To investigate the risk factors involved in radial-femoral artery pressure gradient after cardiac surgery.In this retrospective study, we reviewed 412 cardiac surgeries with both femoral artery pressure and radial artery pressure monitoring before cardiopulmonary bypass. 138 patients had radial-femoral artery pressure gradient after cardiopulmonary bypass (group P) but 263 were not (group N). Their hemodynamic data and other demographic data were analyzed.2 in group P and 2.1±0.3, 2.8±0.5,2.8±0.5, 2.8±0.5 L/min/m2 in group N at baseline, after discontinuation of CPB, at the end of surgery and the first postoperative day (P<0.001).Phenylephrine usage was 1.7±1.1 mg in group N and 2.9±1.2 mg in group P (P<0.001). Total adrenaline usage was 229.2±116.9 µg in group N and 400.6±145.1 µg in group P (P<0.001). SBP gradient was -4±3, 14±9, 10±4, 0±11 mmHg in group P and -3±3, 0±1, -1±9, -6±4 mmHg in group N after induction, during discontinuation of CPB, at the end of surgery and 1 postoperative day respectively. DBP gradient was 3±3, -1±9, 4±5, 0±8 mmHg in group P and 3±3, 5±2, 7±5, 0±8 mmHg in group N after induction, during discontinuation of CPB, at the end of surgery and 1 postoperative day respectively. MAP gradient was 1±2, 4±6, 6±4, 0±8 mmHg in group P and 1±2, 3±1, 1±4, -2±5 mmHg in group N after induction, during discontinuation of CPB, at the end of surgery and 1 postoperative day respectively. Significant arterial pressure gradient emerged during discontinuation of CPB and at the end of surgery, which was more obvious in group P(P<0.01). CI was 2.0±0.3, 2.3±0.4,2.3±0.4, 2.2±0.4 L/min/mDetecting the exact central artery pressure is most important when patients have artery pressure gradients after cardiac surgery. Use inotropic agents to improve cardiac output, avoiding excessive vasoconstriction might reduce artery pressure gradient. Direct intra-radial arterial pressure monitoring is often used in cardiac surgery because of the possible intensity of the hemodynamic changes as well as the need to assess the detailed hemodynamic parameters during the operation. However, a central-radial artery pressure gradient may happen after cardiopulmonary bypass (CPB) surgery, which in some patients may last for a significant long time after CPB. Systemic vascular resistance will be underestimated and therapeutic strategy is possible to be misleading in condition of significant artery pressure gradient . To knowThe exact mechanism of central-radial artery pressure gradient is still to be determined. Some prospective studies reported that there was no relationship between the magnitude of the pressure gradient and type of cardioprotection, bypass duration, temperature and systemic vascular resistance ,3. Howevst, 2002 to Dec 31st 2012 were screened and those patients with both direct femoral and radial artery pressure monitoring were collected in our research.Our research protocol was approved by the institutional review board and institutional review board waived the need for written informed consent from the participants . The cardiac anesthesia case records from Jan 1In this retrospective study, 412 patients received both femoral artery pressure and radial artery pressure monitoring before cardiopulmonary bypass. Of which, 11 patients were excluded because of a prior radial-femoral artery pressure gradient and the rest of patients did not have radial-femoral artery pressure gradient before the surgery. 263 patients did not have radial-femoral artery pressure gradient after CPB (Group N). 138 patients had radial-femoral artery pressure gradient after cardiopulmonary bypass (Group P) , pulmonary vascular resistance index (PVRI), cardiac index (CI), heart rate (HR), central venous pressure (CVP), pulmonary artery pressure (PAP), and pulmonary capillary wedge pressure (PCWP) were also monitored, calculated, and compared.The genders, ages, heights, weights, preoperative cardiac function, history of hypertension or diabetics, echo report about LVEDd (left ventricle end diastolic diameter), EF , pulmonary hypertension, surgical type, clamping time, total bypass time, operation duration, vasoconstriction usage and lowest core temperature during CPB were recorded and compared between the two groups. Systolic, diastolic, and mean artery pressure in radial and femoral arteries were also recorded at baseline, during discontinuation of CPB, at the end of surgery and 12 monitoring. Swan-Ganz catheter was also placed to monitor PVRI, CI, HR,CVP, PAP, and PCWP. Artery blood pressure was about baseline to 20% lower and heart rate was 60-90bpm during the surgery. The target mean artery pressure was set to 60 mmHg and the target heart rate was 90 bpm during the weaning of CPB. Fluid therapy and adrenaline were administered according to hemadynamic data such as MAP, HR, CO, CVP, PCWP, SVRI and so on. During cardiopulmonary bypass, phenylephrine was used if MAP was less than 50 mmHg in condition of adequate flow rate. All patients were regulated to normal homeostasis such as acid–base balance, electrolytes, PaO2 and PaCO2 during the entire surgical period.Every patient received intravenous and inhalational anesthesia. Patients were induced with midazolam (0.1mg/kg), etomidate (0.2-0.3mg/kg), vecuronium (0.15mg/kg), and fentanyl (3-5 μg/kg), and maintained with isoflurane (1-2MAC), atracurium (0.5mg/kg/h) and intermittent fentanyl . Every patient received invasive artery pressure, ECG, SpOP-value less than 0.05 was considered statistical significant.All the data were presented as means ± SD. Statistical analysis was completed by SPSS 13.0 . The qualitative data were compared with Pearson chi-square analysis or Fisher’s exact test and the quantitative data were compared with one way ANOVA. The hemodynamic data were compared with repeated measures variance analysis. With respect to the incidence of central-peripheral pressure gradient was 50% during discontinuation of CPB and permitted error was 5%, 400 patients will be necessary to reach a statistic power of 95%. We chose totally 401 cardiac patients since those patients received operations in a certain period.263 patients without femoral and radial artery pressure gradient were defined as group N. 138 patients with femoral and radial artery pressure gradient were defined as group P. Univariate analysis revealed that there was no significance in the gender, age, height, weight, NYHA function, history of hypertension, history of diabetics, LVEDd, EF value, pulmonary hypertension, surgical types, clamping time, total bypass time, operation time and lowest core temperature in the two groups. However, Phenylephrine usage during CPB was 1.7±1.1 mg in group N and 2.9±1.2 mg in group P(P<0.001). Total adrenaline usage was 229.2±116.9µg in group N and 400.6±145.1µg in group P (P<0.001)  and higher PVRI (P<0.001) immediately after discontinuation of CPB to the first postoperative day. CI was lower in group P right after discontinuation of CPB, at the end of surgery and the first postoperative day compared with group N (P<0.001). CVP was higher in group P than that in group N at baseline and the first postoperative day. There was no difference in HR, PCWP or PAP between the two groups at any time point (In our study, we demonstrated that radial-femoral pressure gradient, especially at the discontinuation of CPB was associated with constriction agents, cardiac index, SVRI, PVRI. There have been a couple of clinical studies investigated artery pressure gradient after cardiopulmonary bypass prospectively. Some of them found that the pressure gradient was associated with bypass time, body temperature, and catecholamine levels –7, but oArtery pressure wave is based on the cardiac output and artery elasticity. Previous studies found that the artery pressure gradient was due to the artery elasticity ,9. In ouIncreased SVRI and PVRI contributed to significant pressure gradient. It was consistent with those studies in which vasodilating therapy was beneficial to artery pressure gradient ,11. NormLiteratures ,18 and oWe regard femoral artery pressure as the central artery pressure because femoral artery pressure is more similar to the central artery pressure than radial artery pressure. And also previous researches have successfully investigated femoral artery to radial artery pressure gradient as central-peripheral pressure gradient after cardiopulmonary surgeries ,16,19,20Our reviewed patients had both femoral artery and radial artery pressure monitoring, which indicated that these patients may have some prior problems such as weaker radial pulsation, poor peripheral vascular perfusion, or difficult radial artery catheter placement. So these patients might be easier to present artery pressure gradient after cardiopulmonary bypass. That’s probably why there were many risk factors in our results and other prospective or retrospective studies did not.In conclusion, when patients have artery pressure gradient after cardiac surgery, detecting the exact artery pressure is most important. Real artery pressure makes it possible to calculate the exact hemodynamic parameter. Inotropic agents, avoiding vasoconstriction might ameliorate artery pressure gradient.


Highly active antiretroviral therapy (HAART) has being impacted significantly therapies for natural human immunodeficiency virus (HIV) infection, which leads to a remarkable decrease in its morbidity and mortality but it is frequently associated with metabolic complications such as dyslipidaemia and cardiovascular complications. HIV reverse transcriptase (RT) inhibitors can be classified into nucleoside and non-nucleoside types. Mitochondrial dysfunction due to the depletion of mt-DNA is partly responsible for various nucleoside RT inhibitors-associated adverse effects including dyslipidaemia. Efavirenz (EFV) is metabolized primarily by cytochrome P450 2B6 (CYP2B6) and the metabolic effects of EFV have been described previously. All patients in this study received the same HAART treatment regime (Stavudine (d4T) + Lamivudine (3TC) + Efavirenz (EFV)). This study aims to assess incidences for dyslipidaemia and atherosclerosis.P<0.001), total cholesterol (4.96 ± 0.46 mmol/L Vs. 6.15 ± 0.83mmol/L) (P<0.001) and LDL cholesterol (2.29 ± 0.33 Vs. 3.11 ± 0.29 mmol/L) (P<0.001). In contrast, the level of HDL cholesterol did not significantly change (1.06 ± 0.01 mmol/L Vs. 1.04 ± 0.01 mmol/L) (P>0.5). The mean IMT of twenty-two patients was (0.86 ± 0.14) mm after HAART, which is higher than the norm age-matched value of (0.7 ± 0.2) mm (P<0.05).This retrospective study was conducted within outpatients of Shanghai Public Health Center. We selected thirty-five HIV-1 infected patients who receiving highly active antiretroviral therapy. Their mean CD4 cell count was 69.5 (±34.6) copies per micro liter before therapy. Fasting total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol values were respectively compared among patients at present and before therapy. Then the data was statistically analyzed. Twenty-two patients had the intima-media thickness (IMT) of their carotid arteries measured by Philips 5000 Color-Doppler ultrasound tests. Results: After therapy, patients achieved significant changes in levels of triglycerides (1.44 ± 0.35mmol/L Vs. 2.07 ± 0.54mmol/L) (P<0.001), total cholesterol (4.96 ± 0.46 mmol/L Vs. 6.15 ± 0.83mmol/L) (P<0.001) and LDL cholesterol (2.29 ± 0.33 Vs. 3.11 ± 0.29 mmol/L) (P<0.001). In contrast, the level of HDL cholesterol did not significantly change (1.06 ± 0.01 mmol/L Vs. 1.04 ± 0.01 mmol/L) (P>0.5). The mean IMT of twenty-two patients was (0.86 ± 0.14) mm after HAART, which is higher than the norm age-matched value of (0.7 ± 0.2) mm (P<0.05).After therapy, patients achieved significant changes in levels of triglycerides (1.44 ± 0.35mmol/L Vs. 2.07 ± 0.54mmol/L) (These data suggest that HAART is potentially dangerous for hyperlipidaemia and maybe an increase in atherosclerosis. Acquired Immune Deficiency Syndrome (AIDS) has become one of the serious challenges in China. As of the end of September 2008, China has an accumulative report of more than 260 thousand cases for HIV-infection, of which 77 thousand cases are AIDS patients and death cases reach 34 thousand. In recent years, the opportunistic infections associated with AIDS have been reduced significantly by virtue of Highly Active Antiretroviral Therapy (HAART). However, dis-regulations on endocrine system and metabolism in patients receiving HAART have been reported frequently . HAART dAll 35 patients were confirmed in Shanghai Center for Disease Control and Prevention and initiated medications according to AIDS Diagnosis Standard issued by the American Centers for Disease Control and Prevention (US-CDC). They all received the same HAART regime (d4T+3TC+ EFV) in our Center as well as a long-term follow-up. The mean age was 38.5 (±15.3), mean length of therapy was 24.6 (±17.5) months from Sept 2001 to Sept 2005 and mean CD4 cell count was 69.5 (±34.6) copies per micro liter before therapy.Fasting blood lipids of every patient pre-therapy and post-therapy were tested: TC by the CHOD-PAP method and TG by the GPO-PAP method; HDL and LDL were tested by deposition. The coefficients of variation for cholesterol and triglyceride values are within 3% and 5%, respectively. Of all patients 22 received Philips 5000 Color-Doppler ultrasound voluntarily in the last assessment in the study.Chinese standard for hyperlipidaemia diagnosis was issued in 1997 . Total TP<0.05. Additionally, single-factor analysis was applied to the blood lipids analysis before and after antiretroviral therapy and t-test was used to compare the mean figures.Data were assessed by mean ± standard deviation and analyzed with SPSS10.0 statistical analysis software. It will be was considered significant for P<0.001). The mean triglyceride level of 35 patients before therapy (d4T + 3TC + EFV) was 1.44 ± 0.35 mmol/L, which was less than the normal level according to diagnosis standard; but it rose to 2.07 ± 0.54 mmol/L exceeding the normal level at the end of this study.Triglyceride: There was significant difference between triglyceride levels before and after HAART (P<0.001). The mean level was 4.96 ± 0.46 mmol/L before therapy, which increased to 6.15 ± 0.83 mmol/L during therapy.Total cholesterol: There was also significant change between cholesterol levels before and after HAART (P>0.5), being 1.06 ± 0.01 mmol/L before therapy and 1.04 ± 0.01mmol/L after.HDL: HDL cholesterol did not significantly change (P<0.001) from 2.29 ± 0.33 mmol/L to 3.11 ± 0.29 mmol/L.LDL: LDL cholesterol changed significantly (P<0.05)  mm, which was thicker than the normal value in age-matched controls (0.7 ± 0.2) mm  are converted to active triphosphate analogues and incorporated into DNA in RT-catalyzed reactions. They act as chain terminators for blocking DNA synthesis since they lack the 3′-OH group required for the phosphor-diester bond formation. Mitochondrial dysfunction due to the depletion of mt-DNA is partly responsible for various NRTI-associated adverse effects including myopathy, cardiomyopathy, peripheral neuropathy, pancreatitis, hepatic steatosis, lipodystrophy and lactic acidosis in severe cases . It has Detection of increased IMT of carotid arteries by ultrasound can predict atherosclerotic coronary artery disease , 8. It i


Vigna angularis) was extracted further with CH2Cl2, EtOAc and n-BuOH to afford four fractions: CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. The EtOAc-soluble fractions showed the highest α-glucosidase inhibitory activity. Two pure flavonoid compounds, vitexin and isovitexin, were isolated (using the enzyme assay-guide fractionation method) from the EtOAc-soluble fractions. We further evaluated the interaction between the flavonoid compounds and α-glucosidase by fluorescence spectroscopy. Vitexin and isovitexin showed high inhibitory activities, with IC50 values of 0.4 mg·mL−1 and 4.8 mg·mL−1, respectively. This is the first study of the active compositions of azuki beans against α-glucosidase.A 70% ethanol extract from azuki beans ( Interest in glucosidase inhibitors is growing because of its implications for the management of diabetes mellitus (DM). DM is a serious metabolic disorder that affects approximately 4% of the population worldwide and is expected to increase to affect 5.4% by 2025 . Acting et al. +.2): yellow powder. 1H NMR  d: 13.55 , 7.93 , 6.93 , 6.78 , 6.51 , 4.58 , 4.03–3.11 . 13C NMR (DMSO d6) d: 181.9 (C-4), 163.5 (C-2), 163.3 (C-7), 161.6 (C-4′), 160.6 (C-5), 158.2 (C-9), 128.4 , 121.1 (C-1′), 116.06 , 108.9 (C-6), 103.4 (C-10), 102.8 (C-3), 93.64 (C-8). Positive ESI-MS: m/z 433 [M + H]+.Isovitexin , followed by isovitexin (IC50 of 4.8 mg·mL−1).To determine the α-glycosidase inhibition ability 0 values . The EtOFluorescence quenching can be divided into two types: dynamic quenching and static quenching. Dynamic quenching stems from the collision between two fluorescent luminophors, while static quenching arises from the formation of a new nonfluorescent complex that forms between the fluorescent luminophors and quencher . DynamicF0 and F are the fluorescence intensities of the fore-and-aft interaction between α-glucosidase and flavonoid, [Q] is the concentration of quencher and flavonoid, and τ0 is the average life of the fluorescent substance without the quencher, valued at approximately 10−8 s. Ksv and Kq are the dynamic quenching constant and rate constant in the process of double molecule quenching [where uenching .Ksv and Kq were obtained with the Stern-Volmer equation from plots of linear equations obtained by F0/F vs. [Q]. The values of Ksv decreased with the increase of temperature, and Kq was greater than 2.0 × 1010  is higher than the isovitexin constant (n) was close to one at 37 °C, which is the most suitable temperature for the flavonoid molecules to bind with α-glucosidase.The vitexin binding constant . Rat intestinal acetone powder was purchased from Sigma-Aldrich . Acarbose was purchased from Bayer Health Care Pharmaceuticals, Inc. (USA). All chemicals used were of analytical grade and were obtained from Beijing Chemical Reagent . Silica gel (200–300 mesh) for column chromatography was purchased from Qingdao Marine Chemical Company . Sephadex LH-20 was purchased from GE Healthcare .2Cl2, EtOAc and n-BuOH to offer four extracts: the CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. Each extract was evaporated to dryness under reduced pressure, while the residual extract fraction was frozen to dryness. Therefore, five extracts were obtained in total. A small amount of each fraction was redissolved in 50% dimethyl sulfoxide (DMSO), and these mixture solutions were subjected to α-glucosidase inhibitory activity assays.Dried Azuki beans (3.0 kg) were crushed and twice extracted with 70% ethanol (3 × 10 L) for 2 h at 60 °C. The extracts were combined and concentrated under vacuum at 50 °C. Then, the concentrated extracts were partitioned with CH2O system as the eluent, and the polarity of the eluent was increased by increasing the ratio of EtOAc during the process. The separation was monitored by TLC, and four fractions were obtained. Fraction 3 [EtOAc:MeOH:H2O = 8:1:0.2 (v:v:v)] showed strong inhibitory activities against α-glucosidase. A further separation was completed using a combination of Sephadex LH-20 column chromatography, with MeOH as the eluent, and reversed-phase TLC to monitor the isolation.The EtOAc-soluble fraction (25 g) was subjected to a silica gel chromatography column, using an EtOAc/MeOH/H−1) was assayed using 50 μL of extracts with varying concentrations incubated with 100 μL of 0.1 M phosphate buffer (pH 7.0) in 96-well plates at 37 °C for 10 min. After preincubation, 50 μL of 5 mM p-nitrophenyl-α-dglucopyranoside solution in 0.1 M phosphate buffer (pH 7.0) was added to each well at varying time intervals. The reaction mixtures were incubated at 37 °C for 5 min. The absorbance readings were recorded at 490 nm on a microplate reader before and after incubation . The results were expressed as a percent of α-glucosidase inhibition and calculated according to the following equation:The α-glucosidase inhibitory activity was determined as previously described with slight modifications ,15. The 50 value was defined as the concentration of bean extracts (acarbose) required to inhibit 50% of the enzyme activity.The ICet al. [−1, pH 6.8, with 0.1 mol·L−1 NaCl), and vitexin (or isovitexin) was dissolved in 60% ethanol. For the FS measurement, a solution of 1.0 mL of α-glucosidase was added to a fluorescence cuvette at a given temperature and titrated with flavonoid for 5 min. Fluorescence spectra of the α-glucosidase and α-glucosidase-flavonoid mixture were recorded in the range from 315 to 500 nm. The slits for both excitation and emission were set at 10 nm with an excitation wavelength of 295 nm and an optical path of 10 mm .The fluorescence spectra were determined using the method reported by Li et al. . The α-gIn conclusion, two major active components, vitexin and isovitexin, were isolated from the azuki bean. There is a static quenching interaction between flavonoid compounds and α-glucosidase, and the most suitable temperature is 37 °C.


SD: 36 ± 8 to 45 ± 11 ms), corresponding to a major drop of z scores for the 2 patients from 7.12 ± 0.69 to 1.28 ± 1.27 (sham procedure: 6.79 ± 0.71). This is the first demonstration that tCCDCS improves upper limb tremor and hypermetria in SCA type 2. In particular, this is the first report of a favorable effect on the onset latency of the antagonist EMG activity, a neurophysiological marker of the defect in programming of timing of motor commands. Our results indicate that tCCDCS should be considered in the symptomatic management of upper limb motor deficits in cerebellar ataxias. Future studies addressing a tDCS-based neuromodulation to improve motor control of upper limbs are required (a) in a large group of cerebellar disorders, and (b) in different subgroups of ataxic patients. The anatomical location of the cerebellum below the skull is particularly well suited for such studies.Cerebellar ataxias represent a very heterogeneous group of disabling disorders for which we lack effective symptomatic therapies in most cases. There is currently an intense interest in the use of non-invasive transcranial DC stimulation (tDCS) to modulate the activity of the cerebellum in ataxic disorders. We performed a detailed laboratory assessment of the effects of transcranial cerebello-cerebral DC stimulation  on upper limb tremor and dysmetria in 2 patients presenting a dominant spinocerebellar ataxia (SCA) type 2, one of the most common SCAs encountered during practice. Both patients had a very similar triplet expansion size in the ATXN2 gene . tCCDCS reduced both postural tremor and action tremor, as confirmed by spectral analysis. Quadratical PSD  of postural tremor dropped to 38.63 and 41.42% of baseline values in patient 1 and 2, respectively. The integral of the subband 4–20 Hz dropped to 46.9 and 62.3% of baseline values, respectively. Remarkably, tCCDCS canceled hypermetria and reduced dramatically the onset latency of the antagonist EMG activity associated with fast goal-directed movements toward 3 aimed targets . Following tCCDCS, the latency dropped from 108–98 to 63–57 ms in patient 1, and from 74–87 to 41–46 ms in patient 2 (mean control values ±  Cerebellar ataxias represent a very heterogeneous group of sporadic and genetic disabling diseases  to modulate cerebellar functions , neurological examination showed impaired horizontal saccades  and decreased tendon reflexes in 4 limbs. Detailed sensory examination  and evaluation of motor strength  were normal in upper limbs. Hoffmann's reflex was negative. Brain MRI showed atrophy of the cerebellum and brainstem, extending to the cervical segments of the spinal cord   they both presented deficits of motor control in upper limbs characterized by a postural/action tremor steady in amplitude and dysmetria , (b) they both exhibited a delayed onset latency of the antagonist EMG activity associated with fast goal-directed movements , and (c) the size of the triplet expansion was very similar.The study was approved by the local ethical committee of ULB and both patients signed a written informed consent following full explanation of the experimental procedures.Data were collected on right upper limb in 3 experimental conditions: (1) at baseline, (2) after a sham stimulation , and (3) after tCCDCS . The rationale for this design baseline-sham-tDCS in cerebellar disorders has been explained elsewhere  and dysmetria were studied at baseline, post-sham and post-tCCDCS. We did not assess kinetic tremor because it was more variable in our patients as compared to postural and action tremor which were both steady.An ataxia rating scale  was applied at baseline, immediately after sham and immediately after tCCDCS.SD) of the 3 recordings performed in each experimental condition.Patients were comfortably seated in an armchair, and equipped with 2 triaxial accelerometers, located at the right index finger and at the right metacarpal region . Patients were asked to keep the manipulandum motionless during 16 s. The manipulandum exerted a constant extensor torque of 0.5 Nm from the neutral position, thus eliciting an action tremor. Patients were allowed to perform 5 practice trials before recordings. This number of practice trials is based on our experience with ataxic patients . Sampling rate for position signal was 2048 Hz. We extracted the short-time Fourier transform (STFT) using Igor Pro 6.01 software . The 16 s time-series were filtered (IIR high-pass 2 Hz) and segmented in 8 segments. Auto-spectra of 8 sequential data epochs of 2 s were averaged. We computed maxPSD, PFr, CF in the 4–20 Hz frequency sub-band, integrals of frequency sub-bands . Because cerebellar action tremor is often more severe at the frequency of 3 Hz, we also extracted the tremor data in the sub-band 2.5–3.5 Hz by applying a corresponding band-pass IIR filter of order 4. The RMS was also computed on the filtered data.Cerebellum is well known to contribute to timing of motor commands and is considered as a “clock” in the brain  signal.qk is the frequency probability of baseline signal.n = 8 right-handed healthy subjects, mean age ± SD: 34.8 ± 10.2 years; 3 women—values of this control group were used to compute z scores in the 3 experimental conditions) . For fast goal-directed movements in the 3 conditions , mean movement amplitudes and onset latencies of antagonist EMG activities were compared to data obtained in a control group reported earlier (see previous section) and expressed as z scores  3, Stance (St) 2, Sitting (Si) 4, Speech (Sp) 3, Finger chase (Fc): right 2 and left 2 (mean 2), Nose-finger test (Nf): right 1 and left 1 (mean 1), Fast alternating movements (Fa): right 1 and left 1 (mean 1), Heel-shin slide (Hs) right 2 and left 2 (mean 2). Total: 18;     -after sham: G3, St2, Si2, Sp3, Fc: right 2 and left 2 (mean 2), Nf: right 1 and left 1 (mean 1), Fa: right 1 and left 1 (mean 1), Hs: right 2 and left 2 (mean 2). Total: 16;     -after tCCDCS: G3, St2, Si2, Sp3, Fc: right 1 and left 2 (mean 1.5), Nf: right 1 and left 1 (mean 1), Fa: right 0 and left 1 (mean 0.5), Hs: right 2 and left 2 (mean 2). Total: 15.     In patient 2, the SARA rating score was:     -at baseline: G3, St2, Si1, Sp3, Fc: right 1 and left 1 (mean 1), Nf: right 1 and left 1 (mean 1), Fa: right 1 and left 1 (mean 1), Hs: right 1 and left 1 (mean 1). Total: 13;     -after sham: G2, St2, Si2, Sp3, Fc: right 1 and left 1 (mean 1), Nf: right 1 and left 1 (mean 1), Fa: right 1 and left 1 (mean 1), Hs: right 1 and left 1 (mean 1). Total: 13;     -after tCCDCS: G2, St1, Si1, Sp3, Fc: right 0 and left 1 (mean 0.5), Nf: right 1 and left 1 (mean 1), Fa: right 0 and left 0 (mean 0), Hs: right 1 and left 1 (mean 1). Total: 9.5.z score: 2.97 as compared to baseline). The analysis of quadratical PSD of all epochs showed a drop of the peak PSD from 20.4 a.u. at baseline (post-sham: 23.0 a.u.) to 7.9 a.u. in the condition post-tCCDCS  and to 29.23 ± 3.92 a.u. post-tCCDCS . CF decreased from 0.47 ± 0.04 to 0.39 ± 0.06 , and to 0.28 ± 0.1 a.u. post-tCCDCS (z score: −3.71 as compared to baseline). Center frequency showed a trend to increase from 7.39 Hz  to 8.6 Hz (post-tCCDCS) (z score: 2.36 as compared to baseline). Quadratical RMS changed from 10.36 ± 2.65 a.u.  to 11.38 ± 0.27 a.u.  and to 4.34 ± 0.48 a.u. post-tCCDCS (z score: −2.22 as compared to baseline)  and to 37.08 ± 4.94 a.u. post-tCCDCS (z score: −3.68 as compared to baseline). PFr, CF, and center frequency showed no significant changes in patient 2. Quadratical RMS changed from 13.0 ± 2.40 a.u.  to 16.10 ± 1.75 a.u. post-sham (z score: 1.28 as compared to baseline) and to 5.19 ± 0.44 a.u. post-tCCDCS (z score: −3.25 as compared to baseline)  to 0.00695 a.u. (post-sham: 90.3% as compared to baseline) and to 0.00361 a.u. (post-tCCDCS: 46.9% as compared to baseline). At baseline, the STFT study showed that the oscillations were predominantly found below 5 Hz Figure . AlthougFor patient 2, the action tremor had a PFr of 7, 7.05, and 1 Hz, respectively, at baseline, after sham and after tCCDCS. The integral in the subband 4–20 Hz changed from 0.01024 a.u. (baseline) to 0.01349 a.u. post-sham (131.7% as compared to baseline) and to 0.00638 a.u. post-tCCDCS (62.3% as compared to baseline). The STFT study also confirmed a favorable effect of tCCDCS on frequencies below 5 Hz, although at a lower extent as compared to patient 1 : 7.12 ± 0.69 at baseline, 6.79 ± 0.71 post-sham (95.5% as compared to baseline), and 1.28 ± 1.27 post-tCCDCS (18.8% as compared to baseline).In both patients, fast goal-directed movements were markedly hypermetric at baseline and following application of the sham procedure . Still, no difference was found for action tremor: median values (±SD) were 0.153 (±0.279) and 0.066 (±0.28) in sham/basal vs. tCCDCS/basal, respectively (p = 0.059). For patient 2, relative entropies were similar for postural tremor  and for action tremor .In both patients, we found a strong overlap in terms of relative entropy of tremor Figure . HoweverOur observation highlights a novel way to modulate cerebellar motor functions in a neurological disorder manifesting with a predominant cerebellar syndrome. This is the first demonstration that tCCDCS could be considered as a symptomatic therapeutic strategy of upper limb deficits in a disabling cerebellar ataxia. In our 2 patients, tCCDCS improved clinically the voluntary movements of the upper limbs, without affecting oculomotor disturbances, dysarthria and ataxia of stance/gait as confirmed by the ataxia rating scale. Interestingly, both patients reported a feeling of motor improvement after tCCDCS.The excitability of the cerebellar circuitry is impaired in numerous cerebellar disorders, as a result of the degenerative process occurring in the cerebellum and its afferences  the critical roles of the cerebellum in the programming of timing and sequencing of motor operations, and (b) the major contributions of cerebellar circuits in the so-called Guillain-Mollaret triangle implicated in tremor genesis  has been reported in a patient with SCA2 (Freund et al., whole cerebellum DCS” strategy in cerebellar ataxias. The mechanisms and duration of the after-effects deserve attention (Márquez-Ruiz et al., Although our results are very encouraging, there are limitations to our study: our number of patients is small, we did not address the pathogenesis of other cerebellar deficits such as gait deficits and we did not investigate specifically the dynamics of the duration of the after-effects. Future studies should be performed in larger groups of ataxic patients not only to confirm our findings in the numerous forms of cerebellar ataxias, but also to identify the categories of ataxic patients which can be considered as potential responders. tDCS is non-invasive and more extensive electrodes covering the skull over the posterior fossa can be envisioned. These electrodes might have the advantage of modulating the entire cerebellar cortex and could be characterized by a spatial map allowing a dynamic application of currents, with the intensity of currents being adapted to the region of the cerebellum targeted by tDCS as well as to the task. Future studies are required to validate this “Cerebellum is part of a distributed cognitive network and its neuromodulation influences the contribution of cerebellar circuits, especially for linguistic processes, working memory tasks and processing of emotions (Ferrucci et al., Overall study design and protocol development: Giuliana Grimaldi, Nordeyn Oulad Ben Taib, Mario Manto. Data analysis: Giuliana Grimaldi, Mario Manto, Florian Bodranghien. Writing of manuscript: Giuliana Grimaldi, Nordeyn Oulad Ben Taib, Mario Manto, Florian Bodranghien. Final version reviewed and approved by all the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptomyces species, i.e. Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T. Chemical, MS and NMR analyses revealed a polyglycerolphosphate backbone substituted with α-glucosaminyl and α-N-acetyl-glucosaminyl residues but devoid of any amino-acid substituent. This structure is very close, if not identical, to that of the wall teichoic acid of this organism. These data not only contribute to the growing recognition that lipoteichoic acid is a cell envelope component of Gram positive Actinobacteria but also strongly support the recently proposed hypothesis of an overlap between the pathways of lipoteichoic acid and wall teichoic acid synthesis in these bacteria. S. hygroscopicus lipoteichoic acid induced signalling by human innate immune receptor TLR2, confirming its role as a microbe-associated molecular pattern. Its activity was partially dependant on TLR1, TLR6 and CD14. Moreover, it stimulated TNF-α and IL-6 production by a human macrophage cell line to an extent similar to that of Staphylococcus aureus lipoteichoic acid. These results provide new clues on lipoteichoic acid structure/function relationships, most particularly on the role of the polyglycerolphosphate backbone substituents.Gram positive bacteria produce cell envelope macroamphiphile glycopolymers, i.e. lipoteichoic acids or lipoglycans, whose functions and biosynthesis are not yet fully understood. We report for the first time a detailed structure of lipoteichoic acid isolated from a  Agromyces species Thermobifida fuscaStreptomyces species would also produce LTA Staphylococcus aureus, lacks clear orthologues in the sequenced actinobacterial genomes Streptococcus pneumoniaeGram positive cell envelopes are characterized by the presence of cell-wall glycopolymers that are attached either to peptidoglycan or to membrane lipids. Lipid-linked glycopolymers are referred to as macroamphiphiles S. aureus, however its amount can be reduced by 90% with only minor phenotypic changes during in vitro cultivation Streptococcus surface plays a role in bacterial interaction with blood-brain barrier endothelium and the pathogenesis of neonatal meningitis S. aureusvia Toll-like receptor 2 (TLR2), a receptor that plays a crucial role in detecting invading Gram-positive bacteria. TLR2-stimulating activity of purified LTA has been a matter of controversy, contamination of LTA fractions by highly active lipopeptides being formally difficult to rule out S. aureus strain lacking lipoproteins The physiological roles of LTA are not yet fully understood. LTA is indispensable in Streptomyces species. Its capacity to induce TLR2 signaling and to stimulate cytokine production was investigated. Altogether, our results provide new clues on LTA biosynthesis in Actinobacteria and LTA structure/function relationships.In the present study, we report for the first time a detailed structural model of LTA isolated from a S. hygroscopicus NRRL 2387 cells were thus extracted by the more gentle butanol procedure introduced by Morath et al.Streptomyces species S. aureus (SaLTA)  and glycerol-phosphate (Gro-P) were detected by gas chromatography (GC)/MS suggesting a polyglycerolphosphate LTA (PGP-LTA). The compound was subsequently termed ShLTA. The predominant fatty acids detected by GC after alkaline hydrolysis were C16:0 (40%), isoC16 (11%), isoC17:0 (13%), anteisoC17:0 (28%), isoC15:0 (2%) and anteisoC15:0 (4%), a profile typical of Streptomyces species whole cells Macroamphiphile glycopolymers are classically extracted by a hot phenol-water procedure  (SaLTA) . Its molrmis LTA . This anm/z 254.1, 336.1, 408.1, 569.2 and 611.2 attributed to (M+H)+ molecular ions that could be assigned to structures containing the motifs Gro-GlcN, P-Gro-GlcN, Gro-P-Gro-GlcN, Gro-GlcN-P-Gro-GlcN, and Gro-GlcNAc-P-Gro-GlcN respectively (m/z 162.0 or a N-acetylated amino sugar (i.e. GlcNAc: N-acetyl-glucosamine) at m/z 204.0 m/z 611.2 corresponded to a fragment containing both N-acetylated and non-N-acetylated amino sugars, suggesting that the PGP backbone of ShLTA was substituted by both types of amino sugars. The control that a GlcNAc standard was not hydrolyzed into GlcN by HF treatment supported the above hypothesis.To get further insights into the distribution of the different structural motifs along the molecule backbone, ShLTA was submitted to 48% hydrofluoric acid (HF) hydrolysis for 48 h at 4°C, a reaction known to hydrolyze phosphodiester bonds. After a chloroform/methanol/water partition, the aqueous phase was analyzed by MALDI-TOF/MS and MS/MS. The data are summarized in ectively . The ass1H-NMR anomeric region exhibited two anomeric signals at δ 5.43 (GI1) and δ 5.10 (GII1) in a ratio 3.7/1  and δ 98.3 (GII1)  and δ 3.94 (GII2)  (NH) (NH) with very low intensity (<10%) was also observed and most probably corresponded to a minor form of N-acetylated amino sugar, whose characterization could not be carried out further.Native ShLTA was subsequently analyzed by NMR. The io 3.7/1 , tentati3 (GII1) . Proton 4 (GII2)  were ind∶1, v/v) . Indeed,/v) (NH)  showed i/v) (NH) . In addi/v) (NH) . Altoget2) and δ 4.05 (gIV2) respectively (NH at δ 8.21 and proton gIV2 (2) and δ 77.3 (gIV2) respectively (not shown) (2 and gIV2) as compared with C2 resonance of an unsubstituted P-1-Gro-3-P unit at δ 70.8 (gI2)  (2), whose resonance at δ 76.6 was also deshielded as compared to that of gI (Δδ 5.8 ppm) , indicating that the C2 hydroxyl of gII was directly substituted by a phosphate group.Interestingly, nOe contacts were observed between anomeric protons of GI and GII and methine protons at δ 4.19  . The desctively) ; Table 35.8 ppm) ; Table 3Integration of the Gro unit H2-C2 cross-peaks on HMQC experiment  allowed via recognition by TLR2 and TLR6 S. pneumonia LTA 3CSK4 . It was recognized by the anti-LTA antibody  wall teichoic acid previously described by Tul'skaia et al.S. aureus or B. subtilis, for which LTA and teichoic acids have unrelated structures and are synthesized by two distinct pathways S. coelicolor and other Streptomyces spp) and of a few Firmicutes, such as S. pneumoniae, lack clear orthologues of the gene encoding LtaS polymerase S. aureus. Consequently, it has been suggested that in S. pneumoniae, LTA synthesis could be achieved by an alternative pathway that overlaps with that of teichoic acid biosynthesis S. hygroscopicus LTA and teichoic acid have a very similar structure further reinforces this hypothesis in which a PGP structure common to both molecules could be synthesized by the same enzymatic machinery and then transferred to peptidoglycan as teichoic acid or to a lipid anchor to yield the PGP-LTA. Accordingly, orthologues of the B. subtilis tagB/tagF teichoic acid biosynthesis genes are present in the genome of S. himastatinicus (a genome sequenced member of the S. hygroscopicus phylogenetic subclade) and other Streptomyces spp. (data not shown), supporting the occurrence of a pathway for teichoic acid biosynthesis in these species.ShLTA was found to contain a PGP backbone, composed of 78% of 1-P-Gro-3-P and 22% of 2-P-Gro-3-P repeating units. A portion of the 1-P-Gro-3-P units were α-glycosylated at position 2 by GlcN or GlcNAc . Not unevia binding to TLR2 on phagocytic cells bona fide TLR2 ligand and they are in agreement with the demonstration by Morath et al.N-acetylated form, as a positively charged substituent, could functionally replace D-alanine in ShLTA. However, it is worth noting that only 22% of the PGP repeating units are substituted by GlcN in ShLTA as compared to 70% by D-alanine in SaLTA et al.2O2. In contrast to what was previously thought, H2O2 and lipoprotein lipase treatments are not selective at all of lipopeptide/lipoproteins LTA is a powerful Gram-positive immunostimulatory component that induces cytokine production et al.Using blocking antibodies experiments, we found ShLTA activity to be dependent on CD14 and partially on TLR6 and TLR1. Interestingly, these data are in agreement with the recent study by Bunk Streptomyces. However, this has now to be definitely proved by genetic manipulation. Finally, our results provide new clues on LTA structure/function relationships, most particularly on the role of PGP substituents.Altogether, our findings contribute to the growing recognition that LTA is a cell envelope component of Gram-positive Actinobacteria S. hygroscopicus subsp. hygroscopicus NRRL 2387 (ATCC27438), S. coelicolor M145 and S. verticillus ATCC 21 890 II6-3 were grown at 27°C for 40 to 50 h under shaking in a culture medium which contained 5 g of yeast extract (BioSpringer), 5 g of bacto soytone peptone (Difco) and 20 g of glucose syrup (Roquette) per liter of deionized water.Cells were delipidated at 50°C by mixing with chloroform/methanol (1∶2 and then 1∶1) overnight. After lyophilisation, delipidated cells were resuspended in a 0.1 M citrate buffer pH 4.7 and disrupted by sonication. The lysate was then extracted twice with 1 volume of butan-1-ol. Aqueous phase, containing LTA, was treated with DNAse, RNAse, α-amylase, pronase, α-chymotrypsin and trypsin. After ultrafiltration, samples were purified by hydrophobic interaction chromatography on an octylsepharose CL-4B column eluted with increasing concentrations of isopropanol  in 0.1 M ammonium acetate. Fractions were collected and analyzed by SDS-PAGE followed by periodic acid-silver nitrate staining. After dialysis and drying, they were submitted to a water/butan-1-ol (1∶1 v/v) partition. The water phases were lyophilized and stored at −20°C. The fraction eluted with 35% isopropanol was used for further analysis.Mycobacterium smegmatis by a phenol/water partition as previously described 2O2 and incubated at 37°C for 24 h. H2O2 was evaporated by lyophilisation.20 µg of LTA or lipoproteins (lipoproteins were extracted from a cleared lysate of Pseudomonas sp. (Fluka) was used at a ratio enzyme/LTA or lipoproteins 1∶2 (w/w) in a 100 mM sodium phosphate buffer, 150 mM NaCl, 0.1% Triton X-100, pH 7.2 and incubated for 16 h at 37°C.Lipoprotein lipase from M. tuberculosis lipoglycan mixture 3CSK4 (InvivoGen)  were adsorbed in 96-wells microtiter plate by air drying and extensively rinsed with washing buffer . Wells were blocked for 2 h at room temperature with TBS, 5% BSA. After washing, 100 µl of 5 µg.ml−1 anti-LTA  or IgG1 isotype control (eBioscience) antibodies in TBS, 1% BSA were added for 2 h at room temperature. After washing, 100 µl of an HRP-conjugated goat anti-mouse antibody 1/3000 (Sigma) was added in TBS, 1% BSA for 1.5 h at room temperature. HRP activity was detected by addition of 100 µl of Sure Blue TMB substrate (BD Biosciences).ShLTA, ScLTA, commercial SaLTA (InvivoGen), 2HPO4 standard solution.Phosphorus content was determined after treatment of 50 µg LTA by 260 µl of perchloric acid at 170°C for 20 min. Then, 400 µl of freshly prepared solutions of 1.25% molybdate and 5% ascorbate were added. After 5 min boiling in a water bath, samples were cooled in ice. Absorbance was read at 660 nm and phosphorus concentration was calculated relatively to a NaAmino sugars were analyzed after hydrolysis of 10 µg LTA by 6N HCl at 110°C, overnight. They were detected either by CE-LIF using a 20 mM sodium borate buffer p-dimethylaminobenzaldehyde in 37% HCl were added and the solution was incubated again for 30 min at 75°C. 2.5 ml of 95% ethanol were further added and after 30 min, OD was read at 520 nm. Amino sugar concentration was calculated relatively to a GlcN standard solution.Amino sugars were quantified spectrophotometrically after hydrolysis of 200 µg LTA as described above. HCl was evaporated by a nitrogen stream and sample was dissolved in 250 µl of water. Then, 500 µl of a solution of 0.625 M sodium carbonate/acetylacetone  were added. After 10 min boiling in a water bath, the solution was cooled and 2.5 ml of 95% ethanol were added. After 5 min at 75°C, 500 µl of 5.3% et al.Amino acids were analyzed after hydrolysis of 100 µg LTA by 6N HCl at 110°C, overnight by LC-LIF according to the method described by Siri Fatty acids were analyzed by GC as their methyl esters after alkaline hydrolysis of 100 µg LTA in 1N NaOH for 2 h at 37°C.−1 in a mixture of ethanol/water . 0.5 µl of ShLTA, at a concentration of 10 µg.µl−1, were mixed with 0.5 µl of the matrix solution. Mass spectra were recorded in the negative mode using a 350-ns time delay with a grid voltage of 90% of full accelerating voltage (25 kV) and a guide wire voltage of 0.15%.LTA molecular mass was determined on a Voyager DE-STR MALDI-TOF instrument (PerSeptive Biosystems) using linear mode detection. The matrix used was 2,5-dihydroxybenzoic acid (Sigma) at a concentration of 10 µg.µl−1 in a mixture of ethanol/water , 0.1% TFA. 0.3 µl of the aqueous phase, at a concentration of 10 µg.µl−1, were mixed with 0.3 µl of the matrix solution. Mass spectra were recorded in the positive mode. Collision-induced dissociation gas type was atmosphere, and the gas pressure was set to medium.ShLTA (1 mg) was depolymerized by 48% HF hydrolysis, 48 h at 4°C. After drying under a nitrogen stream, the sample was submitted to a methanol/chloroform/water  partition. Aqueous phase, containing glycerophosphate motifs, was analyzed by MALDI/MS and MS/MS on a 4700 Proteomics Analyzer  using the reflectron mode. The matrix used was 2,5-dihydroxybenzoic acid at a concentration of 10 µg.µl2O or H2O/D2O   and analyzed in 200×5 mm 535-PP NMR tubes at 298 K. Proton and carbon chemical shifts are expressed in parts per million and referenced relative to internal acetone signals at δH 2.225 and δC 34.00 ppm respectively. All details concerning correlation spectroscopy and homonuclear Hartmann-Hahn spectroscopy sequences used and experimental procedures were as previously described NMR spectra were recorded with an Avance DMX500 spectrometer (Bruker) equipped with an Origin 200 SGI using Xwinnmx 2.6. ShLTA was dissolved in D4 cells per well in 96-wells plates and the different stimuli were added at concentrations indicated in the figure legends in the HEK-Blue Detection medium (InvivoGen) that contains a substrate for alkaline phosphatase. Alkaline phosphatase activity was measured after 18 h by reading O.D. at 630 nm. To investigate the CD14 and TLR dependence of stimuli activity, HEK-TLR2 cells were pre-incubated for 30 min at 37°C, before stimuli addition, with various antibodies: 5 µg.ml−1 of monoclonal anti-CD14 , monoclonal anti-TLR1 , monoclonal anti-TLR6  or an IgG1 isotype control (eBioscience). Anti-LTA  antibody was used at a concentration of 5 µg.ml−1 and pre-incubated with the different stimuli for 30 min at 37°C before HEK-TLR2 cells addition. Pam3CSK4 and FSL-1 were purchased from InvivoGen.The HEK-Blue-2 cell line (InvivoGen), a derivative of HEK293 cells that stably expresses the human TLR2 and CD14 genes along with a NF-κB-inducible reporter system  was used according to the manufacturer's instruction. Cells were plated at 5×105 cells per well in 96-wells plates in the HEK-Blue Detection medium (InvivoGen) or differentiated with 20 ng.ml−1 of PMA for 24 h in RPMI 1640 medium (Lonza) and the different stimuli were added at concentrations indicated in the figure legends. NF-κB activation was measured as described above. IL-6 and TNF-α were assayed in the supernatant by sandwich ELISA using commercially available kits (Diaclone).THP-1-Blue cell line (InvivoGen), a derivative of THP-1 monocyte/macrophage human cells that stably expresses a NF-κB-inducible reporter system  was used according to the manufacturer's instructions. Cells were added at 10t test to determine significant differences between samples.Results are expressed as a mean ± SD and were analyzed using the Student's


T-helper (Th) 22 is involved in the pathogenesis of inflammatory diseases. The roles of Th22 cells in the pathophysiological of ankylosing spondylitis (AS) and rheumatoid arthritis (RA) remain unsettled. So we examined the frequencies of Th22 cells, Th17 cells and Th1 cells in peripheral blood (PB) from patients with AS and patients with RA compared with both healthy controls as well as patients with osteoarthritis.We studied 32 AS patients, 20 RA patients, 10 OA patients and 20 healthy controls. The expression of IL-22, IL-17 and IFN-γ were examined in AS, RA, OA patients and healthy controls by flow cytometry. Plasma IL-22 and IL-17 levels were examined by enzyme-linked immunosorbent assay.Th22 cells, Th17 cells and interleukin-22 were significantly elevated in AS and RA patients compared with OA patients and healthy controls. Moreover, Th22 cells showed positive correlation with Th17 cells as well as interleukin-22 in AS and RA patients. However, positive correlation between IL-22 and Th17 cells was only found in AS patients not in RA patients. In addition, the percentages of both Th22 cells and Th17 cells correlated positively with disease activity only in RA patients not in AS patients.The frequencies of both Th22 cells and Th17 cells were elevated in PB from patients with AS and patients with RA. These findings suggest that Th22 cells and Th17 cells may be implicated in the pathogenesis of AS and RA, and Th22 cells and Th17 cells may be reasonable cellular targets for therapeutic intervention. Ankylosing spondylitis (AS) is a chronic inammatory disease that is characterized by mainly involving bilateral sacroiliitis and axial joints, but sometimes peripheral joints and extra-articular organs are also involved IL-22, a member of IL-10 cytokine family, exerts its effects via a heterodimeric transmembrane receptor complex consisting of IL-10R2 and IL-22R1 + T cells clones have low or undetectable expression of Th1 and Th17 transcription factor T-bet and RORγt, and arylhydrocarbon receptor (AHR) has been considered to be the key transcription factor of Th22 subset Th22 subset is a more recently identified new human T helper subset, which is characterized by abundant secretion of IL-22 but not IL-17 or IFN-γ The roles of both Th22 cells and IL-22 in the pathogenesis of ankylosing spondylitis and rheumatoid arthritis are still unclear and remain to be clarified. Therefore, to investigate their roles in the pathogenesis of AS and RA, we examined the frequencies of Th22 cells in peripheral blood as well as the levels of plasma IL-22 of both AS and RA patients, and assayed their correlations with disease activity in this study.Enrollment took place between May, 2010 and April, 2011 in two centers: Qilu Hospital, Shandong University and Shandong Provincial Hospital, Shandong University, China. Our research has been approved by both the Medical Ethical Committee of Qilu Hospital, Shandong University and the Medical Ethical Committee of Shandong Provincial Hospital, Shandong University. A written informed consent document has been obtained from each participant.A total of 32 patients with AS according to the modified New York criteria +IFNγ–IL17–IL-22+, CD4+IFNγ–IL17+, CD4+IFNγ+ and CD4+IFNγ+IL17+T cells respectively.Intracellular cytokines were studied by flow cytometry to reflex the cytokine-producing cells. Briefly, heparinized peripheral whole blood (400 µl) with an equal volume of Roswell Park Memorial Institute 1640 medium were incubated for 4 h at 37°C, 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 µg/mL of ionomycin, and 1.7 µg/ml Golgiplug. PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity. Monensin was used to block intracellular transport mechanisms, thereby leading to an accumulation of cytokines in the cells. After incubation, the cells were stained with PE-Cy5-conjugated anti-CD4 monoclonal antibodies  at room temperature in the dark for 20 min. The cells were next stained with FITC-conjugated anti-interferon (IFN)-γ monoclonal antibodies , PE-conjugated anti-IL-17A monoclonal antibodies  and APC-conjugated anti-IL22 monoclonal antibodies  after fixation and permeabilization. All the antibodies were from eBioscience, San Diego, CA, USA. Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen). Th22, Th17, Th1 and Th1/Th17 cells were defined as CD4Peripheral blood was collected into heparin-anticoagulant vacetainer tubes. Plasma was obtained from all subjects by centrifugation and stored at –80°C for determination of cytokines. Plasma IL-22 (Cat: BMS2047) and IL-17 (Cat: BMS2017) levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer’s recommendations .BASDAI score of AS patients and disease activity score in 28-joints (DAS28) of RA patients was calculated in our study. At the time of clinical assessment for disease activity, blood samples were collected for the measurement of levels of C-reactive protein (CRP) and ESR.q test) unless the data were not normally distributed, in which case Kruskal - Wallis test (H test) and Nemenyi test were used. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. All tests were performed by SPSS 17.0 system. P value less than 0.05 was considered statistically significant.Results were expressed as mean ± SD or median (range). Statistical significance of Th22, Th17, Th1 and plasma IL-22 as well as IL-17 among patients with AS, RA, OA and HC was determined by ANOVA, and difference between two groups was determined by Newman–Keuls multiple comparison test  of peripheral blood. The number of Th22 cells was significantly increased in AS (204±34) and RA (211±43) patients compared with healthy controls (123±23) after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h .Plasma levels of IL-22 were examined by ELISA. The levels of IL-22 were significantly increased in AS (41.03±16.00pg/ml) and RA (44.53±29.84pg/ml) patients compared to OA patients (24.53±3.45pg/ml) and healthy controls (25.33±3.75pg/ml) .P<0.001; P = 0.027; P = 0.045; P = 0.801)  .P>0.05; RA: 15.02±4.60, P>0.05; OA: 14.80±2.88, P>0.05; HC: 14.39±2.72, P>0.05)  .P>0.05; RA: 11.13±4.09%, P>0.05; OA: 10.73±2.50%, P>0.05; HC: 10.37±2.00, P>0.05)  . +IFNγ-IL17+IL-22+ T cells and CD4+IFNγ+IL17+ T cells. Though most Th17 cells did not simultaneously express both IL-22 and IL-17, the percentage of CD4+IFNγ-IL17+IL-22+ T cells was significantly increased in AS (0.63±0.34%)) and RA (0.65±0.29%) patients compared with OA patients (0.20±0.04%) and healthy controls (0.19±0.05%). In addi19±0.05%).P<0.001) (P = 0.041) in RA patients  . Similarpatients . HoweverP  = 0.008 or r = 0.544, P  = 0.013 respectively)  (P  = 0.105 or P  = 0.205), and plasma level of IL-22 or IL-17 failed to show a statistical correlation with CRP level or DAS28 (P = 0.622 and P  = 0.357 or P = 0.317 and P = 0.872) in RA patients (P = 0.964), CRP (P = 0.393) and BASDAI score (P = 0.226) (P = 0.918), CRP (P = 0.862) and BASDAI score (P = 0.320)    a, b. Coctively)  c, d andpatients , k. In p= 0.226) , b, c. C= 0.320) , h, i. Cctively) , e, f.Th22 cells, a recently defined lineage of T cells distinct from Th1, Th2 and Th17 cells, have been believed to play a complicated and important role in inflammatory and autoimmune diseases. The function of Th22 cells is achieved through the signature cytokine they secreted. Th17 cells have already been proposed to be the primary driver of autoimmune diseases, including rheumatoid arthritis. However, the potential role of Th22 cells in inflammatory arthritis such as AS and RA is far from clear.To determine whether Th22 is involved in AS and RA, the percentages of Th22 cells were examined in the peripheral blood of patients with AS, RA OA, and healthy controls in this study. This is the first study that evaluates the relative abundance of Th22 cells in peripheral blood of RA and AS patients. Our results demonstrated that the percentages of Th22 cells were significantly elevated in the peripheral blood of patients with AS and also in those with RA compared with OA patients and healthy controls, which was consistent with our previous experiments on RA IL-22, a cytokine of IL-10 family, is the most important functional cytokine of Th22 cells. Considerable evidence suggests that IL-22 may be involved in the pathogenesis of many inflammatory diseases, but its pathophysiologic function is not well known. To investigate the potential role of IL-22 in AS and RA patients, we examined the concentrations of plasma IL-22 in our experiments. In line with our previous study, elevated levels of plasma IL-22 were also detected in AS and RA patients in the present study, implicating that IL-22 may be involved in the pathogenesis of both AS and RA. IL-22 has been demonstrated to exert its biological effects via binding to the heterodimeric receptor complex, including IL-22R1 and IL-10R2, and IL-22 activates Jak1 and Tyk2, which further activates STAT-1, STAT-3 and STAT-5 et alConsiderable evidence suggests Th17 cells and Th1 cells have been involved in the development of autoimmune diseases IL-17, the main cytokine of Th17 cells, contributes to the pathogenesis of arthritis as has been demonstrated in many experimental arthritis models. Although elevated levels of IL-17 have been observed in synovial fluid of RA patients, the levels of this T cell cytokine in plasma of these patients is hard to detect. Furthermore, the reports on IL-17 levels in plasma of RA or AS patients are inconsistent et alIn humans, it is not unusual to encounter dual +Th1/Th17 cells. It has previously been demonstrated that T cells from synovial fluid of RA can co-express IL-17 and IFN-γ In conclusion, our experiments showed that the frequencies of Th22 cells and Th17 cells were significantly increased in AS and RA patients compared to OA patients and healthy controls. Plasma IL-22 levels, which correlated positively with Th22 cells, were also demonstrated to be elevated in both AS and RA patients. The increasing percentages of Th22 cells and Th17 cells and the elevation of IL-22 may play important roles in the pathogenesis of AS and RA. Thus, Th22 cells and IL-22 as well as Th17 cells may prove to be a promising therapeutic targets for AS and RA. Further studies are awaited to clarify the pathophysiologic role of Th22 in AS and RA and determine the situation of Th22 cells in AS and RA patients’ joints.Figure S1a, b and c, No positive correlations were found between the percentage of Th22 cells and ESR, CRP as well as BASDAI in AS patients. d, e and f, No positive correlations were found between the percentage of Th17 cells and ESR, CRP as well as BASDAI in AS patients. g, h and i, No positive correlations were found between the levels of plasma IL-22 and ESR, CRP as well as BASDAI in AS patients. j and k, No positive correlations were found between the levels of plasma IL-22 and CRP as well as DAS28 in RA patients. i, No positive correlation was found between the levels of plasma IL-22 and the percentage of Th17 cells in RA patients.(TIF)Click here for additional data file.Figure S2a, The number of Th22 cells was significantly increased in AS and RA patients compared with healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. b, The number of Th17 cells was significantly increased in AS and RA patients compared with healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. (* = P<0.05)The number of Th22 cells and Th17 cells in per volume of peripheral blood in AS patients, RA patients and healthy controls. (TIF)Click here for additional data file.Table S1Major previous treatment of each patient of AS*(DOC)Click here for additional data file.Table S2Major previous treatment of each patient of RA*(DOC)Click here for additional data file.


Leishmania in its life cycle. However, the objective of studying the mechanisms responsible for the differential expression has resulted to be a difficult task due to the complex genome organization of tubulin genes and to the non-conventional mechanisms of gene regulation operating in Leishmania.Alpha tubulin is a fundamental component of the cytoskeleton which is responsible for cell shape and is involved in cell division, ciliary and flagellar motility and intracellular transport. Alpha tubulin gene expression varies according to the morphological changes suffered by α-tubulin genes in the Leishmania braziliensis genome database. The genomic organization of L. braziliensis α-tubulin genes differs from that existing in the L. major and L. infantum genomes. Two loci containing α-tubulin genes were found in the chromosomes 13 and 29, even though the existence of sequence gaps does not allow knowing the exact number of genes at each locus. Southern blot assays showed that α-tubulin locus at chromosome 13 contains at least 8 gene copies, which are tandemly organized with a 2.08-kb repetition unit; the locus at chromosome 29 seems to contain a sole α-tubulin gene. In addition, it was found that L. braziliensis α-tubulin locus at chromosome 13 contains two types of α-tubulin genes differing in their 3′ UTR, each one presumably containing different regulatory motifs. It was also determined that the mRNA expression levels of these genes are controlled by post-transcriptional mechanisms tightly linked to the growth temperature. Moreover, the decrease in the α-tubulin mRNA abundance observed when promastigotes were cultured at 35°C was accompanied by parasite morphology alterations, similar to that occurring during the promastigote to amastigote differentiation.We started this work by analyzing the genomic organization of α-tubulin genes have been reorganized in a drastic manner along Leishmania speciation. In the L. braziliensis genome database, two loci containing α-tubulin sequences were found, but only the locus at chromosome 13 contains the prototypic α-tubulin genes, which are repeated in a head-to-tail manner. Also, we determined that the levels of α-tubulin mRNAs are down-regulated drastically in response to heat shock by a post-transcriptional mechanism which is dependent upon active protein synthesis.Information found in the genome databases indicates that  In unicransport , as manyLeishmania genus comprises at least 20 Leishmania species that infect humans, and the spectrum of diseases that they cause can be categorized broadly into three types: self-healing cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and the often fatal visceral leishmaniasis (VL) [Leishmania, the motile promastigote and the amotile amastigote. Classical studies demonstrated that the promastigote stage synthesizes more tubulin protein than the amastigote stage, and that this biosynthetic change of tubulin was found to correlate with the morphological change of microtubules in leishmanial flagella and cytoskeleton during promastigote-to-amastigote transformation [Leishmania[The sis (VL) . Endemicsis (VL) . There aormation . Studiesormation , but thiormation , in parteishmania.Leishmania species, the genomic organization of α- and β-tubulin genes has been analyzed, showing the existence of multiple copies, both arranged in tandem (forming separate clusters of α- and β-tubulin genes) and dispersed in the genome [Leishmania species [β-tubulin genes in several Leishmania species; these authors suggest that the gene organization has evolved to satisfy a need for innovative expression for β-tubulin genes [In various e genome ,9,10. Th species -13 has aα-tubulin genes in Leishmania braziliensis based on the available, yet incomplete, genome sequence. A comparison with the organization of this gene in L. infantum and L. major is also provided. The 5´ and 3´ untranslated regions (UTRs) for the different α-tubulin genes in L. braziliensis have been determined as well as their mRNA expression levels under different conditions.In this work, we studied the organization of Leishmania major (MHOM/IL/81/Friedlin), the first Leishmania genome that was sequenced [α-tubulin locus exists. According to the data available at the GeneDB database [Leishmania mexicana (MHOM/GT/2001/U1103) genome. In contrast, according to the genome database (GeneDB), the Leishmania infantum (MCAN/ES/98/LLM-877) genome contains two α-tubulin loci, both located at chromosome 13 and separated by a region of 436.6 kb. The more 5´ locus  braziliensis (MHOM/BR/75/M2904) [α-tubulin-like gene . The locus at chromosome 29 .In the genome of equenced , a sole database , the locMHOM/IL/8/FriedlinMHOM/IL/8/FriedlinMHOM/IL/8/Friedlin5/M2904) , also coMHOM/IL/8/FriedlinMHOM/IL/8/Friedlinα-tubulin loci in L. major, L. infantum and L. braziliensis revealed that the chromosomal regions surrounding the α-tubulin genes at chromosome 13 have experienced remarkable reorganizations in these three Leishmania species, and the α-tubulin locus itself appears as the target of DNA deletion and inversion events  and LinJ.13.0340 genes was changed with respect to the equivalent genes of L. major. In L. braziliensis, regarding the L. major chromosomal organization, a region spanning from the LmjF.0200 to the LmjF.0270 gene, corresponding to a string of 8 genes encoding for 7 hypothetical proteins and one putative class 3 lipase .Comparison of the genomic region around the α-tubulin locus at L. infantum chromosome 13, comprising as mentioned above a partial and a complete copy of α-tubulin coding sequence  appeared as the center of the reorganization events , excepting an insertion comprising the LbrM.13.1250 to LbrM.13.1370 gene block .The other α-tubulin gene loci have been reorganized in a drastic manner along Leishmania speciation. However, this fact contrasts with the chromosomal organization of the three β-tubulin loci that is largely syntenic in L. major, L. infantum and L. braziliensis[In summary, these analyses pointed out that the ziliensis.α-tubulin genes among L. major, L. infantum and L. braziliensis are the result of artifacts generated during assembling of contigs into chromosomal scaffolds. The L. infantum and L. braziliensis genome sequences were produced by whole-genome shotgun sequencing to five- and six-fold coverage, respectively [L. infantum and L. braziliensis yielded 470 and 1,031 contigs, respectively. Finally, chromosomal scaffolds were produced by aligning contigs against the reference L. major sequence [L. infantum and L. braziliensis (according to the GeneDB database). Remarkably, the α-tubulin locus at chromosome 29 in L. braziliensis . Tandemly repetition of genes has been proposed as a direct mechanism for increasing the transcript abundance of highly expressed proteins [α-tubulin is an abundant protein in Leishmania[As shown in Figure  L. major and L. eenriettii showed tproteins , and it L. braziliensis total DNA was digested with restriction enzymes lacking cut sites within the α-tubulin loci (PstI and ApoI), and the hybridization pattern was analyzed by Southern blotting, a conspicuous result was obtained  and two (ApoI) large bands was unexpected. An explanation to this finding may be either the existence of allelic sequence polymorphisms in the regions surrounding the α-tubulin locus at chromosome 13 or different copy numbers of α-tubulin genes in each homologous chromosome. Indeed, similar observations have been described for L. braziliensis and other trypanosomatids regarding HSP70, KMP-11 and histone H2A gene loci [PstI digested-DNA would be indicating that L. braziliensis has three homologous for chromosome 13, a very plausible possibility taking into account recent studies showing that L. braziliensis genome is essentially triploid [When d Figure B. The prene loci -19. MoreL. braziliensis α-tubulin genes are encoding for three different amino acids polypeptides: the prototypical α-tubulin (encoded by genes LbrM.13.0190 and LbrM.13.0200) and two α-tubulin variants, dubbed α-tubulin-A (LbrM.13.0210) and α-tubulin-B (LbrM.29.2700)  between the divergent region of gene LbrM.13.0210 and a genomic region of chromosome 28, located at the intercoding region of genes LbrM.28.2580 and LbrM.28.2590 , or in related trypanosomatids (Trypanosoma brucei and T. cruzi). Finally, an additional finding pointing to a possible assembly error in gene LbrM.13.0210 is the fact that the region conserved with α-tubulin would extend further (35 amino acids) if the nucleotide at position 245 is deleted (data not shown).As mentioned above, if assembly artifacts exist in the -workers  when anae genome , but it 3 strain , in otheLeishmania genes are transcribed into polycistronic RNA precursors that need to be further processed into individual mRNAs by trans-splicing and polyadenylation, post-transcriptional regulation represents the main level of controlling gene expression in these parasites [L. braziliensis α-tubulin gene, RT-PCR reactions using specific oligonucleotides were performed. Thus, it was possible to determine the 5´ UTR region of the α-tubulin genes LbrM.13.0190 and LbrM.13.0200, which was 69 nucleotides in length and identical for both genes. This sequence was deposited in the GenBank database under the accession number FJ750454, and it was incorporated to the GeneDB database [L. braziliensis α-tubulin locus at chromosome 13 should be composed by 8–15 copies of the prototypic α-tubulin gene, but they collapsed to only two (LbrM.13.0190 and LbrM.13.0200) during assembly process, it is reasonable concluding that the 3´ UTR for the rest of genes would be identical or near-identical to the 3´ UTR-I. Similar situation has been reported in β tubulin array from L. major[HSP70 genes [α-tubulin protein under different conditions as occurs with the HSP70 protein [Given that arasites ,21. Consarasites ,21. To ddatabase . Regardi70 genes  in which protein . Remarka protein , the 3´  protein .α-tubulin gene expression in L. braziliensis has not been addressed to date. Early studies in Leishmania enriettii demonstrated that in agreement with the greater tubulin content in the promastigote stage, the levels of α- and β-tubulin mRNAs in the promastigotes were significantly higher than those found in amastigotes [L. major, using microarray hybridizations, it was found that α-tubulin mRNAs are 6.3-fold more abundant in promastigotes than in amastigotes [L. mexicana, similar amounts of tubulin mRNAs were found in both promastigotes and amastigotes [T. cruzi, the regulation of tubulin genes has been studied in some extent [α- and β-tubulin transcripts are found in amastigotes and trypomastigotes compared to epimastigotes. Experimental evidence suggests tubulin mRNA half-lives are negatively affected by the amount of free, unpolimerized tubulin proteins. The regulatory mechanism would operate through the recognition of sequence elements located at the beginning of the coding region and in the 3´ UTR of the tubulin genes [Analysis of stigotes . In L. mstigotes . Howeverstigotes ,29. In te extent ,31. In tLeishmania[L. braziliensis α-tubulin mRNA stability. For this purpose, parasites were cultured at 35°C during several periods of time, determining the parasite morphology and α-tubulin mRNA abundance at each time culture-point. As shown in Figure α-tubulin mRNA abundance was also observed, suggesting a down-regulation of α-tubulin mRNAs levels during adaptation of the parasite to the mammalian host temperature.Temperature shifts trigger differential gene expression and stage transformation in eishmania. Thus, wL. braziliensis α-tubulin genes, we used inhibitors of transcription (actinomycin D), trans-splicing (sinefungin) and protein synthesis (cycloheximide). The effects of these drugs were analyzed at both 26°C and 35°C for several periods of time , whereas the last copy of the tandem possesses a different sequence (named 3´ UTR-II). Finally, we demonstrated that mRNA expression of L. braziliensis α-tubulin genes is controlled by a temperature-dependent post-transcriptional mechanism.The analysis of the genomic organization and genome distribution of L. braziliensis MHOM/BR/75/M2904 were cultured in vitro either at 26°C in Schneider’s insect medium supplemented with 20% heat-inactivated fetal calf serum , and 0.1 μg/mL of 6-biopterin  or at 35°C with 5% CO2 by several time periods. Total DNA and RNA from parasite cells were isolated using the phenol-chloroform-isoamilic alcohol method [Promastigotes of l method , and theL. braziliensis α-tubulin coding region was amplified from genomic DNA by PCR with Lb-Tub-F (5´ GGATCCATGCGTGAGGCTATCTGC 3´) and Lb-Tub-R (5´ CTGCAGCTAGTACTCCTCGACGTCCT 3´) primers, which contain the BamHI and PstI restriction sites (underlined in the sequence), respectively. Amplification of the UTRs was performed from poly-T primed-cDNA using the following primers: LbSL (5´ CGCTATATAAGTATCAGTTTC 3´) and LbαT-106 (5´ GTGAGGCTATCTGCATTCA 3´) for the 5´ UTR and Lbαt1312 (5´ TCGTGCACTGGTACGTTG 3´) and poly-T EcoRI (5´ CGGAATTCTTTTTTTTTTTTTTTTTTT 3´), for the 3´ UTR.All oligonucleotides were synthesized by IDT, Inc. . The L. braziliensis total RNA using an oligo-dT primer and the Transcriptor first strand cDNA synthesis kit . The following PCR mix reaction was used for all amplifications in a final volume of 20 μl: 1× reaction buffer , 1.5 mM MgCl2, 0.4 mM of dNTP mix, 0.5 μM of each primer, 1 M betain, 0.06 units per μl of expand high fidelity enzyme  and 15 ng/μl DNA or 1 μl of cDNA product. An MJ Research PTC-100 DNA thermocycler was used for the reaction with the following amplification profile: 95°C/5 min , 35 cycles at 92°C/0.5 min, annealing at 52-58°C/0.5 min and extension for 72°C/1 min, with a final incubation at 72°C for 10 min. All the amplified fragments were resolved in agarose gels and visualized under UV exposure after ethidium bromide staining. RT-PCR products were excised from gels, purified using Wizard® SV Gel and PCR Clean-Up System  and cloned into the pCR®II  or pGEM®-T Easy plasmids . The following clones were obtained and submitted to the Addgene web site [αtub-B , containing the α-tubulin ORF, pLb5αTub-D , the 5´ UTR and pLb3αTub-C6 , the 3´ UTR. The sequences from each plasmid insert were determined using the Big Dye Terminators v3.1 kit  by automatic sequencing at the Servicio de Genómica . In order to deduce the α-tubulin mRNA UTRs from other Leishmania species, LALIGN [α-tubulin variants was performed by BLAST/N analysis [α-tubulin loci synteny and comparison among the three Leishmania species was carried out by BLAST/N analysis.First-strand cDNA synthesis was carried out from web site  pTOLbαtuα-tubulin locus (PstI and ApoI) or inside it (Csp451), sequences of α-tubulin loci from the three Leishmania species were retrieved from GeneDB database [in silico restriction analysis through NEBcutter V2.0 tools [α-tubulin ORF as probe. For probe synthesis, the L. braziliensis α-tubulin gene was released from clone pTOLb α tub-B by BamHI/PstI cutting, excised from gel, purified using Wizard® SV Gel and PCR Clean-Up System , and finally labeled with digoxigenin by randomly primed synthesis using the DIG High Prime DNA Labeling kit . Hybridizations were performed using the Detection Starter kit II  according to manufacturer's instructions. Afterwards, membranes were washed twice under the following conditions: 2× sodium citrate solution (SSC) and 0.5% (p/v) sodium dodecyl sulfate (SDS) at room temperature for 5 min, 0.5 × SSC, 0.1% SDS solution for 15 min at 65°C and 0.1 × SSC, 0.1% SDS solution for 15 min at 65°C, under constant shaking. Finally, after the immunological detection according to the above mentioned kit recommendations, membranes were exposed on Curix RP2 plus medical X-Ray film .With the aim of choosing endonucleases cutting at the boundaries of the database  and used.0 tools . DNA fro.0 tools  and hybrα-tubulin expression levels, L. braziliensis promastigote cultures were treated as follows: maturation of mRNA were inhibited by addition of 10 μg/ml sinefungin to the L. braziliensis promastigote cultures at logarithmic phase (5–9 × 106 promastigotes per ml), synthesis of RNA were inhibited by addition of 10 μg/ml actinomycin D five min later [2, with or without inhibitors for several time periods . Afterwards, parasites were harvested for RNA extraction and 8 μg of total RNA per line were separated on 1.5% (w/v) low electroendosmosis agarose/MOPS/formaldehyde gels  and transferred to nylon membranes . Probes, hybridization and immunological detection were performed as previously mentioned.To investigate the in later . ProteinThe authors declare that they have no competing interests.CAR, JMR, and CJP conceived and designed the experiments. CAR performed the experiments. CAR, JMR, and CJP analyzed the data. CAR, JMR, and CJP wrote the paper. All authors revised and approved the final version of the manuscript.L. braziliensis there is a 457 nucleotide sequence that is identical to the final 355 nucleotides of the LbrM.13.0210 ORF and the first 102 downstream nucleotides. Boxes in red comprise ORFs. (B) Alignment of the LbrM.13.0210 entry with the intergenic region between the LbrM.28.2580 and the LbrM.28.2590 entries (28IGR). A 100% of identity in 457 nt overlapped, showed in red, was found. The ATG and stop codons of the LbrM.13.0210 entry are indicated in blue.(A) The 3′ end of LbrM.13.0210 gene is derived from chromosome 28. In the intercoding region of LbrM.28.2580 and LbrM.28.2590 entries at chromosome 28 from Click here for file


The effects of long-term alcohol consumption on the mitogen-activated protein kinases (MAPKs) pathway and N-methyl-D-aspartate-type glutamate receptor 1 (GluN1) subunits in the mesocorticolimbic system remain unclear. In the present study, rats were allowed to consume 6% (v/v) alcohol solution for 28 consecutive days. Locomotor activity and behavioral signs of withdrawal were observed. Phosphorylation and expression of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38 protein kinase and GluN1 in the nucleus accumbens, caudate putamen, amygdala, hippocampus and prefrontal cortex of these rats were also measured. Phosphorylation of ERK, but not JNK or p38, was decreased in all five brain regions studied in alcohol-drinking rats. The ratio of phospho/total-GluN1 subunit was reduced in all five brain regions studied. Those results suggest that the long-term alcohol consumption can inhibits GluN1 and ERK phosphorylation, but not JNK or p38 in the mesocorticolimbic system, and these changes may be relevant to alcohol dependence. To differentiate alcohol-induced changes in ERK and GluN1 between acute and chronic alcohol exposure, we have determined levels of phospho-ERK, phospho-GluN1 and total levels of GluN1 after acute alcohol exposure. Our data show that 30 min following a 2.5 g/kg dose of alcohol , levels of phospho-ERK are decreased while those of phospho-GluN1 are elevated with no change in total GluN1 levels. At 24 h following the single alcohol dose, levels of phospho-ERK are elevated in several brain regions while there are no differences between controls and alcohol treated animals in phospho-GluN1 or total GluN1. Those results suggest that alcohol may differentially regulate GluN1 function and ERK activation depending on alcohol dose and exposure time in the central nervous system. Alcohol dependence is a complex neuropsychiatric disorder characterized by chronic drinking, abstinence, relapse and behavioral impairments. Long-term consumption of alcohol has been reported to modify a multitude of molecular events such as the function of neurotransmitter receptors, intracellular signal transduction systems and biochemical processes in the central nervous system Drugs of abuse enhance the activity of the dopaminergic mesocorticolimbic pathway, which arises from the ventral tegmental area (VTA) and projects to the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC) The mitogen-activated protein kinases (MAPKs) pathway represents a converging point for many signaling pathways and can be activated by chronic drug treatments 2+-dependent signaling pathway. Increasing evidence also shows that glutamate receptor-dependent activation of the MAPK pathway is critical for the development of striatal neuronal plasticity and is an important molecular mechanism for the long-lasting behavioral plasticity The N-methyl-D-aspartate-type glutamate receptors (NMDARs) are heteromeric complexes that incorporate the NMDAR1 (GluN1), NMDAR2, and NMDAR3 subunits. Without GluN1, NMDAR complexes are not functional Although the association between drug dependence and dysfunction of mesocorticolimbic systems is well documented, the molecular mechanisms and particularly the effects of long-term alcohol consumption on the phosphorylation of MAPKs and GluN1 subunits have not been elucidated. In the present study, we hypothesized that alcohol would alter the phosphorylation of ERK, JNK, p38 and GluN1 in the mesocorticolimbic system of the rat. To test this hypothesis, rats were exposure to a 6% (v/v) alcohol solution for 28 d and a single dose of alcohol . The phosphorylated MAPKs and GluN1 in the NAc, CPu, Amy, Hip and PFC was examined in rats.ad libitum. All experiments were carried out between 9∶00 and 16∶00. All experiments were approved by the Animal Care and Use Committee of Xi’an Jiaotong University, and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines have been followed. Efforts were made to minimize animal suffering and to reduce the number of animals used.Sixty-four male Sprague-Dawley rats  that were 16 weeks old and weighed 260–280 g at the beginning of the experiment were habituated for 7 d before the experiments. Rats were individually housed in a temperature- and humidity-controlled room  under a 12 h light/dark cycle (lights on at 8∶00). Food was available Anhydrous ethanol  was dissolved in sterile water at a concentration of 6% (v/v). Rabbit anti-phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-GluN1 (Ser897), ERK, JNK, p38 and GluN1 polyclonal antibodies were purchased from Cell Signaling Technology . Mouse anti-β-actin monoclonal antibody and horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Santa Cruz Technology .ad libitum, respectively, as the only liquid source during the experimental period. In order to induce alcohol consumption, an alcohol solution was administered at a concentration from 0.5% v/v to 6% v/v for adaptation in the first 12 d and 6% v/v in the following 28 d, as described by Turchan J. et al. Rats were assigned randomly to two groups: a control group (n = 10) and an alcohol group (n = 10), receiving water and alcohol solution To minimize the effects of handling and behavioral assessments, a separate cohort of rats was allowed to drink the 6% alcohol solution or water (n = 10 per group) for 28 d. Those rats neither undergo any behavioral tests nor alcohol withdrawal. The rats were sacrificed by decapitation at the end of the 28-d treatment, brains were quickly removed and stored at −80°C until use.Blood samples (40 µl) were taken via tail vein at 8∶00–9∶00 after 7, 14, 21 and 28 d of alcohol drinking. Ten µl of plasma was used to determine blood alcohol concentration using an EnzyChrom™ Ethanol Assay Kit .et al. According to the method of Erden, At the end of the 28-d exposure to 6% alcohol solution, alcohol was withdrawn from the drinking water at 9∶00. The rats were then observed for 10 min at the 0 , 2, 6, 24, 48 and 72 h of the withdrawal period. At each observation time, rats were assessed simultaneously for the following behavioral signs: stereotyped behavior, agitation (irritability to touch), tail stiffness, abnormal posture and gait. In the study, grooming, sniffing, head weaving, gnawing, and chewing were observed as major stereotypes behaviors during the alcohol withdrawal. Stereotypic behaviors, abnormal posture and gait, agitation, and tail stiffness were scored using a rating scale as previously described  [29]. Thet al.For acute alcohol exposure, rats were infused intragastrically with 2.5 g/kg ethanol in sterile water, volume administered was equivalent to 0.015 ml/g of body weight of a 21% ethanol solution , and at 30 min, 2 h, 6 h and 24 h after alcohol administration. Blood alcohol levels (BALs) were determined. To reduce the number of animals used, animals were decapitated when an acute effect was expected and disappared, i.e. 30 min (n = 6) and 24 h (n = 6). Controls (n = 12) were administered with water and killcd with the alcohol-treated rats. Brains were quickly removed and stored at −80°C until use.The NAc, CPu, Amy, Hip, PFC were dissected on ice using a rat brain atlas . Brain tp<0.05.Data analyses were performed using SPSS . Alcohol consumption and BALs were determined using a one-way analysis of variance (ANOVA) with repeated measures. Weight gain, total fluid intake, locomotor activity, global EWS scores were compared between the two treatment groups using two-way ANOVA with repeated measures. Immunoreactive protein bands were quantified by densitometry using the Quantity One . Proteins levels in the water-drinking control rats were set at 100%. Independent Student’s t-tests were conducted to analyze immunoreactivity changes in MAPKs and GluN1. All data are presented as means ± the standard deviation (SD). Statistical significance was accepted at  = 2.54, p = 0.1128]  = 179, p<0.0001]. The mean body weight gain for the period was 121 g in the alcohol-drinking group and 125 g in water-drinking control group.No significant differences in weight gain between the alcohol- and water-drinking rats were noted [treatment: F 0.1128] , A. All  = 0.11, p = 0.7454]  = 14.37, p<0.0001]. Rats stably consumed an average of 4.85±0.79 g/kg/day of alcohol in their home cages for 28 d   = 0.51, p = 0.7276]  = 11.23, p<0.0001] in total distance traveled, which increased over the 28 days period in both groups  . Repeated measures ANOVA analysis of rearing behavior revealed a significant treatment–time interaction  but no time effect  .Repeated measures ANOVA revealed a significant treatment–time interaction [F<0.0001] , E. The  0.3293] , F. Diff = 111.8, p<0.0001; time: F = 20.66, p<0.0001]  = 19.14, p<0.0001] in those rats. The sum of the 5 observation scores  progressively increased from 7.7±0.6 at 2 h  to 5.1±0.9 at 24 h (p<0.05), with a peak score around 6 h , confirming the presence of significant overall withdrawal severity. All individual withdrawal ratings (compared with water-control rats), measured at 6 h after withdrawal (p<0.0001 ), agitation (p<0.0001), tail stiffness (p<0.0001), abnormal posture (p<0.05) and gait (p<0.01).Alcohol-drinking rats showed significant withdrawal signs (compared with water-control rats) at different withdrawal time points [treatment: F<0.0001] . Moreovethdrawal  inset, wp<0.0001), CPu (p<0.01), Amy (p<0.01), Hip (p<0.01) and PFC (p<0.05) of rats as compared with the water-control rats (Following 28 d of 6% (v/v) alcohol drinking, phospho-ERK (Thr202/Tyr204) was attenuated in the NAc , CPu (p<0.0001), Amy (p<0.0001), Hip (p<0.01) and PFC (p<0.01) showed significantly higher levels in the alcohol-drinking rats compared to the control rats  vs. control rats.Western blots analysis of total-GluN1 and phospho-GluN1 the NAc  = 57, p = 0.0029]  = 20.72, p<0.0001; post hoc: p<0.0001), CPu  = 76.6, p<0.0001; post hoc: p<0.0001), Amy  = 60.0, p<0.0001; post hoc: p<0.0001), Hip  = 54.2, p<0.0001; post hoc: p<0.0001) and PFC  = 42.0, p<0.0001; post hoc: p<0.0001) of rats as compared with the water-controls  was significantly attenuated in the NAc  = 0.98, p = 0.39), CPu  = 0.74, p = 0.63), Amy  = 0.68, p = 0.51), Hip  = 0.26, p = 0.76) and PFC  = 0.02, p = 0.97) in the alcohol rats compared to the controls  = 18.33, p<0.0001; post hoc: p<0.01), CPu  = 54.46, p<0.0001; post hoc: p<0.0001), Amy  = 20.42, p<0.0001; post hoc: p<0.00001), Hip  = 73.21, p<0.0001; post hoc: p<0. 01), PFC  = 16.54, p<0.01; post hoc: p<0.05) in the alcohol rats /total-GluN1 was found in NAc . Shah et al. reported that the blood alcohol levels (BALs) in Swiss-Webster mice reached 20.06±8.32 mg/dl on the first day, and 14.75±3.98 mg/dl on the 7th day, by administrating a 7% v/v alcohol solution for 7 d Alcohol consumption inhibits locomotor activity et al. wherein they found only minor and mostly non-significant changes v.s. n = 10) used or the duration of the induction periods (12 days v.s. 28 days). Although drugs of abuse possess diverse neuropharmacological profiles, activation of the mesocorticolimbic system, particularly the NAc, Amy, CPu, PFC and Hip via dopaminergic and glutamatergic pathways, constitutes a common pathway by which various drugs of abuse mediate their reinforcing effects 9-tetrahydrocannabinol, nicotine, morphine and alcohol) Activated ERK phosphorylates cellular targets or translocates into the nucleus where it activates specific gene transcription factors et al. study, they observed that chronic alcohol exposure lead to an increase in expression of GluN1 subunit in the insoluble postsynaptic density fraction, but this increase was more transient and was no longer observed after 1 week of withdrawal The GluN1 subunit forms the molecular backbone of functional NMDA receptors and has been hypothesized to play a role in chronic alcohol consumption. To address this issue, we examined the expression and phosphorylation levels of the GluN1 subunit. Long-term alcohol consumption induced significant up-regulation of GluN1 subunits in all five brain regions. Enhanced GluN1 subunit mRNA expression was evident in samples from chronic ethanol-exposed animals in amygdala neurons et al. and the present study, it is also possible that neuronal NMDA receptors exist in a complex intracellular environment characterized by a wide array of signaling proteins that are presumably missing in a heterologous cell expression system like human embryonic kidney cells. Our results confirmed previous observations that continuous alcohol drinking greatly increases GluN1 in both total protein and its phosphorylated version et al. have demonstrated that decreased phospho/total-GluN1 is consistent with decreased NMDAR function Protein phosphorylation has been recognized as a major mechanism for the regulation of NMDA receptor function In conclusion, the findings of the present study indicate that alcohol can affects phosphorylation of ERK and GluN1 in brain reward circuits. JNK and p38 phosphorylation in mesocorticolimbic areas were not significantly influenced by this treatment. Our findings support previously reported associations between GluN1-ERK and dependence to alcohol and other substances, and emphasize the relevance of decreased phosphorylation of GluN1 and ERK in the mesocorticolimbic system for chronic alcohol exposure.Figure S1Schematic representation showing the approximate location of the brain regions excised and used for analysis. NAc: nucleus accumbens; CPu: caudate putamen; Amy: amygdala; Hip: hippocampus; PFC: prefrontal cortex .(TIF)Click here for additional data file.Table S1Rating scale for some behavior signs induced by ethanol withdrawal in rats.(DOCX)Click here for additional data file.