the 2.1–2.5 ppm range and 1.33 ppm, respectively, and their relationship with Cr (Glx/Cr and Lac/Cr) was calculated. The association between Glx-Cr ratios and lesion size was also assessed.t-test for paired samples was used to compare mean metabolic ratios obtained in the groups, and the chi-square test was used to correlate differences in the relationships, followed by Yates' correction or Fisher's exact test for cell values below five.Data were analyzed using Excel and BioEstat 4.0 software for the metabolic characterization of the four types of masses or nodules analyzed, namely, adenomas, carcinomas, pheochromocytomas, and metastases. Central tendency (mean and mode) and dispersion values (standard deviation and range) were calculated for each group. Student's P ≤ 0.05. The association between Glx-Cr ratios and nodule size was assessed using Pearson's linear correlation. Also, receiver operating characteristic (ROC) curves were used to determine cutoff points, sensitivity, specificity, positive predictive value (PPV), and accuracy were calculated for the cutoff points established. Significance was set at 1H MRSI was successfully performed in 127 (96.2%) of the 132 adrenal masses and nodules assessed. P < 0.05), however, with a low PPV. The ROC curve calculated did not reveal a cutoff point that could allow adequate differentiation between these two types of lesions   . In the P < 0.05), but not for the other two lesions. A cutoff point of 1.44 was established for the differentiation between carcinomas larger versus smaller than 4 cm . Conversely, we did observe a tendency toward increased Lac concentrations in nonadenomas.Lac/Cr was also calculated for the different types of lesions in an attempt to define a cutoff point for differentiation. However, no significant differences were found in the comparison of adenomas versus nonadenomas , despite a trend toward differentiation. The ROC curve for adenomas and carcinomas   , namely 1H MRSI protocol in differentiating between adrenal masses and nodules  based on the previously tested Lac/Cr and Glx/Cr ratios.The present study tested the effectiveness of an 1H MRSI to assess the adrenal gland in vivo were aimed at locating the region of interest using spectroscopic images [The first studies using c images . Althougc images . Again, In an attempt to overcome such limitations, two-dimensional multivoxel systems started to be used to locate and select the spectroscopic region of interest, starting from the sagittal plane of the adrenal gland . In mult1H MRSI acquisition and analysis protocol originally proposed [The present study describes some changes made to the proposed . Among iWhole-body scanners with a magnetic field strength of 3 T are becoming more and more available to the scientific and clinical communities . The advWith regard to image analysis, changes made to the protocol reflect the technological improvements incorporated by the spectroscopic systems themselves . In particular, the possibility to use different scales, from higher (0.5–8.5 ppm) to lower (0.5–4.7 ppm) amplitude values, allowed to better adjust the base line, taking into consideration peak amplitude measurements calculated for water and other metabolites in the automatic and manual determination of spectral phase and frequency. Whenever necessary, amplitude values were lowered to 2.5–3.5 ppm to improve the visualization of Cho and Cr peaks, and voxel size was increased or decreased according to lesion size. All these strategies contributed to a faster and safer selection of eligible voxels.Moreover, adrenal oncocytoma does not follow the rule of four: (a) 4% diagnosed on CT, (b) 4% of these tumors are pheochromocytoma or adrenocortical carcinoma, (c) ≥4 cm in size is an indication for surgery, and (d) 4 years of followup is required . High Glx and Lac peaks were observed, and therefore their relationships with Cr were assessed. Both metabolites showed a great variance across the different lesions analyzed. The analysis did not reveal statistically significant associations between Glx/Cr and different types of masses/nodules, but rather with lesion size: a cutoff point of 1.44 was established for the differentiation between carcinomas larger versus smaller than 4 cm; no differences were observed in other lesions. Although the number of carcinomas included in our analysis was small (only 10 lesions), our results gain importance if we take into consideration the very low prevalence of this type of tumor in the general population . P = 0.063). Notwithstanding, the following cutoff points could be established: adenomas versus carcinomas (−4.25), pheochromocytomas versus carcinomas (−6.56), and carcinomas versus metastases (−5.34) (P < 0.05). A cutoff point of −6.5 for Lac/Cr was established for the diagnosis of carcinomas. These findings therefore add to the existing literature, and especially to our previous study [Conversely, Lac/Cr did not show statistically significant differences in the differentiation between adenomas and nonadenomas , preventing the inclusion of this category in the statistical analyses. Another limiting factor is the reduced number of patients using a 3.0 T scanner, rendering impossible comparisons between the curves generated by different fields. Finally, our two examiners had experience in spectroscopy, and maybe readings made by less experienced examiners could yield different results. This potential limitation is important because spectroscopic analysis of adrenal masses is still gaining space, especially if compared with areas in which it is highly consolidated, such as prostate cancer.Future studies are warranted to investigate the possibility of differentiation between functional and nonfunctional adenomas, still a major difficulty in the management of these patients, as well as to compare in vivo and in vitro spectroscopic findings with the aim of identifying metabolites present in different masses.1H MRSI sequences adopted in the present study proved effective for the differentiation between adrenal masses and nodules and data analysis was effective and reliable. Other studies are currently underway that focus on lipid concentrations as observed on spectral graphs compared with laboratory tests.In sum, the Our main findings can be summarized as follows: Glx levels are directly proportional to lesion size in carcinomas, and Lac/Cr allows to differentiate between carcinomas and noncarcinomas.


Male reproductive toxicity induced by exposure to bisphenol A (BPA) has been widely reported. The testes have proven to be a major target organ of BPA toxicity, so studying testicular metabolite variation holds promise for the discovery of mechanisms linked to the toxic effects of BPA on reproduction.Male Sprague-Dawley rats were orally administered doses of BPA at the levels of 0, 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites. Two n-6 fatty acids, linoleic acid (LA) and arachidonic acid (AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. According to these suggestions, testicular antioxidant enzyme levels were detected. Testicular superoxide dismutase (SOD) declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase (GSH-Px) as well as catalase (CAT) also showed a decreasing trend in BPA treated group.BPA caused testicular n-6 fatty acid composition variation and decreased antioxidant enzyme levels. This study emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity. Bisphenol A (BPA) is a chemical with one of the highest volume of production in the world, and in the U.S. the volume of BPA was estimated to be 2.4 billion pounds in 2007 in vitro and in vivo studies have clearly demonstrated that exposure to BPA is a causal factor of spermatogenesis impairment BPA is one of many endocrine disrupting compounds (EDCs) and its health risk has aroused public concern recently Metabolomics has been proven to be a useful tool in the discovery of new biomarkers for mechanisms of toxicity Legdig cells. In addition, the hypothalamic-pituitary-testes (HPT) axis is critical in the development and regulation of male reproduction. Testes have also been proven to be the key target organ of BPA toxicity Testes are an important part of both the reproductive system and the endocrine system. The testes act to produce sperm and to produce androgens, primarily testosterone in the Here we used a non-targeted liquid chromatography-quadrupole time-of-flight (HPLC–QTOF) based metabolomic technique to study testicular toxicity of BPA using a model of adult rats exposed to BPA. This unbiased analysis allowed us a hypothesis-free exploration of endogenous compound’s metabolic perturbation cased by BPA exposure. The metabolomic analysis workflow is shown in BPA and corn oil were purchased from Sigma Aldrich Laboratories, Inc. . BPA dosing solutions for rats were prepared in corn oil. Fresh solution of BPA in corn oil was made each week. Commercial assay kits for detection of superoxide dismutase (SOD) , glutathione peroxidase (GSH-Px)  and catalase (CAT)  were bought from Nanjing Jiancheng Biotech Ltd., China. Arachidonic acid  and linoleic acid  were purchased from Aladdin reagent company . Methanol was purchased from Merck Inc. . Acetonitrile was purchased from Fisher Scientific. Water was purified with a Milli-Q purification system . The structures of chemicals are shown in Male Sprague-Dawley rats were purchased from Slaccas . Twelve male Sprague-Dawley rats  were housed under controlled humidity (40–60%) and temperature (20–24°C) with a 12 h light/dark cycle, and were randomized into two groups (six rats per group). Animals had free access to food and water. The animals were acclimated to the laboratory for 1 week prior to the start of the experiments. The rats in non-exposed group and exposed group were given daily gavage administration 0.5 mL of corn oil containing 0, 50 mg/kg BPA for 8 weeks, respectively (6 days/week). Using this gavage method, none of the rats died, and no injuries in the stomach, esophagus or trachea were found. After the last treatment, urine was collected using a metabolic cage that was placed under an ice bath so as to avoid the degradation of metabolites and urine was stored at −30°C. Several testes sections from each rat were placed into cryo-vials. After snap freezing in liquid nitrogen, they were preserved at −80°C. This study was carried out strictly in accordance with international standards on animal welfare and the guidelines of the Institute for Laboratory Animal Research of Nanjing Medical University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University (Permit Number: BK2006576). All efforts were made to minimize animal suffering and to reduce the number of animals used.The testes sample preparation procedure for LC-QTOF analysis was according to previous report with minor modification All supernatant from testes and urine precipitated extract was transferred and then dried in a vacuum. The dried supernatant extract was reconstituted with 200 µL 0.1% formic acid in 50/50 acetonitrile/water (V/V). All reconstitutions were centrifuged at 12000 g at 4°C for 10 min before LC injection. To avoid potential contamination, a blank sample for testes, which was prepared without adding testes, was analyzed in parallel with testicular samples.LC-QTOF was carried out using an Agilent (1260) HPLC  with an electrospray ionization source (ESI) coupled to a micrOTOF-Q II mass spectrometer . The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The reconstitutions of urine and testes were loaded onto a ZORBAX SB-C18 Column  maintained at 30°C, and gradient eluted  directly into the mass spectrometer at a flow rate of 0.3 mL/min. The injection volume was 10 µL.m/z for positive ion scan, 100–1500 m/z for negative ion scan; for urine: 50–1000 m/z for both positive and negative ion scan. Solution of sodium trifluoroacetate was used for the external calibration. The injection order of samples from the different treatment groups was completely randomized.The parameters for QTOF were as follows: ESI source positive and negative full scan mode, capillary voltage −4500 V for positive ion scan and 3500 V for negative ion scan, offset for end plat 500 V, capillary exit voltage 120–160 V, Nebulizer 1 bar, Dry Gas 6 L/min, Dry Heater 220°C. Mass range collected for testes: 50–1500 Bucket tables for statistical comparison of urinary BPA-glucuronide and unknown testicular analytes levels between groups were generated by ProfileAnalysis 2.0 software  using FindMolecularFeatures (FMF) compounds and advanced bucketing .http://www.hmdb.ca/). This comparison matched experimentally derived data to the library database of molecular weight, molecular formulas and related biological information created from known chemical entities. Finally, potential biomarker structure identification was confirmed by comparing retention times with standards using the same LC separation condition.To chemically define the structures of the analytes selected for further investigation, multiple approaches were used. First, by using SmartFormula software (Bruker Corporation), possible molecular formulas of metabolites were provided. This software combines accurate mass (<4 mDa) and isotopic patterns (<25 mSigma) for enhanced confidence of molecular formulas identification. Second, potential biomarkers in testes were preliminarily identified by detailed information comparison with Human Metabolome Database . Briefly, SOD was assayed based on its ability to decrease the oxidation of hydroxylamine to form a red product. GSH-Px was measured by detecting absorbance of a yellow product formed in the reaction between reduced glutathione and dithiobisnitrobenzoic acid. CAT was determined based on the fact that ammonium molybdate could rapidly terminate the degradation reaction of hydrogen peroxide catalyzed by CAT and react with the residual hydrogen peroxide to generate a yellow product. Total protein concentration in testicular homogenate samples was determined using the Coomassie blue method.p-values acquired from permutation). The correlation between levels of unknown analytes was tested with Spearman correlation test. Statistical significance was assumed to p<0.05.Urinary BPA-glucuronide, unknown testicular analytes and antioxidant enzymes levels between groups were compared with a two-sided Wilcoxon rank-sum test   . No detem/z showing data zero. Then, we removed the candidate compounds did not show an apparent chromatographic peak in the extracted chromatograms. Next, for improving structure identification confidence, we removed those candidate compounds when their formula predictions had mSigma>25 or accurate mass >4 mDa with SmartFormula software. Subsequently, we compared information of the remaining potential analytes with data in HMDB. Finally, we focused on those with m/z 279.2330 in negative ion mode and 305.2462 in positive ion mode. Those analytes with m/z 279.2330 and 305.2462 showed a significant difference between non-exposed and exposed groups . Moreover, these two analytes demonstrated significant  negative correlation among each sample, suggesting a potential relationship via a common biochemical pathway and an important biological significance  when compared with corresponding standards . So the p = 0.0148), while AA was contrary (p = 0.0042) (p = 0.0021) (The testicular LA and AA relative levels between BPA exposed and non-exposed groups are shown in  0.0042)  A,B. Sin 0.0021)  C.Leydig cells p = 0.0268) (p = 0.0914) (Polyunsaturated fatty acid (PUFA) is very susceptible to peroxidation  0.0268) . The GSH 0.0268)  B,C. The 0.0914)  B. TheseIn this study, we validated an unbiased metabolomics system and a rat model of exposure to BPA through finding and analyzing known urinary BPA metabolite, and used this metabolomics system to discover unknown metabolite changes in testes. With multiple identification approaches, we identified AA and LA which might be potential biomarkers of BPA exposure. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were found in BPA exposed group. Because this kind of varied fatty acid composition might biologically adapt to oxidative stress Sertoli cell showed significant increase of LA (18∶2 n-6), decrease of AA (20∶4 n-6), decrease in AA/LA ratio and drop in desaturase activities Lipids are abundant in testicles. Phospholipid is the largest component, while triglyceride is present in smaller quantities Leydig cell steroidogenisis Oxidant/antioxidant imbalance in the testes may induce oxidative stress and thereby hamper testicular function Metabolomic analysis of urinary known analyte showed great amounts of BPA-glucuronide excreted in rat urine. This finding agreed with a previous report Within the toxicology community, although metabolomics has been widely used in exploring renal In conclusion, using metabolomic analysis with LC-QTOF, we found and structurally identified two potential biomarkers, LA and AA.The BPA-induced testicular toxicity showed decreased LA levels, as well as increased AA levels and AA/LA ratio. Based on the suggestion of fatty acid composition variation, an imbalance of the antioxidant enzyme system was found. This study highlights the application of metabolomics to the discovery of chemical reproductive toxicity.


Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue. Aedes aegypti or Aedes albopictus mosquito [Dengue virus (DENV) infection is an important arthropod-born viral infection infecting about 2.5 billion people worldwide, of which approximately 975 million belong to large and small cities of tropical and sub-tropical countries in Southeast Asia, the Pacific and the America ,2. The pmosquito ,7. Theremosquito ,9. Dengumosquito . Predomimosquito . Pakistamosquito . To dateTo date, many research studies have been conducted to understand the life cycle of dengue virus, especially the viral protein processing and the genome replication. Dengue virus binds to its receptor, and this process is mediated by envelop protein (E). In mammalian cell, DEN 1–4 serotypes bind with Heparan sulfate, nLc4Cer, DC-SIGN/L-SIGN and Mannose receptors. DEN-2 serotype also binds with HSP70/HSP90, GRP78, CD14-associated protein and two unknown proteins having trypsin resistance and trypsin sensitive properties. DEN 1–3 serotypes as well bind with Laminin receptor. DEN 2–4 serotypes also bind with an unknown protein having the property of serotype specific binding , a membrane associated protein (M) and Envelop protein (E). These proteins are involved in defining the structure of the virus.DENV core protein is a highly basic protein responsible for assembly of nucleocapsid through interaction with RNA but only a little is known about C protein. Structure of the protein revealed that the C protein dimer had high net charge and there is asymmetric distribution of basic residues over the surface of protein . Anti DEMembrane associated protein is a membrane glycoprotein works as a part of nucleocapsid and assists to envelop protein to form mature virions. Antibodies against prM protein were developed for Japanese encephalitis virus (JEV), West Nile virus (WNV) and dengue virus. Antibodies against the prM protein of JEV do not react with the prM of dengue viruses or West Nile virus. prM protein can be used to investigate differentiating antibody responses to difference flaviviruses . C-prM wEnvelop protein is present on the surface of the virus and is extremely involved in virus attachment with host cell through cell receptors like heparin sulphate DC-SIGN. It is most important protein for the entry of virus in cell. This protein has three domains, domain I , domain II (dimmers) and domain III (binding domain). Dimmer domain links structural and binding domain ,27. The DENV genome encodes seven non-structural proteins, including NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5. These proteins are responsible for viral replication and other cellular functions.st day to day 9 of infection. This was even detected when viral RNA was negative in RT-PCR. Its level differed in individuals ranging from a couple of nanograms per milliliter to several micrograms per milliliter. Thus, detection of NS1 protein may allow diagnosis of dengue infection in early stages [NS1 is expressed on the surface of the infected cell and can serve as an antibody target for dengue infection . NS1 proy stages . In a sty stages . NS1 medy stages .NS2A protein is majorly involved in RNA replication and viral assembly. A mutation in NS2A protein blocked virus replication. NS2A also inhibits the IFN-β promoter-driven transcription, thus serves as interferon (IFN) antagonist ,35. A siNS2B works as a cofactor in NS2B-NS3 serine protease. NS2B is required for the cleavage of NS2A/NS2B and NS2B/NS3.NS2B is also required for NS3/NS4A cleavage and for other internal cleavages .Wang andNS3 of DEN-2 is second largest non-structural protein having serine protease at N-terminus and NTPase helicase RNA triphosphate (RTPase) at C-terminus end. . NS3 is NS4A is the least characterized protein. At its C-terminus end, it is highly hydrophilic and serves as a signal to translocate NS4B to the endoplasmic reticulum lumen. In membrane bound viral replication complex (RC), NS4A forms components that help in maintaining this complex . NS4A haNS4B interacts with NS3 to regulate RNA replication. When treated with IFN-β or IFN-γ, NS4B expressing cells did not exhibit signal transducer and activator of transcription (STAT 1), thus indicated that NS4B may be involved in blocking IFN signaling and can be implicated as IFN-signaling inhibitor . FunctioNS5 has a methyltransferase (MTase) domain at N-terminus end and a polymerase domain at C-terminus end. NS5 has a role in RNA replication due to presence of RNA dependent RNA polymerase activity. It is also involved in de novo initiated RNA synthesis .Charge-tDengue has a worldwide history of about 200–400 years, mostly causing infection in tropical and sub-tropical areas. Dengue virus is believed to come in Pakistan with tyres at Karachi sea port carrying eggs of infected mosquitoes. To date, dengue virus infection has caused several outbreaks in Pakistan ,58. Untihttp://www.paperpk.com/news/index.php/new-dengue-cases-punjab-2012/). The year 2007 and 2011 has been worst years in regard of dengue virus infection in Pakistan. Dengue virus circulates in Pakistan throughout the year with a peak incidence in the post monsoon period. Recent flood in Pakistan made the situation worse [During 2011, the disease rapidly assumed the proportions of epidemic, specifically in Punjab and particularly Lahore where, in September, more than 250 people were reported dead and, according to the Punjab Health Department, over 12,000 people were infected during January to September 2011. Sindh, too, was hit by the dengue virus. According to the Sindh Provincial Dengue Surveillance Cell, nearly 400 people were infected with the virus in few months with over 300 people in Karachi alone, and the death toll stood at five at the end of September 2011. In February 2012, 73 confirmed cases of dengue in Lahore and 13 other regions of the state of Punjab were reported (Dengue virus can cause mild fever to Dengue fever (DF) or more severely DHF/DSS. DF causes fever in 3–5 days of infection, in which patient can feel headache, joint pain and rashes on the body with thrombocytopenia and relative leucopenia. Both DHF and DSS have same symptoms as of DF but they also have increased vascular permeability and hemorrhage, can lead to death. Dengue infection is mostly clinically diagnosed through symptoms. However, diagnosing dengue infection through symptoms is not reliable, and it needs laboratory studies to confirm the presence of dengue virus ,72. DiffLaboratory diagnosis of dengue virus infection can be made by the detection of particular virus, viral antigen, genomic sequence, and/or antibodies. IgM captured ELISA, virus isolation in mosquito cell lines and live mosquitoes, dengue specific monoclonal antibodies and detection of viral RNA by nucleic acid amplification tests (NAAT) have all represented major advances in dengue diagnosis. There are two stages of diagnosing dengue infection. In stage 1, fever is accompanied by NS1 antigens in blood and in the stage 2, post-febrile period in which IgM and IgG antibodies are in excess. In acute phase of dengue, patients generally suffer from flu-like fever and seek medical attention in first two days. During this phase, diagnosis is possible by detecting viral RNA/proteins in patient blood. If after two days fever is not recovered, then patient should be advised for a complete blood checkup (CBC). After the checkup, if platelet count is below normal range , and WBC count is below normal range  of blood) then patient should go for dengue antigen test. Patients with DHF/DSS should go for serological test. Using serotype specific NS3 primers, infection was identified in 80% patients . Dengue http://en.wikipedia.org/wiki/2011_dengue_outbreak_in_Pakistan). The Second is vaccination, which is currently not available and the Third is drug therapy but no antiviral drugs are available to target dengue virus [http://www.denguevirusnet.com/treatment.html).To date, there is no specific treatment available for dengue virus infection. There are three ways to solve the problem of dengue infection. The First is to use a preventive measure by avoiding contact with infected mosquitoes. Aedes mosquitoes bite during daytime and its contact can be avoided by: properly managing waste and improving storage of water, removing all sources of stagnant water, using household pesticides to kill mosquitoes, using mosquito coils and nets, wearing long-sleeved shirts, socks and trousers and using insect repellent to avoid mosquitoes. Insecticide treated nets (ITNs) are available to protect young children, pregnant women, old people, in addition to others who may rest during the day . Governmue virus . HoweverPlants have been traditionally used to cure a number of human diseases. To date, few plants derivatives due to their medicinal properties have successfully been tested against viral diseases. The first step of DENV cycle is to attach with host via host receptors. Envelop protein (E) is involved in viral and host attachment. Thus, dengue infection can be inhibited by targeting envelop protein/inhibiting host-viral interactions. NS2-NS3 protease and NS5 also serve as important antiviral drug targets due to their role in viral replication and other cellular processes. To date, many medicinal plants have been tested against DENV and some of them showed significant inhibition effects in the DENV replication cycle.3/μL to 168x103/μL, White blood cells from 3.7x103/μL to 7.7x103/μL and neutrophils from 46% to 78%. Thus carica papya can be used to target dengue fever [6/36  cells. Aqueous neem extracts at its maximum nontoxic concentration of 1.897 mg/ml, completely inhibited 100–10,000 TCID50 of virus as indicated by the absence of cytopathic effects [50) value of 55 μg/mL and selectivity index (SI) of 4.49. In Vero cells, rutin and naringenin did not inhibit DENV-2 replication [Antiviral effects of methanolic extracts of Andrographis paniculata, Citrus limon, Cymbopogan citrates, Momordica charantia, Ocimum sanctum and Pelargonium citrosum on dengue virus serotype 1 (DENV-1) were investigated by Tang and colleagues. A. paniculata showed the most antiviral inhibitory effect followed by M. chrantia in in-vitro assays. Both these plants can be advantageous in developing novel antiviral compounds . Carica ue fever . Extract effects . Talaric effects . Rehman  effects . DEN-2 i effects . Kiat an effects . A serie effects . Zandi a effects . Three flication . Recentllication .Dengue infection has emerged as a major health concern in Southeast Asia, the pacific and America. Dengue virus has become a serious issue in Pakistan as it has caused many endemics starting from 1994 to 2011. Developing tetravalent vaccine against all four dengue serotypes is quite challenging. To date, there is no licensed vaccine available for dengue virus. Therefore, there is an urgent need to develop an alternative solution to combat this endemic infection. Currently, there are no antiviral compounds available against dengue virus and there is a need to develop antiviral compounds that can target all four serotypes of dengue with same efficiency. Dengue virus enveloped protein is involved in virus cell entry; NS3 and NS5 are involved in viral replication and other cellular processes; therefore, these can serve as an important drug target to combat this life-threatening disease. Several medicinal plants have been tested against dengue virus entry and replication; many of them showed significant inhibitory effects. Nevertheless, it will be very exciting to see these medicinal plants as potential DENV inhibitors to progress through clinical developments and, hopefully, provide dengue patients with much needed, more effective therapies.All authors have no kind of institutional and financial competing interests.UAA designed the study and SI wrote the manuscript. Both the authors read and approved the final manuscript.Sobia Idrees (MPhil student), Usman A Ashfaq (PhD molecular Biology and Group leader, Human Molecular Biology Group, Department of Bioinformatics and Biotechnology, GCU, Faisalabad.


Idiopathic juvenile osteoporosis (IJO) is a rare condition of poorly understood etiology and pathophysiology that affects otherwise healthy children. This condition is characterized clinically by bone pain and vertebral fractures; spontaneous recovery is observed after puberty in the majority of cases. Although decreased trabecular bone turnover has been noted previously, cortical and trabecular bone characteristics as determined by quantitative computed tomography (QCT) and their relationship to bone histomorphometry are unknown.All children with a clinical diagnosis of IJO who were followed in our center since 1995 and who had undergone at least one diagnostic bone biopsy were included in this cross-sectional analysis.Fifteen patients  with median ages of 5.8 and 10.2 years at first symptoms and at referral, respectively, were included in the analysis. Histomorphometric analysis demonstrated decreased trabecular bone turnover (BFR/BS) in the majority of patients with heterogeneous parameters of trabecular mineralization and volume. QCTresults demonstrated that bone mineral density (BMD) was reduced in both trabecular/lumbar and cortical/femoral bone: Z score: -2.1  and −0.9 in the two compartments, respectively. In the eight patients who underwent both bone biopsy and QCT, cortical BMD was associated with trabecular separation and with trabecular bone formation rate .This series confirms that IJO is characterized by impaired trabecular architecture that can be detected by both bone biopsy and QCT. The association between bone biopsy and QCT results may have implications for diagnosis, treatment, and follow-up of these children. Osteoporosis, a well-known and frequent condition in postmenopausal women, rarely affects children but may be seen in primary genetic conditions involving the skeleton or the connective tissue, such as for example OsteogenesisImperfecta, Bruck syndrome, Osteoporosis Pseudoglioma Syndrome, Ehlers-Danlos syndrome, Marfan syndrome and Homocystinuria, or may be secondary to chronic inflammation, glucocorticoid therapy, and prolonged immobilization . In contThe diagnosis of IJO is made clinically. Children of both genders present two to three years before the onset of puberty with an insidious onset of pain in the back and lower limbs in association with vertebral compression fractures, trouble ambulating and long bone fractures, especially around the weight-bearing joints , in the 2) are affected by bone size, body size and composition [Osteopenia is evident on radiographs; however, the true extent of osteoporosis and osteopenia in patients with IJO is unknown. Indeed, previous studies have evaluated bone density using dual energy X-ray absorptiometry (DXA), a technique that does not discriminate between cortical and trabecular bone density and whose assessment of bone mineral density as an area BMD , evidence of low bone mass on BMD assessed by QCT and/or of osteoporosis on bone histomorphometry, without any evidence for a secondary osteoporosis or other primary bone disease. A clinical score was developed which accounted for the subjective severity of disability (moderate or severe restriction of daily activities) and the objective number of fractures. Children were divided into two groups based on number of fractures: moderate disability was defined as fewer than five fractures  while severe disability was defined as 5 or more fractures .3for cortical BMD in all children, and 250 ± 45 and 285 ± 45 mg/cm3 for cancellous BMD in pre-pubertal and pubertal children, respectively. These local reference values are routinely used by Dr. Gilsanz, and trabecular BMD details have been described from a cohort of 1222 White healthy subjects aged 5–21 years [QCT examination was performed at three sites (both femora and the spine) using the same scanner  in all patients. Measurements of the size  and the density of cortical bone were obtained from a 1.5 mm-thick sections at the midshaft of the femora at 120 kVp and 70 mA; the final results consisted in the mean of both femora. Measurements of the size  and the density of cancellous bone were obtained from 10 mm-thick imaging sections at the mid-portions of the first four lumbar vertebrae at 80 kVp and 70 mA; the final results consisted in the mean of L1, L2, L3 and L4 values. Reference values assessed with QCT are 2000 ± 60 mg/cmThe full thickness bone biopsy, performed at the iliac crest 2 to 5 days after the end of a double tetracycline labeling , was performed as previously reported . Biopsy Statistical analysis was performed using the SPSS software® 18.0 for Windows. Comparisons between groups were performed using the nonparametric Wilcoxon signed rank test. Bivariate correlations were computed with the Spearman test. Comparisons of percentages were made with a chi-square test. All statistical tests were performed at the two-sided 0.05 level of significance. Results are presented as median (range).Fifteen patients  with at least one bone biopsy were included in the analysis; only 12 initial bone biopsies were interpretable as one biopsy contained growth plate and two were technically unreadable. Eleven patients had an initial QCT. Eight patients underwent both baseline QCT and bone biopsy.th percentile for height at diagnosis. Twelve patients were White, two were Hispanic and one was White by her father and Hispanic by her mother.The median (range) for age at first symptoms and for age at referral were 5.8 (1.5–14) and 10.2 (5.5-15.4) years, respectively. The median time between first symptoms and the diagnosis of IJO was 3.5 (0.2-9.4) years. The median (range) percentile of height at referral was 88 (15–98); 11 patients were at or above the 50Most of the patients had a family history of bone disease or disorder of calcium/phosphate metabolism, including post-menopausal osteoporosis in four, scoliosis in one, and parathyroid adenoma in one. Two patients were siblings and two others were homozygous twins. Ten patients underwent fibroblast analysis or direct gene analysis to rule out Osteogenesis Imperfecta. All but one patient had a history of fractures occurring with minimal trauma; five complained of back pain and four of leg pain. Two patients had kyphosis or scoliosis, two had vertebral compression fractures and one developed avascular necrosis and osteomyelitis. Of note, one patient received calcitonin prior to referral; even though calcitonin is known to have inhibitory effects on osteoclast activity ,15, it iTrabecular bone histomorphometric parameters of turnover, mineralization and volume parameters are summarized in Tables Biochemical abnormalities were found in five patients: one displayed transient hypercalciuria, two had increased levels of urinary N-telopeptide, one was 25 (OH) vitamin D deficient (defined by a 25 (OH) vitamin D level of 22 ng/mL) and one had a moderate but transient increase in PTH (120 pg/mL). The median (range) for PTH and 25 (OH) vitamin D at the time of the bone biopsy were 26 (8–120) pg/ml and 44 (22–93) ng/ml, respectively. Serum PTH levels were directly related to trabecular bone volume , and 25 (OH) vitamin D levels were associated with the rate of bone mineralization (osteoid maturation time (OMT), r = −0.786, p = 0.036 with a similar trend for mineralization lag time (MLT), r = −0.714, p = 0.07), although not with osteoid accumulation (as measured by osteoid volume (OV/BV), osteoid thickness (O.Th), or osteoid surface (OS/BS)).3 and 1946 (1506–2081) mg/cm3, respectively, with a Z-score of −2.1  and −0.9 , respectively. Trabecular (lumbar) and cortical  cross sectional area were 11.35 (4.72–12.69) cm2 and 4.81 (1.86–6.61) cm2, respectively; Z-score according to age and body weight for these two parameters were 2.30  and −0.7 , respectively. There was no significant association between cortical and trabecular bone mineral densities .Trabecular (lumbar) and cortical  bone mineral density were both low at 187 (124–224) mg/cmA comparison of the eight patients who had both bone biopsy and QCT within a median time frame between the two examinations of 0.4 years 0.1–2.5) revealed a relationship between cortical bone mineral density and parameters of trabecular bone volume (trabecular number (Tb.N): r = 0.898, p = 0.02; trabecular separation (Tb.Sp): r = −0.838, p = 0.009), with a trend between cortical bone mineral density and cortical bone volume  and external porosity, r = 0.754 and −0.754, respectively, both p = 0.084). Trabecular bone mineral density was associated with trabecular bone turnover (trabecular bone formation rate (BFR/BS): r = −0.881, p = 0.004). Figure .5 revealThe proportion of boys with clinically severe idiopathic juvenile osteoporosis was higher than the proportion of boys with clinically moderate disease. There were no imaging differences between the two groups to account for the clinical findings; however, patients with severe osteoporosis had significantly lower mineralization lag time (MLT): the median (range) for MLT was 36.3 (28.8–105.1) and 12.4 (4.6–58.3) days in moderate and severe osteoporosis, respectively (p = 0.028).This study is the first to compare QCT, which allows separate measurements of cortical and trabecular bone mineral density, with bone biopsy, the reference standard for evaluating bone, in a cohort of 15 children with idiopathic juvenile osteoporosis. Major findings include the description of decreased bone turnover in many patients, a decrease in both trabecular and cortical Z-scores for bone mineral density, and a relationship between both cortical and trabecular bone density with parameters of bone histology, thus allowing a non-invasive evaluation of cortical and vertebral bone volume in these children.Two large clinical cohorts of IJO patients have been previously reported, containing 21 and 61 patients respectively ,17. The et al. have described the presence of decreased bone formation, in the absence of increased bone resorption or cortical damage, in trabecular bone of patients with idiopathic juvenile osteoporosis [Histomorphometric data from Rauch oporosis . They thoporosis . In the Published data on bone mineral density in children with idiopathic juvenile osteoporosis have been obtained from DXA. Using this technique, Lorenc et al. reported that total body bone mineral density was lower than the normal for age . AlthougIn the current study, which used QCT to discriminate cortical and cancellous bone compartments, a decrease in both trabecular and cortical BMD Z-scores was observed, with greater impairments in trabecular BMD, confirming previous reports which highlighted the greater importance of impaired trabecular bone in patients with idiopathic juvenile osteoporosis. However, the low Z-score for both BMD and cross-sectional area in the cortical compartmentmay explain the important frequency of cortical fractures also observed in this cohort; indeed, even though children with IJO are usually known to present both vertebral compression fractures and long bone fractures, in our series, only two patients presented with vertebral compression fractures whereas long bone fractures and extremity fractures  were much more frequent; however, due to the retrospective design of the study, compression fractures were not systematically screened in the patients, but only in case of clinical symptoms. Furthermore, the association between histomorphometry and QCT parameters highlights the potential interest of this non-invasive three-dimensional imaging technique for the follow-up of children with IJO. In some patients the biopsy and QCT were not obtained at the same age due to the retrospective design of the study and may have lessened the association between QCT and bone biopsy; however, to the best of our knowledge, this association between QCT and bone biopsy has never been evaluated in pediatrics and further prospective studies are warranted to evaluate its potential clinical and therapeutic ramifications. Moreover, after the new definition in 2000 of osteoporosis by the National Institute of Health (NIH), adding ‘quality’ criteria in addition to the quantitative evaluation of BMD, new bone imaging techniques have been developed, including High-Resolution peripheral Quantitative Computed Tomography (HR-pQCT), and therefore leading to an improvement in bone evaluation. Indeed, HR-pQCT can assess both compartmental  volumetric BMD and trabecular microarchitecture . This teRecent studies have reported an association between mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) and low bone mass in children ; howeverCurrently, no evidence-based guidelines exist for the management of idiopathic juvenile osteoporosis. Some case reports have described the potential benefit of bisphosphonate therapy -26; howeIn conclusion, idiopathic juvenile osteoporosis is a rare bone disease affecting pre-pubertal children, with both cancellous and cortical impairments. Its etiology and pathophysiology are not well understood and its diagnosis remains one of exclusion. The current study brings new insights into this rare disease, confirming bone impairment as assessed by bone biopsy and QCT, and therefore allowing the description of a promising association between bone histology and QCT results that could be of relevance in daily practice.BFR/BS: Bone formation rate/Bone surface; BMD: Bone mineral density; BV/TV: Bone volume/Tissue volume; DXA: Bone density using dual X-ray absorptiometry; ES/BS: Eroded surface/Bone surface; IJO: Idiopathic juvenile osteoporosis; LRP5: Lipoprotein receptor-related protein 5; MAR: Mineral apposition rate; MLT: Mineralization lag time; O.Th: Osteoid thickness; Ob.S/BS: Osteoblast surface/Bone surface; Oc.S/BS: Osteoclast surface/Bone surface; OMT: Osteoid maturation time; OS/BS: Osteoid surface/bone surface; OV/BV: Osteoid volume/bone volume; PTH: Parathyroid hormone; QCT: Quantitative computed tomography; Tb.N: Trabecular number; Tb.Sp: Trabecular separation; Tb.Th: Trabecular thickness.The study sponsors had no role in the study design; the collection, analysis, and interpretation of data; the writing of the report; and the decision No honorarium, grant, or other form of payment was given to anyone to produce the manuscript and to submit the paper for publication. The authors declare no conflicts of interest.The first draft of the manuscript was written by JB and KWP. RP read the bone biopsies and VG performed the QCT analyses. BG was involved in the data’s collection. IS designed the study and revised the manuscript. All authors approved the last version of the manuscript.


MYLIP region in the Mexican population in order to fine-map the actual susceptibility variants of this locus. The p.N342S polymorphism was identified as the underlying functional variant accounting for one of the previous signals of genome-wide association studies and the N342 allele was associated with higher cholesterol concentrations in Mexican dyslipidemic individuals. To date, there is no further evaluation on this genotype-phenotype association in the literature. In this scenario, and because of a possible pharmacotherapeutic target of dyslipidemia, the main aim of this study was to assess the influence of the MYLIP p.N342S polymorphism on lipid profile in Brazilian individuals.A recent study investigated the MYLIP rs9370867  polymorphism were detected by high resolution melting analysis.1295 subjects of the general population and 1425 consecutive patients submitted to coronary angiography were selected. General characteristics, biochemical tests, blood pressures, pulse wave velocity, and coronary artery disease scores were analyzed. Genotypes for the MYLIP rs9370867 genotypes with lipid profile, hemodynamic data, and coronary angiographic data was found. Analysis stratified by hyperlipidemia, gender, and ethnicity was also performed and the sub-groups presented similar results. In both general population and patient samples, the MYLIP rs9370867 polymorphism was differently distributed according to ethnicity. In the general population, subjects carrying GG genotypes had higher systolic blood pressure (BP), diastolic BP, and mean BP values  compared with subjects carrying AA genotypes  , even after adjustment for covariates. However, in analysis stratified by ethnicity, this finding was not found and there is no evidence that the polymorphism influences BP.No association of the MYLIP variant can present distinct results according to the studied population. In this moment, further studies are needed to reaffirm if the MYLIP p.N342S polymorphism is functional or not, and to identify other functional markers within this gene.Our findings indicate that association studies involving this  Lipid profile disorders have been significantly associated with risk of cardiovascular disease (CVD), which is also influenced by genetic factors, hypertension, type 2 diabetes mellitus, obesity, and smoking. CVD are the main cause of morbidity and mortality in developed countries and the financial cost is enormous. Thus, guidelines from the National Cholesterol Education Program (NCEP) rely on low-density lipoprotein cholesterol (LDL-C) for the prevention of CVD -6.A conventional lipid panel reports several parameters, including total cholesterol (TC), LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Of these, the NCEP and the American Heart Association recommend using LDL-C as a primary target of therapy in conjunction with assessing cardiovascular risk factors. The Third Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults  updated clinical guidelines for cholesterol testing such as high TC (≥ 240 mg/dL), high LDL-C (≥ 160 mg/dL), and low HDL-C (< 40 mg/dL) ,7,8.MYLIP gene that were associated with LDL-C concentrations [MYLIP functional variant and the mechanistic basis of these associations were recently postulated by Weissglas-Volkov et al. [Population genetic and epidemiological studies could help to assess the etiologic role of lipid profile in CVD and, novel genetic determinants of blood lipids can also help to provide new insights into the biological pathways and identify novel therapeutic targets. In this way, current genome-wide association studies (GWAS) have identified genetic loci contributing to inter-individual variation in serum concentration of lipids -12. Sometrations . The MYLv et al. .MYLIP gene encodes a regulator of the LDL receptor pathway for cellular cholesterol uptake called MYLIP . Weissglas-Volkov et al. investigated the MYLIP region in the Mexican population in order to fine-map the actual susceptibility variants. They identified the rs9370867 non-synonymous polymorphism (p.N342S) as the underlying functional variant accounting for one of the previous GWAS significant signals and associated N342 allele with higher TC concentrations in Mexican dyslipidemic individuals [ividuals .MYLIP p.N342S – lipid profile). In this scenario, the main aim of this study was to assess the influence of the MYLIP polymorphism on lipid profile in Brazilian individuals.To date, there is no further evaluation on this genotype-phenotype association , Sao Paulo, Brazil. All patients had a clinical diagnosis of angina pectoris and stable angina. No patient enrolled in this study was currently experiencing an acute coronary syndrome. Patients with previous acute ischemic events, heart failure classes III–IV, hepatic dysfunction, familiar hypercholesterolemia, previous heart or kidney transplantation, and in antiviral treatment were excluded -19. PatiPardo in Portuguese), Black, Amerindian or Oriental descent [Weight and height were measured according to a standard protocol, and body mass index (BMI) was calculated. Individuals answered a clinical questionnaire that covered questions regarding smoking status and current medical treatment. Individuals who had ever smoked more than five cigarettes per day for the last year were classified as smokers ,20. Ethn descent ,21,22.Triglycerides (TG), TC, HDL-C, LDL-C, and glucose were evaluated by standard techniques in 12-h fasting blood samples. Diabetes mellitus was diagnosed by the presence of fasting glucose ≥ 126 mg/dL or the use of antidiabetic drugs . HyperliBlood pressure was measured in the sitting position with the use of a standard mercury sphygmomanometer on the left arm after 5 min rest. The first and fifth phases of Korotkoff sounds were used for systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively. The SBP and DBP were calculated from two readings with a minimal interval of 10 min apart. Hypertension was defined as mean SBP ≥140 mmHg and/or DBP ≥90 mm Hg and/or antihypertensive drug use . The meaCarotid-femoral pulse wave velocity (PWV) was analyzed with an automatic device  by an experienced observer blinded to clinical characteristics. Briefly, common carotid artery and femoral artery pressure waveforms were recorded non-invasively using a pressure-sensitive transducer . The distance between the recording sites (D) was measured, and PWV was automatically calculated as PWV = D/t, where (t) means pulse transit time. Measurements were repeated over 10 different cardiac cycles, and the mean was used for the final analysis. The validation and its reproducibility have been previously described, and increased arterial stiffness was defined as PWV ≥ 12 m/s ,25.Twenty coronary segments were scored: each vessel was divided into three segments , except for the secondary branches of the right coronary artery (posterior ventricular and posterior descending), which were divided into proximal and distal segments. Stenosis higher than 50% in any coronary segment was graded 1 point and the sum of points for all 20 segments constituted the Extension Score. Lesion severity was calculated as follows: none and irregularities, 0 points; <50%, 0.3 points; 50–70%, 0.6 points; >70–90%, 0.8 points; and >90–100%, 0.95 points. The Severity Score was calculated through the sum of points for all 20 coronary segments .MYLIP rs9370867  polymorphism was detected by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor Gene 6000® instrument  [Genomic DNA from subjects was extracted from peripheral blood following standard salting-out procedure. Genotypes for the  France) ,27. The  France) .2; 200 μM of each dNTP; 0.5 U Taq DNA Polymerase; 200 nM of each primer; 10 ng of genomic DNA template) with addition of fluorescent DNA-intercalating SYTO9® .Amplification of the fragment was performed using the primer sense 5’- TTGTGGACCTCGTTTCAAGA -3’ and antisense 5’- GCTGCAGTTCATGCTGCT -3’ (80 pairs base) for the rs9370867. A 40-cycle PCR was carried out with the following conditions: denaturation of the template DNA for first cycle of 94°C for 120 s, denaturation of 94°C for 20 s, annealing of 53.4°C for 20 s, and extension of 72°C for 22 s. PCR was performed using a 10 μL reactive solution  or type 2 diabetes mellitus were excluded. The subjects were classified as high-TC if their serum TC levels were ≥ 240 mg/dL and normal TC if their serum TC levels were < 240 mg/dL in the absence of lipid lowering medication. The subjects were also classified as combined hyperlipidemia if their serum TC and TG levels were ≥ 240 mg/dL and > 200 mg/dL, and as controls if TC and TG levels were < 240 mg/dL and < 150 mg/dL, respectively [The analysis stratified by hyperlipidemia was performed according to Weissglas-Volkov et al.‘s inclusion and exclusion criteria ,15. Fastectively ,15. All Table MYLIP polymorphism was found. Only hypertension frequency presented a trend (p = 0.05) in the general population  was higher in Whites (45.8% and 22.1%) compared with Blacks (15.2% and 3.1%) . In the patients submitted to coronary angiography, the frequencies of the MYLIP rs9370867 A variant allele and of the homozygous genotype (AA) was higher in Whites (44.6% and 20.7%) compared with Blacks (21.3% and 5.9%) . The genotypic distribution for the MYLIP rs9370867 polymorphism was in Hardy–Weinberg equilibrium according to ethnic groups.In both general population and patient samples, the ty Table . In the Table MYLIP rs9370867 genotypes with TC, LDL-C, HDL-C, triglycerides, and glycemia values in both samples  compared with subjects carrying AA genotypes  , even after adjustment for age, gender, and ethnicity  or with angiographic data  was observed Table .The analysis stratified by hyperlipidemia was performed according to Weissglas-Volkov et al.‘s inclusion and exclusion criteria (see Methods section for details) ,15. In tMYLIP rs9370867 polymorphism was associated with TC levels in a Mexican dyslipidemic sample and this genetic data was supported by functional data, which demonstrated that rs9370867 influences plasma cholesterol levels by modifying the degradation of the LDL-receptor [A recent study reported that receptor . In thisHere, the frequency of the A was much higher in Whites compared with Blacks. Previous studies reported that in African and Asian groups, the frequency is relatively low at 2% - 8%, whereas in European descent, the frequency is much higher at 49% - 60% ,29,30. TMYLIP studied polymorphism influences blood pressures.In the same way, this variation of the allele frequency among ethnicity influenced blood pressures data in general population samples. SBP, DBP, and MBP values were higher in GG genotype group (major frequency of Blacks) while lower values were observed in AA genotype group (minor frequency of Blacks). The adjustment for covariates plus ethnicity was not able to exclude the participation of the ethnicity variable in the observed results. But, in analysis stratified by ethnicity, this finding was not found in any ethnicity group. Corroborating with this observation, our group demonstrated in a recent study that SBP, DBP, and MBP values were higher in Black individuals than in the other ethnic groups in the Brazilian general population (p < 0.001) . Thus thMYLIP genetic loci contributing to variation in serum lipids [MYLIP region in Mexican individuals in order to restrict the associated region and identified the variant p.N342S (rs9370867). They associated this substitution with cholesterol levels in a Mexican dyslipidemic study sample [In this study, the replication of the previously identified association was not found in both general population and patient samples. Two GWAS of populations of European descent have identified m lipids ,13. Weisy sample .MYLIP functional polymorphisms could be differently distributed leading to distinct findings.It is important to report that the mentioned study have only observed the association in Mexican dyslipidemic individuals. Our Brazilian general population sample allowed a first assessment of this association in a general population and, our second sample, patients submitted to coronary angiography, allowed an analysis with major frequency of dyslipidemic individuals. In both samples, even after performing analysis according to Weissglas-Volkov et al.‘s criteria (see Methods section), no association was observed. The patterns of linkage disequilibrium vary across populations and ethnicities according to previous studies ,29,30; tMYLIP variant can present distinct results according to the studied population. In this moment, further studies are needed to reaffirm if the MYLIP p.N342S polymorphism is functional or not, and to identify other functional markers in this locus.Our findings indicate that association studies involving this The authors declare that they have no competing interests.PCJLS carried out the molecular genetic studies, statistical analysis and drafted the manuscript. TGMO carried out the molecular genetic studies. ACP participated in the design of the study, statistical analysis and manuscript preparation. ACP, PAL, JGM, JEK participated in the design of the study and were responsible for individual selection and characterization. All authors contributed critically to the manuscript, whose present version was read and approved by all.