Nosema ceranae, and trypanosomatids. Our results indicated that pathogen prevalence and abundance were associated with both sampling date and beekeeping operation, that prevalence was highest in honey bee samples obtained immediately after almond pollination, and that weak colonies had a greater mean pathogen prevalence than strong colonies.Honey bees are important pollinators of agricultural crops. Since 2006, US beekeepers have experienced high annual honey bee colony losses, which may be attributed to multiple abiotic and biotic factors, including pathogens. However, the relative importance of these factors has not been fully elucidated. To identify the most prevalent pathogens and investigate the relationship between colony strength and health, we assessed pathogen occurrence, prevalence, and abundance in Western US honey bee colonies involved in almond pollination. The most prevalent pathogens were Black queen cell virus (BQCV), Lake Sinai virus 2 (LSV2), Sacbrood virus (SBV), The online version of this article (doi:10.1007/s13592-015-0395-5) contains supplementary material, which is available to authorized users. Apis mellifera) are the primary pollinators of US crops valued at over $17 billion per year  prevalence and abundance compared to unaffected colonies  concern growers, beekeepers, scientists, and policy makers  and eukaryotic parasites including the microsporidia Nosema spp. and trypanosomatids . Herein, we present data from an observational cohort study at the colony level that provides current information regarding pathogen prevalence and abundance in Western US commercially managed honey bee colonies involved in almond pollination. Enhanced understanding of the dynamics of pathogenic infections in bee colonies across different geographies and in independent beekeeping operations will inform ongoing and future epidemiologic studies aimed at investigating the potential relationships between colony health and pathogen occurrence, prevalence, and abundance on a larger scale , pathogen prevalence, and pathogen abundance, before, during, and after the 2013–2014 almond pollination season  commercial beekeeping operations that transport their honey bee colonies ~1,200 miles to California (Merced and Stanislaus counties) each winter for the almond bloom provided honey bee samples before (October–December 2013), during (February 2014), and after (March/April and June 2014) almond pollination Figure . Colony N = ln(1 − D) / ln(1 − P) , predicts that with a sample size of five bees, pathogenic infections affecting 45 % or more of the individuals within a colony would be detected with 95 % probability  according to the manufacturer’s instructions  and a TissueLyzer (Qiagen) at 30 Hz for 2 min. Samples were centrifuged for 12 min at 12,000cDNA synthesis reactions were performed by incubating 1,000–2,000 ng total RNA, Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega), and 500 ng random hexamer primers (IDT) for 1 h at 37 °C, according to the manufacturer’s instructions ; reaction conditions and equations for determining the relative abundance based on standard curves are provided in supplemental methods , beekeeping operation, colony strength, and pathogen prevalence. Observations with average strength rating were not included in some analyses to simplify the inferences between strong (S) and weak (W). The natural logarithm (ln) of the pathogen prevalence data was used in comparisons between each beekeeping operation and time period combination  and 0 otherwise.Si = 1 if observation i was strong (colony strength) and 0 otherwise.In all, our model can be expressedγj(i) is the random effect for colony. We assume γj(i) ∼ N, ϵi ∼ N, and γj(i) and ϵi are independent for all j = 1, 2, …, 60, i = 1, 2, 3, …, 180. Variables are defined as they were in Section β0, β6, and β7. Estimated means and standard errors for these coefficients of interest were obtained using the regression output. Models were fit using the software R  sense RNA viruses , we utilized virus copy number as an indication of infection severity, though the relationship between virus copy number and virus associated disease or affects on the honey bee host are largely unknown . Colony population size was utilized as a proxy for colony health and monitored at each sampling event. At the onset of the study, each beekeeping operation selected weak, average, and strong honey bee colonies that were monitored throughout the study. These colonies were located in Montana before and after almond pollination and in California during the almond pollination season (February 2014)  and eukaryotic parasites including the microsporidia Nosema spp. and trypanosomatids . In order to identify the most prevalent pathogens associated with each sample , five bees per sample were utilized for pathogen analyses , LSV2, LSV3, LSV4, LSV5, Black queen cell virus (BQCV), DWV, SBV, ABPV, CBPV, IAPV, and KBV. In addition, we tested for bacterial pathogens ; however, pathogen occurrence also varied for each beekeeping operation  transformed after adding 1  .Figure The overall colony mortality in this study was 14.8 %. Colony loss varied with the time of sampling and beekeeping operation Table . Colony Honey bees play an important role as pollinators of numerous agricultural crops. The majority of commercially managed honey bee colonies in the US are transported throughout the year to meet the pollination demands of crops including California almond production. Commercial beekeepers in the US and some parts of Europe have experienced high annual average losses, though the factors most responsible for these losses are not well understood. The objectives of this study were to identify the pathogens currently circulating in Western US-based commercial honey bee colonies involved in almond pollination and to examine the relationship between colony strength, pathogen prevalence, and abundance. Pathogen incidence and abundance have been associated with CCD-affected colonies and colony losses in the US   accounted for a large proportion of the positive tests. One of the first US-based longitudinal monitoring studies involved 20 colonies managed by a single large-scale commercial beekeeping operation , though the reason(s) for low colony mortality remain unknown. Future studies that closely monitor additional factors, such as foraging success and diversity via pollen traps, agrochemical exposure via chemical analysis, and mite abundance, in conjunction with colony health and pathogen prevalence and abundance will help elucidate the factors that most correlate with colony health and survival.ESM 1(DOCX 40 kb)ESM 2(PDF 598 kb)ESM 3(PDF 159 kb)ESM 4(PDF 123 kb)ESM 5(XLSX 65 kb)ESM 6(PDF 1.64 mb)Below is the link to the electronic supplementary material.


Phenobarbital is an old antiepileptic drug used in severe epilepsy. Despite this, little is written about the need for dose adjustments in renal replacement therapy. Most sources recommend a moderately increased dose guided by therapeutic drug monitoring.A 14 year old boy with nonketotic hyperglycinemia, a rare inborn error of metabolism, characterized by high levels of glycine, epilepsy, spasticity, and cognitive impairment, was admitted to the emergency department with respiratory failure after a few days of fever and cough. The boy was unconscious at admittance and had acute renal and hepatic failure.Due to the acute respiratory infection, hypoxic hepatic and renal failure occurred and the patient had a status epilepticus.The patient was intubated and mechanically ventilated. Continuous renal replacement therapy was initiated. Despite increased phenobarbital doses, therapeutic levels were not reached until the dose was increased to 500 mg twice daily. Therapeutic drug monitoring was performed in plasma and dialysate. Calculations revealed that phenobarbital was almost freely dialyzed.Correct dosing of drugs in patients on renal replacement therapy may need a multidisciplinary approach and guidance by therapeutic drug monitoring. There is no treatment apart from low protein diet, to keep the levels of glycine low. NMDA receptor antagonists such as dextromethorphan have been tried. The epilepsy is treated with antiepileptic drugs, and ketogenic diet has also been tried.10The patient was a 14-year-old boy  with nonketotic hyperglycinemia (NKH), a rare autosomal recessive inborn error of metabolism, which leads to neurological deficits and severe epilepsy. The disease can present itself either in the newborn infant or as late onset NKH. All patients show signs of hypotonia, which later evolves into spasticity. The cognitive development is delayed. The primary lesion is a defect in the glycine cleavage system, a mitochondrial enzyme complex, which leads to increased concentrations of glycine in plasma, urine, and cerebrospinal fluid. The pathophysiology remains obscure; the increased levels of glycine may increase the QD) 1400 mL/h. Regional citrate anticoagulation was used with an infusion rate of 119 mL/h of sodium citrate 0.13 mmol/L and 11 mL/h of calcium glubionate 9 mg/mL. Net ultrafiltration rate (UFR) was 50 mL/h. Total convective flow can thus be calculated to be 50 + 119 + 11 mL/h = 180 mL/h.The patient was treated with ketogenic diet and several anticonvulsants  including an oral daily dose of 135 mg phenobarbital. In February 2013, he was admitted to the intensive care unit due to a severe infection with respiratory syncytial virus. He had acute hepatic and renal dysfunction, probably due to respiratory failure. His renal function deteriorated and he was started on continuous veno-venous hemodialysis the day after admission. Blood flow was 60 mL/min, dialysate flow /(Cl × τ), where Css is steady-state concentration, F is bioavailability, and τ is the dose interval.Systemic clearance of phenobarbital at baseline can be calculated from the relationship Cl) can be calculated to be By rearrangement, clearance  were calculated according to the trapezoidal rule to be 1718 μmol × h/L in plasma and 1103 μmol × h/L in dialysate.Areas under the concentration–time curves  could be calculated according to the formula Total extracorporeal clearance  × t, Since 7 and the total UFR would be the sum of the infusion rates of calcium and citrate and the net ultrafiltration. Theoretical convective clearance would, therefore, be 144 mL/h = 2.4 mL/min. Total theoretical clearance would then be 11.7–16.4 mL/min. This is very close to the observed clearance in our case.Phenobarbital clearance was dramatically increased during continuous veno-venous hemodialysis. Extracorporeal clearance alone was 6-fold higher than baseline systemic clearance and systemic clearance during renal replacement therapy was 7.5-fold higher than at baseline. During complete equilibration between blood and dialysate, theoretical dialysis clearance can be calculated as the product between dialysate flow and unbound fraction, that is, 1400 mL/h × (0.4 − 0.6) = 9.3 − 14 mL/min. Theoretical convective clearance can similarly be calculated as the product of sieving coefficient and UFR. In the case of phenobarbital, the sieving coefficient has been measured to be about 0.8,This case illustrates that although a drug has a relatively low renal clearance, its clearance by continuous dialysis may be considerably higher. The difference may be explained by tubular reabsorption that decreases renal clearance and that may be amplified in ketogenic conditions.5 Other sources recommends supplementation doses after hemodialysis,8 and a fourth differs considerably between adult and pediatric patients in recommended dosing during CRRT,4 where children are recommended an up to 5-fold higher weight-adjusted dose in CRRT, whereas adults are not.Sources for dose recommendations in renal disease differ considerably regarding their recommendation in continuous renal replacement therapy (CRRT). One source even states that phenobarbital is not dialyzed by CRRT.This case description illustrates that it may be difficult to decide a priori the pharmacokinetic consequences of initiating renal replacement therapy. Proper dose adjustments will depend on an adequate estimation of the individual patient and the intended parameters of the renal replacement therapy. We would like to stress the need for therapeutic drug monitoring and a multidisciplinary approach to patient care.


The raphe nuclei provide serotonergic innervation widely in the brain, thought to mediate a variety of neuromodulatory effects. The mammalian olfactory bulb (OB) is a prominent recipient of serotonergic fibers, particularly in the glomerular layer (GL), where they are thought to gate incoming signals from the olfactory nerve. The dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) are known to densely innervate the OB. The majority of such projections are thought to terminate in the GL, but this has not been explicitly tested. We sought to investigate this using recombinant adeno-associated viruses (rAAV)-mediated expression of green fluorescent protein (GFP)-synaptophysin targeted specifically to neurons of the DRN or the MRN. With DRN injections, labeled fibers were found mostly in the granule cell layer (GCL), not the GL. Conversely, dense labeling in the GL was observed with MRN injections, suggesting that the source of GL innervation is the MRN, not the DRN, as previously thought. The two raphe nuclei thus give dual innervation within the OB, with distinct innervation patterns. J. Comp. Neurol. 523:805–813, 2015. © 2015 Wiley Periodicals, Inc. Caenorhabditis elegans , is a major recipient of serotonergic innervation  both provide serotonergic inputs to the OB  for anterograde tracing. Specifically, we expressed enhanced green fluorescent protein (EGFP) fused to synaptophysin to augment visualization of presynaptic terminals , with the start codon Met removed. Note that the expression is not required to be conditional.Animal experiments were performed in compliance with the German animal welfare law. rAAV was prepared as previously described  were anesthetized with ketamine/xyalzine  and placed in a stereotaxic frame . Lambda and bregma were leveled using an eLeVeLeR  to 12° with bregma lower and a small craniotomy was made at the following coordinates (relative to the bregma): X = 0 mm; Y = –4.525 mm; Z = –2.230 mm for DRN and X = 0 mm, Y = –4.780 mm; Z = –3.350 mm for MRN; 7.9–18.4 nl of rAAV was dispensed through a capillary glass (tip diameter 19–25 μm) using a Nanoject II  at 2.3 nl every 10 seconds.2PO4 (1.5), Na2HPO4 (8.1) with pH adjusted to 7.4. All chemicals listed were from Sigma-Aldrich  or Merck  unless otherwise stated. Fourteen days after the virus injection, animals were sacrificed and perfused transcardially with 4% formalin (pH = 8.9). The whole brain was dissected out, postfixed overnight in 4% formalin at room temperature, washed in PBS, embedded in gelatin (10%), and further fixed overnight in 4% formalin before sagittal sections 50–75 μm thick were cut using a vibratome .Solutions used for histology were in phosphate-buffered saline (PBS) that contained (in mM): NaCl (137), KCl (2.8), KHAlternate sections were treated for Nissl staining with cresyl violet or immunohistochemistry  with a ×63 glycerol immersion objective . Laser wavelengths of 405, 458, or 561 nm were used for DAPI, Alexa Fluor 488, or Cy3 signals, respectively, and recorded separately. The pictures were stitched with Leica Confocal Software . Nissl-stained sections were imaged with a brightfield microscope . All images were analyzed in Fiji . Colocalization measure of EGFP and serotonin signals was based on a method previously described . Confocal fluorescent images taken at high resolution (×63) were tiled together to reconstruct the entire coronal section. Ten samples (120 × 120 μm each) were taken from each OB layer, based only on the structures visible in the DAPI channel in order not to be biased by the locations of labeled fibers. Samples covered all mediolateral and dorsoventral positions equally, where approximately one sample was placed randomly within each 36° sector. Different sampling coordinates were used for each animal, as the shape of the OB differed between animals, ensuring that samples placed in distinct OB layers did not sample other layers. Each sample was denoised ("despeckle" function) and the background subtracted ("subtract background" function with radius set to 30 μm) in Fiji. The mean and standard deviation of pixel values from all samples were calculated for each animal . Fluorescent signal was defined to be pixel values above the global mean + 6 standard deviations. Our overall findings are not sensitive to the exact cutoff values. Signal density was defined as the number of detected pixels normalized by the total number of pixels analyzed, and calibrated by the image resolution (∼7 pixels/1 μm) to obtain the units reported . EGFP labeling was hardly present in the glomerular or the external plexiform layer (0.001 ± 0.001 μm2/100 μm2 and 0.003 ± 0.001 μm2/100 μm2 in GL and EPL, respectively, P = 0.04, one-way ANOVA across layers; Following the DRN injections, numerous brightly labeled fibers were visible in the OB C. The maThe above results suggest that the DRN does not project to the GL, raising the question as to the source of the serotonergic innervation in the glomerular layer. The MRN is a potential source, as this nucleus also contains serotonergic neurons (Steinbusch, 2/100 μm2, 73.4 ± 12.4% of all label; P < 0.01 paired t-test for significant difference from the rotated image sets, n = 3; r = 0.84, P = 0.017, n = 7 animals; Abundant labeling in the OB was observed following MRN injections. In particular, in stark contrast to the pattern observed with the DRN injections, the majority of the labeling was now in the GL (0.013 ± 0.004 μmOur localized injections in the two distinct raphe nuclei suggest that the DRN and the MRN have markedly different innervation patterns in the OB: the former projects mainly to the GCL, whereas the latter to the GL. Innervation in the EPL were relatively low in both DRN- and MRN-injected animals.The lack of labeled fibers in the GL following DRN injections is particularly notable given the previous studies that linked the DRN directly to the serotonergic innervation and neuromodulation in the GL (McLean and Shipley, 1A receptors (Pompeiano et al., EGFP signals following the MRN injections clearly colocalized with serotonin at least partially in the GL C–E, makiThe nucleus-specific innervation pattern we observe here is reminiscent of largely nonoverlapping projection patterns from the raphe to other brain areas (Steinbusch,


In Parkinson disease (PD), long‐term treatment with the dopamine precursor levodopa gradually induces involuntary “dyskinesia” movements. The neural mechanisms underlying the emergence of levodopa‐induced dyskinesias in vivo are still poorly understood. Here, we applied functional magnetic resonance imaging (fMRI) to map the emergence of peak‐of‐dose dyskinesias in patients with PD.Thirteen PD patients with dyskinesias and 13 PD patients without dyskinesias received 200mg fast‐acting oral levodopa following prolonged withdrawal from their normal dopaminergic medication. Immediately before and after levodopa intake, we performed fMRI, while patients produced a mouse click with the right or left hand or no action (No‐Go) contingent on 3 arbitrary cues. The scan was continued for 45 minutes after levodopa intake or until dyskinesias emerged.p < 0.001) as well as severity of their day‐to‐day symptomatic dyskinesias (p < 0.001).During No‐Go trials, PD patients who would later develop dyskinesias showed an abnormal gradual increase of activity in the presupplementary motor area (preSMA) and the bilateral putamen. This hyperactivity emerged during the first 20 minutes after levodopa intake. At the individual level, the excessive No‐Go activity in the predyskinesia period predicted whether an individual patient would subsequently develop dyskinesias (PD patients with dyskinesias display an immediate hypersensitivity of preSMA and putamen to levodopa, which heralds the failure of neural networks to suppress involuntary dyskinetic movements. Ann Neurol 2014;75:829–836 L‐dopa is the most effective drug for the treatment of Parkinson disease (PD).Recent studies have evidenced a substantial progress in understanding the cellular and molecular mechanisms underlying dyskinesias.A previous fMRI study has demonstrated abnormal neural activity in prefrontal areas in PD patients with dyskinesias after withdrawal of medication.We enrolled 36 patients fulfilling a clinical diagnosis of PDTen of the 36 patients were not able to undergo the MRI scan because they did not tolerate the withdrawal of dopaminergic medication (n = 8) or developed claustrophobia in the scanner (n = 2). Thirteen of the remaining 26 PD patients had clinically diagnosed choreiform peak‐of‐dose dyskinesias without off‐dyskinesias, or biphasic dyskinesias (L‐dopa–induced dyskinesia [LID] group), whereas the other 13 patients had no dyskinesias (No‐LID group). The presence and severity of dyskinesias were quantified using the Unified Dyskinesia Rating Scale (UDysRS). Dose of dopaminergic medication was higher in LID patients compared to No‐LID patients. However, the groups were closely matched with regard to disease duration and severity off and on medication. All clinical characteristics are listed in the All PD patients underwent the same experimental procedures Fig A. A firsIn each session, a motor task (see below), which lasted 9 minutes, was followed by a 5‐minute pause to avoid fatigue. A physician (D.M.H.) was always present inside the scanner room during MRI acquisition to visually observe whether dyskinesias emerged after L‐dopa intake. As soon as patients developed dyskinesias, MRI measurements were stopped.Prior to the OFF‐fMRI scans, we acquired T1‐weighted structural brain scans. We additionally recorded arterial spin labeled (ASL) MRI to rule out putative differences in cerebral perfusion underlying L‐dopa–induced changes in the blood oxygenation level–dependent (BOLD) signal measured in fMRI.During fMRI, participants continuously performed a stimulus–response mapping task see Fig B. Particp = 0.902, Levene test). Accuracy rates were compared using a nonparametric Kruskal–Wallis test, because Levene test showed that distribution of error variance was not equally distributed (p < 0.001). Between‐group differences in accuracy were assessed for the 3 conditions  and sessions. All group data are given in mean ± standard deviation (SD), and the significance threshold was set to p < 0.05 after Bonferroni correction.Reaction times (RTs) and accuracy rates were analyzed to test for between‐group differences in task performance. Using RT as dependent variable, we computed a 3 × 3 × 2 analysis of variance (ANOVA) with the factors group , run, and task laterality  after testing equality of distribution of error variance  with a 32‐channel head coil. MRI data were preprocessed and analyzed using statistical parametric mapping software .3, vessel suppression with bipolar gradients, b = 10 s/mm2, 36 slices, whole brain coverage). We used Fast ASL and BOLD Bayesian Estimation Routine (FABBER) to analyze ASL‐based perfusion measurements.19A T1‐weighted structural image of the brain was acquired using a magnetization prepared rapid acquisition gradient echo sequence . T1‐weighted images were segmented to create individual gray matter, white matter, and cerebrospinal fluid masks for ASL analysis. ASL was used to map regional cerebral perfusion before and after L‐dopa intake.Task‐related BOLD signal changes were mapped using an echo planar imaging sequence . A single fMRI volume consisted of 36 slices covering the entire brain . Preprocessing comprised realignment, normalization, and spatial smoothing , and high‐pass filtering (1/128Hz).Analysis of task‐related BOLD signal changes was performed using the general linear model. The first‐level model specified 6 regressors of interest for each session, comprising Right, Left, and No‐Go and their first‐order temporal derivative. The influence of head movement artifacts was modeled by including 24 nuisance regressors derived from realignment.21t tests to assess between‐group differences in task‐related activity at the group level. Separate t tests were computed for Right, Left, and No‐Go after withdrawal of medication (OFF‐scan) to test for differences in task‐related activation at baseline.We performed independent samples We expected a gradual change in task‐related neural activity following L‐dopa intake. Therefore, using the contrast maps reflecting the temporal derivative of each regressor , we compared L‐dopa–induced linear changes in task‐related activity in LID and No‐LID patients. Here, we included the first post–L‐dopa scan, which was available for all patients (see Results). This test enabled us to identify rapidly emerging changes in task‐related activity before the clinical manifestation of dyskinesias.p < 0.05 using the familywise error correction method. Given our a priori hypothesis that PD24All fMRI results were thresholded at a cluster‐corrected threshold of p < 0.05 after Bonferroni correction.Using fMRI data from the first post–L‐dopa scan, we performed a post hoc regression analysis to assess whether the gradually increasing neural response to L‐dopa correlated with severity of the day‐to‐day symptomatic dyskinesias as reflected by the objective UDysRS scores. In each patient, we extracted the first eigenvariate of regions, which displayed abnormal modulation by L‐dopa in the LID group . We then entered the individual values into a linear regression model with severity of dyskinesias as dependent variable using SPSS v20 . The same regression analysis was repeated using the individual Unified Parkinson Disease Rating Scale (UPDRS)‐III as dependent variable. Results were thresholded at We used the same regional eigenvariates as in the regression analysis to predict whether an individual PD patient was diagnosed with LID. We entered the parameters of each region (n = 3) and patient n = 26) as predictors in a binary classifier to see whether the parameters could classify a given patient as dyskinetic or nondyskinetic. For classification analysis, we applied a linear support vector machine  as prediAdditional post hoc analyses included voxelwise comparisons of the mean BOLD signal between groups to test whether differences in task‐related BOLD signal changes could be explained by regional reduction in mean BOLD signal, for instance due to regional iron accumulation.p = 0.009, Pearson correlation). There was no correlation between dyskinesia severity and LEDD (p > 0.1). Three of the 13 LID patients did not develop dyskinesias during the fMRI experiment, but showed mild dyskinesias after the fMRI experiment. These patients had the lowest objective UDysRS scores (6 points) in the LID group and received average levels of LEDD (mean = 883mg). None of the patients in the No‐LID group developed dyskinesias.The intake of 200mg L‐dopa provoked mild to severe peak‐of‐dose dyskinesias in 10 of 13 LID patients. Dyskinesias first developed in the foot on the side that was most affected by PD. Four LID patients developed dyskinesias after the first post–L‐dopa scan ∼20 minutes after L‐dopa intake. Patients with severe dyskinesias developed dyskinesias more rapidly than patients with mild dyskinesias , session (p = 0.204), or response condition (p = 0.222), and no significant interactions among the 3 factors. Likewise, we found no differences in accuracy rates between groups in any task or session (lowest p = 0.33).Mean RT during the task was 667 ± 72 (SD) milliseconds in the control group, 684 ± 92 milliseconds in the No‐LID group, and 673 ± 103 milliseconds in the LID group. Error rates were <4% in all 3 groups and fMRI runs regardless of response condition. There was no difference in RT between group (Zmax = 3.4), whereas the magnitude of activation in LID patients was in‐between these two groups , left putamen , and right putamen , which gradually emerged during the first 20 minutes after L‐dopa intake.The first post–L‐dopa fMRI scan yielded differences in the responsiveness to L‐dopa between LID and No‐LID patients Fig A. ComparThe increased activity was only found in the No‐Go condition, where patients had to actively suppress a motor response. By contrast, left or right button presses were not associated with any between‐group differences in changes of task‐related activity. ASL measurements did not demonstrate significant differences in regional perfusion in bilateral putamen and preSMA between the LID and No‐LID groups, indicating that the observed changes in neural activity were not induced by perfusion differences.R2 = 0.70, adjusted R2 = 0.67, β = 0.84, p < 0.001), explaining ∼70% of the variance of individual dyskinesia scores .The L‐dopa–induced linear increase of No‐Go activity in preSMA was a strong predictor of LID severity  was 74 ± 12/min and mean respiration frequency was 16 ± 4/min. Separate ANOVAs yielded no differences between groups and sessions with respect to head movements , pulse rate, or respiration frequency.Using the BOLD signal as index of regional neural activity, we show for the first time that exposure to L‐dopa leads to an excessive increase in activation of the putamen and preSMA in PD patients with peak‐of‐dose dyskinesias. Hyperactivity of the putamen and pre‐SMA in the predyskinesia period was only observed in the behavioral context of response inhibition and was highly predictive of emergence and day‐to‐day severity of dyskinesias.Within the basal ganglia, excessive activation in PD patients with dyskinesias was restricted to the bilateral putamen. This part of the basal ganglia is most strongly affected by dopaminergic denervation in PD29In the present study, putaminal hyperactivity emerged rapidly within the first 20 minutes after L‐dopa intake. This predyskinesia response indicates that abnormal dopaminergic stimulation is already present before the threshold for dyskinesias has been reached and demonstrates that excessive activity is not simply a consequence of involuntary movements, which could be the case when comparing a dyskinetic with a nondyskinetic state.12In the LID group, the putamen only became overactive in a context where subjects had to block automatic response tendencies (No‐Go). Current models postulate that the basal ganglia play a key role in motor control.At the cortical level, the preSMA was the only area showing increased responsiveness to L‐dopa in the LID group. Moreover, the L‐dopa–induced increase in No‐Go activity in preSMA, but not in putamen, predicted individual severity of dyskinesias. Interestingly, activation of preSMA has been linked to internally generated movements and the intention to act,Previous studies using transcranial magnetic stimulation (TMS) have shown that interrupting function of the caudal SMA alleviates dyskinesias, but only for a limited duration.In conclusion, we used a novel fMRI approach to assess the dynamic response of neural regions to a standard dose of fast‐acting soluble L‐dopa. We demonstrate that a rapidly emerging hypersensitivity of putamen and preSMA in the context of movement suppression can be used to predict the onset and severity of dyskinesias in individual PD patients.D.M.H., M.S.C., K.H.M., A.L., and H.R.S. designed the experiment. D.M.H. and A.L. recruited the participants. D.M.H., B.N.H., and A.L. acquired the data. D.M.H., B.N.H., K.H.M., J.B.R., and H.R.S. analyzed the data. D.M.H., B.N.H., M.S.C., K.H.M., J.B.R., A.L., and H.R.S. wrote the article.NeuroImage; royalties, Springer; speaking fees, Biogen Idec, Genzyme; travel support, MagVenture; grants, Lundbeck Foundation, John and Birthe Meyer Foundation, Fri Forskningsråd for Sundhed og Sygdom, Region Hovedsteden Forskningsfond, Fri Forskningsråd for Samfund og Erhverv Ludiomaniprogrammet.A.L.: speaking fees, Lundbeck Pharma, Medtronic; advisory board, UCB; nonfinancial support, Abbott, UCB, Medtronic. H.R.S.: advisory board, Lundbeck; editor,


Most hemodialysis patients present with chronic systemic inflammation characterized by the elevation of serum C-reactive protein (CRP) levels and/or the production of proinflammatory interleukins by the immune system in response to the hemodialysis process. Plasma ferritin(PF) is one of the parameters used to correct anemia. An PF level of >500 ng/mL is not recommended for correction of anemia because of the uncertainty of whether these levels are elevated because of anemia or a mere reaction to inflammation. we aimed to study the effects of inflammation on seminal ferritin (SF) levels and hypothesized that SF is not affected because of the testicular immune privilege.A prospective prevalence study was conducted at the Department of Hemodialysis of the University Hospital of Brasília (HuB) between June 2010 and July 2011. The sample included 60 chronic renal patients undergoing hemodialysis and 20 control subjects from the health promotion general outpatient clinic. All participants were males aged 18–60 years. Inflammation was assessed through serum CRP levels, and the testicular condition was determined by measuring sex hormone levels. In the patient group, inflammation was considered to be present when CRP was >5 mg/L (n = 27) and absent when CRP was ≤5 mg/L (n = 33). Control group (n = 20) CRP was ≤1 mg/L. Blood and semen were collected via arm venoclysis and after voluntary masturbation, respectively. CRP was measured by turbidimetry; PF, SF, and sex hormone levels by immunochemoluminescence. Statistical significance was set at p < 0.05.There was no significant difference in mean SF levels among patients with inflammation (295.34 ± 145.39 ng/mL), those without inflammation (324.42 ± 145.51 mg/mL), and controls . There was no correlation between mean SF and PF levels in the patients with and without inflammation). All participants were eugonadal with mean serum FSH, LH, and testosterone levels of 3.76 ± 2.17 mUI/mL, 7.00 ± 3.53 mUI/mL, and 454.18 ± 173.08 ng/dL, respectively.Systemic inflammation did not significantly alter SF levels in eugonadal hemodialysis patients. Ferritin (Fn) is an intracellular protein that stores iron in the atoxic form. It is present in most cells and is found in the extracellular environment as a result of cellular synthesis and secretion . In clin2, without clinical comorbidities that interfere with iron metabolism were included. All subjects were aged between 18 and 60 years and were assessed for inflammation via serum CRP measurements and for testicular function via serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) measurements. The study was approved by the Research Ethics Committee of the Faculty of Medicine of the University of Brasilia under protocol 024/2009. In the patient group, inflammation was considered to be present when CRP was >5 mg/L (n = 27) and absent when CRP was ≤5 mg/L (n = 33) according to the recommendation of National Kidney Foundation [This prospective prevalence study was conducted at the Department of Hemodialysis of the University Hospital of Brasilia (HUB) between June 2010 and July 2011. A total of 60 voluntary chronic renal male patients undergoing three weekly sessions hemodialysis for >6 months and without receive iron intravenous there at least 15 days. 20 control subjects from the health promotion general outpatient clinic of the same hospital, had normal glomerular filtration rate (GFR), this is, over 90 mls/min/1.73 mThe inclusion criteria were as follows: patients aged between 18 and 60 years, patients undergoing hemodialysis for >6 months, and the absence of acute or chronic liver disease, neoplasia and clinical/laboratory signs of acute or chronic infection/inflammation, positive serology for hepatitis B, C, and HIV, vascular access infection, leucocytosis, fever, hypoproteinemia, hypogonadism, infection of the urinary tract, orchitis and vasectomy.g for 20 min, and the supernatant was collected in a centrifuge tube and maintained at −20°C until Fn, FSH, LH, T, and CRP measurements. On the same day of blood collection, seminal plasma was collected from the ejaculate after voluntary masturbation in the proper environment at 37°C. After 30 min of liquefaction, the resultant fluid was centrifuged at 3500 × g for 20 min, and the supernatant was collected in a centrifuge tube and maintained at −20°C for the measurement of SF levels. Serum CRP levels were measured by turbidimetry in the automatic analyzer BN II . PF, SF, and sex hormone levels were measured by enzyme immunochemoluminescence using the automatic analyzer Immulite 2000/Siemens. Specific kits were used for quantification in addition to calibrators and controls recommended by the manufacturer. All statistical analyses were performed using SPSS® for Windows, version 20.0. After the analysis of sample distribution using the Shapiro–Wilk normality test, differences between three independent quantitative variables were evaluated using the Kruskal–Wallis test, while those between 2 independent quantitative variables were evaluated using the Mann–Whitney U test. Statistical significance was set at p < 0.05 to reject the null hypothesis.Blood was collected before a hemodialysis session in the patient group and on a previously scheduled day in the control group, always between 8:00 a.m. and 10:00 a.m. in the clinical laboratory of the same hospital. Five milliliters of blood were collected by arm venoclysis without an anticoagulant. The blood was centrifuged at 3500 × The mean SF levels were comparable between patients and controls . Likewise, the mean SF levels according to the serum CRP levels were as follows: CRP > 5 mg/L (295.34 ± 145.39 ng/mL) vs CRP ≤ 5 mg/L (324.42 ± 145.51 ng/mL) vs CRP ≤ 1 mg/L (335.70 ± 075.90 ng/mL) as per the Kruskal–Wallis test (p = 0.49). There was no correlation between SF and PF levels in the patients with serum CRP > 5 mg/L and those with serum CRP ≤ 5 mg/L .The sample under study comprised eugonadal patients with mean serum FSH, LH, and testosterone levels of 3.76 ± 2.17 mUI/mL, 7.00 ± 3.53 mUI/mL, and 454.18 ± 173.08 ng/dL, respectively, in the patient group.SF is found in abundance in the seminal plasma, and Sertoli cells (SCs) produce approximately 70% of its contents .SCs haveA few studies have investigated SF in humans and have focused mainly on its potential role as an antioxidant in populations with impaired fertility ,10.The effects of systemic inflammation on intratesticular Ferritin levels were investigated in an experimental study using an animal model in which Ferritin exhibited a behavior similar to that of PF, i.e., the intratesticular Ferritin levels increased in response to systemic inflammation induced by intraperitoneal injection of a lipopolysaccharide, thereby increasing the synthesis of intratesticular IL-6 .To the best of our knowledge, this was the first study to investigate this effect in humans. Our results indicated that the mean SF values Table  in the pThe difference in the reactivity of PF to various commercial kits has been reported by , who fouSystemic inflammation, according to the definition (CRP > 5 mg/L), did not cause a significant change in the mean SF levels in the patients. Moreover, there was no significant difference in the mean levels between the patients and controls Table . This reMechanisms of endocrine and paracrine  control lead to a fine control of Ferritin intratesticular levels, thus protecting germ cells from the toxic effects of excessive iron and ensuring optimal iron supplies to the various enzyme systems that produce the energy necessary for germ cell maturation .On the other hand, the small sample used in our study may have contributed to the results. This sample was primarily a consequence of the inclusion criteria, particularly the frequent presence of chronic viral infection and participants’ fear of having their intimacy exposed during the study.The lack of a linear correlation between SF and PF levels Table  in the pCompared with the use of serum IL-6 levels for the same purpose, the use of serum CRP levels for the investigation and quantification of the inflammatory condition in the population under study may be questioned by some because of the intrinsic characteristics of the protein; however, it does not invalidate our results because other comparative studies have indicated a strong correlation between serum CRP and IL-6 levels during inflammation ,23.Systemic inflammation does not cause significant changes in mean SF levels in eugonadal chronic renal patients undergoing hemodialysis, with the SF levels being similar to those observed in normal controls. To the best of our knowledge, this is the first report on this specific topic in relation to humans, and further investigations are needed to confirm the results.CRP: Reactive-C protein; CS: Célua de Sertoli; Fn: Ferritin; FSH: Follicle stimulating hormone; HuB: University Hospital of Brasília; IL: Interleucin; LH: Luteinizing hormone; PF: Plasma ferritin; SF: Seminal ferritin; T: Testosterone; TNF: Tumor necrosis factor.The authors declare that they have no competing interests.GPS drafted the manuscript. CDVE, FPC and JPRV critically reviewed it and makes addition. All authors declared the final version of manuscript.


Following prolonged perforant pathway stimulation (PPS) in rats, a seizure-free “latent period” is observed that lasts around 3 weeks. During this time, aberrant neuronal activity occurs, which has been hypothesized to contribute to the generation of an “epileptic” network. This study was designed to 1) examine the pathological network activity that occurs in the dentate gyrus during the latent period, and 2) determine whether suppressing this activity by removing the main input to the dentate gyrus could stop or prolong epileptogenesis. Immediately following PPS, continuous video-EEG monitoring was used to record spontaneous neuronal activity and detect seizures. During the latent period, low frequency oscillations (LFOs), occurring at a rate of approximately 1 Hz, were detected in the dentate gyrus of all rats that developed epilepsy. LFO incidence was apparently random, but often decreased in the hour preceding a spontaneous seizure. Bilateral transection of the perforant pathway did not impact the incidence of hippocampal LFOs, the latency to epilepsy, or hippocampal neuropathology. Our main findings are: 1) LFOs are a reliable biomarker of hippocampal epileptogenesis, and 2) removing entorhinal cortex input to the hippocampus neither reduces the occurrence of LFOs nor has a demonstrable antiepileptogenic effect. Epilepsy is a common neurological disorder that is characterized by recurrent, unprovoked seizuresAfter a potentially epileptogenic brain injury, a so-called Initial Precipitating Incident (IPI), such as a traumatic brain injury or a febrile seizure, there is often a silent or “latent” period lasting months or years, during which seizures do not occur56Although aberrant electrographic activity is a common finding in both epilepsy patients and animal models91011121415During the course of a previous experiment, we noticed that perforant pathway stimulation (PPS)-based rat models of TLE exhibit spontaneous, large-amplitude electrographic activity in the dentate gyrus during the latent period. These spontaneous events did not appear to be fast ripples (FRs)1820The present study was designed to characterize electrographic activity in the hippocampus during epileptogenesis and test our hypothesis. Immediately following 8 h PPSContinuous EEG recordings obtained from dentate granule cell layer revealed spontaneous events that began immediately following PPS  and persHere we tested the hypothesis that removing entorhinal cortex input to the hippocampus alters epileptogenesis either by prolonging the latent period, i.e. duration to first spontaneous seizure, or reducing the severity of epilepsy, e.g. seizure duration. Continuous video-EEG monitoring revealed that bilateral PPT did not significantly alter the time between 8 h PPS and the first spontaneous seizure . The aveContinuous monitoring of electrographic activity, with recording electrodes positioned in the hippocampal granule cell layer, was used to detect LFOs. LFO frequency was not significantly different in animals that received PPT (p > 0.05). An average of 14332.8 ± 5535.9 and 15045.2 ± 6796.7 LFOs were detected per day in Sham PPT and PPT groups, respectively .In order to determine whether PPT, directly following pro-epileptogenic PPS, affects hippocampal atrophy, a characteristic of this animal model, we measured and compared hippocampal area in 21 rats that received 8 h of PPS with and without PPT.2 + 1.39 mm2 for Sham PPT (n = 8) and 3.20 mm2 + 1.09 mm2 for PPT rats (n = 13).Hippocampal area was essentially the same for both groups (p > 0.05), thereby demonstrating no effect of PPT on the development of hippocampal sclerosis. Mean values were 3.59 mmTo quantify the extent of neuron loss after 8 h perforant pathway stimulation, we analyzed ventral hippocampus sections obtained from untreated control, Sham PPT, and PPT groups (n = 5 per group) that were immunostained for NeuN22There are two principal, original findings in this study. First, that LFOs recorded from the dentate gyrus are a reliable biomarker of hippocampal epileptogenesis. Such events began immediately after 8 h PPS, occurred with seemingly no predictability, e.g. frequency, duration, time of day , were occasionally accompanied by population spikes, and persisted into chronic epilepsy. LFOs occurred at a rate of around one per second with a frequency of 13–17 Hz and a peak intensity of 15 Hz . To the et al. found up to 2,000 such spikes/day in control animalsThe LFOs described in the present manuscript have not, to the best of our knowledge, been described in previous studies. The closest thing to LFOs was presented by White and colleaguesLFOs are similar to fast ripples (FRs), a high-frequency phenomenon described by Bragin and colleaguesOur second main finding is that removing entorhinal cortex input to the hippocampus after a pro-epileptic insult is not antiepileptogenic. This was done in an attempt to silence the aforementioned aberrant electrographic activity, i.e. LFOs, that we hypothesized to be epileptogenic. Transection of the perforant pathway, thereby interrupting the main fiber tract connecting the entorhinal cortex to hippocampus, did not prolong the latency to the first spontaneous seizure. Nor did PPT affect either seizure semiology or duration 3234In summary, 1) LFOs recorded from the dentate gyrus are a reliable biomarker of hippocampal epileptogenesis and 2) transecting the perforant pathway immediately after an epilepsy-inducing injury did not affect epileptogenesis in any of the measured electrophysiological or histological parameters. Therefore, we conclude that the direct EC-DG connection has a limited causal role in epileptogenesis and it is unlikely that a kindling effect, at least one induced by aberrant input from the EC, is important for either epileptogenesis or the development of epilepsy-associated neuroanatomical changes, e.g. hippocampal sclerosis.84 Male Sprague–Dawley rats  were treated in accordance with the European Communities Council Directive (2010/63/EU). All experiments were approved by the local regulation authority (Regierungspräsidium Gieβen). Animals were housed in an on-site animal facility  under a 12:12 light/dark cycle and with ad libitum access to food and water.Under ketamine (120 mg/kg i.p.) and xylazine (5 mg/kg i.p.) anesthesia, bipolar stainless-steel stimulating electrodes  were placed in the angular bundles of the perforant pathway and custom unipolar recording electrodes  were lowered into each dorsal dentate gyrus. Electrode locations were determined by optimizing the potentials evoked by low-frequency PPS. Dental cement fixed the electrodes to anchor screws and the skull. Plastic connectors  joined the electrodes with stimulation/recording equipment.All stimulation protocols utilized a paradigm designed to evoke and maintain hippocampal seizure activity throughout the stimulation, but not convulsive status epilepticus42424As described previously, stimulation for 30 minutes (on two consecutive days) required only isoflurane to terminate seizuresEEG activity during and after PPS was amplified and recorded digitally at 10 kHz utilizing a PowerLab 16/35 and LabChart software versions 7 and 8 . Spontaneous granule cell layer activity was recorded continuously (24 h/d) and stored digitally and automatically in 3-hour epochs. Each day, the preceding 24 h of recordings were analyzed and all events with amplitudes exceeding 120% of average baseline (free of LFOs and seizures) were analyzed. Potential seizures were related to behavior on the time-stamped video recordings. Continuous (24/7) video monitoring utilized AirCam OD-325HD infrared cameras . Video files were captured at 15 frames/sec and time-stamped for integration with the electrophysiological data using SecuritySpy surveillance software  and stored digitally. Confirmed seizures were scored according to the Racine scale2/Hz) of frequencies ranging from 0–50 Hz was plotted.As mentioned above, all EEG events with amplitude that exceeded threshold (120% of baseline amplitude) were analyzed. This was confirmed to include all spontaneous seizures as well as other events, such as LFOs. The Spike Histogram feature in LabChart v7 was used to quantify LFOs and measure their amplitude. The Spectrum feature, built-in to LabChart v8, generated Power Spectrum Density Plots using Welch’s method. Briefly, a Fast Fourier Transform size of 32 K (32768) was applied to the 10 kHz signal with a window overlap of 50% and the Power  and xylazine (5 mg/kg i.p.) and placed in a stereotaxic apparatus. Electrodes were removed and a crescent blade microknife  was used to cut in the frontal plane between the entorhinal area and the hippocampus , which resulted in the destruction of the entorhinal afferents to the fascia dentata. The following coordinates measured from the interaural line were used with the knife angled backward against the frontal plane by 10°: AP 0; L 3 to 7; V down to the base of the skullAt least two months after stimulation, in order to allow for completion of macroscopic anatomical changes, e.g. hippocampal sclerosis, rats were sacrificed in order to evaluate hippocampal neuropathology. Animals were deeply anesthetized with ketamine (200 mg/kg i.p.) and xylazine (5 mg/kg i.p.) and perfused through the aorta with 0.9% saline for 2 min followed by 10 min of 4% paraformaldehyde . Brains were immediately removed from the skull and placed in 4% paraformaldehyde. Following post-fixation at 4° for at least 48 h, 30-μm-thick transverse sections were cut on a freezing microtome. Free-floating sections were incubated overnight in primary mouse anti-NeuN  at room temperature. On the following day, sections were incubated in biotinylated secondary antibody solution  for 2 h, washed twice in PBS, and then incubated for 2 h in avidin–biotin–HRP (horseradish peroxidase) complex  at room temperature. Finally, sections were washed three times in PBS and developed in a solution containing hydrogen peroxide and diaminobenzidine.Images of Nissl-stained sections were acquired with an Axio Imager M2 microscope . Equally spaced (every 5th section throughout the entire hippocampus), transverse sections were analyzed at 1.6× magnification. The area of the hippocampus was calculated for each section with Adobe Photoshop CS5 To estimate the extent of neuron loss after 8 h PPS, we analyzed ventral hippocampus sections obtained from untreated control, Sham PPT, and PPT animals that were immunostained for NeuN22Statistical analyses were performed using BIAS version 10.12 . Statistical disparity between groups was tested using the Wilcoxon-Mann-Whitney-Test. Histograms and calculations were created with Excel for Mac 2011 . All data are presented as mean ± standard deviation.How to cite this article: Meyer, M. et al. Removing entorhinal cortex input to the dentate gyrus does not impede low frequency oscillations, an EEG-biomarker of hippocampal epileptogenesis. Sci. Rep.6, 25660; doi: 10.1038/srep25660 (2016).


The prevalence and burden of atherosclerotic (AS) diseases are increasing during the last twenty years. Some studies show a close relationship between overweight and AS, but influence on AS diseases of different weight loss methods are still studying. The purpose of the research was to study the effectiveness of a weight loss program in AS patients in randomized controlled trial, and to develop a conception of evolution of AS.A randomized controlled prospective clinical trial including 97 people, from them 71 patients with various AS manifestations. Patients were divided in 2 subgroups for non-drug weight loss program, and conventional drug therapy. The weight loss program included calorie restriction with 100–150 kcal/day, fat-free vegetables, salt diet, and optimum physical activity. Statistical analysis was performed using SPSS for Windows version 17.0.P = 0.016). Weight loss was achieved due to fatty mass reduction only (P = 0.005). Hemoglobin levels (P < 0.001), bone mineral density (P < 0.001), percentages of water (P = 0.006) and muscle masses (P = 0.0038) were increased in weight loss subgroup. Ejection fraction (P < 0.0001), systolic output (P < 0.0001) were increased in patients with coronary artery disease. The weight loss program led to a decrease in symptomatic drugs doses up to total abolition. A conception of AS was developed.The weight loss subgroup lost ranging between 7-20 % from an initial weight (The weight loss program treated the AS diseases; improved laboratory and instrumental parameters, decreased symptomatic drugs doses. AS development is a logical way of ontogenetic ageing of body fat.NCT01700075.ClinicalTrials.gov State registration is # 0109RK000079, code is O.0475 at the National Center for Scientific and Technical Information of the Republic of Kazakhstan. The prevalence and disease burden of Atherosclerosis (AS) are increasing. During the last twenty years morbidity and mortality form AS is globally increasing . AS is aMany "Civilization Diseases" such as metabolic syndrome, coronary artery disease (CAD), hypertension, diabetes mellitus (DM), allergic diseases, non-alcoholic fatty liver disease, psoriasis, gout, etc., have similar metabolic disorders such as hyperlipidemia/dyslipidemia, hyperglycemia, hyperinsulinemia, hypercortisolemia, hyperuricemia, microalbuminuria, which can be signs of AS , 5. In tHowever, none of them has been universally recognized and proved in practice up to date . Last stOpen randomized controlled prospective clinical trial with subsequent development of a conception of evolution of AS based on systematic review of scientific databases.2 (patient group), and 26 healthy volunteers  aged 48.5 ± 3.7 years .We enrolled a total of 97 adult people of Asian and European ethnicities, from them 71 were patients  with various clinical manifestation of AS aged 46.5 ± 2.3 years with body mass index (BMI) 29.7 ± 0.8 kg/m2) with various AS diseases were randomly recruited for the weight loss program . The weight loss patient subgroup included next types of AS diseases: Leriche disease  in 4 patients, CAD with family history more than 10 years was present in 12 patients, CAD with postinfarction cardiosclerosis (PICS) in 6 patients, cerebral stroke in 7 patients, and Alzheimer's disease in 2 patients. All the 31 patients had hypertension, from them 19 patients had DM and the rest 12 patients had an impaired glucose tolerance. All enrolled AS patients had abdominal obesity and before weight loss they took a conventional drug therapy.The patient group was divided in two subgroups. The weight loss patient subgroup consisted from 31 patients  who were receiving a conventional drug therapy including hypoglycemic , lipid lowering (atorvastatin 40 mg per day), antihypertensive , anti-inflammatory , and symptomatic therapy.The second subgroup for comparison included 40 patients with AS  and at Republic scientific center for emergency medicine at National medical holding .Inclusion criteria: written informed consent form for participation in the study; dyslipidemia ; body fat% > 21; waist circumference in male > 94.0 cm or in female > 80.0 cm; blood pressure (BP) > 140 mmHg of systolic blood pressure (SBP) and > 95 mmHg of diastolic blood pressure (DBP), or ongoing treatment with antihypertensive drugs, fasting glucose > 6.1 mmol/l or treatment with glucose-reducing drugs; absence of contraindications to weight loss; the possibility of treatment for 6 months and dynamic observation for 1 year.The primary efficacy endpoint of the study was the complete recovery from AS diseases. The secondary efficacy endpoint of the study was data imaging methods  and measurement of clinical status presence.P = 0.199) and baseline BMI .An independent statistician unconnected with clinical practice used computer generated random numbers  to prepare randomization lists. The block randomization was two  with stratification by sex, age . The weight loss intervention study period was between 2–6 months duration depending on individual patient clinical situation, severity and stages of disease. Physical activity was assessed as the number of steps taken by patients, as determined by the individual pedometer from Hoffmann-La Roche, Ltd . Mental status was defined by the test of numbers binding by Reitano .2). We also evaluated body composition parameters including as fat mass , visceral fat rating (units), fat free mass (kg), total body water (in % and kg), muscle mass (in % and kg), bone mass (in % and kg), metabolic age (years), basal metabolic rate , and bioimpedance (Ohms) by using Tanita-SC330S Body Composition Analyzer . General clinical study of blood and urine chemistry, liver and kidneys function tests, and imaging methods , bone densitometry , and computed tomography scans  were performed.We defined anthropometrical indicators including age (years), weight (kg), BMI , and optimum physical activity , 15. TheEthical Committee of the Scientific research institute of cardiology and internal diseases  approved the study. Approval Number is in the protocol #9 from 06.02.2009. Board Affiliation: Health Ministry of the Republic of Kazakhstan.t-test and Odds ratios (ORs) with 95 % Confidence intervals (CIs) were used. The study data were tested on the normal distribution. The study data are presented in Tables as Mean ± Standard Error of the Mean (M ± SE). The correlation analysis (r) and multinomial logistic regression model ORs with CIs were used. P-values of < 0.05 was set as significant. Statistical analysis was performed using SPSS for Windows version 17.0  and Microsoft Excel-2013. Databases of WEB OF SCIENCE, SCOPUS, PUDMED, MEDSCAPE, GOOGLE SCOLAR were used for systematic review analysis of scientific data for working out of development conception of AS.The two-sample Student’s We compared the patients and control  groups concerning metabolic age, basal metabolic rate, anthropometrical data, and body composition Table .Table 1A2 compared with the control group. Fat mass was a significantly higher in the patient group compared with the control group for ≈ 12.1 % or ≈ 10.0 kg. Table P = 0.0023).As seen in Table P = 0.0004), Muscle mass +11.5 % (P < 0.0001), and bone mass +0.3 % (P = 0.04) than the patient group. The regression linear analysis strongly inverse correlated the relationships between fat mass and muscle/water/bone masses in the patient group (n = 71) , metabolic age , basal metabolic rate , and bioimpedance  than the control group have. Increased body fat mass is associated with accordingly increased bioimpedance of the body.The patient group also have significantly raised visceral fat rating . This data analysis can provide evidence that Fat mass could be the main risk factor for AS patients.As seen in Fig. n = 71) .We studied the regression correlation between the level of obesity  and the metabolic age in the patient group (71) Fig. . As seenn = 31) who were randomly assigned into this category. As a result of the treatment average weight lost varied from 6 to 18 kg (or 7-20 %). No differences in weight loss were found between patients with Asian and European ethnicities. The weight loss led to positive changes of the cardiovascular diseases symptoms: SBP and DBP decreased in 94.4 % of patients (P < 0.001) for more than 19 % from the initial status (Table We began the weight loss program in the patient group ( kg or 7-0 %. No dP < 0.001). However, the conventional treatment in AS patients did not lead to significant improvement in DBP (P = 0.29), Blood hemoglobin levels (P = 0.28), and Bone mineral density (P = 0.4). Noteworthy, in the weight loss subgroup we observed more significant declines in SBP, DBP, glucose, cholesterol, and triglyceride levels (P < 0.001). There were a significant increase in Hemoglobin levels (P < 0.001) and Bone mineral density (P < 0.001).Table P = 0.016). Importantly, all of the 31 patients weight lost due to reduction of fat mass only . The percentages of total body water and muscle masses had also significant tendency to increase in the study endpoint . Lean body mass did not significantly change with fat mass loss (P = 0.36).The data shown in the Table During the first 2–3 days of the treatment the most of the patients complained of an intense feeling of hunger, slight dizziness, weakness, lower extremity and abdominal muscle tremor, a feeling of warm in the umbilical and/or solar plexus area, and psychogenic fear due to changed eating behavior. All of these uncomfortable feelings were disappearing on subsequent days.On the 3–5 day after start of the weight loss program the urine of the patients was getting more turbid, muddy and intensively colored (dark) which were not marked before. The urine symptoms were persisted for several days. Microscopy of the urine revealed the turbidity and muddiness were due to the organic salts mainly, such as oxalates, urates, phosphates, and carbonates of calcium and magnesium. An increase in erythrocyte sedimentation rate, leukocyte count in blood samples, and body temperature were observed between 4–10 days after start of the weight loss program.P = 0.034), improved an exercise tolerance, improved objective electrocardiography indicators of cardiovascular function, increased in ejection fraction from 56.3 ± 1.1 % to 72.1 ± 1.3 % (P < 0.0001), and increased in systolic output from 65.4 ± 1.8 ml to 89.6 ± 1.7 ml (P < 0.0001). All patients with cerebral stroke and with Alzheimer's disease noticed a decrease in mental fatigue and improvement of memory .Regression of AS symptoms were also gradually observed in the weight loss patient subgroup. The patients noticed a physical relief, and an increase in physical and mental workability. Doppler-ultrasound imaging data revealed that blood flow in the lower extremities was restored and the affected gastrocnemius muscle sizes were also restored in all of the 4 patients with the Leriche disease. In CAD patients including with PICS disappeared the angina pectoris (1.52, 95 % CI: 1.24–1.81; As clinical symptoms were improved, as the doses of previous symptomatic drugs were adequately decreased by the 2–3 day after the treatment started. By the 7–10 day after the beginning of the treatment the drugs were abolished. The observational period for the patients was up to 1 year, and there was no recurrence of clinical symptoms of the AS diseases. The weight usually was not regained during the time period. If a person regained overweight again, the clinical symptoms of AS diseases have been gradually manifesting. However, these clinical symptoms were reversed if the weight has been lost again.Obesity/Overweight is a global public health problem. Overweight is widely acknowledged to be a risk factor for a wide spectrum of cardiovascular, metabolic, neoplastic and musculoskeletal disorders and is estimated to reduce life expectancy by as much as 10 years . The impOne of the first theories of AS development was gerontological, and many researchers consider AS of vessels as one of the determining factors of the ageing process , 11. TheHyperinsulinemia , dyslipiBays H. calls the unused adipose tissue as Adiposopathy or "sick fat" which contributes to the emergence of general metabolic disorders and CVD accidents . AdiposoDue to “super-nutrient” meal regime a contemporary human begins to accumulate more fat than he is able to use it . PermaneThe more overweight the more metabolic burden for the body’s organs and systems is , 29. OveOur weight loss program is directed to metabolize of “old lipids” , 15. WeiGender and race can influence for intention to lose weight , but ourIn last decade the importance role of adipose tissue in storing of organic pollutants was considered and highlighted . AdiposeHypertension, hyperthermia, insulin resistance, inflammation, allergy can be directed on the maintenance of metabolic rate in the body of patients with overweight/ obesity , 40. PerAbility to accumulate of adipose tissue is one of the most important adaptive mechanism for survive. However, nowadays, we are eyewitnesses of another side of the survival mechanism when obesity-related diseases increase steadily . "Since As represented in the Fig. Published studies about possible role of overweight in cause of AS are limited in scope and number. We acknowledge that the randomized clinical trial had a small sample size, and data of comparative or control groups are not completely shown. The clinical results were validated by our usual scientific methods. We should perform the prospective randomized controlled clinical trial with a numbers of patients for more statistical power.Thus, the more fat mass is in the body the less muscle, bone, and water masses are. Increased fat mass leads to the increase of bioimpedance and metabolic age. We have developed the weight loss program including a caloric restriction, fat-free vegetables and salt diet with optimum physical activity is effectual treatment method of the AS diseases. The physical activity model was important part for cure of endogen metabolic intoxication of the weight loss program. The program leads to positive change of disease symptoms, improvement in laboratory and instrumental parameters, and competent reduction of previous symptomatic drugs doses up to total abolition. The main conclusion of the study is AS development is a logical output of ontogenetic ageing of body fat. The “ageing lipids” could be used during the weight loss program. The conception of evolution of AS is developed.


Pulmonary hypertension (PH) is frequently associated with an increase in sympathetic tone. This may adversely affect cardiac autonomic control. Knowledge about the clinical impact of autonomic dysfunction in patients with PH is limited. We aimed to assess whether parameters of heart rate variability (HRV) are related to disease severity in children with PH.n = 11), patients with severe PH (PAP/SAP ratio > 0.75) (n = 6). An additional group of five adolescents with Eisenmenger syndrome (PAP/SAP ratio 1.13 ± 0.36) was included.Parameters of HRV  were determined from Holter electrocardiograms of 17 patients with PH without active intracardial shunt . Patients were allocated to two groups according to their disease severity: patients with moderate PH [ratio of pulmonary to systemic arterial pressure (PAP/SAP ratio) < 0.75] . SDNN inversely correlated with ratio of PAP/SAP of PH patients without shunt . Eisenmenger patients showed no significant difference of HRV  compared to patients with moderate PH without shunt .Children with severe PH had significantly lower values of HRV  compared to patients with moderate PH (According to our results, children with severe PH may have alterations in HRV. Since HRV appears to be related to disease severity, it may therefore serve as an additional diagnostic marker of PH. Remarkably, although Eisenmenger patients have systemic pulmonary arterial pressures, they seem to have preserved HRV, which might reflect a more favorable autonomic adaptation. Pulmonary hypertension (PH) is characterized by progressive pulmonary vascular remodeling and consecutive elevation of pulmonary vascular resistance and pressure, which may ultimately result in right ventricular failure and death. Chronic right heart failure is associated with an increase in sympathetic tone, which may adversely affect cardiac autonomic control , 2. LittSince PAH is a progressive disease and shows a high mortality, despite new developments in the treatment of PAH in children , markersThe aim of our study was therefore to assess whether sympathetic parameters of HRV are related to disease severity in children with PH.Seventeen patients with PH  were included in this prospective study after written informed consent was obtained. Twelve of the 17 patients had idiopathic PAH, 3 patients had associated chronic lung disease, 2 had “out of proportion” PH due to left heart disease . An additional group of five adolescents with Eisenmenger syndrome was studied. A cohort of 10 healthy children, adolescents, and young adults  served as a control group.®CM 3000, GETEMED, Teltow, Germany) was performed for 48 h during daily activities. None of the patients had a history of palpitations or syncope. Data were analyzed by an operator-controlled analysis software package . Parameters of HRV  were determined from the recordings.Holter-ECG recording (CardioMem2), cardiac index (CI) (using Fick method), and pulmonary vascular resistance index (PVRI). Analysis of invasive hemodynamic data has been performed retrospectively, time interval between cardiac catheterization and Holter-ECG was 18 ± 14 months. Cardiac catheterization was performed free-breathing under sedation with midazolam and propofol.The clinical data obtained included WHO functional class, echocardiographic assessment of tricuspid annular plane systolic excursion (TAPSE), and right atrial area indexed for body surface area, and at cardiac catheterization, right atrial pressure (RAP), mean pulmonary arterial pressure (mPAP), ratio of mPAP to mean systemic arterial pressure (mSAP), pulmonary capillary wedge pressure (PCWP), mixed venous oxygen saturation  and severe PH (mPAP/mSAP ratio >0.75) were analyzed separately. The study was approved by the local Ethics Committee.t-test and non-parametric Mann–Whitney U-test as appropriate. Correlations were tested using linear regression analysis. Analysis was performed using GraphPad statistical software package . A p value ≤0.05 was considered statistically significant.The data are presented as mean and SDs. Differences between groups were examined for significance by the use of unpaired n = 11) and (group 2) patients with severe PH (PAP/SAP ratio >0.75) (n = 6). There were no significant differences in age, time interval since diagnosis, height, weight, and body surface area between both groups  patients with moderate PH (mPAP/mSAP ratio <0.75) ( patientsZ-score < −2) in three patients in group 1 (27%) and five patients in group 2 (83%), resulting in significantly worse TAPSE Z-score in patients with severe PH   compared to 7.1 ± 3.0 mmHg in group 1, which can be explained by the fact that both patients with “out of proportion” PH due to left heart disease were in group 2. CI and mixed venous sO2 was lower in group 2, however, did not reach statistical significance  and was not significantly different between both subgroups. Mean PAP for all children was 48.4 ± 17.3 mmHg, mPAP/mSAP 0.62 ± 0.26, and PVRI 14.0 ± 7.9 WUxm.9 mmHg ap = 0.004). Mean HR was also higher in patients of group 2 compared to group 1, but did not reach statistical significance. In contrast, maximum HR tended to be lower in patients of group 2 .Minimal heart rate (HR) was significantly higher in children with severe PH (75.7 ± 12.2 bpm) compared to children with moderate PH (56.5 ± 10.3 bpm)  were significantly lower in patients with severe PH compared to healthy controls Table .Supraventricular premature beats were recorded in five patients of group 1 and two patients of group 2, whereas only two patients had more than three premature beats per hour. There was no occurrence of atrial tachycardia or atrial flutter. Ventricular premature beats were recorded in four patients of group 1 and one of group 2, whereas only one patient had more than three premature beats per hour. There was no occurrence of higher degreed ventricular arrhythmias. No relation between the occurrence of arrhythmias and disease severity could be seen.2, and CI , the ratio of PAP/SAP , PVRI , and PCWP   and indexed right atrial area was 11.01 ± 3.38 cm2/m2. RAP was 9.0 ± 5.7 mmHg, mPAP 91.3 ± 30.6 mmHg, ratio of mPAP/mSAP was 1.13 ± 0.36 and PVRI 33.8 ± 15.9 WUxm2.Patients with Eisenmenger syndrome were significantly older (25.1 ± 5.2 years) compared to patients of group 1 and 2. All of them were in WHO functional class II and III and all of them were on PAH specific medications. Right ventricular function was preserved   (All of the patients with Eisenmenger syndrome had ventricular premature beats [four of them (80%) more than 3/h]. About 40% of the patients had higher degreed ventricular arrhythmias, one had couplets of premature ventricular beats, and another patient had non-sustained ventricular tachycardia. One patient had frequent supraventricular premature beats and a non-sustained supraventricular tachycardia.The results of our presented study showed for the first time that there is a strong relation between HRV and disease severity in children with PH. Except for patients with the Eisenmenger syndrome, HRV correlated with mPAP, mPAP/mSAP, PVRI, and PCWP. HRV may therefore serve as an additional marker of disease severity in children with PH without intracardiac shunt to identify patients at risk, to detect early clinical worsening, and to guide medical therapy.There are different mechanisms discussed in the literature, which may contribute to impaired HRV in patients with PH. Stretching of the sinoatrial node may result in a reduction of HRV . ChildrePrevious authors were able to demonstrate an impact of impaired left ventricular function on HRV  in patieRemarkably, minimal heart rate was significantly higher in children with severe PH compared to children with moderate PH, and also mean heart rate tended to be higher, which might be a physiological response to achieve an adequate cardiac output despite of imminent RV failure. However, those patients with more accelerated heart rate may show a relevant shortening of diastole, and thus, impaired ventricular filling during diastole, which may result in progressive RV failure. β-Blockers may be a reasonable treatment option of this condition. β-Blockers can improve ventricular filling by decreasing heart rate and extending the duration of diastole. Concern about the use of β-blockers in PH was formerly based primarily on their potential to produce negative inotropic effects , 14. In The prevalence of serious supraventricular or ventricular arrhythmias in our PH-patients was low, and there was no relation of arrhythmias to disease severity. Previous studies on arrhythmias in patients with PH were focused on adult patients, and primarily revealed supraventricular arrhythmias  often leading to clinical deterioration –18. AccoOf particular interest are also the results of our adolescent patients with Eisenmenger syndrome. Eisenmenger patients are known for their more physiological adaptation to right ventricular afterload . As theyThe small number of patients studied and the heterogeneity of the underlying etiology of PH may limit the interpretation of the data. Although there was a longer time interval between the HRV measurement and the invasive assessment of hemodynamics, the patients’ clinical condition remained stable and medical treatment unchanged, suggesting that the severity of PH did not change significantly during that time. While digoxin and beta blockers are known to alter heart rate and myocardial inotropy, which may affect the results of the HRV measurement, only three patients in this study received such medication.According to our results, children with severe PH may have alterations in HRV. Since HRV appears to be related to disease severity, it may therefore serve as an additional clinical marker of disease in PH. Remarkably, patients with the Eisenmenger syndrome seem to have preserved HRV despite of systemic PA pressures, which might reflect a more favorable autonomic adaptation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Failure to meet an adequate dietary intake is involved in the pathogenesis of sarcopenia and osteoporosis, which in turn increase the risk for falls and fractures, respectively. Older people with hip fracture are often protein-malnourished at hospitalization. Whether low protein-energy intake is associated with muscle atrophy in hip-fractured patients is presently unknown. This information is necessary for the development of novel strategies to manage this especially vulnerable patient population. The aim of this study was, therefore, to explore the relationship between dietary intake and muscle mass in older hip-fractured patients.Methods: Analyses were conducted in hip-fractured elderly admitted to an orthopedic and trauma surgery ward . Muscle mass was estimated by bioelectrical impedance analysis within 24 h from admission. Dietary information was collected via 24-h dietary recall and nutrient intake calculated by a nutrition software.Results: Among 62 hip-fractured patients , the average energy intake was 929.2 ± 170.3 Kcal day−1, with higher values reported by men  relative to women . Absolute and normalized protein intake was 50.0 ± 13.5 g day−1 and 0.88 ± 0.27 g kg (body weight)–1 day–1, respectively, with no gender differences. A positive correlation was determined between total energy intake and muscle mass . Similarly, protein and leucine consumption was positively correlated with muscle mass .Conclusion: A low intake of calories, protein, and leucine is associated with reduced muscle mass in hip-fractured elderly. Given the relevance of sarcopenia as a risk factor for adverse outcomes in this patient population, our findings highlight the importance of a comprehensive dietary assessment for the detection of nutritional deficits predisposing to or aggravating muscle atrophy. Hip fracture is a devastating event for elderly people, with over 25% per-year mortality and incomplete recovery of pre-fractural conditions in more than 50% of survivors  emerges as serious candidate for interventions. Indeed, declines in muscle mass and strength are associated with poor functional recovery following hip fracture repair  of the Teaching Hospital “Agostino Gemelli,” Catholic University of the Sacred Heart . Exclusion criteria were age < 65 years, presence of peripheral edema, bone metastasis, cognitive impairment , presence of pacemaker or implantable cardioverter defibrillator, and unwillingness to take part to the study. The study was approved by the Institutional Review Board of the Catholic University of the Sacred Heart, and all participants signed a written consent before enrollment.Information pertaining to demographic, clinical, functional, and lifestyle characteristics were collected by attending physicians upon admission to the Orthopedic and Trauma Surgery ward using the interRAI Acute Care instrument .Whole-body fat-free mass was measured by bioelectrical impedance analysis (BIA) using a Quantum/S Bioelectrical Body Composition Analyzer  with an operating frequency of 50 kHz at 800 μA, as previously described .All data are expressed as proportions (%) or mean ± standard deviation (SD). Differences between continuous variables were assessed by ANOVA comparisons or the Kruskal–Wallis test, as appropriate. Distributions of categorical variables were compared by the Fisher exact test. The Pearson’s correlation test was used to assess the strength of association between variables. All tests were two sided, with significance set at −2 and 8.83 ± 1.73 kg m−2, respectively), but the difference did not reach the statistical significance (p = 0.08) –1 day–1, with no differences between genders , albeit slightly above the RDA for protein, may not still be sufficient to sustain optimal muscle protein synthesis. Notably, over 75% of participants did not reach the protein intake currently recommended for older adults (1.0 g kg–1 day–1), and only 8% reported a protein consumption between 1.2 and 1.5 g kg–1 day–1, which is the amount recommended by the Society for Sarcopenia, Cachexia, and Wasting Disease to maximize muscle health in advanced age  improves the clinical outcomes of older hip-fractured patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Artemisia afra which was collected from its natural habitat in Bale Zone, around Goba town, 455 km southeast of Addis Ababa. Air dried aerial parts of Artemisia afra were separately extracted with both distilled water and 95% methanol. Oral acute toxicity test was conducted on healthy Swiss albino mice. Antidiabetic effect of the aqueous and methanolic extracts of Artemisia afra was separately evaluated on alloxan induced diabetic mice at doses of 500, 750, and 1000 mg/Kg body weight orally. The results indicate that mean lethal dose (LD50) for aqueous extract of Artemisia afra was 9833.4 mg/Kg. Blood glucose level was significantly decreased by 24% (p < 0.005) and 56.9% (p < 0.0004) in groups that received aqueous extract of Artemisia afra at dose of 500 mg/Kg and 750 mg/Kg, respectively. The methanolic extract of Artemisia afra also significantly lowered blood glucose by 49.8% (p < 0.0001) at doses of 1000 mg/kg on the 5th hr. Aqueous extract of Artemisia afra was regarded as nontoxic and safe since its LD50 was found above 5000 mg/Kg. Aqueous extract showed higher effect at relatively lower dose as compared to methanolic extract. The aqueous extract was screened positive for phytochemicals like flavonoids, polyphenols, and tannins that were reported to have antioxidant activity.Diabetes mellitus is metabolic syndrome that causes disability, early death, and many other complications. Currently insulin and many synthetic drugs are used in diabetes treatment. However, these pharmaceutical drugs are too expensive particularly for sub-Saharan population in addition to their undesirable side effects. The present study was aimed to evaluate antidiabetic effect and toxicity level of  There is a global increase in the prevalence of diabetes mellitus predominantly, related to life styles and the resulting surge in obesity . Diabeteα-glycosidase inhibitors. Many of these oral antidiabetic agents have a number of serious adverse effects. Thus, the management of diabetes without any side effects is still a challenge [Management of diabetes without any side effect is still a challenge to the medical community. The use of the drugs is restricted by their pharmacokinetic properties, secondary failure rates, and accompanying side effects . Despitehallenge . Therefohallenge .Artemisia afra, is locally called “chikugn” also known as African wormwood. It belongs to family Asteraceae. It is one of the best known and widely used plants in traditional medicine. Various preparations such as infusions, decoctions, molasses, and alcohol extracts of Artemisia afra are used for the treatment of coughs, colds, chills, stomachache, and dry dyspepsia and as a purgative and for the cure of smallpox and malaria. The herbaceous leaves of this plant contain between 0.3% and 1.4% v/w of a bluish-green essential oil, with a strong fragrance [ Artemisia afra in comparison with each other and standard hypoglycemic agents like glibenclamide.ragrance . This st Artemisia afra were collected during their flowering period when plants give highest yield [Fresh aerial parts ofst yield . The pla Artemisia afra were cleaned with distilled water and air dried at room temperature. It was ground into powder using grinding mill. Powdered aerial part of Artemisia afra weighing 220 gm was sorely mixed with 450 mL distilled water in flat bottom flask on orbital shaker for 24 hours. Distilled water was added until completely wet and the dried plant powder was sufficiently covered. After gentle maceration for 24 hours plant materials were filtered separately through mousseline cloth into 1000 mL beakers. The obtained filtrate was deep frozen at −70°C and lyophilized (freeze dried) using lyophilizer to collect crude aqueous extract [ Artemisia afra provided yield of 21 gm (9.5% w/w).Fresh aerial parts of Artemisia afra were cleaned with distilled water and air dried at room temperature. A dried and powdered aerial part of Artemisia afra weighing 165 gm was sorely mixed with 400 mL of 95% methanol in flat bottom flask on orbital shaker. After gentle maceration for 24 hours methanolic extracts were filtered through Whatman filter paper chart. The filtrate from Artemisia afra was concentrated under reduced pressure using rotary evaporator at 50 rpm and 40°C bath temperature. Finally concentrated methanolic extract was collected in vials and placed on water bath at 40°C to evaporate methanol completely. The crude methanolic extract of Artemisia afra had a yield of 13 gm (7.8% w/w).Fresh aerial parts ofDiabetic condition was induced to Swiss albino mice of both sexes, weighing 25–35 g after overnight fasting, by intraperitoneal injection of freshly prepared alloxan  dissolved in distilled water . Three d ad libitum with 12-hour light/dark photoperiod. The experiment was conducted according to the ethical norms approved by Animal Ethics Committee Guidelines of the EHNRI.Healthy Swiss albino mice weighing 25 g–35 g were obtained from Ethiopian Health and Nutrition Research Institute (EHNRI) animal unit and kept in laboratory animal house of Drug Research Department during the experiment. The animals were provided standard pellet food and tap water Artemisia afra at dose of 500 mg/Kg, 750 mg/Kg, and 1000 mg/Kg PO. Groups-4–6 included diabetic mice treated with crude methanolic extract of Artemisia afra at dose of 500 mg/Kg, 750 mg/Kg, and 1000 mg/Kg PO. Group 7 included diabetic mice treated with standard drug glibenclamide (standard group) 10 mg/kg PO [Alloxan induced experimental diabetic Swiss albino mice (FGB ≥ 250 mg/dL) were divided in to 9 test groups with 5 mice in each group that match with weight and fasting blood glucose level . Groups-mg/kg PO . Group 8 Artemisia afra was aseptically suspended in distilled water and administered at a single doses of 0, 1000, 2000, 3000, 4000, 5000, 7500, 10000, and 12000 mg/Kg body weight by gavages perorally (PO) to the test groups. The control groups were given an equal volume of water. The change in general behaviors of the mice was continuously monitored [50) value. All surviving animals were euthanized with diethyl-ether at day 14 as described by [The mice were divided into 10 groups with 6 animals in each group matched for weight and sex. These animals were housed 3 mice per sex per cage in a well-ventilated room with 12 hr cycle of day and night light conditions and temperature was maintained at around 25°C. Food was withdrawn for the 12 hr fasting period before and for 24 hr fasting period after oral administration of the single doses crude aqueous extract . Aqueousonitored . The numribed by . Artemisia afra aerial parts was tested qualitatively for presence of major secondary metabolites. The qualitative test was done according to procedure described by [Aqueous extract ofribed by . The metn = 5) animals in each group. The data was analyzed, using Microsoft Office Excel 2007, by Student's t-test at 95% CI.All the results of blood glucose were expressed as mean ± standard deviation for  of the Artemisia afra aqueous extract is higher than 5000 mg/kg. The expected median lethal dose (LD50) is between 7500 mg/Kg and 12000 mg/Kg. The actual median lethal dose (LD50) of Artemisia afra aqueous extract calculated from 50) is greater than 5000 mg/Kg, aqueous extract of Artemisia afra is nontoxic. Substances with LD50 between 5000 mg/kg of body weight and 15000 mg/kg of body weight are regarded as practically nontoxic [As shown in the nontoxic . Artemisia afra was administered to alloxan induced diabetic mice orally at single doses of 500, 750, and 1000 mg/kg. Blood glucose level was significantly decreased in diabetic groups which received aqueous extract of Artemisia afra at dose of 500 mg/Kg and 750 mg/Kg in hours after treatment relative to untreated diabetic control with p < 0.01 and p < 0.001, respectively . A sharp decline in blood glucose level at dose of 750 mg/Kg occurred in the first two hours after treatment.The aqueous extractofectively . Among t Artemisia afra showed a remarkable blood glucose lowering effect in alloxan induced diabetic mice at doses of 750 and 1000 mg/kg as compared to the untreated diabetic control group  as compared to the group receiving the vehicle.In contrast the methanolic extract ofol group . Though  Artemisia afra which showed maximal effect at 750 mg/kg dose level the methanolic extract of Artemisia afra at this level did not significantly decrease blood glucose at this dose level (p > 0.05),  Artemisia afra at 750 mg/kg dose level. Thus blood glucose level of diabetic mice did not come below 263.5 mg/dL throughout the 5 hr posttreatment period for this dose.Unlike the aqueous extract of Artemisia afra aerial part was screened positive for presence of chromophores, saponins, phosphosteroid and withanoids, flavonoids, tannins, and anthraquinone but showed negative result for presence of polyphenols, cyanogenic glycoside, anthranides, carotenoids, phenolic glycoside, cardiac glycoside, and alkaloids  than the methanolic extract at dose of 1000 mg/kg. This finding manifests the presence of the active ingredients in the aqueous extract at higher concentration than in methanolic extract, indicating the most active portions are polar.As compared to methanolic extracts of