Achyranthes bidentata radix (AB) extract for the treatment of spasticity has been evaluated. Rats were pretreated with distilled water (DW), OY, or AB extract by oral administration every day for 7 days. After 10 min of the final dose of DW or each herbal medication, baclofen (1 mg/kg) was given by oral administration and plasma concentrations of baclofen were determined by LC/MS/MS. The plasma baclofen concentration-time profiles were then analyzed by noncompartmental analysis and a population PK model was developed. Baclofen was rapidly absorbed, showed biexponential decline with elimination half-life of 3.42–4.10 hr, and mostly excreted into urine. The PK of baclofen was not affected by AB extract pretreatment. However, significantly lower maximum plasma concentration (Cmax) and longer time to reach Cmax (Tmax) were observed in OY pretreated rats without changes in the area under the curve (AUC) and the fraction excreted into urine (Furine). The absorption rate (Ka) of baclofen was significantly decreased in OY pretreated rats. These data suggested that repeated doses of OY might delay the absorption of baclofen without changes in extent of absorption, which needs further evaluation for clinical significance.The potential pharmacokinetic (PK) interaction of conventional western drug, baclofen, and oriental medications Oyaksungisan (OY) and  Spasticity is a muscle control disorder characterized by an abnormal increase in muscle tone or muscle stiffness due to the impaired nerve pathways. It may occur in association with cerebral palsy, traumatic brain injury, stroke, multiple sclerosis, spinal cord injury, and so forth. Symptoms of spasticity may include abnormal posture, carrying the shoulder, arm, wrist, and finger at an abnormal angle, exaggerated deep tendon reflexes, repetitive jerky motions, scissoring, and fixed joints , which mγ-aminobutyric acid (GABA), has been used as the reference treatment for spasticity. Other management options such as therapeutic exercise, physical modalities, complementary/alternative medicine interventions, and chemodenervation are also available [Baclofen , the first synthesized structural analogue of vailable , 3. Citrus unshiu peel, lindera root, cnidii rhizoma, Angelica dahurica root, Bombyx batryticatus, Aurantii fructus immaturus, Platycodon root, Zingiberis rhizoma, Glycyrrhizae radix et rhizoma, Zingiberis rhizoma crudus, and Zizyphi fructus [ Achyranthes bidentata radix (AB) is another traditional medicinal plant which has been used for spasm. AB is derived from the roots of Achyranthes bidentata Blume and Achyranthes japonica Nakai in the Korean Pharmacopeia, A. bidentata Blume in the Chinese Pharmacopoeia, and A. bidentata Blume and A. fauriei Leveille et Vaniot in the Japanese Pharmacopoeia [A variety of herbs are another option for treating spasticity which can help alleviate tight and sore muscles. Oyaksungisan (OY) is a traditional prescription of oriental medicine which is a water extract of twelve herbs including ephedra herb, fructus . OY has  fructus  and appr fructus , and ant fructus . Achyranacopoeia . AB has acopoeia , 8, arthacopoeia . It has acopoeia , cardiopacopoeia , and antacopoeia  effects These herbal medicines are often used as self-medication and available as over-the-counter drugs without prescription. Thus, they may be used in combination with conventional drugs, and the potential of their interactions is raising concern . ConcurrTherefore, we aimed to evaluate the potential PK interaction of baclofen with OY and AB, which are commonly used for the treatment of spasticity. Our study examined the effects of OY and AB extract on baclofen PK in rats by evaluating the changes in baclofen PK following multiple oral administration of each herbal medicine. The noncompartmental PK parameters were assessed and a population PK model was developed to describe and evaluate the alterations of baclofen PK. Achyranthes bidentata radix was obtained from Daegu Haany University .Baclofen, gabapentin , glycyrrhizin, hesperidin, and ferulic acid were purchased from Tokyo Chemical Industry Co., Ltd. . Naringin and 6-Gingerol were purchased from Sigma-Aldrich Co. LLC. . Neohesperidin was purchased from Santa Cruz Biotechnology, Inc. . Ecdysterone and R,S-Inokosterone were obtained from analytical chemistry laboratory in College of Pharmacy at Catholic University of Daegu. Acetonitrile, methanol, and distilled water  were purchased from Mallinkrodt Baker, Inc. . Perchloric acid and formic acid were purchased from Aldrich Chemical Co. . Oyaksungisan Extract Granule was obtained from Hankook Shinyak Corp. .μm) to obtain homogeneous particles. The obtained herbal specimen was then extracted in distilled water  at 100°C for 2-3 hours and filtered out. Following evaporation of the remaining water by using a rotary evaporator, the final herbal extracts were obtained by freezing at −80°C and lyophilization. The extraction yield of AB extract was 30.4%.Extraction of AB was performed in accordance with the Notification number 2003-17 of the Ministry of Food and Drug Safety, Korea (2003). Herbal materials were crushed and separated by passing through a standard #35 sieve  is glycyrrhizin, naringin, hesperidin, and neohesperidin; 6-Gingerol and ferulic acid were selected based on the literature [To guarantee the consistent quality of the herbal formula extract, the constituents of AB extract and OY were determined by HPLC coupled with UV detector  and diodterature . All theThe animal study was approved by the Ethics Committee for the Treatment of Laboratory Animals at the Catholic University of Daegu (IACUC 2012-11) and conducted following the standard operating procedures (SOPs). Male Sprague-Dawley rats  were kept in plastic cages with free access to standard diet . The animals were maintained at a temperature of 23 ± 2°C with a 12 hr light-dark cycle and relative humidity of 50 ± 10%.n = 6) or herbal medicine, that is, OY  and AB extract , was administered orally for 7 consecutive days. The doses of OY and AB extract were selected based on the human doses and animal toxicity studies. Considering fast metabolic processes of rats compared to human, 8–10-fold higher doses than clinically recommended doses of 15 g/day (=214.3 mg/kg/day) for OY and 10 g/kg/day (=142.8 mg/kg/day as AB) for AB extract were administered. The AB extract dose was calculated considering the yield of AB extract (30.4%). Based on the literature, the no-observable-adverse-effect level (NOAEL) was considered over 5000 mg/kg in mice for OY [μL of the venous blood samples was collected at 5, 15, and 30 min and 1, 2, 4, 8, 12, and 24 hr after dose from jugular vein. Plasma samples were harvested by centrifugation of the blood samples at 16,060 g for 10 min and stored at −20°C until analysis. Urine sample was collected for 24 hr.Distilled water  and diluted perchloric acid (4 μL of 2 M solution for plasma samples and 40 μL 0.2 M solution for urine samples) were added to 20 μL of each sample as precipitation solvent. The mixture was vortexed and centrifuged for 10 min at 16,060 g. The supernatant was collected and 2 μL of the sample was injected onto the LC/MS/MS.Baclofen concentrations were determined by the LC/MS/MS assay as reported previously . BrieflyC18 column  with a KrundKatcher ultra column inline filter  and the mobile phase was composed of a mixture of acetonitrile and 0.05% of formic acid (10 : 90 v/v). Urine samples were separated on Agilent Zorbax SB-Aquasil Column  with SecurityGuard Cartridge  and the mobile phase was a mixture of acetonitrile and 0.05% of formic acid (5 : 95 v/v). The flow rate was set at 0.2 mL/min and the column oven temperature was 30°C for all samples. The electrospray ionization (ESI) source was operated in positive mode with the curtain and turbo-gas  set at 20 and 6 psi, respectively. The turbo-gas temperature and the ion spray needle voltage were set at 450°C and 4500 V, respectively. The mass spectrometer was operated at unit resolution for the first quadrupole (Q1) and low resolution for the third quadrupole (Q3) in the multiple reaction monitoring (MRM) mode with a dwell time of 300 ms per MRM channel. The selected precursor/product ion pairs were m/z 214.3 → 151.1 for baclofen and m/z 172.2 → 154.2 for IS. The collision energy was set at 23 and 19 eV for baclofen and IS, respectively. Data acquisition was performed with Analyst 1.4 software .LC/MS/MS system consisted of API 4000 triple quadrupole mass spectrometer  coupled with an Agilent 1100 HPLC system . Plasma samples were separated on a Kinetex The assay was linear over a concentration range of 0.25–500 ng/mL for rat plasma and 2–5000 ng/mL for urine with correlation coefficients >0.999. The mean intra- and interday assay accuracies were 94.6–104.6 and 96.0–103.6%, respectively. The mean intra- and interday precisions were ≤5.71 and ≤5.70%, respectively.t1/2), area under the serum concentration-time curve from time zero to the last observation time point  and infinity (AUCinf), apparent volume of distribution during the terminal phase (Vz), systemic clearance (CL), and fraction of dose excreted into urine . The peak serum concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from the observations. The absolute bioavailability (F) of baclofen after oral dosing was calculated as F = /, where AUCiv was obtained from the previous study [For descriptive PK analysis, PK parameters were determined by the noncompartmental analysis using WinNonlin . These parameters included terminal half-life  wascontrol, BOLUSFOY, and BOLUSFAB, the initial condition for Xgut was BOLUSFcontrol · Dose for the DW, BOLUSFOY · Dose for the OY, and BOLUSFAB · Dose for the AB extract pretreated group. The absorption rates for DW, OY, and AB extract pretreated group, that is, Ka,control, Ka,OY, and Ka,AB, respectively, were estimated separately. The central compartment received drug from the gut compartment. The differential equation for the amount of drug in the central compartment (X1) contained terms for the drug transfer rates into and from the peripheral compartment and for the drug elimination rates into the urine  and other processes (Kel) :K12 and K21 are the intercompartmental rate constants between the central (X1) and peripheral (X2) compartments for baclofen. The differential equation for the peripheral compartment was The structural model was designed to capture the absorption, disposition, and elimination of baclofen. We considered a PK model with two compartments and first-order absorption of baclofen. The absorption process after oral administration of baclofen was described as a first-order rate constant, All plasma concentrations were simultaneously fitted by the population pharmacokinetic (POP-PK) modeling using the importance sampling version of the Monte Carlo parametric expectation maximization (MC-PEM) algorithm in the parallelized S-ADAPT software (version 1.57)  supportet-test between the two means for unpaired data or one-way ANOVA followed by Scheffe's post hoc test among more than two means for unpaired data. P values < 0.05 were considered as statistically significant.The obtained parameters were compared by unpaired Cmax) within 1 hour, and declined thereafter. The PK parameters of baclofen following AB extract pretreatment were not significantly different from those of the control group. Although the overall concentration versus time profile looks similar to other groups, baclofen Cmax was found to be significantly lower in OY pretreated rats compared to that in DW pretreated rats  and the time to reach Cmax (Tmax) was also longer in OY pretreated rats . Nevertheless, all other PK parameters including AUC and Fe,urine after OY pretreatment were similar to control  was estimated to be lower than that in DW  or in AB extract pretreatment group , suggesting slower absorption of baclofen after repeated doses of OY  and the fraction excreted into urine (Furine), was not affected by OY administration (Our data clearly suggested that the absorption of baclofen might be significantly delayed by repeated doses of OY . The nonstration .Ka) was significantly delayed, while the bioavailability of baclofen in OY pretreatment group was comparable with that in the control group. Based on the estimated Ka values for each treatment group, baclofen was expected to take more than twice of time to be absorbed after OY dose compared to control  and to achieve lower peak concentration.These findings were further analyzed by a POP-PK modeling to quantitatively evaluate the alterations in baclofen PK. POP-PK analysis is known to be valuable to investigate potential drug-drug or drug-herb interactions . The preThe slower rate of absorption of baclofen following repeated doses of OY may be associated with changes in gastrointestinal physiology, such as increased gastric pH, reduced intestinal blood flow, and diminished gastrointestinal motility. OY consists of twelve herbs and some of the components are known to have gastrointestinal effects. Among these, ephedra is well known to diminish gastrointestinal motility , which mThe PK alteration of baclofen, that is, slower rate of absorption with similar systemic exposure caused by coadministration of OY, allowed sustained exposure of baclofen as a result. Common side effects of baclofen include drowsiness, dizziness, weakness, confusion, and fatigue. More severe side effects are associated with abrupt withdrawal of baclofen, which include hallucinations, confusion, manic psychotic episodes, seizures, autonomic dysreflexia, hyperthermia, and rebound severe spasticity , 31. TheThe potential pharmacokinetic interaction of conventional western drug and oriental medication for the treatment of spasticity has been evaluated by noncompartmental and POP-PK modeling analysis. Following the repeated doses of OY, sustained systemic exposure of baclofen was observed caused by the slower rate of absorption without alteration in the extent of absorption.Supplementary Material includes analytical conditions of determination of the representative standard compounds and their contents in Oyaksungisan and Achyranthes bidentata radix extract as well as the fingerprint chromatograms.


It has been suggested that exercise training (ET) protects against the pathological remodeling and ventricular dysfunction induced by myocardial infarction (MI). However, it remains unclear whether the positive adjustments on baroreflex and cardiac autonomic modulations promoted by ET may afford a cardioprotective mechanism. The aim of this study was to evaluate the effects of aerobic ET, prior to MI, on cardiac remodeling and function, as well as on baroreflex sensitivity and autonomic modulation in rats.Sham surgery (C); trained rats submitted to Sham surgery (TC); sedentary rats submitted to MI (I), trained rats submitted to MI (TI). Sham and MI were performed after ET period. After surgeries, echocardiographic, hemodynamic and autonomic  evaluations were conducted.Male Wistar rats were divided into 4 groups: sedentary rats submitted to Prior ET prevented an additional decline in exercise capacity in TI group in comparison with I. MI area was not modified by previous ET. ET was able to increase the survival and prevent additional left ventricle dysfunction in TI rats. Although changes in hemodynamic evaluations were not observed, ET prevented the decrease of baroreflex sensitivity, and autonomic dysfunction in TI animals when compared with I animals. Importantly, cardiac improvement was associated with the prevention of cardiac autonomic impairment in studied groups.Prior ET was effective in changing aerobic capacity, left ventricular morphology and function in rats undergoing MI. Furthermore, these cardioprotective effects were associated with attenuated cardiac autonomic dysfunction observed in trained rats. Although these cause-effect relationships can only be inferred, rather than confirmed, our study suggests that positive adaptations of autonomic function by ET can play a vital role in preventing changes associated with cardiovascular disease, particularly in relation to MI. Coronary artery disease, together with myocardial infarction (MI), is the most prevalent cardiovascular disease (CVD), and commonly progresses to heart failure in affected individuals . It has Metabolic, cardiovascular, autonomic and anti-inflammatory benefits of a physically active life-style have led many researchers to suggest exercise training (ET) as an important non-pharmacological tool in the prevention and treatment of CVD -10. The On the other hand, a few studies have evaluated how ET undertaken prior to a MI affected aerobic capacity, cardiac function and morphometry, as well as mortality rate. Despite distinct experimental designs and ET regimens, the data found in the literature suggest that ET protects against the pathological remodeling and ventricular dysfunction induced by MI in rodents -23. MechTherefore, the present study was undertaken to investigate 1) whether ET prior to MI prevents cardiac dysfunction and morphometric derangements; 2) whether ET prior to MI changes mortality rate; and 3) whether the potential cardiac benefits of ET could be associated with preserved baroreflex sensitivity and cardiac autonomic modulation.Experiments were performed in adult male Wistar rats (275-300 g) from the Animal House of the São Judas Tadeu University, São Paulo, Brazil. Rats were fed standard laboratory chow and water ad libitum. The animals were housed in collective polycarbonate cages in a temperature-controlled room (22-23°C) and under 54-55% humidity with a 12-h dark–light cycle (light 07:00-19:00 h). The experimental protocol was approved by the institutional animal care and use committee of the São Judas Tadeu University (008/2013), and this investigation was conducted in accordance with the Principles of Laboratory Animal Care formulated by the National Institutes of Health .Sham surgery ; trained control rats submitted to Sham surgery ; sedentary rats submitted to MI surgery , trained rats submitted to MI surgery .The rats were randomly assigned to four groups: sedentary control rats submitted to Sham or MI surgeries. Our group previously demonstrated that maximal treadmill exercise test can detect differences in aerobic performance; since that the maximal running speed achieved in the test presented a good correlation with the maximum oxygen consumption [Sedentary and trained rats were adapted to the treadmill  for 5 days. All animals were submitted to a maximal treadmill exercise test to determine aerobic capacity and exercise training intensity at the beginning of the protocol , after 4 weeks , after ET protocol , and 2 days after sumption .ET was performed on a motorized treadmill at low-moderate intensity  for 1 hour a day, 5 days a week for 8 weeks, with a gradual increase in speed from 0.3 to 1.2 km/h .Sham surgery. The chest was closed with a silk suture.Anaesthetized rats  underwent surgical occlusion of the left coronary artery, which resulted in MI as described previously ,17. BrieSham or MI surgeries, echocardiographic evaluations were performed by a blinded observer, under the guidelines of the American Society of Echocardiography. Rats were anaesthetized , and images were obtained with a 10-14 mHz linear transducer in a SEQUOIA 512  for measurements of parameters: left ventricular mass (LVmass); left ventricular end-diameter during diastole (LVDD); relative wall thickness (RWT); fractional shortening (FS); ejection fraction (EF); E wave A wave ratio (E/A); left ventricular isovolumetric relaxation time (IVRT); myocardial performance index (MPI), as described in detail elsewhere [One day after lsewhere ,17.post mortem histological analysis [The MI area was delimited taking into account the movement of LV walls during initial and final echocardiographic evaluations by a blinded observer. MI was defined by echocardiography as any segmental wall motion abnormality such as hypokinesis, akinesis and dyskinesis, as described previously ,16,17. Oanalysis ,16,17,29Twenty-four hours after echocardiographic evaluation, 2 catheters filled with 0.06 mL of saline were implanted into the femoral artery and femoral vein of the anesthetized rats . On the next day, the arterial cannula was connected to a strain-gauge transducer , and arterial pressure (AP) signals and pulse intervals (PI) were recorded over a 30-minute period in conscious animals, as previously described ,17.Sequential bolus injections (0.1 mL) of increasing doses of phenylephrine (0.25-32 mg/kg) and sodium nitroprusside (0.05-1.6 mg/kg) were given to induce increases or decreases in mean AP responses (for each drug), ranging from 5 to 40 mmHg. Baroreflex sensitivity was expressed as bradycardic response (BR) and tachycardic response (TR) in beats per minute per millimeter of mercury, as described elsewhere ,17.The overall variability of the pulse interval (PI) was assessed in the time and frequency domains by spectral estimation in the 30-minute recorded basal period. Fluctuations in PI were further assessed in the frequency domain by means of autoregressive spectral estimation, as described elsewhere ,30. BrieSham/MI surgeries were assessed using repeated-measures ANOVA. Pearson’s correlation was used to study the association between variables. The survival analyses were estimated by the Kaplan–Meier method and compared by the log-rank (Mantel–Cox) test. All tests were two sided and the significance level was established at P < 0.05.Statistical analyses were performed with SPSS software . Data are reported as mean ± SEM. After confirming that all continuous variables were normally distributed using the Kolmogorov–Smirnov test, statistical differences between the groups in all evaluations were obtained by two-way  analysis of variance (ANOVA) followed by the Bonferroni post-test. Statistical differences between the data from maximal treadmill exercise test measured at the beginning of the protocol , after ET or following protocols , and 2 days after Sham surgery, no death was registered in experimental groups. After MI surgery, mortality rate was higher in I animals  when compared to TI (no deaths).During exercise training protocol or after Body weight was similar among all studied groups at the beginning of the protocol (~289 ± 6 g). At the end of the protocol, all experimental groups increased body weight when compared to their initial values ; however, TC and TI groups had reduced body weight when compared to C and I groups, respectively. Similarly, retroperitoneal adipose tissue weight was decreased in TC (2.4 ± 0.2 g) and TI (2.2 ± 0.2 g) rats when compared to C (5.2 ± 0.1 g) and I (4.9 ± 0.1 g) rats.Sham or MI surgeries are presented in Table Sham surgery, physical capacity remained similar in C and CT animals when compared to their final evaluations; however, after MI, I animals presented decreased physical capacity when compared to C animals, and to their initial and final evaluations. On the other hand, ET prevented an additional decrease in maximal running speed in TI rats , as well as increased E/A ratio and myocardial performance index when compared to C rats. It should be stressed that ET was able to prevent systolic, diastolic and global dysfunction in TI animals, as observed by normalization of fractional shortening, E/A ratio and myocardial performance index. Although ET did not prevent low ejection fraction in TI group, these values remained higher when compared to the I group Table .Hemodynamic parameters can be observed in Table Values of PI variability parameters are presented in Table Positive correlations were observed between LF/HF ratio and relative wall thickness  Figure A, and wiIn recent years, pharmacological therapies and changes in lifestyle, including weight control, physical activity, smoking cessation and reduced consumption of calories in the diet, have contributed to the primary prevention of CVD. ET plays a key role in health promotion and has become a unanimous approach in cardiology -34. The Reduced exercise capacity after MI has been considered an important predictor of mortality ,36. In tLeft ventricular remodeling is an indicator of great impact on cardiac mortality after ischemia ; howeverLikewise, Dayan et al.  have shoThere are multiple mechanisms by which moderate to vigorous ET may decrease mortality rates associated with CVD, including antiatherosclerotic, antithrombotic, anti-ischemic, antiarrhythmic, and antioxidant effects ,40. In oIt is well known that after MI catecholamine production and release from adrenal glands and from cardiac sympathetic nervous system nerve endings are enhanced . In the In the present investigation, we demonstrated that moderate ET, when performed prior to MI, prevented baroreflex sensitivity impairment, sympathetic modulation increase and parasympathetic modulation decrease, as observed in TI rats. Furthermore, improvements in autonomic balance (represented by LF/HF ratio), and in parasympathetic modulation (HF band) were strongly correlated with structural, systolic, diastolic and global left ventricle function. Since the later development of left ventricular dysfunction has been associated with further increment in neurohumoral excitation, due to arterial and cardiopulmonary baroreceptors ,52, our This work has some limitations that deserve comments. Although our group have previously showed strong correlations between the MI area assessed by echocardiogram and post mortem histological analysis ,16,17,29In summary, we have demonstrated that prior exercise training was effective in aerobic capacity, left ventricular morphology and function in rats undergoing myocardial infarction. Furthermore, these cardioprotective effects were associated with attenuated cardiac autonomic dysfunction observed in trained rats. Although these cause-effect relationships can only be inferred, rather than confirmed, our study suggests that positive adaptations of autonomic function by exercise training can play a vital role in preventing changes associated with cardiovascular disease, particularly in relation to MI. However, other systemic and local mechanisms cannot be ruled out. Thus, these findings encourage enhancing baroreflex sensitivity and cardiac autonomic function as a therapeutic strategy for the prevention of cardiac abnormalities triggered by myocardial ischemia.CVD: Cardiovascular Disease; MI: Myocardial Infarction; ET: Exercise Training; C: Sedentary Control Rats Submitted To Sham Surgery; TC: Trained Control Rats Submitted To Sham Surgery; I: Sedentary Rats Submitted To Myocardial Infarction Surgery; TI: Trained Rats Submitted To Myocardial Infarction Surgery; LV: Left Ventricle; LVDD: Left Ventricular End-Diameter During Diastole; RWT: Relative Wall Thickness; FS: Fractional Shortening; EF: Ejection Fraction; E/A: E Wave A Wave Ratio; IVRT: Left Ventricular Isovolumetric Relaxation Time; MPI: Myocardial Performance Index; AP: Arterial Pressure; SAP: Systolic Arterial Pressure; DAP: Diastolic Arterial Pressure; MAP: Mean Arterial Pressure; HR: Heart Rate; PI: Pulse Interval; BR: Bradycardic Response; TR: Tachycardic Response; SD: Standard Deviation Of The Pulse Interval Variability; RMSSD: Root-Mean Square Of Differences Of Successive Rr Intervals; LF: Low Frequency Band; HF: High Frequency Band; LF/HF: Autonomic Balance.The authors have no conflicts of interest to declare.FR: Data acquisition, analysis and interpretation; DJF: Data acquisition, analysis, interpretation; CAB: Data acquisition and analyze; MEVA: Data acquisition and analysis; LYR: Data acquisition and analysis; NMC: Data acquisition; CM: Data analysis and interpretation; DF: Data acquisition and analyze; GIHS: Data analysis and interpretation; KDA: Statistical analysis and helped to draft the manuscript; MCI: interpretation of data and helped to draft the manuscript; BR: conception and design of the work and draft the manuscript. All authors approved final version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/84/prepub


Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei.Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge  The marine nitrogen cycle controls the availability of nitrogenous nutrients and the biological productivity in oceanic systems234Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosovibrio); while all known NOB belong to some genera of the phyla Proteobacteria , Nitrospinae (e.g. Nitrospina), Nitrospirae (e.g. Nitrospira) and Chloroflexi (e.g. Nitrolancea)amoA gene (encoding the subunit of the ammonia monooxygenase in AOA and AOB) and nxrA or nxrB gene (encoding the subunit of the nitrite oxidoreductase in NOB) have served as useful phylogenetic markers for investigating nitrifying communities in various ecosystems891011121314Nitrification describes the oxidation of ammonia to nitrite and subsequently to nitrate for energy purposesSponges are sessile invertebrates that grow widely in virtually all aquatic ecosystems with high filtration ratesNitrosospira AOB and Planctomycetes anaerobic bacteria in the sponges Ircinia strobilina and Mycale laxissima were confirmed by amoA gene and 16SrRNA gene assaysamoA gene detection resultsNitrosopumilus lineage in the sponge holobiontsNeamphius huxleyi has demonstrated the genomic potentials, including Nitrosopumilus ammonia oxidation, at the functional-population levelDidiscus oxeata and Scopalina ruetzleri has uncovered the symbionts’ functions at the whole-community level, including the nitrification process that might be performed by a diverse set of archaeal and bacterial nitrifying lineages, including Nitrosopumilus, Nitrosococcus, Nitrosomonas and NitrosospiraLike many other marine invertebrates, sponges excrete ammonia as a metabolic waste2022in situ, whereas RNA-based strategies would provide valuable hints on the activity of microbial assemblage313334in situ active functions, such as archaeal ammonia oxidation and photosynthetic carbon fixation, carried out by sponge symbionts3637amoA and nxrA/nxrB genes, has not been performed in sponge metagenome/metatranscriptome so far.In many niches, such as sponges, the presence of functional genes is not the direct evidence of their corresponding metabolic activity Theonella swinhoei  is widely distributed in tropical and subtropical oceansNitrosopumilus, Nitrosospira and Nitrospira lineages in T. swinhoei near Caroline Islands, Palau 21T. swinhoei from the South China Sea using a DNA and RNA combined approach targeting the nitrifying genes, 16S rRNA genes and their transcripts.The sponge amoA, nxrA and nxrB genes from T. swinhoei DNA and cDNA templates. Cloning and sequencing results showed that archaeal and bacterial 16S rRNA gene, archaeal amoA gene and Nitrospira nxrB gene were positively amplified from both DNA and cDNA templates; while bacterial amoA gene, Nitrobacter nxrA gene, Nitrococcus nxrA gene, Nitrolancea nxrA gene and Nitrospina nxrB gene were not detected from either DNA or cDNA templates (data not shown). After quality check and trimming, archaeal 16S rRNA fragments of 657 ± 3-bp length, bacterial 16S rRNA fragments of 694 ± 14-bp length, archaeal amoA fragments of 532-bp length and Nitrospira nxrB fragments of 469-bp length were used for further analysis. Rarefaction curve analysis showed that the amount of sequenced clones in each clone library, shown in The archaea and bacteria were detected by PCR amplification using the16S rRNA primers  and nitrT. swinhoei was detected by 16S rRNA gene and transcript amplification. The 69 16S rRNA gene amplicons and 65 16S rRNA transcript amplicons were grouped into five OTUs (accession no. KM010247–KM010250 and KT380844) belonging to the Nitrosopumilus cluster, according to phylogenetic analysis and RDP Classifier identification (Luffariella sp. (accession no. EU049816 and EU049817) and T. swinhoei (accession no. AF186424); meanwhile, these OTUs shared 93–94% identity with the sequences of Nitrosopumilus spp. (accession no. KR737579 and HQ331116) and 90–92% identity with the sequences of Cenarchaeum spp. (accession no. AF083071 and AF083072). Each OTU took different proportions of the sequences in the corresponding clone library ranging from 9–48%  (amoA OTU accounted for 5–41% of the sequences in the respective clone library (Luffariella sp. (accession no. EU049831- EU049833) and Xestospongia muta (accession no. GQ485791). Meanwhile, these OTUs showed 79–81% similarity with the amoA sequences of Nitrosopumilus spp.  and 77–79% similarity with the amoA sequences of Cenarchaeum spp. (accession no. DQ397569 and DQ397580)  and 92–93% similarity with the AmoA sequences of Cenarchaeum spp. (accession no. DQ397569 and DQ397580), which indicated that they were more likely the Nitrosopumilus-like amoA sequences.Furthermore, PCR assays of archaeal M010255) . Nucleic library . These OQ397580) . On the Nitrosopumilus species in the South China Sea T. swinhoei; particularly, Nitrosopumilus-like AOA with transcriptional activity might predominantly contribute to the aerobic ammonia oxidation in this sponge.Altogether, the phylogeny of archaeal 16S rRNA genes and transcripts strongly demonstrated that the active archaeal community was dominated by T. swinhoei, since 66 bacterial 16S rRNA OTUs were revealed  and Xestospongia muta (accession no. HQ270303 and HQ270307). Meanwhile, none of the Proteobacteria and Chloroflexi 16S rRNA OTUs was ascribed into the AOB genera Nitrosomonas, Nitrosococcus, Nitrosospira or Nitrosovibrio, or the NOB genera Nitrobacter, Nitrospina, Nitrococcus, Nitrospina, Nitrotoga or Nitrolancea, according to the phylogenetic analysis and RDP classification  and one unique 16S rRNA OTU at the DNA level (accession no. KT121415) fell into the rospirae . These Ofication . This reT. swinhoei was verified by positive PCR detection of Nitrospira nxrB genes and their transcripts. All the Nitrospira nxrB gene and transcript amplicons were clustered into one shared OTU at DNA/transcript levels (accession no. KM010256) belonging to the Nitrospira cluster (nxrA or nxrB PCR assays. This OTU was most similar (98% sequence identity) to the sequences from the sponge Hyrtios proteus (accession no. KC884911), and it shared 85% identity to the sequence of Nitrospira marina strain ATCC 43039 (accession no. KC884902) (The presence and activity of NOB in the South China Sea  cluster . No otheC884902) .T. swinhoei. Particularly, Nitrospira species dominated the nitrifying bacterial community in this sponge. The revealed Nitrospira nxrB genes/transcripts and Nitrospira16S rRNA genes/transcripts demonstrated that the Nitrospira NOB species had transcriptional activity, and they were predominantly responsible for nitrite oxidation in the South China Sea T. swinhoei.Collectively, our results on 16S rRNA genes and transcripts suggested that a fraction of bacteria interspersing among 12 reported phyla had transcriptional activity in the South China Sea Nitrosopumilus-like amoA and Nitrospira nxrB genes and their transcripts in the South China Sea T. swinhoei. Copy numbers of Nitrosopumilus-like amoA genes and their transcripts were (6.59 ± 1.21) × 106 and (5.41 ± 2.57) × 105 per reaction, respectively; while copy numbers of Nitrospira nxrB genes and their transcripts were (8.55 ± 2.24) × 103 and (2.20 ± 0.91) × 104 per reaction, respectively  in T. swinhoei, which was similar to the transcript-level revelation of this sponge that the abundance of Nitrosopumilus-like amoA transcripts was higher than that of Nitrospira nxrB transcripts  (Nitrosopumilus-like AOA than those of Nitrospira NOB in the South China Sea T. swinhoei.qPCR assays were employed to quantify the abundance of ectively . At a si t-test) . This fiNitrosopumilus, Nitrosococcus and Nitrospira nitrifiers, in the sponge Rhopaloeides odorabile, showed significant shifts under changed thermal stresses by 16S rRNA assaysNitrosospira AOB amoA genes but no transcript counterparts in the sponges Ircinia strobilina and Mycale laxissimaAlthough some lineages, like AOB, retain an appreciable abundance of rRNAs even in the dormant period when their activity is expected to be minimalamoA OTUs, one nxrB OTU, four archaeal 16S rRNA OTUs and 14 bacterial 16S rRNA OTUs, indicated the existence of community-wide transcriptionally active prokaryotic populations, especially the nitrifying microbes in the South China Sea T. swinhoei. The unique OTUs at the DNA level, including one archaeal 16S rRNA OTUs and 28 bacterial 16S rRNA OTUs, implied that the microbes represented by these OTUs might be during their dormant period or be transcribed at low level which was below the PCR sensitivity threshold. Meanwhile, 26 bacterial 16S rRNA OTUs were uniquely revealed at the transcript level, indicating that these bacterial members may have very low genomic abundance but high transcriptional activity in the South China Sea T. swinhoei. Similar DNA- and transcript-level discrepancies of rRNA or functional genes have also been discovered in various sponge species2432Nitrosopumilus-like AOA and Nitrospira NOB were revealed by functional transcript-based assays. This study provides direct molecular evidences for the inhabitancy of nitrifying community with transcriptional activity in the South China Sea T. swinhoei.The shared OTUs at DNA/transcript levels, including five T. swinhoeiT. swinhoei in this study, suggesting that archaeal not bacterial ammonia oxidizers dominate the first step in nitrification in this sponge, which is consistent with the previous reports on the sponge Rhopaloeides odorabile adults and larvae, and other sponges5Nitrospira bacteria, known to be able to catalyze the nitrite oxidation step, were active in the South China Sea T. swinhoei, which is consistent with the revelation of the sponges Stylissa carteri and Geodia barretti37nxrA/nxrB targeting, indicating the strict nitrifying bacteria in the South China Sea T. swinhoei.In contrast to Palauan amoA genes/transcripts has indicated that AOA greatly outnumbered AOB in the sponges Halisarca caerulea, Higginsia thielei and Nodastrella nodastrellaamoA transcripts would be significantly affected by the physiological status of sponge Xestospongia mutaNitrosopumilus-like AOA than those of Nitrospira NOB in the South China Sea T. swinhoei. Within the nitrification process, the oxidation of ammonia to nitrite is the rate-limiting step, and archaeal ammonia oxidation rates have been reported to be correlated with AOA abundance in the oceanT. swinhoei samples.Quantitative assays on nitrifying genes or transcripts have reflected the abundance and transcriptional activity of nitrifying communities in sponges. For example, quantification of T. swinhoei-mediated nitrogen cycle has been partially predicted by the genomic information of the bacterial isolates from T. swinhoei. For example, genomic analyses of T. swinhoei-derived candidatus Synechococcus spongiarum and candidatus Entotheonella sp. have revealed the genes related with nitrogen fixation, nitrate reduction, ammonia/nitrite/urea assimilation and ammonia remineralization47Nitrosopumilus and Nitrosospira, and that of nitrite oxidation of Nitrospira have been suggested for Palauan T. swinhoei by amoA and 16S rRNA genes assays21amoA/nxrB/16S rRNA genes with their transcripts, we are able to provide a community-wide profile of the in situ active microbes, including Nitrosopumilus-like AOA and Nitrospira NOB in the South China Sea T. swinhoei, thus gaining a picture of the active nitrification potentials in this sponge.As a ubiquitous sponge species in the Indo-Pacific OceanT. swinhoei21T. swinhoei and the South China Sea T. swinhoei indicated that, archaeal community in these allopatric T. swinhoei were dominated by the Nitrosopumilus genus. ClustalW sequence alignment analysis  indicated that, not all of the archaeal 16S rRNA sequences from these allopatric T. swinhoei were overlapped, as multiple alignment similarity of these archaeal 16S rRNA fragments is 95.6%    . Meanwhiis 100%) . These rT. swinhoei. These methods are acceptable in conducting censuses of environmental microbes at the current time. For example, PCR and qPCR analyses of archaeal amoA gene have revealed the AOA diversity and abundance in Qinghai LakeamoA transcript in the deep seaT. swinhoei.In this study, PCR cloning and Sanger sequencing methods have been used to reveal the putatively active nitrifying community in the South China Sea T. swinhoei were revealed by targeting 16S rRNA genes, amoA and nxrB genes and their transcripts. Our investigation demonstrated the inhabitancy and transcriptional activity of Nitrosopumilus-like AOA and Nitrospira NOB that dominate the nitrifying community in this sponge host. Meanwhile, quantitative assessments indicated relative higher abundances of nitrifying gene and transcript of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. These results replenished an growing data pool of associations between sponge and nitrifiers, and further contributed to the understanding of the ecological functions of so far uncultivated functional community in sponges.In summary, the composition and activity of nitrifying community in the South China Sea T. swinhoei was collected by Scuba diving near Xisha Archipelago  in the South China Sea at a depth of ca. 20 m in May, 2009. The sponge species was identified by morphological characterization and 28S rRNA Sequencing. The 28S rRNA gene from this sponge showed 99% similarity to T. swinhoei voucher NCI218 28S rRNA gene (accession no. KC884844) and was submitted to GenBank under the accession number JF506040. Samples from three different individuals were cut into 15 mm thick slices, then immediately preserved in RNAfixer  and stored at −70 °C before DNA or RNA extraction.T. swinhoei RNA was extracted using the RNApure Plant Kit , and DNA was extracted using the DNeasy Plant Mini Kit , respectively, according to the manufacturer’s instructions. RNA and DNA were extracted from the samples of three individuals, respectively; for each individual, several specimens were homogenized to minimize the sampling bias. RNA was treated with DNase (CoWin Biotech) at 37 °C for 30 min and purified with RNA Cleanup Kit (CoWin Biotech). RNA and DNA were accurately quantified by Qubit 2.0 Fluorometer using Qubit RNA BR Assay Kit  and Qubitds DNA BR Assay Kit (Invitrogen), respectively. For reverse-transcription, purified RNA was converted into single strand cDNA with random hexamers primer using the SuperScript First-Strand Synthesis System  according to the manufacturer’s protocol. DNA and cDNA products were stored at −70 °C for subsequent experiments.amoA, nxrA, nxrB and 16S rRNA gene fragments were selected for PCR-screening from T. swinhoei DNA and cDNA templates. A novel primer pair NpnnxrBF/NpnnxrBFR specially targeting the Nitrospina nxrB gene was designed using the CODEHOP toolnxrB sequences of Nitrospina gracilis3/211 (accession no. KC262217), Nitrospina sp. AB-629-B18 (accession no. A3QC_RS0100660) and an uncultured Nitrospina sp. clone (accession no. KJ571877) were selected for primer design to cover a 425 bp region. Forward primer NpnnxrBF and reverse primer NpnnxrBR target the 685–707 and the 1089–1109 nucleotide regions of nxrB2 gene in Nitrospina gracilis3/2, respectively. Specificity of this primer pair targeting Nitrospina nxrB was tested by BLASTn (www.ncbi.nlm.nih.gov/blast/) searches against the GenBank database. PCR mixture (40 μl) consists of 2 μl cDNA or 4 ng DNA templates, 0.1 μM of each primer and 20 μl TaqMaster Mix (CoWin Biotech). PCR amplifications were carried out on a Thermocycler  according to the following procedures: 95 °C for 5 min; followed by 30 cycles consisting of 95 °C for 40 s, annealing (temperature referring to Escherichia coli DH5α competent cells (CoWin Biotech) according to the standardized protocols. For each gene marker, individual triplicates-derived clones were screened based on ampicillin resistance; while inserts were identified by PCR with vector-specific M13 primers and read on an ABI 3100 capillary sequencer .Reported primers  targetinamoA sequences, KM010256 for nxrB sequence, KM010247 - KM010250, KM010257 - KM010299, KM121407 - KM121435 and KT380844 for 16S rRNA sequences.The obtained 16S rRNA sequences were checked for chimeras by DECIPHER’s Find Chimeras web toolet al.Nitrosopumilus-like amoA gene, Nitrospira nxrB gene, or their transcripts, the PCR was performed in a total volume of 25 μl containing 12.5 μl of SYBR Premix Ex Taq™ II , 2ng of DNA template or 1μl of cDNA template and 0.1 μM of each primer (amoA) or 56 °C (for nxrB) for 45 s, and 72 °C for 45 s. Cycling was followed by a final elongation step at 72 °C for 10 min. Standard curves (log-linear R2 > 0. 99) were generated using purified and quantified plasmids containing amoA sequence (168bp) or nxrB sequence (179bp) in a dilution series that spanned from 101 to 107 gene copies per reaction. Plasmid DNA was extracted with the PurePlasmid 96 Kit (CoWin Biotech), and the plasmid concentration was measured with a Qubit 2.0 Fluorometer using Qubitds DNA BR Assay Kit (Invitrogen). Since the sequences of the vector and PCR insert are known, we calculated the copy numbers of amoA or nxrB directly from the concentration of extracted plasmid DNA according to the reported formula−1 = (A × 6.022 × 1023) × (660 × B)−1, where A is the plasmid concentration (g μl−1), B is the recombinant plasmid length (bp) containing the amoA or nxrB sequence, 6.022 × 1023 is the Avogadro’s number and 660 is the average molecular weight of one bp. For negative control, similar procedure was carried out using purified RNA to ensure that there was no genomic DNA contamination. After real-time PCR assay, the specificity of amplification was verified by generation of melting curves and agarose gel electrophoresis. Data acquisition and analysis of the Real-time PCR assay were performed using the 7500 System SDS Software Version 1.2 (Applied Biosystems). Data statistics and independent-sample t-test were performed using corresponding commands in Microsoft Excel 2007.qPCR were performed using an ABI 7500 Fast Real-time PCR platform , according to the procedures used by Radax h primer . The qPCHow to cite this article: Feng, G. et al. Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei. Sci. Rep.6, 24966; doi: 10.1038/srep24966 (2016).


Hepatitis B virus (HBV) infection is a critical global health issue and moderately epidemic in Western China, but HBV molecular epidemiology characteristics are still limited. We conducted this study to investigate HBV genotypes and antiviral resistant mutations in this multi-ethnic area. A total of 1316 HBV patients were recruited from four ethnic groups from 2011 to 2013. Genotypes and resistant mutations were determined by Sanger sequencing. Four genotypes  were identified. Genotype B and C were common in Han population, while genotype D was predominant in Uygurs. Genotype C was the major genotype in both Tibetans and Yis, and recombinant C/D was found in Tibetans only. Lamivudine resistance was common in all populations, especially in Hans with prevalence of 42.8%. Entecavir resistance was barely observed regardless of ethnicity. Genotype C isolates had higher rates of rtA181T/V than genotype B , in accordance with higher prevalence of resistance to adefovir . While incidence of resistant mutations to other drugs and clinical factors showed no difference among different genotypes. HBV genotypes and resistance-conferring mutations had different geographic and demographic distributions in Western China, which provided molecular epidemiology data for clinical management. Hepatitis B is one of the major public health priorities with more than 350 million chronic hepatitis B virus (HBV) carriers over the world and causing 686, 000 deaths per year1HBV has an incomplete circular double-stranded DNA genome. According to more than 8% genetic variability in the full-length nucleotide sequence or 4%–8% divergence in the S gene, at least 10 HBV genotypes (A to J) and their subtypes have been defined5687In China, genotype B is predominant in Northern areas, and genotype C is more common in Southern areas1113141417Currently, seven antiviral agents are approved for management of CHB infection including two formulations of IFN-α , and five nucleos(t)ide analogues (NAs), namely lamivudine (LMV), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF)20As depicted before, CHB patients carrying different HBV genotypes could have diverse outcomes. The response to NAs and prevalence of NAs-induced resistance between different HBV genotypes also evoke much altercation. Two large-scale clinical studies from China found that rtM204I with or without rtL180V was more frequent in genotype C than genotype B252829Western China is a multi-ethnic area, including Han residing in most provinces, Tibetan mainly in Tibet and Sichuan province, Uygur in Xinjiang, Yi and Miao in Yunnan province, and so on. These diverse populations have distinct lifestyles, socio-economy, culture and genetic backgrounds. HBV prevalence in Western China is a bit higher than that in Eastern China, however, data of HBV molecular epidemiology from Western China is rather scarce. Up to now, there is very limited information about HBV genetic discrepancy, including genotype distribution, HBV-DNA load and prevalence of antiviral resistant mutations between different ethnic minorities in Western China. Therefore, we conducted a retrospective study to investigate HBV genotypes and drug resistant mutations in Sichuan, Tibet, Xinjiang and Qinghai to provide detailed molecular epidemiology information of HBV for evaluating the potential future disease burdens and the management strategies.A total of 1316 HBV HBsAg carriers were recruited from five hospitals in four provinces, and HBV reverse transcriptase fragment was amplified successfully from 1221 samples consist of 657 Hans, 472 Tibetans, 46 Uygurs and 46 Yis. The median age of total patients was 36  years, and 74.53% of them were male. The median serum HBV DNA was 6.27  log10 copies/ml. All of the patients were positive for HBsAg and antibody to hepatitis B core antigen (anti-HBc), and 97.38% of them were HBeAg positive .In total, four HBV genotypes  were identified in the 1221 samples. Among them, 429 (35.1%) patients were infected with genotype B, 559 (45.8%) with genotype C, 204 (16.7%) with genotype D, and 29 (2.4%) with recombinant C/D. The geographic distribution of HBV genotypes in five provinces from Western China varied extraordinarily. Genotype B and C were common in Sichuan with comparative prevalence of 45.4% and 45.1%, respectively. Genotype D was predominant in Xinjiang, followed by genotype C and B. In Tibet and Qinghai, the main prevalent HBV isolates were genotype C and D, with a little higher rate of genotype C than genotype D . Recombinant C/D was only found in Sichuan and Tibet .In the subgroup analysis stratified by ethnic population, significant differences of HBV genotype distribution were observed among Hans, Tibetans, Uygurs and Yis . The maiThen we conducted the phylogenetic analysis to confirm the genotyping method. We randomly selected 10 of the HBV isolates in each genotype from all of the ethnic groups by using the random number table to do the phylogenetic tree analysis. If the number of HBV isolates in the group was less than 10, we chose all of them. For further analysis of the association between HBV genotypes and clinical characteristics, 639 Han HBV subjects in Sichuan were included to compare their demographic information and clinical laboratory markers . The disHBV antiviral resistant mutations were determined for all of the 1221 patients, and analysis was carried out to evaluate four widely used NAs in China, namely LMV, LdT, ADV and ETV. The overall resistant rate showed variability among different areas. Sichuan had the highest prevalence of antiviral drug resistance of 34.6%, followed by Qinghai and Xinjiang with the pooled prevalence of 19.0% and 10.9% respectively, and Tibet with the lowest resistant rate of 4.4%. In ethnic-specific subanalysis, the resistant rate in Hans reached up to 46.7%, compared with 23.9% in Yis, and less than 10% resistant rate was observed both in Tibetans and Uygurs .In Han patients, resistant rate to LMV ranked the first with the prevalence of 42.8%, followed by 37.0% resistance to LdT. However, Han patients in different areas had different distributions of resistance to four antiviral agents. HBV isolates in Sichuan Hans showed a high resistant rate to all of the NAs, while in Xinjiang Hans, relatively high resistance to LMV (16.7%) was only observed. As to Tibetans and Uygurs, resistance to LMV was also the major source of antiviral resistant rate with the prevalence of 4.2% and 8.7% respectively, and resistance to ADV and ETV were seldom found, except Qinghai Tibetans with a resistant rate of 4.8% to ADV. In Yi patients, the predominant drug resistance occurred in LMV and LdT (both of 17.4%) compared with 6.5% in ADV, and no resistance to ETV was observed as well .Based on 639 Han patients in Sichuan Province, the correlation between HBV genotypes and antiviral drug resistance were estimated. Patients infected with genotype C had a much higher ADV-resistant rate than patients carrying genotype B . However, no difference was found when comparing the resistant rate of LMV, LdT, ETV and the total resistant incidence between genotype B and C .Next, we took a further step to study the percentage of mutant sites in different HBV genotype strains in these 639 Han patients to comprehend the molecular mechanism of antiviral resistance. As shown in In addition, subgroup analysis by NAs was performed in Sichuan Han population to evaluate the effect of HBV genotypes on resistant mutation rate. In patients resistant to LMV and LdT, mutation rtM204V and rtM204I showed no difference between genotypes, while mutant rtL180M had a higher frequency in genotype C isolates than genotype B isolates in both subgroups  . InteresBesides the different distribution of single mutant between genotype B and C, we further analyzed the correlation of certain mutation models and HBV genotypes . The mosIn this study, we analyzed the geographic and demographic distribution of HBV genotypes and the prevalence of antiviral resistant mutations of 1221 HBV isolates from four ethnic groups in Western China. We determined four HBV genotypes in all, namely genotypes B, C, D and recombinant C/D, and each ethnic population had different major genotypes. LMV-resistance was common in all populations, and ETV resistance was hardly observed. ADV and LdT resistance was more predominant in Han and Yi groups than Uygurs and Tibetans. Patients infected with genotype C had a higher rate of mutant rtA181T/V and resistance to ADV than genotype B, while the incidence of resistant mutations to other NAs and clinical factors had no difference statistically among different genotypes.et al.Obviously, we found that the predominant genotypes in Han population were genotype B and C, although the distribution frequency varied among different geographic regions. Genotype C was dominant in Xinjiang Hans, while genotype B predominated in Sichuan Hans, which was in accordance with previous studies about HBV genotypes in Northern and Southern China1614et al.30Consistent with previous studies, LMV-resistance was the most common resistance in Western China, with the highest resistant rate of 34.6% in Sichuan Province. In the subgroup analysis of ethnicity, LMV-resistance was the most common in Han population with the prevalence as high as 42.8%. Currently, conflicting results exist in whether HBV genotypes have an impact on LMV resistance. Damerow et al.HBV mutation rtA181T/V and rtN236T could lead to drug resistance of ADV, and the influence of HBV genotypes on the resistance to ADV has not been clarified. Yuen ETV is a potent antiviral drug with higher genetic barrier and was recommended to be used as the first line agent in naïve CHB patients. The occurrence of resistance to ETV in drug-naive patients is rather rare during the first year and remains low even after more than 6 years of treatment (approximately 1%)373839et al. found 3.83% (37/966) untreated CHB patients and asymptomatic carriers harboring natural YMDD mutations in a city of Sichuan Province in Western China, and genotype C strains had a higher rate of spontaneous YMDD mutations than genotype BAs is known to all, NAs-experienced HBV patients have a risk of developing drug resistant mutations. In addition, the occurrence of spontaneous resistant mutations in treatment-naïve patients was reported over the world. In China, the incidence of natural resistant mutations varied greatly among different areas. Yang This cross-sectional study provided an integrated landscape of HBV genotypes and resistant mutations in West China, however, there were some limitations in our study. First, clinical features of CHB patients from Tibet, Xinjiang and Qinghai were not available and the analysis of HBV genotypes and clinical characteristics was only taken in Sichuan Han population. Second, we determined HBV genotypes and resistant mutations by direct sequencing, which has a lower sensibility than Ultra-deep pyrosequencingIn conclusion, our study described the HBV genotypes prevalent in Western China and analyzed the prevalence of antiviral drug resistant mutations in different geographic and ethnic subgroups. The different predominant genotypes in every ethnic population revealed the complex and diversified distribution of HBV genotypes in China. NAs-resistance was found in all of the regions and ethnicities, and Sichuan Han group had more severe resistance than the other minorities. LMV had the highest resistant rate among the four NA agents, while resistance to ETV was the lowest. The influence of HBV genotypes on the development of resistant mutations was weeny, and only more frequent ADV-resistance was found in genotype C patients than genotype B patients in Sichuan Han population. Although HBV genotype detection was not demanded in current guidelines of HBV management, many studies have demonstrated that different genotypes have different clinical features and outcomes. A comprehensive understanding of the molecular epidemiology of HBV will provide evidence for clinical determination. Besides, resistance to NAs could lead to treatment failure and even cross-resistance. The selection of initial therapy and subsequent rescue treatment should be based on the knowledge of resistance rate, and combinations of NAs have been highlighted to refractory patients.This was a retrospective study on patients with chronic HBV infection from four areas of Western China: Sichuan, Tibet, Xinjiang and Qinghai. A total of 1316 serum samples from four ethnic groups were recruited, and HBV RT region was successfully amplified from 1221 samples consisting of 639 Sichuan Hans, 18 Xinjiang Hans, 249 Tibet Tibetans, 202 Sichuan Tibetans, 21 Qinghai Tibetans, 46 Xinjiang Uygurs and 46 Sichuan Yis. All the subjects were collected from West China Hospital of Sichuan University, People’s Hospital of Tibet Autonomous region, Tibetan Hospital of Tibet Autonomous region, First Affiliated Hospital of Xinjiang Medical University and People’s Hospital of Qinghai Province from June 2011 to May 2013. All enrolled CHB patients were characterized as HBsAg-positive >6 months, no antibody to hepatitis B surface antigen (anti-HBs) existed, persistent or intermittent elevation of ALT and (or) AST levels, and with no evidence of cirrhosis or carcinoma by imaging and laboratory testing. Patients co-infected with hepatitis A, C, D virus or human immunodeficiency virus (HIV) were excluded. Serum was separated by centrifugation at 4 °C in local hospitals, and stored at −20 °C in West China Hospital of Sichuan University, Chengdu, Sichuan.Demographic information such as age, ethnicity, family members and residential address were recorded by interview. Since Tibetan patients in Tibet had no family members from other ethnicities, their genetic backgrounds were pure. So did the Uygurs in Xinjiang. This study was approved by the Ethics Review Committee of all participating hospitals, and consistent with the guidelines of the Helsinki Declaration. All participants provided written informed consent for participation in this study.3 viral genome copies/ml. The handling procedures were carried out in strict accordance with the HBV DNA PCR-Fluorescence Quantitative Diagnostic Kit . The reaction volume was 35 μl, and the reaction condition was at 50 °C for 2 min, 94 °C for 5 min, and 40 cycles at 94 °C for 10 s and 60 °C for 45 s, then final extension at 72 °C for 5 min.Serum HBV viral load was assessed by using real-time fluorescent quantitative polymerase chain reaction  on Roche Light Cycler 480II  with a lowest limit of detection of approximately 10Serology markers of HBV including HBsAg, anti-HBs, HBeAg, antibody to hepatitis B e antigen (anti-HBe), and anti-HBc were analyzed using Elecsys Modular E170 immunoassay  or I2000 immunoassay  in accordance with manufacturers’ instruction.The HBV nucleic acid was extracted from 200 μl patient serum samples using NucliSENS easyMAG system  following the operators manual. The RT coding region was amplified by designed primers as follows: F: 5′-GTTGCCCGTTTGTCCTC-3′, R: 5′-GACAAACTTTCCAATCAATAGG-3′. A typical amplification was performed in a 20 μl reaction volume containing 1 μl HBV DNA and 0.2 μl Ex Taq HS  at 95 °C for 5 min, followed by 40 cycles at 95 °C for 45 sec, 58 °C for 45 sec, and 72 °C for 30 sec, and final extension at 72 °C for 5 min. PCR products were identified using agarose gel electrophoresis and digested by the mixture of shrimp alkaline phosphatase (SAP) and exonuclease I  to eliminate the redundant single-strand DNA.Sample processing, PCR, products identification and digestion was done in separate laboratory rooms which were certified for molecular diagnostics using standard precautions to prevent contamination.PCR products of RT region was 529 bp. HBV genotypes and resistant mutations were determined by direct Sanger sequencing the RT region of viral genome on ABI 3130 Genetic Analyzer .Sequencing primers was: 5′-ACTTTCCAATCAATAGG-3′. Sequencing reactions were performed in a 5 μl reaction volume containing 1 μl SAP digested PCR products, 0.75 μl BigDye® Terminator v3.1 5xSequencing Buffer, 0.5 μl BigDye® Terminator v3.1 cycle Sequencing RR-100,  and 0.5 μl primers. The sequencing amplification was performed at 96 °C for 1 min, and then 25 cycles at 96 °C for 10 sec, 50 °C for 5 sec, and final extension at 60 °C for 4 min. PCR products were purified by using Bigdye XTerminator Purification Kit  according to the manufacturer’s instructions.http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi). Resistant mutations were analyzed by Stanford University HBV RT drug resistance database (http://hivdb.stanford.edu/HBV/DB/cgi-bin/MutPrevByGenotypeRxHBV.cgi)Purified PCR products were sequencing on ABI 3130 Genetic Analyzer by capillary electrophoresis. The results were analyzed by using software Chromas 2.23 . HBV genotypes were determined according to National Center for Biotechnology Information Genotyping Database (http://sray.med.som.jhmi.edu/SCRoftware/). The bootscan window sizes were 200 bases, the step size was 20 with 100 replicates..To confirm the genotyping method, phylogenetic analysis was constructed with the MEGA 5.0 , using the Kimura two-parameter matrix model and the Neighbor-Joining method with 1,000 bootstrap replicates. A total of 24 HBV reference sequences were obtained from GenBank, representing nine HBV genotypes A to H. Recombination signals were detected by the SimPlot 3.5.1 (2 test) or Fisher’s exact test where cell numbers were small. Mann-Whitney’s U test or Student’s t test was used for continuous variables as appropriate. A two-tailed P < 0.05 was considered to be statistically significant.All statistical analyses were performed using SPSS 13.0 . Categorical variables, like the percentages of HBV genotypes, were compared by Chi-square test .


Background. Electroacupuncture (EA) has been reported to treat functional constipation (FC). The aim of this study was to investigate the efficacy and safety of EA with different needle insertion method for FC. Methods. Sixty-seven participants were randomly assigned to control  and EA (with deep puncture) groups. Every patient received 5 treatments per week in the first two weeks, then 3 treatments per week during the following six weeks. Complete spontaneous bowel movements (CSBM), spontaneous bowel movements (SBM), Bristol stool scores (BSS), and Patient Assessment of Constipation Quality of Life (PAC-QOL) were assessed. Results. Both shallow and deep EA significantly increased CSBM frequency compared to the baseline. CSBM was increased from 0.50 ± 0.59/wk to 2.00 ± 1.67/wk with deep EA and from 0.48 ± 0.59/wk to 1.33 ± 1.09/wk with shallow EA . Similar finding was noted in SBM. Deep EA was more potent than shallow EA (P < 0.05) during the treatment period. No difference was found on BSS and PAC-QOL between two groups. Conclusion. It is effective and safe with EA to treat FC. Studies with large sample size and long-term observation are needed for further investigation. According to Rome III diagnostic criteria , functioConstipation may cause disorders in perianal, such as perianal abscess and anal fistula; anorectal lesions, such as hemorrhoids and colorectal cancer; digestive system diseases, such as bloating, indigestion, and diverticulosis; psychiatric symptoms, such as headache, insomnia, and irritability, aggravating the symptoms, even threatening the life, such as increasing blood pressure, inducing acute cerebral vascular disease, and even sudden death , 8. ConsMore and more constipation patients prefer alternative and complementary treatment because of worry from drug side effect and deficiency of long-term effect , despiteThe recruitment of subjects took place from October 2012 to September 2013. The study was approved by Medical Ethics Committee and completed in the Outpatient Department of Guo Yi Tang in Nanjing, China.As shown in Participants were excluded from the study if they had a diagnosis of irritable bowel syndrome (IBS), or constipation caused by other diseases or medicine, or other significant diseases and medicine that may interfere with completion of the study. Pregnant or breastfeeding women were also excluded.Patients had the rights to decide to whether participate in or withdraw the study at any time. Their decisions did not affect their deserved treatments.Participants recruited through advertisements in hospitals and schools were randomized by stochastic systems in computer and decided to receive control or EA treatment. All participants were blinded to the type of treatment method received until completion of the study.The total study period was shown in Each patient received total 28 treatments, including 5 times per week for the first two weeks and 3 times per week for the following six weeks.Patients in EA group received EA at 6 acupoints, ST25 (Tianshu) and SP14 (Fujie) and ST37 (Shangjuxu), bilaterally. The physician inserted into ST25 and SP14 with HuaTuo 0.30 × 75 mm needles, deep to the parietal peritoneum without lifting and twisting. The two needles at ST25 and SP14 unilaterally were connected to an electric stimulator  for 30 min. The frequency was 2/15 Hz alternately. The current was strong enough (0.1 mA–1.0 mA) to produce a slight tremor in patients' abdominal muscles. HuaTuo 0.30 × 40 mm needles were inserted into ST37 with depth of 1 cun, lifted and twisted slightly three times to Deqi every 10 minutes for a total of 30 minutes. Patients in the control group received EA with same techniques and parameters, but with shallow puncture with depth of 2 mm and at points located one cun away from those 6 acupoints (on the median between Stomach Meridian of Foot Yang-ming and Spleen Meridian of Foot Tai-yin), respectively, without lifting and twisting, for 30 minutes.The primary outcome was CSBM (complete spontaneous bowel movements); the secondary outcomes consisted of spontaneous bowel movements (SBM), Bristol stool scores (BSS), hard defecation score, and Patient Assessment of Constipation Quality of Life (PAC-QOL). The participants filled the defecation diary every day during the entire experimental period.P < 0.05 was considered as significance.All of statistical analysis was performed in both ITT analysis  and PP analysis . The data are expressed as the mean ± standard error (SEM) in each group. SPSS Win. Ver.14.0 software was used and n = 37) or EA group (n = 35) randomly; 67 participants completed all treatments and the follow-up visits. In control group, two participants lost contact, and the other two failed in blinding. One participant in EA group received another treatment of constipation , there were no significant differences between the two groups, including gender, age, and disease course .n = 34, 2.00 ± 1.67/week and 4.10 ± 2.29/week, resp.), compared to control group , CSBM and SBM were increased significantly in EA group ; however, no differences were found between the two treatment methods (P > 0.05)   .There were no serious adverse events reported. Local subcutaneous congestion appeared in two participants; one participant reported mild abdominal pain.Electroacupuncture (EA) is based on acupuncture, an ancient Chinese traditional medicine therapy, in which electric current is transmitted to needles inserted acupoints on skin. During the past decade, EA has been reported to treat constipation by acupuncturists. However, evidences to efficacy and safety are deficiency because of less randomized controlled clinic trails reported.In this study, EA showed effective on constipation. Times of spontaneous bowel movements per week were increased; properties of stool were improved so that evacuation became smooth; qualities of life of patients with constipation were taking a turn for the better.Nonacupoints were active in control group, despite the fact that they locate at one cun away from normal acupoints and the middle of two meridians. In the literature, opinions on nonacupoints were controversial, especially the distance between nonacupoint and normal acupoint. Some researchers consider that acupoint is not located at a point on skin but in a field ; therefoThe technique of deep puncture performed on acupoints ST25 and SP14 caused that EA group acted better than control group. Taking needles perpendicularly and slowly into skin of abdomen until penetrating the peritoneum had been proved effective for constipation . OperatiThe mechanism of EA for treating constipation could be attributed to the improvement of colonic motility. It was reported that EA promotes contractility of distal colon in rats . EA was In conclusion, it is effective and safe with EA to treat FC. There are deficiencies in this study, including small sample sizes and single blind. More rigorous studies with larger sample sizes are required.


Cardiovascular Diseases (CVD) represent about 30% of all causes of death worldwide. The development of CVD is related in many cases with the previous existence of metabolic syndrome (MS). It is known that apple consumption has a cardiovascular protecting effect, containing phenolic compounds with antioxidant effect, which are concentrated in the fruit peel. The objective of this study was to test the effect of apple peel consumption in a murine model of MS and apoE−/− mice. Apple supplemented diets reduced the biochemical parameters ) of MS model in CF1 mice significantly. The model apoE−/− mouse was used to evaluate the capacity of the apple peel to revert the progression of the atherogenesis. FD with HAP reverts cholesterol significantly and slows down the progression of the plate diminishing the cholesterol accumulation area. With these results, it can be concluded that the consumption of apple peel reduces several MS parameters and the atherogenic progression in mice. Cardiovascular diseases (CVD) are the main cause of death in the world  and therThe rate of atherothrombotic progression is influenced by the exposition to risk factors which increase the probability to suffer from a cardiovascular event. Such factors can be classified as modifiable  and nonmodifiable . For this reason, genetically modified experimental animals have been generated which have contributed to the knowledge of the atherothrombotic phenomenon: the formation of the atheroma plaque, the induction of its formation, and the cellular elements involved . in vitro [ in vitro have demonstrated that apple flavonoids improve the availability of nitric oxide and protect the endothelial cells from apoptosis preventing the process of endothelial dysfunction and in vivo reduces blood pressure and oxidative stress [Epidemiologic studies have demonstrated that fruit and vegetable consumption contributes to decreasing the cardiovascular risk . For insin vitro , 8, antiin vitro , 10, andin vitro . Studiese stress . Furthere stress . In the e stress .The ApoE deficient mice are an idoneous experimental model for the study of atherosclerosis , becauseFour-week male CF-1 mice and C57BL6 mice obtained from the Public Health Institute, Santiago, Chile, and ApoE−/− mice  were used in the experiments. Animals were maintained at 22 ± 2°C, at a regular darkness-light 12:12 h cycle (light 08:00 to 20:00 h). Weight, food intake, and water consumption were measured daily. Animal handling was carried out in conformity with the regulation for the use of laboratory animals of the National Commission for Scientific and Technologic Research (CONICYT), Chile. The protocol was approved by the bioethics committee of Universidad de Talca. After an adaptation period of 10 days, animals were randomized into 5 groups.At the end of the experimental period, mice were anesthetized with an intraperitoneal injection of ketamine 50 mg/kg  and xylazine 5 mg/kg . Animals were euthanatized by blood collection from the abdominal aorta and later rupture of the diaphragm. Plasma and tissues were harvested and stored at −70°C until the analysis.Initial and final weight of each animal, as well as systolic arterial blood pressure (SAP) at the tip of the mice tails, were obtained by using ultrasound equipment .CF-1 mice were administered five types of diet: normal diet (ND), fat diet (FD), fat diet plus healthy apple peel (FD + HAP), fat diet plus dehydrated healthy apple peel (FD + DAP), and fat diet plus sun damaged apple peel (FD + SDAP) coming from Fuji apples, during 43 days.ApoE−/− mice were administered ND, FD, and FD + HAP during 10 weeks using peel from Granny Smith apples as a supplement. C57BL6 mice were administered ND. Ingredients used and the preparation of different diets were performed as described by Moore-Carrasco et al., 2008 . DiffereTotal phenolics were determined by the Folin-Ciocalteu method . Briefly−5 M DPPH solution and incubated for eight min at room temperature and the absorbance measured at 515 nm. Ethanol was used as blank. Chlorogenic acid in different concentrations was used as a standard and the capture of the DPPH free radicals was expressed as mg of chlorogenic acid equivalents (CAE) g-1 FW.The capture of the free radical 2,2-diphenyl-1-picrylhydrazyl  was measured by the method described by Von Gadow et al.,  with modμm filter) extracts were injected. To identify the compounds, different standards of specific phenolics were used with the UV-VIS spectra. The chromatogram was monitored at 256 nm. The solvents of the mobile phase were A: 1% formic acid in H2O quality HPLC, B: 40% acetonitrile in H2O, and C: acetonitrile. The elution parameters were time 0–10 min: A (70), B (30), and C (0) flow 1 mL min−1; time 45 min: A (25), B (75), and C (0) flow 0.5 mL min−1; time 52 min: A (0), B (0), and C (100) flow 1 mL min−1; and time 55 min: A (70), B (30), and C (0) flow 1 mL min−1. The results were expressed in μg of samples in g of FW-1.Specific phenolics (quercetins glycosides) and lipid profile in the samples (diets) were determined using a HPLC-DAD Merck Hitachi  equipment consisting of a LaChrom L-7100 pump and a diode array detector, L-7455 LaChrom, and a 100-5 C18 Kromasil column of 259 mm × 4.6 mm with a precolumn of the same characteristics, maintained at 20°C. Briefly, 0.02 mL previously filtered  and asymmetric dimethylarginine, ADMA (ADMA Direct (mouse/rat) ELISA Kit, Immundiagnostik), were determined by ELISA. Measurements were obtained following the instructions of each manufacturer.μm. Sections of hydrated and deparaffinized tissues were stained with hematoxylin and eosin (HE) and Masson's Trichrome, following the appropriate methods for histologic observation. From each aorta description, 10 sections were analyzed by 3 independent observers .Thoracic aorta obtained from ApoE−/− and C57BL6  mice were fixed in 10% formalin at pH 7.4. Once the samples were fixed, the dehydration, clarification, and inclusion protocol were carried out. After blocks were obtained, sections were obtained using a microtome (Microm HM325), with a thickness of 5 © CANON INC, USA, 2008) was used for image capturing. Later, they were quantified to determine the indirect presence of lipids as well as the degree of inflammation of the lesions, in terms of the presence of inflammatory cells; determining representative areas in each artery lesion with a dimension of 50 μm2, the software AxioVision Release 4.8.1 (Carl Zeiss version 11. 2008) was used for this purpose, and the software Micrometrics SE Premium  was used for counting inflammatory cells. The lesion surface was quantified by using the ImageJ software  to determine the degree of fibrosis. Each image was analyzed through the Colour Deconvolution Plugin, selecting the vector for Masson's Trichrome, determining the quotient (density/area), and allowing the characterization of each lesion [After the staining procedures, photographs (40x) were obtained with a Primo Star Carl Zeiss trinocular microscope used for this purpose and a Canon EOS Rebel XSI camera with the software EOS Utility . Comparisons between groups were done by the analysis of variance (ANOVA) using Graphpad software. A statistical difference was evaluated using a confidence interval of 95%, with Dunnett's and Newman-Keuls as post hoc tests.First, we prepared diets supplemented with apple products. P < 0.01). On the other hand, mice subjected to FD + HAP, FD + DAP, and FD + SDAP presented a lower weight gain when compared to the FD group.Next, we tested our diets in an animal model of MS, for 43 days. P < 0.01) than mice subjected to ND (79.14 ± 10.1 mmHg) (P < 0.01).Regarding blood pressure, the group of mice that received FD presented significantly higher SAP  . On the P < 0.05 versus ND), triglycerides , and glycemia  when compared to mice subjected to ND  (P < 0.001) was the one that presented lower levels of glycemia and the group FD + SDAP apple was the one which presented lower levels of triglyceridemia , when compared to FD group.Mice that received FD presented high levels of blood cholesterol  . Mice suThe group of mice that received FD (2.34 ± 0.37 ng/mL) presented significantly higher insulin levels than mice fed with ND (1.23 ± 0.25 ng/mL) . On the P < 0.001) , the FD + HAP group being the one that presented the lower ADMA levels.The group of mice subjected to FD presented higher serum ADMA levels than mice subjected to ND (< 0.001) , while mP < 0.01), FD (P < 0.001), and FD + HAP (P < 0.001) was higher than control group (WT). Only glycemia of FD group had a significant increase with respect to the control group (P < 0.05).Next, we tested our diet with apple peel supplement in the ApoE−/− mouse, a model of dyslipidemia and atherosclerosis. Total cholesterol, triglycerides, and basal glycemia were determined in mice fed with the three types of diets, and control group of C57BL6 (WT) mice were incorporated for this analysis. Total cholesterol of ApoE−/− mice fed on a ND (P < 0.01), and only basal glycemia of the FD group had a statistically significant difference compared to the ND group (P < 0.05)  .P < 0.05 . Also, mice on FD had higher body weight than mice on FD + HAP (P < 0.05).Besides, using ANOVA to compare the initial and final body weight of all groups, it could be determined that the increase in body weight of mice on FD and mice on FD + HAP compared to the initial body weight of mice on ND was statistically significant  . in vivo studies that have demonstrated that flavonoids present in apple decrease blood pressure [Mice that received FD show higher SAP than mice fed with ND. Mice subjected to FD+ HAP showed lower SAP compared to FD. These results are in agreement with otherpressure  and withpressure . Recentlpressure .Mice that consumed a FD showed an increase in glycemia, triglycerides, and cholesterol when compared to the ND group, while mice subjected to FD + HAP, FD + DAP, and FD + SDAP showed significantly lower levels of these parameters compared to the FD group. These results agree with the study by Zhao et al. (2004) , using tThe group of mice that received FD showed insulin levels significantly higher than the control group, and mice with FD + HAP showed insulin values significantly lower compared to the FD group. Gao et al. (2010)  demonstrStudies have shown that the intake of polyphenol-rich foods influences cardiovascular health and decreases the expression of P-selectin . Also, iWe found that mice subjected to FD showed higher ADMA levels when compared to the ND group, which indicates that endothelial dysfunction is associated with MS. Mice subjected to FD + HAP, FD + DAP, and FD + SDAP showed lower plasma concentrations of ADMA compared with the FD group, specially the FD + HAP group. Some authors have stated that there is a reciprocal relation between ADMA and insulin resistance, suggesting that when there are metabolic alterations leading to insulin resistance, plasma levels of ADMA increase and vice versa , 37. In With the data obtained in this study, it can be concluded that an apple peel enriched diet decreased significantly several MS criteria in CF-1 mice.It is well established that raw or processed fruit and vegetable consumption decreases the incidence of CVD . The mecIn the advanced atherosclerosis model obtained in ApoE−/− mice fed with FD + HAP, we observed a significant decrease of the fat content at the atheroma plaque, expressed as a lipid infiltration area. With these results, we can postulate that the apple peel supplement in diet may have a protecting effect in the advance of the atherosclerotic disease, directly at the fat content level of the plaque, reducing endothelial dysfunction and serum cholesterol. This effect is possibly due to stimulation of cholesterol catabolism, or inhibition of its intestinal absorption, correlating with previous reports in ApoE−/− mice , and SprAs a conclusion, apple peel consumption improves metabolic alterations associated with a fat-rich diet and also slowed the atherogenesis development, one of the most lethal consequences of a hypercaloric diet. Our results contribute to the concept of having a functional food from apple products with beneficial effects on risk factors for cardiovascular disease .


Neurocognitive assessment in HIV-infected patients in Regional Center Mureş was held between 01.01.2013-31.12.2013 on a group of 26 patients. Out of the 26 patients recruited and evaluated, all were included in the national database and 6 patients were recommended for comprehensive assessment at the “Victor Babeş” Hospital - Bucharest. Inclusion criteria: patients born between 1987-1991, at least 8 years of education, who had not been institutionalized, without psychiatric conditions.We used the following methods for neurocognitive screening of patients included in our study: International HIV Dementia Scale, Simioni questionnaire, Beck Depression Inventory, Patient’s Assessment of Own Functioning Inventory (PAOFI).Preliminary psychological evaluation in 26 patients showed no evidence of neurocognitive deficit. Out of the 6 patients who underwent comprehensive neurocognitive testing, 1 patient was diagnosed with neurocognitive deficit, obtaining lower values than the group’s average for most tested areas.It is necessary to validate a tool to identify the neurocognitive changes caused by HIV. The final results will be given by the National Screening Program.


Background & Aims. The currently available treatment for chronic hepatitis C (CHC) in children is costly and with much toxicity. So, predicting the likelihood of response before starting therapy is important. Methods. Serum adiponectin, vitamin D, and alpha-fetoprotein (AFP) were measured before starting pegylated-interferon/ribavirin therapy for 50 children with CHC. Another 21 healthy children were recruited as controls. Results. Serum adiponectin, vitamin D, and AFP were higher in the CHC group than healthy controls . In univariate analysis, serum adiponectin was significantly higher in responders than nonresponders (p < 0.0001) and at a cutoff value ≥8.04 ng/mL it can predict treatment response by 77.8% sensitivity and 92.9% specificity, while both AFP and viremia were significantly lower in responders than nonresponders, p < 0.0001 and p = 0.0003, respectively, and at cutoff values ≤3.265 ng/mL and ≤235,384 IU/mL, respectively, they can predict treatment response with a sensitivity of 83.3% for both and specificity of 85.7% and 78.6%, respectively. In multivariate analysis, adiponectin was found to be the only independent predictor of treatment response (p = 0.044). Conclusions. The pretreatment serum level of adiponectin can predict the likelihood of treatment response, thus avoiding toxicities for those unlikely to respond to therapy. Although chronic hepatitis C (CHC) in children is usually asymptomatic, it is often persistent and is a possible cause of morbidity in later life. Moreover, progressive liver disease, including cirrhosis, has been reported during childhood . Althoug interleukin-28B (IL28B) polymorphism in pediatric patients with CHC, one study suggested that IL28B genotype was not a strong predictor of SVR [ IL28B genotype as a predictor of treatment response [In spite of the extensive researches in this field, knowledge is still defective. On defining predictors of treatment response, many studies concentrated on virus genotype, gender, age, race, and fibrosis stage, among others. Nevertheless, all of these are nonmodifiable factors but only give a prediction about the likelihood of sustained virologic response (SVR). Moreover, some of them are expensive and invasive . On inver of SVR , while oresponse –12.Virological responses during therapy, such as rapid virologic response (RVR) and early virologic response (EVR), are widely used for predicting end of treatment response (ETR) and SVR . NonetheWe aimed to investigate some inexpensive, easy to perform, and noninvasive modifiable (as adiponectin and vitamin D) and nonmodifiable  factors which may have a relation to treatment response in children with CHC. If we found a significant relation to successful treatment, we can use them not just to predict the likelihood of treatment response but also to improve the SVR rates by modulating the modifiable ones. This knowledge can also constitute a base for later on researches when DAA became approved in pediatric age.This prospective cohort study included fifty children with CHC recruited from Pediatric Hepatology Department, National Liver Institute (a tertiary level institute), Menofiya University, Egypt, between June 2012 and June 2014. Their mean age was 11.46 ± 3.48 years and thirty three (66%) of them were males. Another 21 healthy children with comparable age  and sex  to the disease group were enrolled as controls. CHC was diagnosed on the basis of positive anti-HCV antibodies and positive HCV-RNA for more than 6 months duration, together with the pathological picture of CHC , was excAlanine transaminase (ALT), aspartate transaminase (AST), prothrombin time (PT), complete blood count (CBC), thyroid stimulating hormone (TSH), and serum autoantibodies  were performed for every patient.Viral markers  surface antigen, HBV core immunoglobulin (Ig)M, and IgG antibodies) were performed for all patients using chemiluminescence immunoassay . Real-time PCR for HCV-RNA was performed using Abbott m2000 real-time system  for every patient before starting therapy and at week 12 of therapy. For those who continued therapy, PCR was tested at weeks 24 and 48 of treatment and lastly 6 months after the end of treatment. The detection limit was 15 IU/mL.Abdominal ultrasound was performed using 2–5 MHz curved linear and 4–8 MHz linear transducers .An ultrasonographic-guided liver biopsy was performed for all patients using a true-cut needle, size 16 G. Biopsy specimens were fixed in formalin, embedded in paraffin, and finally the slides obtained were stained by hematoxylin and eosin (H&E), perls, and orcein for routine histopathological evaluation.Histological evaluation of chronic hepatitis was performed using Ishak et al.  scoring α-2b  at a dosage of 60 μg/m2/week subcutaneously and RBV orally at a dosage of 15 mg/kg/day on two divided doses. The duration of therapy for those who completed the course was 48 weeks. Virological responses of therapy were defined as reported by Ghany et al. [All children with CHC received Peg-IFNSerum samples were collected from every patient before starting Peg/RBV therapy and from healthy controls then stored at −80°C until used. Serum adiponectin level was determined by the Human Adiponectin (Acrp30) enzyme-linked immunosorbent assay (ELISA) kit . Serum vitamin D was measured using 25-OH Vitamin D Enzyme-Immunoassay (EIA) Kit  intended for the quantitative determination of the 25-OH vitamin D in plasma or serum. Lastly, serum AFP was measured by Quantikine, a human AFP EIA kit . Serum levels of the three test parameters were expressed as nanograms per milliliter (ng/mL).t-test or nonparametric Mann-Whitney U test according to the nature of the data. For qualitative data, significance was tested by Chi-square test or Fisher exact test. A multivariate analysis was performed using a binary logistic regression analysis for factors that significantly associated with treatment response on univariate analysis. Correlation was tested by Spearman test. The cutoff values for optimal clinical performance of adiponectin, vitamin D, AFP, and level of HCV-RNA for differentiation between responders and nonresponders were determined from the receiver-operating characteristic (ROC) curves. The diagnostic performance was presented as sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and accuracy. Results were considered significant if p value was <0.05. Statistical analysis was performed using SPSS, version 13 .Values were expressed as mean ± standard deviation (range) or number (percentage) of individuals with a condition. For quantitative data, statistical significance was tested by either independent samples 2. Level of hepatitis C viremia ranged from 8.04 × 103 to 6.23 × 106 with a mean of 6.28 × 105 ± 1.23 × 106 IU/mL. Forty-five (90%) patients with CHC showed mild stage of fibrosis and 5 (10%) cases showed moderate fibrosis. Most of (86%) CHC children had mild activity, while 7 (14%) cases had minimal activity. Laboratory parameters are shown in Body mass index of children with CHC was within average with a mean of 18.56 ± 2.79 kg/mp < 0.0001). Also, in spite of being insignificant, serum vitamin D and AFP were higher in the CHC group  than healthy controls   .p < 0.0001). On the other hand it was found that both AFP and viremia were significantly lower in the treatment responders  than in nonresponders  with p < 0.0001 and p = 0.0003, respectively. Lastly, vitamin D was found to be higher in the treatment responders (77.2 ± 46.6 ng/mL) than nonresponders (57.2 ± 53.9 ng/mL), with borderline significance, p = 0.076. Other studied pretreatment parameters showed no difference between responders and nonresponders (p = 0.044) (Pretreatment factors that could be associated with response to peg/RBV therapy were compared between treatment responders (SVR) and treatment nonresponders . It was sponders , while i= 0.044) .Cutoff points for variables showing significant associations with treatment response were analyzed by the ROC curves . For adir = −0.29 and p = 0.043) and level of viremia (r = −0.39 and p = 0.005), with no significant correlation with other studied parameters. Also, there was no significant correlation between AFP, vitamin D, and level of viremia and all other studied parameters . This may indicate the benefit of the anti-inflammatory role of adiponectin [α and ameliorates hepatic steatosis [In this work, the pretreatment serum level of adiponectin was significantly higher in the treatment responders (SVR) than nonresponders, and at a cutoff value of >8.04 ng/mL it can predict the treatment response by a sensitivity of 77.8% and a specificity of 92.9%. Zografos et al.  found thponectin  in thoseteatosis .Moreover, the significant negative correlation found in this work between serum adiponectin and viremia may indicate an antiviral role of adiponectin. On the other hand, it may suggest that HCV may directly affect adiponectin. This later concept was suggested by the study of Zografos et al.  who founAbdel Latif et al.  found thAccording to the previous results, not only can adiponectin be used as a reliable pretreatment predictor of treatment response in combination with other defined parameters but also it can be tried as an adjuvant therapy with peg/RBV especially for those with pretreatment lower serum levels. To prove this, it needs a well controlled clinical trial.p = 0.071). This difference from Lange et al. [Beside its action in calcium homeostasis, vitamin D has a significant immunomodulatory action and is an important mediator of innate and adaptive immune systems . In spite et al.  study mae et al.  who demoPetta et al.  found thVitamin D concentration has emerged recently as a new predictor of HCV treatment response. This novel predictor is of great interest because it is easily modifiable by supplementation . HoweverIn the present work, a higher vitamin D level was found in HCV treatment responders than nonresponders but with borderline significance. Similar to our results Petta et al. , BitettoHypothetically, in the presence of vitamin D deficiency, it might be preferable to correct the deficiency before starting antiviral therapy. Nevertheless, to date there are few published reports on the role of vitamin D supplementation in patients with CHC. Bitetto et al.  found thThe reported higher vitamin D among treatment responders in the present study together with the known immunomodulatory action of vitamin D  and prevSerum AFP is a fetal glycoprotein produced by the yolk sac and fetal liver. Following birth, AFP levels decrease rapidly to less than 20 ng/mL and increase significantly in certain pathologic conditions. Serum AFP is a routinely used marker for hepatocellular carcinoma . Yet, siIn agreement with the above reports, we found that AFP was higher in children with CHC than healthy controls with statistically insignificant difference. The absence of a significant correlation between AFP and stage of fibrosis in the present work may be due to the milder liver affection with low stages of fibrosis in reported cases when compared to the adult series.Some studies found significant correlation of pretreatment low AFP serum level and treatment response in adults with CHC –39. In a per se with selective transcriptional activation of the AFP gene [p = 0.043) is supporting this hypothesis and may give an explanation to the link between the baseline serum AFP and the treatment response.The link between AFP and treatment response needs further studies. Increased production of AFP in hepatitis and cirrhosis was first thought to reflect the process of surviving hepatocytes, but this hypothesis has been refuted by other reports , 40. MorAFP gene . In the AFP gene  and AFP In the present study viral load in univariate analysis was found to be a significant pretreatment predictor of treatment response as it was shown in previous reports . However IL28B genotype could be considered another limitation of this work.The emergence of the new DAAs for the treatment of CHC , 42 may In conclusion, serum adiponectin can be added to the list of the pretreatment determinants of SVR in children with CHC, with the advantage of being easy to perform, noninvasive, and modifiable. In spite of its significant prediction for the likelihood of response, we cannot state to screen and select the patients to be treated or not based on their pretreatment levels of this predictor. Instead, it can be used to prioritize patients to treatment when resources are limited, thus avoiding toxicities and cost for those unlikely to respond to treatment. Also as it is a modifiable factor, supplementation for deficient cases could be considered before starting treatment if this could be proved in further studies.