As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in Alzheimer’s disease, efforts are being made in search of new molecules with anti-AChE activity. The fact that naturally-occurring compounds from plants are considered to be a potential source of new inhibitors has led to the discovery of an important number of secondary metabolites and plant extracts with the ability of inhibiting the enzyme AChE, which, according to the cholinergic hypothesis, increases the levels of the neurotransmitter acetylcholine in the brain, thus improving cholinergic functions in patients with Alzheimer’s disease and alleviating the symptoms of this neurological disorder. This review summarizes a total of 128 studies which correspond to the most relevant research work published during 2006-2012 (1st semester) on plant-derived compounds, plant extracts and essential oils found to elicit AChE inhibition. For the sake of brevity and in order to focus our attention on the most relevant findings, only those research papers reporting quantified results (IC50 and/or percentage of inhibition at a given concentration) were included. Extracts or essential oils with IC50 > 0.5 mg/ml were considered weakly active and were therefore not taken into account in the present review. With a few exceptions, only molecules with IC50 < 50 µM have been considered. Furthermore, unless otherwise stated, those results on AChE inhibition included in the present review refer to in vitro assays carried out with AChE from electric eel.Several reviews on the newly discovered AChEi obtained from plants, fungus and marine organisms have also been published over the last years -10. The H-pyrano quinoline-5-one (6), ribalinine (7) and methyl isoplatydesmine (8) isolated from the aerial parts of Skimmia laureola (Rutaceae) were found to be linear mixed inhibitors of AChE with iK = 110.0, 30.0 and 30.0 µM, respectively  were obtained. Some of these alkaloids were analyzed to determine whether they are AChEi or not.. Ungeremine (72) (IC50 = 3.85 µM), norsanguinine (73) (IC50 = 1.43 µM) and 8-demethoxy-10-O-methylhostasine (69) (IC50 = 2.32 µM) were all found to be potent AChE inhibitors.Although benzylphenethylamine alkaloids were considered to belong exclusively to the Amaryllidaceae, some of them have been found to belong to other families . A new eliaceae) . Sevente5) from Huperzia serrata (Lycopodiaceae), several plants belonging to the genus Lycopodium have been investigated in an attempt to find alkaloids with unusual skeletons that could have AChE inhibitory activity  (186) was observed to inhibit AChE with an IC50 = 20.5 µM [On the other hand, a pterocarpan with moderate AChE inhibition was isolated from the polar extract of  20.5 µM . InteresMansonia gagei (Sterculiaceae) proposed a novel class of cholinesterase inhibitor, mansonones or 1,2-naphtoquinones [187) was observed to be the most active AChE (IC50 = 23.5 µM) and BChE inhibitor.A study conducted on AChE and BChE inhibitory activity of coumarins and naphtoquinones obtained from quinones . The lev188, IC50 = 5.85 µM) and ellagic acid  [190), which was identified as a new compound isolated from Hopea hainensis, was observed to elicit a notable AChE inhibition (IC50 = 4.33 µM) with respect to huperzine A (IC50 = 1.6 µM), as a reversible mixed-type inhibitor [In several studies published during the period covered in the present review various phenolic compounds with different structural characteristics were reported as AChEi. Some of them are structurally simple such as gallic acid (5.63 µM) . Hopeahanhibitor .Terminalia chebula (Combretaceae) fruits allowed the isolation of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (191) which demonstrated to be a significant AChE inhibitor (IC50 = 29.9 µM) [The bioassay-guided fractionation of the extract from 29.9 µM) . This gaKnema laurina (Myristicaceae) yielded two active fractions (dichloromethane and hexane) which were subjected to chromatographic separation. That latter yielded five alkenyl phenol and salicylic acid derivatives 192 - 196, of which 192 and 193 were new compounds [192, 195 and 196, all having salicylic acid moiety, were observed to strongly inhibit AChE with an IC50 = 3.182, 2.172 and 0.573 µM, respectively. Compounds 193 and 194, with no carboxyl moiety, were observed to be good AChE inhibitors . These findings suggest that the acidic group is key to good AChE inhibition. It was also observed that anti-AChE activity dramatically decreased when the acidic and the phenolic hydroxy group were methylated. Two catechol alkenyls were isolated from the fruits of Semecarpus anacardium (Anacardiaceae), a species used in Ayurvedic medicine for retarding and treatment of memory loss [197 and 198 were identified as active components of the dichloromethane extract through a fractionation guided by the detection of AChE inhibition. Microplate assay revealed that these catechol alkenyls are moderate and weak selective AChEi. Compound 197, with a double bond in the aliphatic chain, was identified as a stronger inhibitor (IC50 = 39.7 µM) with respect to compound 198, with two double bonds in the aliphatic chain (IC50 = 108 µM).The bioassay-guided extraction of the stem bark of ompounds . Compounory loss . CompounNelumbo nucifera (Nelumbonaceae) stamens [199), p-hydroxybenzoic acid (200), vanilloloside (201) and nuciferoside (202) were found to elicit good and noncompetitive inhibition against AChE with an IC50 = 11.89, 20.07, 4.55 and 3.2 µM, respectively. In the same study, compounds 199, 200 and 202 were observed to exert moderate BChE inhibition and compounds 199 - 202 were found to show no inhibition against BACE1.On the other hand, four structurally diverse AChEi were isolated from the polar extract of  stamens . Cycloar1 summarizes the studies published from 2006 to 2012 on plant extracts, fractions and essential oils that have been found to be good AChE inhibitors (IC50 < 500 µg/mL). Those plants included in other recent reviews were omitted [50 values were chosen instead of inhibition percentages when both were available.Table  omitted , 77. Ext


These findings suggest that acupuncture stimulation can suppress bladder overactivity, and regulate the excitability of bladder interstitial cells of Cajal in treatment of overactive bladder myogenic mechanism.It is well known that acupuncture treatment has an effect on patients with an overactive bladder, but the mechanism of its action remains to be clarified. This study was aimed to investigate the effects of acupuncture on bladder overactivity, and the excitability of interstitial cells of Cajal of the bladder in a rat model of partial bladder outlet obstruction. Electroacupuncture  was applied to stimulate the Ciliao point (BL32) and the Huiyang point (BL35) of rats for 20 min, 3 days. Results showed that acupuncture suppressed detrusor unstable contraction frequency and decreased detrusor maximum pressure in the bladder filling period. Compared with the normal control rats, HCN2 mRNA and protein expression within the bladder were upregulated and were reversed by electroacupuncture in overactive bladder rats as determined by RT-PCR, western blotting and immunohistochemistry. Moreover, in-vitro cell-cultured OAB rats bladder interstitial cells of Cajal intracellular Ca Overactive bladder (OAB) syndrome is characterized by urinary frequency and urgency with or without urge incontinence, and is often accompanied by nocturia. Acupuncture, as one of the traditional Chinese therapeutics, provides a nondrug therapy for OAB. It has been reported that the acupuncture stimulation to the sacral vertebrae has a suppressive effect on bladder activity, improves the symptom of nocturnal enuresis, decreases mean urge frequency, and diminishes voiding symptoms –5. Our pMany studies have found that interstitial cells of Cajal (ICCs) in bladder, like in gastrointestinal tract, act as primary pace maker cells that generate depolarizing currents into neighboring smooth muscles and coordinate muscle contractions, playing a fundamental role in signal transmission from bladder nerves to smooth muscle cells . BladderAll experiments were performed on female Wistar rats, obtained from the Experimental Animal Center, Shanghai University of Tranditional Chinese Medicine, weighing 180–200 g at the beginning of the experiment. Animals were housed in a 12 h light/dark cycle with food and water available ad libitum. The room temperature was maintained at 22 ± 1°C and relative humidity at 45–50%. Five animals were housed in each cage. All experiments were performed complying with international ethical standards in the care and use of animals, and passed the examination of animal ethics committee of Shanghai University of TCM.n = 100) firstly were randomly divided into two groups: normal control (n = 15), model (n = 85). Rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). The normal control group underwent a sham operation. Partial bladder outlet obstruction (PBOO), the classical method to induce OAB i concentration. Intensity ratios at 340 and 380 nm excitation (F340/F380) were calibrated using an in vitro method and converted into Ca2+ concentration according to the equation, [Ca2+]i = Kd ((R − Rmin)/(Rmax − R)), where Kd represents the dissociation constant with a value of 224 nM, R represents F340/F380, and Rmin and Rmax represent intensity ratios in 0 Ca2+ (added 0.1 M EGTA) and saturating Ca2+ (0.1% Triton X-100 and 0.1 M CaCl2). Preparations were loaded in DMSO containing 5 P < 0.05 considered significant.All data were expressed as mean ± SD. Statistical significance was tested using one-way analysis of variance (ANOVA) and post hoc tests, with values of P = 0.227 before treatment, P = 0.77 after treatment). No significant differences in body weight were observed among four groups before and after treatment. (n = 13), the detrusor unstable contraction frequency (time/period) in bladder filling period was significantly increased in the model group  , acupuncture group  , and Glivec group  , but no significant difference among model group, acupuncture group, and Glivec group . Comparison before and after treatment, there was no difference in the normal control group  and model group respectively, , and decreased in the acupuncture group , and Glivec group respectively, . Comparison among groups after treatment , normal control group (0.27 ± 0.60) acupuncture group (0.57 ± 0.92), Glivec group (1.31 ± 1.29) were less than model group (3.20 ± 2.68) , and there were no significant difference among the normal group, acupuncture group and Glivec group  , the detrusor unstable contraction maximum pressure (cmH2O) in bladder filling period was significantly raised in the model group  , acupuncture group  , and Glivec group  , but no significant difference among model group, acupuncture group, and Glivec group . in a Comparison before and after treatment, there was no difference in the normal control group  and model group respectively , and decreased in the acupuncture group  and Glivec group respectively . Comparison among groups after treatment , normal control group (7.68 ± 4.69) and acupuncture group (14.29 ± 10.94), Glivec group (18.24 ± 18.10) were lower than model group (45.69 ± 23.55) , and there were no significant difference among normal group, acupuncture group, and Glivec group  . Model group  is significantly higher than normal control group  , acupuncture group  , and Glivec Group ,  (The expression of HCN2 mRNA in bladder was significantly different among groups ( < 0.05) .F = 3.394, P = 0.05). Model group  was increased compared with normal control group  , and significantly decreased in the acupuncture group   and Glivec group    . F = 3.086, P = 0.039, P < 0.05). Model group (2.70 ± 2.41) was more than normal control group (0.40 ± 0.84) , but there were no difference compared with acupuncture group (1.30 ± 0.95)  and Glivec group (1.60 ± 2.07)   were found in rat urothelium, suburothelium, and muscle layer by fluorescence microscope. Bladder ICCs were found in detrusor muscle layers that were mainly located along the boundary of smooth muscle bundles and between muscle bundles. The quantity of ICCs was counted in every visual field and 10 visual fields were observed by random in every group. The result showed a significant difference among groups ( > 0.05) .F = 6.756, P = 0.00, P < 0.05), the quantity of HCN2 positive ICCs in model group (10.50 ± 8.53) was significantly increased compared with normal control group (0.40 ± 0.97) , and was less distributed in acupuncture treatment (4.20 ± 4.64)  and Glivec group (4.60 ± 2.80)   . Cultured cells were observed under inverted microscope. The morphology of ICCs was spindle-shaped, with several branches emanating from a central soma, and connected with neighboring cells, showing networks . The ICC2+ indicator fura-2 AM. Varioskan flash (ThermoFisher Scientific) was used for measuring [Ca2+]i concentration, every well in 96-well plates was determined on 5 regions of interest, and 40 regions were selected in each group. According to the equation, [Ca2+]i = Kd ((R − Rmin)/(Rmax − R)), to convert into Ca2+ concentration. The intracellular free Ca2+ concentration in bladder ICCs of model group (10.73 ± 3.05) was higher as compared to normal control group (5.24 ± 2.80), ; acupuncture stimulation (5.57 ± 5.31) and Glivec bladder irrigation (6.21 ± 2.60) was significantly lower the OAB rats bladder ICCs intracellular Ca2+ concentration   .2+ concentration. This is the first evidence that acupuncture stimulation regulates the excitability of  bladder ICCs in treatment of overactive bladder myogenic mechanism.This study demonstrated that acupuncture stimulation suppressed bladder overactivity, down-regulated the expression of HCN2 mRNA and protein in bladder, reduced the quantity of HCN2 channel in bladder ICCs, and also decreased bladder ICCs intracellular free Ca2 level and coccyx, which belongs to the Bladder Meridian of Foot Taiyang in Traditional Chinese Medicine [The points of stimulation in this study are the classical points BL32 and BL35 corresponding to sacral vertebrae SMedicine . The cliMedicine , 23. In −5 M, improved bladder capacity, compliance, voided volumes, urinary frequency, and reduced contraction thresholds and spontaneous activity [−5 mol/L bladder irrigation. ICCs have been widely recognized as playing key roles in the initiating contractile activity, mediating neurotransmission, conducting electrical impulses, and acting as a stretch sensor . The spoactivity . Our res−5 M bladder irrigation. The results from RT-PCR and western blotting analysis revealed that HCN2 subtype was detected in bladder, and its mRNA and protein expression were increased in OAB rats, suggesting that there are some changes in the HCN2 channel in the pathologic conditions, with the excitability enhancement of HCN2 channel in bladder. HCN2 involved in the suppressing effect of acupuncture stimulation on bladder unstable contraction, acupuncture stimulation and Glivec 10−5 M bladder irrigation down-regulated the HCN2 mRNA and protein expression, suggesting that HCN channel in ICCs may be the new therapeutic target for OAB. HCN channels are active at resting membrane potential and thus are believed to participate in autorhythmicity and excitability. Immunofluorescence and Western blotting revealed that HCN1, HCN2, HCN3, and HCN4 channels were all present in the ICCs of the human bladder . HCN1, 22+ concentration is a mechanism common to signal transduction pathways regulating many cellular phenomena, the role of intracellular Ca2+ is of particular importance, as it is the main determinant of detrusor contractile activity. Spontaneous, autonomous cellular activity-Ca2+ and membrane potential oscillations, originate from detrusor smooth muscle in human bladders, mediated by extracellular Ca2+ influx and intracellular release [2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal that triggers contraction through opening L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction [2+ concentration was elevated in Bladder ICCs from overactive bladders and was significantly decreased through acupuncture stimulation and Glivec, which suggest that acupuncture might have affected the membrane potential and made it difficult to induce Ca2+ influx, and the L-type calcium channel opened to trigger the action potential.Modulation of cytoplasmic Ca release . Meanwhitraction . In this2+ concentration. The result may help to provide a scientific foundation to build the clinical acupuncture treatment in OAB.A systematic profile of the effects of acupuncture stimulation on bladder overactivity and the excitability of bladder ICCs were shown in the present study. The results show that acupuncture stimulation suppressed bladder overactivity, down-regulated the expression of HCN2 mRNA and protein in bladder, and reduced the quality of HCN2 chanel in bladder ICCs. Furthermore, acupuncture stimulation also decreased bladder ICCs intracellular-free Ca


Recently, a new model, the zQ175 knock-in (KI) mouse, was developed  is an autosomal neurodegenerative disorder, characterized by severe behavioral, cognitive, and motor deficits. Since the discovery of the  Our work highlights significant, robust, dose-dependent, progressive, and early-onset alterations in these endpoints, which may prove useful to further investigate biological pathways affected by mHtt and to evaluate pharmacological or genetic interventions to modify disease onset and progression.Existing genetically-modified HD mouse lines use different Experiments were conducted at Charles River Discovery Research Services, Kuopio, Finland, except the immunohistochemical measurements which were performed at MBF Labs, Williston, VT, USA, and the brain slice physiology conducted at Neuroservice SARL, France. All animal experiments were conducted according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals, and approved by the State Provincial Office of Southern Finland. For behavioral experiments, 10 female heterozygous (HET), 9 female homozygous (HOMO) zQ175 mice and 10 female wild-type littermate controls (WT) were acquired from Psychogenics Inc. . zQ175 mice, originating from the CAG 140 mice (from germline CAG expansion) were generated by Psychogenics Inc. Homozygous, heterozygous and wild-type mice were generated by crossing heterozygous zQ175 mice on a C57B/L6J background. Genotyping and CAG repeat count were determined by Laragen Inc.  at 10–15 days of age by PCR of tail snips. The average CAG repeat length was 178.3 (range 173 to 182) in heterozygous mice and 180.7 (range 175 to 185) in the homozygous mice. All the mice were housed in groups of up to 5 per cage, in a temperature (22±1°C) and humidity (30–70%) controlled environment with a normal light-dark cycle (7:00–20:00). All mice were housed in cages (dimensions: length 35 cm×width 19 cm×height 13 cm) with clean bedding covering the ground changed as needed to provide animals with dry bedding. In addition, a red mouse igloo was placed in each cage to provide environmental enrichment and shelter. Food (Purina Lab Diet 5001) and water were available ad libitum to mice in home cages. Water spouts were fitted with extensions to allow easy access from floor level. Mouse body weight was recorded weekly.The tests were conducted during the dark phase of the light-dark cycle (between 23:00–5:00).Open field test was performed at the age of 2 months, 4 months, 8 months and 12 months. The mice were tested at approximately 10–30 lux of red light. The mice were brought to the experimental room for at least 1 h acclimation to the experimental room conditions prior to testing. The open field behavior was measured by automated Open Field Activity System and the data analyzed by Activity Monitor software . The activity chambers (27.3 cm×27.3 cm×20.3 cm) were equipped with infrared beams. Mice were placed in the center of the chamber and their behavior was recorded for 30 min in 5-minute bins.Rearing climbing test was performed at the age of 2 months, 4 months, 8 months and 12 months. Each animal was placed into custom made pencil holder with wire mesh walls (height 15 cm×diameter 10.5 cm), and was tested for 5 minutes. Each test session was videotaped and the behavior was analyzed later from the videotapes by an experimenter blinded to the groups The swim tank was (dimensions: length 70 cm×width 30 cm×height 30 cm) filled with water (25±1°C) no higher than 1 cm from the top of a hidden escape platform. A hidden cylindrical platform  was placed either at right or left end of the tank. During testing only red lights were used in the room. During acquisition of the task, each mouse was given 8 training trials per day to swim towards or away from the right end  to escape onto the platform. On each trial, mice were released in the center of the tank facing the experimenter and allowed to swim for up to 60 seconds or until they found the platform. If the mouse chose first the end of the tank with the platform “correct choice” was recorded, if the end without the platform “incorrect choice” was recorded. All animals were trained for 5 consecutive days. In all trials, choice and latency were recorded. The test was first commenced at 10 months of age and again at 12 months of age. The platform location was randomly placed on either the left or right side for each mouse and the platform location was the same throughout the testing. Each animal had the platform on the same end of the tank at both 10 and 12 months of age during testing.4 2, NaH2PO4 1.25, NaHCO3 26, D-glucose 11.1, kynurenic acid 1. Slices containing the dorsal striatum were transferred into an incubation chamber containing artificial cerebrospinal fluid (ACSF) with a composition of (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, NaHCO3 25, NaH2PO4 1.25, D-glucose 25 at room temperature (RT) for at least 30 min to recover. All solutions were continuously bubbled with carbogen gas . For recordings, the slices were transferred into a 1 ml volume chamber and continuously superfused with ACSF (∼1.5 ml/min) at RT.250 µm thick horizontal corticostriatal brain slices from mixed gender 2 – 9 month old zQ175 wild-type, heterozygous and homozygous mice were acutely prepared on a VT1200S vibrotome (Leica) in ice-cold sucrose solution composed of (in mM): sucrose 248, KCl 2, MgSOa) , were discarded. Average Ra was 9.5±0.28 MΩ, (n = 115), no significant difference between genotypes or age groups was noted.Medium spiny neurons (MSNs) within the slice were visualized using an upright microscope equipped with infrared differential interference contrast (IR-DIC) and a 60× water-immersion objective . Cells had an average membrane capacitance of 57.8±0.7 pF, n = 115, with no significant differences between genotypes or age groups noted. Recordings with an unstable or poor access resistance . Under voltage clamp control, cells were held at −80 mV and membrane capacitance (Cm) and membrane resistance (Rm) were calculated in response to a −10 mV hyperpolarizing pulse using the Membrane Test function of Clampex. Resting membrane potential (RMP), rheobase  and resultant action potential amplitude of the neurons were evaluated by switching to current clamp mode, prior to returning to voltage clamp control. Cortical stimulation to evoke glutamatergic excitatory post synaptic currents (eEPSCs) in the striatal MSNs were performed via a custom-built bipolar tungsten electrode that was placed between layer V of the cortex and deeper cortical layers. In a given MSN recording, cortical stimulation at a range of intensities (0.2–20 V) was used to determine the strength of glutamatergic transmission. Stimulation was repeated 20 times at a frequency of 0.1 Hz for each intensity level and the resultant eEPSCs were averaged per neuron. To determine the paired-pulse ratio, two stimulating pulses with a specific inter stimulus interval (ISI) were applied to the cortex . Each individual cell was stimulated 5 times at a frequency of 0.1 Hz. The paired-pulse ratio was then calculated based on the average of these responses by dividing the amplitude of the second postsynaptic current recorded from the MSN with the first. Three age groups at 3–4 months, 6–7 months and 8–9 months of age were evaluated for recordings of MSN membrane parameters and evoked corticostriatal transmission. Whole-cell patch clamp experiments were performed in both voltage-clamp and current clamp mode using a software-controlled MultiClamp 700B amplifier in combination with a Digidata 1440A digitizer (Molecular Devices). Patch pipettes were pulled from borosilicate glass capillaries (electrode resistance = 2–4 MΩ). For these experiments the intracellular solution contained (in mM): K-gluconate 105, KCl 30, MgCl2 1, EGTA 10, MgATP 5, Na3GTP 0.5, pH 7.2. All recordings were performed with the addition of tetrodotoxin  and picrotoxin (50 µM) to the ACSF. Recordings were made at −80 mV for 20 min after observing stable activity for 10 min. The experiments were terminated by application of 10 µM CNQX to confirm that the events recorded were entirely AMPA receptor mediated. 23 neurons/genotype were recorded in the 2 month age group, and between 10–35 neurons/genotype recorded in the 7–9 month age group.A separate set of experiments was performed in 2 month (n = 4 mice/genotype) and 7–9 month old mice  to isolate miniature excitatory postsynaptic currents (mEPSCs). In this instance, the intracellular solution comprised (in mM): Cs-methanesulfonate 100, Na-methanesulfonate 10, QX314-Cl 5, TEA-Cl 10, HEPES 10, CaClData were analyzed with Clampfit 10 (Molecular Devices), Prism 5 (GraphPad) and Igor Pro 6 (Wavemetrics). mEPSCs recordings were analyzed for amplitude and frequency using Minianalysis (Synaptosoft). After the automatic detection of events, each trace was manually checked for false positives and false negatives.MRS and MRI measurements were performed for all mice at the age of 3 months, 4 months, 8 months and 12 months in a horizontal 7T magnet with bore size 160 mm  equipped with Magnex gradient set  interfaced to a Varian DirectDrive console . Linear volume coil was used for transmission and surface phased array coil for receiving . Isoflurane-anesthetized mice were fixed to a head holder and positioned in the magnet bore in a standard orientation relative to gradient coils. Rectal temperature  and respiration  were monitored throughout the study and the body temperature was kept at 36–37°C. For determination of volume of brain, striatum and cortex T2-weighted multi-slice (17 continuous slices) images were acquired using fast spin-echo sequence with TR/TEeff = 3000/36 ms, matrix size of 256×128, FOV of 20×20 mm2, slice thickness of 0.7 mm and 8 averages.3 was placed in the striatum of the mouse based on T2-weighed images collected as described above. Automatic 3D gradient echo shimming was initially used to adjust B0 homogeneity in the voxel and further manually shimmed to average water line-widths of 10.8±0.8 Hz. The water signal was suppressed using variable power RF pulses with optimized relaxation delays (VAPOR) to obtain B1 and T1 insensitivity. A PRESS sequence (TE = 19 ms) combined with outer volume suppression (OVS) is used for the pre-localization. Three OVS blocks were used interleaved with water suppression pulses. Data were collected by averaging 512 excitations (frequency corrected 16×32 blocks) with TR of 4 s, number of points 3000 and spectral width 3 kHz. In addition a reference spectrum without water suppression is collected from the identical voxel using the same acquisition parameters. Peak concentrations for major metabolites  were analyzed using LCModel  and results are given relative to water content in tissue.1H-MRS data were collected using the same experimental setup. Voxel of 2.5×2.5×3 mmTotal of 3 female and 3 male mice per genotype per age  were used for stereological analysis. The brains were perfusion-fixed, frozen and sectioned at 60 µm sections at 360 µm intervals . One series of sections per animal was processed for NeuN immunohistochemistry using overnight incubation with Millipore antibody MAB377, lot LV1634819, dilution 1∶100 000. Secondary antibody  and Vectastain ABC-kit (Vector) were used before visualization by DAB.The stained sections were shipped to MBF Labs for stereologic analysis. All analyses were performed using a Olympus BX51 modified light microscope  with Plan 2×  objective for delineation, and UPlanApo oil 100× (N.A. = 1.35) objective for counting, motorized specimen stage for automated sampling , CCD color video camera  and Stereo Investigator stereology software .The number of NeuN-positive neurons was quantified in bilateral striatum (Caudate-putamen and Nucleus Accumbens) using the Optical Fractionator stereological method. Sampling parameters were optimized to return an average coefficient of error of 0.05 (Gunderson (m = 1), The statistical analyses were performed using IBM SPSS Statistics 19 and StatsDirect software unless otherwise specified. Behavioral and MRI measures were analyzed with Linear Mixed Models, analyzing the interactions for genotype, age and genotype×age interaction. If significant genotype effect was found One-way ANOVA followed by Dunnett's post-hoc test (comparison to wild-type mice) was used for each time point separately. The MRS data were analyzed with One-way ANOVA for each time point and metabolite separately. Stereology data were analyzed using 2-way ANOVA. All values are presented as mean ± Standard Error of Mean (SEM), and differences are considered to be statistically significant at the p<0.05 level. For the electrophysiological recordings, statistical analysis was performed using Prism 5 (GraphPad) and Igor Pro 6 (Wavemetrics). One-way ANOVAs with post hoc Tukey's test was used to assess statistical significance of the membrane parameters Rm, Cm, RMP, rheobase and spike amplitude, mean mEPSC frequency and amplitude values. A Two-way ANOVA with Bonferroni multiple comparison test was used to assess statistical significance of evoked corticostriatal transmission and paired pulse ratios. For mEPSCs, statistical differences in genotype were additionally assessed using D'Agostino and Pearson omnibus normality test to test for Gaussian distribution, followed by one sample t-test, to test whether means were significantly different than WT mean values from the frequency distribution analysis.The homozygous zQ175 mice had decreased body weight on the first measurement point at 8 weeks of age compared to wild-type mice. The body weight of the homozygous mice remained significantly decreased until the end of the study, at 56 weeks of age, except on age weeks 19 and 21 compared to wild-type mice. The heterozygous mice had decreased body weight at 52, 53 and 56 weeks of age compared to wild-type mice  = 359.0, p<0.001; age main effect, F = 43.9, p<0.001; genotype×age interaction, F = 1.6, p = 0.033; One-way ANOVA, p<0.05) .The homozygous mice showed decreased horizontal activity compared to wild-type mice, i.e. they traveled shorter total distance already at 2 months of age, but only at the later phase of the test session, from 15 to 30 min. Furthermore, both the homozygous and heterozygous mice traveled a shorter total distance at 4 and 8 months of age, and the homozygous mice also at 12 months of age  = 20.4, p<0.001; age main effect, F = 65.0, p<0.001; One-way ANOVA, p<0.05) . AlthougThere was a significant main genotype effect in the rearing frequency in the rearing climbing test but no significant differences at separate time points were observed between the different genotypes  = 4.6, p = 0.013; age main effect, F = 22.6, p<0.001) . HoweverThe procedural two-choice swim test was performed at 10 and 12 months of age. At 10 months of age the homozygous mice had a decreased percentage of correct choices on each test day  = 30.0, p<0.001; test day main effect, F = 44.6, p<0.001; One-way ANOVA, p<0.05)  and on tWhole cell patch clamp recordings of medium spiny neuron (MSN) membrane properties were taken from acutely prepared brain slices from zQ175 wild-type, heterozygous and homozygous mice between 3 and 9 months of age, and were pooled into ‘early’ (3–4 months) and late (6–9 month) time-points for analysis . The resImpaired corticostriatal connectivity and dysfunctional information processing in corticostriatal circuitry is a key feature of Huntington's disease To further attempt to identify the basis of this impairment in evoked glutamatergic transmission, a paired-pulse stimulation paradigm  was employed to specifically assess alterations in glutamate release probability in the stimulated corticostriatal synapses The homozygous zQ175 mice had decreased whole brain  = 66.8, p<0.001; age main effect, F = 3.3, p<0.001; One-way ANOVA, p<0.05), striatal  = 107.8, p<0.001; age main effect, F = 30.7, p<0.001; One-way ANOVA, p<0.05) and cortical volumes  = 83.8, p<0.001; age main effect, F = 6.2, p = 0.001; One-way ANOVA, p<0.05) at 3, 4, 8, and 12 months of age . The hetVolumetric changes were most pronounced in the striatum (down to approximately −21% at 12 months) in homozygous mice, followed by cortical atrophy (down to approximately −15% at 12 months) and whole brain atrophy (down to approximately −11% at 12 months) . HeterozVarious brain transmitter and metabolite changes are also observed in the striatum of zQ175 mice over time  and 7. FA power analysis of the MRI volumetry data at 12 months of age  indicated that a group size of 8–12 mice per genotype would be sufficient to detect a 50% improvement in whole brain, striatal and cortical volumetric loss in homozygous zQ175 mice  and Nucleus Accumbens (NA), was analyzed for changes in regional volume, neuron number and neuronal cross-sectional area due to genotype, age and interactions of genotype and age, using stereology .The CPu volume increased with age in wild-type mice  = 13.9, p<0.001). No age-related changes in CPu volume were observed in heterozygous or homozygous mice. No significant age-related difference was detected in the NA in wild-type mice. However, in heterozygous mice, the NA was larger at 10 months than at 4.5 months  = 4.7, p = 0.038). Similarly, the volume of both CPu and NA were significantly reduced at both 4.5 and 10 months old in homozygous animals, when compared to wild-type and heterozygous animals . Total striatal volume of homozygous mice was significantly changed  = 19.2, p<0.001), when compared to age-matched wild-type and heterozygous mice . MRI vol6 vs. 2.60×106) and 10 months of age (1.97×106 vs. 2.31×106). Neuronal cross-sectional area was not significantly different within the striatum or the caudate putamen,  = 1.1–1.7; p = 0.16–0.37). While no change in cell size was observed in homozygous mice, a significant increase in cell size was observed in the nucleus accumbens for heterozygous and wild-type mice at 10 months as compared to genotype-matched 4.5 months groups  = 2.8; p = 0.03).Stereological analysis of NeuN-immunoreactive cells showed that neuronal number was highest in 4.5 month old wild-type animals (wild-type>heterozygous>homozygous) in all regions examined. In the CPu, homozygous mice had significantly fewer neurons when compared to wild-type animals at both 4.5 and 10 months of age  = 4.8, p = 0.003). Homozygous mice also had significantly fewer neurons in the NA at 4.5 months  = 8.5, p<0.001) compared to age-matched wild-type mice. No significant differences were seen in CPu or NA between heterozygous mice and wild-type at either age. At 10 months of age, NA neuronal number was decreased in all genotypes  when compared to genotype-matched young (4.5 month) mice  = 6.4–14.7, p = 0.017-0.001). While wild-type mice showed the greatest decrease with age, this is likely due in part to the early loss of neurons in transgenic animals by 4.5 months. When the CPu and NA are considered together as the striatum, total striatal neuron number was significantly reduced in both 10 month-old homozygous and wild-type mice when compared to genotype-matched 4.5 month-old mice  = 5.2–6.1, p = 0.02–0.03; Because of concomitant changes in volume and cell number, neuronal density was reduced in wild-type and homozygous mice at 10 months when compared to genotype matched mice 4.5 months of age in CPu  = 6.8–9.7; p = 0.004–0.01). The neuronal density in NA was also significantly reduced as function of age in all genotypes  = 4.7–17.8, p = 0.001). In the striatum, each group had also significantly reduced neuronal density at 10 months of age than in their younger cohorts  = 5.8–11.7, p = 0.009–0.022; We have evaluated the onset, progression and magnitude of behavioral, electrophysiological, morphological and brain metabolic changes in the new zQ175 KI mouse model of HD. Body weight decrease is a commonly observed manifestation of progressive disease in many HD mouse models and also occurs in HD patients Changes in voluntary and involuntary movement are apparent in HD; chorea, followed by bradykinesia and dystonia, are common. Changes in motor function have been reported in other HD mouse models; YAC 128 mice show open field hyperactivity at 3 months followed by decreased activity at 12 months Cognitive function was measured in a procedural two-choice swim test; this kind of procedural stimulus-response task presumably depends on intact striatal circuitry HTT insult and dramatic loss of this neuronal population in HD patients is a hallmark of the disease Medium spiny neurons (MSNs) are particularly vulnerable to mHtt expression from both alleles in zQ175 mice. In contrast, we saw no early MRI changes in brain regional volumes in heterozygotes, followed by a slowly progressing decline over a 12-month period; consistent with this, stereological changes in brain regional volume were not evident at early ages in heterozygotes. However, in contrast to MRI, stereology indicated only trends toward decreased brain regional volumes. This difference may be due to smaller N values and larger variation, possibly due to use of both sexes in the stereology studies.In pre-manifest and manifest HD patients, brain atrophy is one of the most consistent and early pathological changes Given that the CAG repeat in the zQ175 mice is in the high juvenile HD range, a comparison of the time course and magnitude of changes to that seen in early stage juvenile HD may be instructive. Furthermore, in regard to brain regional changes, the heterozygous zQ175 mice may be more suitable in studies evaluating this outcome measure as they do not show the very early  changes reported in the homozygotes.We report here a decrease in neuronal number, measured by stereology, as early as 4.5 months in homozygotes, remaining constant at 10 months. At 4.5 months the striatal neuronal number was decreased 5% in heterozygotes and 14% in homozygotes compared to WT. Striatal neuronal loss has been reported in some transgenic HD mouse models; N171-82Q mice expressing 82 CAG repeats have as much as 39% fewer NeuN-positive neurons in striatum compared to WT MRS data are normally given either as concentration ratio relative to total creatine or as absolute concentration . Increased creatine levels are common in HD mouse models while clinical MR studies indicate decreased striatal creatine levels. Although the direction of creatine change is intriguing in an HD context, creatine ratios should be avoided since changes in concentration ratio of a metabolite may be a result of either true change or from a change in creatine levels. While brain water homeostasis is very efficient, increased metabolite levels reported here raise the possibility that there is a significant contribution from altered NMR visible water content in both hetero- and homozygotes as a function of age, HD pathology, or combination of the two. Interestingly, the metabolic changes in HD and hypernatremia are similar In summary, the behavioral, electrophysiological, histological, and metabolic imaging measurements reported here indicate that the progressive phenotypes in the zQ175 mice up to 12 months of age recapitulate some aspects of HD. The robustness of the phenotypes, their dosage sensitivity, and their progressive nature makes this new mouse line a promising model for pharmacological or genetic testing to identify mechanisms that can modulate HD progression.Table S1MRI volmetry power analysis in zQ175 and WT mice. Summary of the sample size needed to detect a 50% effect in the MRI volumetry for whole brain, striatum and cortex, and in the concentrations of selected MRS striatal metabolites with an alpha of 0.05 and a power of 0.8 for the heterozygote (HET) and homozygote (HOMO) mice at 12 months of age. N/A, not applicable.(DOC)Click here for additional data file.


HIV infection has commonly been found to affect lipid profile and antioxidant defense.To determine the effects of Human Immunodeficiency Virus (HIV) infection and viral subtype on patient’s cholesterol and oxidative stress markers, and determine whether in the absence of Highly Active Antiretroviral Therapy (HAART), these biochemical parameters could be useful in patient’s management and monitoring disease progression in Cameroon. For this purpose, we measured total cholesterol (TC), LDL cholesterol (LDLC), HDL cholesterol (HDLC), total antioxidant ability (TAA), lipid peroxidation indices (LPI), and malondialdehyde (MDA) in HIV negative persons and HIV positive HAART-naïve patients infected with HIV-1 group M subtypes.We measured serum TC, LDLC, HDLC, plasma MDA, and TAA concentrations, and calculated LPI indices in 151 HIV-positive HAART-naïve patients and 134 seronegative controls. We also performed gene sequence analysis on samples from 30 patients to determine the effect of viral genotypes on these biochemical parameters. We also determined the correlation between CD4 cell count and the above biochemical parameters.We obtained the following controls/patients values for TC (1.96±0.54/1. 12±0. 48 g/l), LDLC (0. 67±0. 46/0. 43±0. 36 g/l), HDLC (105. 51±28. 10/46. 54±23. 36 mg/dl) TAA (0. 63±0. 17/0. 16±0. 16 mM), MDA (0. 20±0. 07/0. 41±0. 10 µM) and LPI (0. 34±0. 14/26. 02±74. 40). In each case, the difference between the controls and patients was statistically significant (p<0.05). There was a positive and statistically significant Pearson correlation between CD4 cell count and HDLC , TAA  and a negative and statistically significant Pearson correlation between CD4 cell count and LPI . Pearson correlation between CD4 cell count and TC, CD4cell count and LDLC was positive but not statistically significant while it was negative but not statistically significant with MDA. The different subtypes obtained after sequencing were CRF02_AG (43.3%), CRF01_AE (20%), A1 (23.3%), H (6.7%), and G (6.7%). None of the HIV-1 subtypes significantly influenced the levels of the biochemical parameters, but by grouping them as pure subtypes and circulating recombinant forms (CRFs), the CRF significantly influenced TC levels. TC was significantly lower in patients infected with CRF (0.87±0.27 g/l) compared to patients infected with pure HIV-1 subtypes (1.32±0.68 g/l) (p<0.017). MDA levels were also significantly higher in patients infected with HIV-1CRF01_AE (0.50±0.10 µM), compared to patients infected with CRF02_AG (0. 38±0. 08 µM) (p<0.018).These results show that HIV infection in Cameroon is associated with significant decrease in TAA, LDLC, HDLC and TC, and increased MDA concentration and LPI indices which seem to be linked to the severity of HIV infection as assessed by CD4 cell count. The data suggests increased oxidative stress and lipid peroxidation in HIV-infected patients in Cameroon, and an influence of CRFs on TC and MDA levels. Human immunodeficiency virus type 1 (HIV-1) is the pathogen responsible for acquired immunodeficiency syndrome, a disease which has spread throughout the world and which affects immune cells, especially CD4+ lymphocytes and macrophages HIV-1 is divided into four groups: M for major, O for outlier, N for non M non O Dyslipidemia is a clinical condition which often leads to alterations in lipid profile: total cholesterol (TC), low density lipoprotein cholesterol (LDLC) and high density lipoprotein cholesterol (HDLC) Cameroon is a country with a high diversity of HIV-1 subtypes Informed consent was obtained from all subjects according to the guidelines of the Cameroon National Ethics Committee that approved the study. After obtaining informed consent, we enrolled 285 individuals who met our inclusion criteria: (1) for control subjects, exclusion criteria were pregnancy, serological evidence of hepatitis B/C, diabetes, hypertension, current intake of drugs, alcohol, tobacco, malaria and other known parasitic infection and inclusion criteria were HIV negative with none of the above conditions, and be able to read and sign an informed consent; (2) for patients, the exclusion criteria were the same as for control subjects; in addition, HIV-positivity was confirmed.The 285 individuals included 151 patients (thirty were taken for genotypic studies) and 134 control subjects.Following a 10 hour fast, 10 ml of blood was collected from each participant into a labeled dry tube (5 ml) and a tube containing EDTA anticoagulant (5 ml). Following clotting, the dry tubes were centrifuged at 1200 g for 15 min to collect serum which was aliquoted and used for the assay of the different biochemical parameters while EDTA tubes were centrifuged at the same conditions to collect plasma which was also aliquoted and used for RNA extraction and measurement of oxidative stress markers. All samples were stored at −20°C and processed within four days after collection.3+ to Fe2+ at pH 3.6 in the presence of 2.4.6 tri (−2 pyridyl-s-triazine); the reaction produces an intense blue color that absorbs at 593 nm. LPI was obtained by calculating the ratio MDA/TAA.Serum TC, HDLC, LDLC concentrations were determined using commercially available kits . Plasma TAA was determined using the method of Benzie and Strain 2. Results were calculated using the formula:TC concentration was determined using colorimetric enzymatic techniques based on the successive action of cholesterol oxidase and peroxidase; HDLC concentration in the serum supernatant was determined by the same process after the precipitation of VLDL cholesterol, LDL cholesterol and chylomicrons in the presence of phosphotungstic acid and MgCl500nm sample/OD500nm standard) × Concentration of standard .TC (g/l) or HDLC (mg/dl) concentrations =  = TC (mg/dl)-[HDLC (mg/dl)-Triglycerides (mg/dl)/5].RNA was extracted from patient’s plasma using the QIAmp Viral RNA kit , according to the manufacturer’s instructions.TCACCTAGAACTTTGAATGCATGGG) sense (nucleotide 777 to 801 of HIV-1 genome) and H1P202 (CTAATACTGTATCATCTGCTGCTCCTGT) antisense (nucleotide 1874 to 1898 of HIV-1 genome). Nested PCR was performed using H1Gag1584 (AAAGATGGATAATCCTGGG) sense and g17 (TCCACATTTCCAACAGCCCTT) antisense primers to enable amplification of the 460 bp encoding amino acid 132 of p24 to amino acid 40 of p7 from the gag gene. The amplification conditions of the RT-PCR were as followed: 50 min at 42°C (cDNA reaction) followed by 3 min at 94°C and the addition of 0, 2 µl Taq polymerase (5UI/ µl); then the reaction continued with 40 cycles of 30s, 30s, and 90s at 94°C, 50°C, and 72°C, respectively, and 1 cycle of 7 min at 72°C. For Nested PCR, cycling conditions were 1 cycle of 2 min at 94°C; 35 cycles of 30s, 30s, and 60s at 94°C, 50°C, and 72°C respectively; and 1cycle of 7 min at 72°C. The PCR amplification products were detected by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining under UV light.RNA and cDNA samples were then amplified in a one tube, two steps RT- PCR using a thermal cycler  with the following primers: H1G777  or Spearman (non parametric) correlations were used to establish the correlation between the different parameters. Logistic regression and ANOVA were used to study the association of the different subtypes with biochemical parameters.Data were analyzed using PASW STATISTICS version 18 software. We obtained means, standard deviation and percentages. Two-group comparisons were done with the parametric Student t test or the non parametric Mann Whitney test, and ANOVA was used when more than two series of data were compared. Kruskal Wallis test was used for quantitative variables while Results were considered statistically significant at p<0.05.Participant’s demographics characteristics are summarized in Samples from 50 HIV+ patients were used in genotypic studies, and we successfully sequenced the viral genome in samples from 30 patients, all of which belonged to the CDC category B Results in Results for TC, LDLC, HDLC, TAA, MDA, and LPI are summarized in 1) (p = 0.017); there was a lower mean value in CRFs patient group (0.87±0. 27 g/l) compared to patients carrying pure subtypes group (1. 32±0. 68 g/l). Patients carrying CRFs had lower LDLC, HDLC, TAA mean values compared to patients carrying the pure subtypes although the results were not statistically significant , high density lipoproteins  and by very low density lipoproteins  al.This study showed statistically significant (p<0.05) lower mean values of TC, HDLC and LDLC in HIV-infected patients compared to serologically negative controls even though their mean ages and sex distribution were somewhat different . Age is −OH, HO., O−2, H2O2) produced during chronic inflammation react with antioxidants and contribute to the reduction of their plasma concentration TAA was evaluated using the FRAP test which expresses the antioxidant potential of the organism. It measures its capacity to neutralize through antioxidant molecules the oxidant  damage on various substrates . Our results showed about a threefold reduction of TAA plasma concentration in patients compared to controls. This may be linked to the high level of free radicals production due to the antigenic (virus) activation of lymphocytes, phagocytes and chronic inflammatory processes induced by viral replication Chronic inflammation in HIV infection increase free radicals formation; these free radicals induce lipid peroxidation which leads to MDA formation 1) are thought to be implicated in the decrease of TAA, LDLC, HDLC and TC and the increase of MDA and LPI Lipid peroxidation indice (LPI) is the ratio MDA/TAA; it estimates the degree of free radical aggression due to HIV infection. When the plasma total antioxidant ability decreases or when the plasma MDA concentration increases, LPI increases and this is associated with increased oxidative stress in patients Our patients were infected by different HIV-1 subtypes, as determined by the sequencing experiments . All HIVOur results , in spitThe CRF01 _AE subtype seems to induce high lipid peroxidation , probablThese results, in spite of some limitations like mean age and sex distribution differences, and small sample size for genotyping studies, show a significant reduction in TAA, LDLC, HDLC, TC and an elevated MDA concentration and LPI in HIV-positive patients compared to serologically negative controls. This may be due to chronic inflammation caused by HIV replication which produces free radicals. These free radicals may be responsible for the lipids peroxidation, CD4 cell reduction, low TAA, and high LPI and MDA observed in our study. The differences in biochemical parameters in patients infected with different HIV subtypes may be due to their replication velocities as HIV-1 CRF01 _AE has been shown to have a faster replication velocity


Pharmaceuticals and their metabolites are inevitably emitted into the waters. The adverse environmental and human health effects of pharmaceutical residues in water could take place under a very low concentration range; from several µg/L to ng/L. These are challenges to the global water industries as there is no unit process specifically designed to remove these pollutants. An efficient technology is thus sought to treat these pollutants in water and waste water.A novel chemical, ferrate, was assessed using a standard jar test procedure for the removal of pharmaceuticals. The analytical protocols of pharmaceuticals were standard solid phase extraction together with various instrumentation methods including LC-MS, HPLC-UV and UV/Vis spectroscopy. Ferrate can remove more than 80% of ciprofloxacin (CIP) at ferrate dose of 1 mg Fe/L and 30% of ibuprofen (IBU) at ferrate dose of 2 mg Fe/L. Removal of pharmaceuticals by ferrate was pH dependant and this was in coordinate to the chemical/physical properties of pharmaceuticals. Ferrate has shown higher capability in the degradation of CIP than IBU; this is because CIP has electron-rich organic moieties (EOM) which can be readily degraded by ferrate oxidation and IBU has electron-withdrawing groups which has slow reaction rate with ferrate. Promising performance of ferrate in the treatment of real waste water effluent at both pH 6 and 8 and dose range of 1–5 mg Fe/L was observed. Removal efficiency of ciprofloxacin was the highest among the target compounds (63%), followed by naproxen (43%). On the other hand, n-acetyl sulphamethoxazole was the hardest to be removed by ferrate (8% only).Ferrate is a promising chemical to be used to treat pharmaceuticals in waste water. Adjusting operating conditions in terms of the properties of target pharmaceuticals can maximise the pharmaceutical removal efficiency. Pharmaceuticals such as antibiotics, anti-inflammatory drugs, β-blockers and X-ray contrast media are widely used in our daily life. These pharmaceuticals and their metabolites cannot be fully utilized by human beings or animals and are inevitably emitted into the waters by execration The presence of emerging micro pollutants and their potential toxicity are challenge to the global water industries as there is no unit process specifically designed to remove these pollutants; activated sludge and secondary sedimentation in most wastewater treatment works (WWTWs) seems to be inefficient to eliminate them VIO42−, Fe (VI)). Ferrate (VI) exhibits many advantages because of its dual function as an oxidant and a coagulant Another promising technique which can address the concerns on pharmaceutical residues is ferrate (VI) . The dose of ferrate, test water pHs and the physical and chemical properties of pharmaceuticals are major factors to influence the overall treatment efficiency and these were studied in this work.Ciprofloxacin , ibuprofen , ibuprofen sodium salt (IBU-Na) and potassium ferrate (VI) (>90%) were purchased from Sigma-Aldrich; ciprofloxacin hydrochloride BioChemica (CIP·HCl) was purchased from VWR; other chemicals and reagents used were obtained from Fisher Scientific. All chemicals and reagents were used without further purification. Deionised water used was generated by Elgacan B114 deioniser.2FeO4 to 0.005 M Na2HPO4/0.001 M borate buffer solution at pH 9.0, the pH at which the ferrate solutions are most stable The ferrate (VI) working solution (1 g/L) was prepared by the addition of solid KBoth CIP and IBU stock solutions were prepared separately in deionised water with the concentration of 10 mg/L. Several types of model test solution samples  were preThe wastewater samples were taken from the second sedimentation effluent at Shieldhall WWTW, Glasgow, and the major properties of the samples were COD = 35 mg/L, turbidity = 2 NTU and pH was 7.37.X-ray contrast media: Iopamidol, Ammonium diatrizoate; 2) Antibiotics: Ciprofloxacin, Sulphamethoxazole, N-acetyl sulphamethoxazole, Erythromycin-H2O; 3) Non-steroidal anti-inflammatory druds (NSAID): Naproxen, Ibuprofen; 4) β-blockers: Atenolol; 5) Antineoplasic: Cyclophosphamide, Ifosfamide; 6) Antiepileptics: Carbamazepine; 7) Blood Lipid Lowering: Bezafibrate and 8) Local anesthetic: Lidocaine.Two litres of raw samples were analysed for pharmaceutical concentrations, and the sample was then spiked with selected pharmaceuticals; each with concentration of 10 µg/L. The spiked pharmaceuticals are as follows, 1) 2SO4 or 0.05 M NaOH to 7.0–7.5. All experiments were duplicated. For the real wastewater effluents, the jar test protocol was the same as the above stated but ferrate dose sued was 0, 1, 2, 3, 4, and 5 mg/L as Fe and working pH was pH 6 and 8.A series of jar test experiments was carried out with a six-unit stirrer  under the following protocol: fast mixing for 1 min at 400 rpm; slow mixing for 20 min at 40 rpm; and then sedimentation for 60 min. The ferrate dose applied was 0–4 mg/L as Fe, and pH of solutions was adjusted by 0.1 M HAs shown in 2O; (5) dry: 15 min under gentle nitrogen flow; and (6) elute: 2×2 mL 2∶49:49 (v/v/v) formic acid/MeOH/ACN. Elutes were either re-constituted to 5 mL for UV/Vis spectrophotometric measurement or dried down to 1 mL for HPLC-UV detection.The treated model test solution samples were filtered through 0.45 µm cellulose nitrate membrane filters (Milipore) and then enriched by solid phase extraction (SPE). Two types of SPE cartridges were employed in the experiment: Strata-X  and Oasis HLB . Oasis HLB cartridges were only used for those model water samples containing IBU only. Generally, for Strata-X 200 mg/6 mL cartridges, the extraction method was: (1) condition: 4 mL methanol (MeOH); (2) equilibrate: 4 mL deionised water; (3) loading samples: desired amount of model water samples under vacuum at a flow rate of 5–10 mL/min; (4) wash: 2 mL 50∶50 (v/v) MeOH/H2O; (2) loading samples: desired amount of water samples at 10 mL/min; (3) wash: 0.5 mL water; (4) dry: 10 min under a gentle stream of nitrogen gas; and (5) elute: 2×2 mL methanol. Then, samples elutes were evaporated to less than 1 mL using nitrogen gas by Barkey sample evaporation system , and reconstituted with methanol to 1 mL for LC/MS analysis. Prior to extraction, selected samples were spiked with 25 µL of thidiazuron solution (20 ng/µL) as an internal standard.On the other hand, IBU only model test solutions were extracted by Oasis HLB cartridges with the use of SPE preparation system Gilson GX-271 ASPEC. The programmed SPE procedure was as follows: (1) condition: 3 mL methanol and 3 mL H2SO4, spiked with 1 mL deuterated internal standards; 2) 10 mL methanol +10 mL water were added for the conditioning; 3) flow rate was 10 mL/min for loading samples; 4) 10 mL water was used for washing samples and 5) Drying the cartridges under N2 flow. After extraction, the SPE cartridges were labelled and kept in the freezer for future elution.For the real wastewater treatment, the samples were filtered by 1.2 µm glass fibre filter papers (Fisher), the filtrates were then filtered by 0.45 µm membrane filters (Milipore). The Tandem SPE cartridges used were Strata-X 1 g/12 mL+ENV+500 mg/6 mL, and the extraction procedures were: 1)Adjusted the pH of solution to 2 by 2 M H2.The elution was conducted on a Phenomenex SPE 24-position vacuum manifold. The solvents used for the elution are shown in The concentrations of CIP in the 5-mL Strata-X SPE elutes could be measured by a UV/Vis spectrophotometer (Jenway 6505 with 10 mm light-path) at the wavelength of 280 nm. A six-point calibration curve was generated based on absorption of standard CIP solutions in 2∶49:49 (v/v/v) formic acid/MeOH/ACN at 280 nm. Concentrations of CIP in model wastewater samples were calculated based on the absorbance reading and the established calibration curve 18 column (Phenomenex). The column was kept at 25°C and eluted with 0.1% formic acid in deionised water and acetonitrile (ACN) at a flow rate of 0.2 mL/min. The elution started with 20% ACN and then with a linear gradient from 20% to 30% ACN over the next 3 min. Then the percentage of ACN was raised to 55% in 2 min, held at this percentage for 9 min and finally lowered to 20% in 1 min. Before the next injection, the system was allowed to equilibrate for 10 min. The DAD wavelengths for IBU and CIP detection were set at 220 nm and 280 nm, respectively.The HPLC separations were performed on the Agilent 1100 system (Agilent Technologies) consisting of a degasser, a binary pump, a thermostated column oven and a diode array detector (DAD). 50 µL of samples was manually injected to a 2.6 µm, 100 mm×2.10 mm reversed phase Kinetex XB-C−) were monitored at m/z 221.0497.The LC-MS instrument employed to analyse IBU was Acquity LCT Premier XE system which consists of ACQUITY ultra performance liquid chromatography (UPLC) and orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer (MS) . The UPLC contained ACQUITY UPLC High Strength Silica (HSS) T3 Column . The mobile phase was a mixture of two solvents . For the analysis of IBU, the elution of the column started from initially 5% B, then a with a linear gradient increased to 90% B over the course of 7 min, then consistent till 8 min, and finally lowered back to 5% B at 8.1 min. The injection volume of sample was 250 µL, with the running time of 10 min and a flow rate of 0.3 mL/min. After LC separation, the MS analyser for IBU was ESI- mode, with the following parameters: capillary 1823.0 V, sample cone 30.0 V, desolvation temperature 350.0°C, source temp 150.0°C, and MCP detector 2600.0 V. Parent ions  0.997. In terms of CIP, an equation y = 28.11x−4.35 could fit the seven-point calibration well with the coefficient of correlation (r2) 0.998.The calibration of IBU and CIP were carried out simultaneously with standard solutions containing IBU and CIP. The curve covered the concentration range of 0.5–6 mg/l, and was conducted four times. The IBU calibration equation with the linear regression is y = 80.18x +0.05, where x means the concentration of IBU and y means the response of instrument, with a coefficient of correlation (rRecovery studies of standard solutions (n = 4) showed 78.0±14.7% of CIP and 139.1±2.2% of IBU recovered by the instrument, respectively.−) vs. linear regression equation y = 57.76x, where x means the concentration of IBU and y means the response of the instrument, with the coefficient of correlation 0.998. Recovery was obtained by running standard IBU solutions (5 µg/L) through SPE and LC-MS process, with the mean recovery 117.7±30.6% (n = 4).A seven-point calibration curve for the concentration range of 0.04–1.2 mg/L was generated based on MS response of standard IBU solutions in methanol. Quantification of IBU was based on the response at m/z 221.0497  dose ranging from 0.02 to 0.34 mg/L as Fe . As incrFurther studies were carried out to investigate if relatively high ferrate doses could improve CIP removal. However, the treating performance of IBU by ferrate (VI) was not as good as that of CIP, less than 25% of IBU was removed from the model water when dosed ferrate (VI) was 0.5–4 mg Fe/L . The higCompared with the case of 100 µg/L CIP model test solutions, when the initial concentration was 10 µg/L, the overall treating performance of CIP by ferrate (VI) decreased to less than 60% . SpecifiOn the other hand, the overall treating performance of IBU improved when the initial concentration was 10 µg/L. As shown in Further study was carried out to investigate the treating performance of mixed IBU and CIP model test solutions by ferrate. The initial concentrations of both compounds were 10 µg/L, and the ferrate doses applied were 0.5–2.5 mg/L. Results show that ferrate salts possess more capability to remove CIP than IBU . AverageFerrate (VI) has shown much more capability in the degradation of CIP than that of IBU. This can be explained that CIP belongs to electron-rich organic moieties (EOM) which can be easily transformed during ferrate oxidation. However, the carboxylic group in IBU is an electron-withdrawing group with which the reactivity of ferrate is usually slow Preliminary studies showed that comparing with initial pH, final solution pH after dosing ferrate has much more influence on the removal of CIP and IBU. As shown in pKa values. The pKa of IBU is 4.91, therefore when the pH was <5, the major species would be IBU, which was relatively easy to be oxidised. For CIP, its pKa is 6.09 and 8.2, respectively, and when the pH of solution was >8, the major species of CIP would be dissociated CIP-O−, which was relatively difficult to be oxidised −1 s−1 at pH 7.5 then slipped sharply to 0.01 M−1 s−1 at pH 9.0 −1 s−1 at pH 6 but only 10 M−1 s−1 at pH 9.5 The effect of final solution pH on the IBU or CIP removal might be understood by considering compounds 3FeO4+, H2FeO4, HFeO4−, and FeO42−, and the corresponding dissociation constants are pK1 1.6±0.2, pK2 3.5, and pK3 7.23, respectively 42− is the dominant species in alkaline conditions, and HFeO4− predominates in mildly acidic conditions. Ferrate(VI) has a higher oxidation potential at low pH (2.2 V) than in the alkaline condition (0.72 V) 4− predominates) although the stability of ferrate(VI) decreases at low pH solutions.Strong pH effect on the IBU and CIP removal by ferrate(VI) can also be considered from ferrate(VI) speciation against solution pH. There are four ferrate(VI) species in aqueous solution that depend on pH: H2O, lidocaine and bezafibrate were also detected in the effluent, with the concentrations 100–120 ng/L.Six compounds were detected in the raw effluent samples, with concentrations ranging from 100 to 320 ng/L. The concentrations of naproxen, carbamazepine and atenolol were over 200 ng/L, among which naproxen exhibited the highest concentration with an average concentration 317.3 ng/L. In addition, erythromycin-HAs shown in Removal efficiencies of ciprofloxacin, naproxen and ibuprofen were 20–60% for ferrate doses 1–5 mg Fe/L. However, the elimination of n-acetyl sulphamethoxazole was only less than 10% for any ferrate dose. Besides, except ciprofloxacin and ifosfamide, raising ferrate dose did not improve the pharmaceutical removal significantly. As for ifosfamide and ciprofloxacin, when the ferrate dose was raised from 3 mg/L to 5 mg/L, the removal efficiency increased from 9% to 38% for ifosfamide and from 44% to 63% for ciprofloxacin, respectively .Results of treatment of CIP by ferrate show that ferrate can remove at least 60% of CIP from model test solutions even at very low ferrate doses (<0.3 mg/L). Besides, increasing ferrate dose up to 1 mg Fe/L as Fe can achieve more than 80% removal efficiency of CIP. However, ferrate did not show similar treating performance for IBU degradation, with only 30% IBU removal at ferrate dose of 2 mg Fe/L.The final solution pH affects the treating performance significantly for both pharmaceuticals. When final pH of solution was greater than 8, CIP removal efficiency by ferrate drops significantly. In the case of IBU, pH 4 was the optimum pH for IBU removal by ferrate.Ferrate has shown higher capability in the degradation of CIP than IBU. This is due to that CIP belongs to electron-rich organic moieties (EOM) which can be readily degraded by ferrate oxidation. Instead, the reactivity of ferrate with electron-withdrawing groups (carboxylic groups) in IBU is slow and consequently, lowers ferrate removal efficiency for the IBU.Ferrate has shown promising performance at both pH 6 and 8 and dose range of 1–5 mg Fe/L in the treatment of real waste water effluent. Removal efficiency of ciprofloxacin was the highest among the target compounds, with an average removal of 63%, followed by naproxen (43%). On the other hand, n-acetyl sulphamethoxazole was the hardest to remove by ferrate, with an average removal of 8%. Furthermore, the removal efficiencies of other compounds by ferrate were between 20% and 40%.


The hepatic transcriptional level of G6Pase, FAS and mtGPAT were reduced. The transcription of genes involved in fatty acid oxidation and the mitochondrial biogenesis of muscle tissue were elevated. The ACC phosphorylation was increased in muscle and liver. This study provides a novel allosteric AMPK activator for functional study in vitro and in vivo and demonstrates that AMPK allosteric activators could be a promising therapeutic approach for type 2 diabetes mellitus and metabolic syndrome.AMP-activated protein kinase (AMPK) is an energy sensor of metabolism that is an attractive therapeutic target for type 2 diabetes mellitus and metabolic syndrome. Using a homogeneous scintillation proximity assay (SPA), we identified a new small-molecule AMPK activator, ZLN024, which allosterically stimulated active AMPK heterotrimers and the inactive α1 subunit truncations α1 (1–394) and α1 (1–335) but not α1 (1–312). AMPK activation by ZLN024 requires the pre-phosphorylation of Thr-172 by at least one upstream kinase and protects AMPK Thr-172 against dephosphorylation by PP2Cα. ZLN024 activated AMPK in L6 myotubes and stimulated glucose uptake and fatty acid oxidation without increasing the ADP/ATP ratio. ZLN024 also activated AMPK in primary hepatocytes, decreased fatty acid synthesis and glucose output. Treatment of  Type 2 diabetes mellitus and metabolic syndrome have become an increasing health risk. Novel agents that ameliorate insulin resistance and hyperglycemia need to be explored. AMPK activators are emerging as a promising therapeutic target for type 2 diabetes mellitus and metabolic syndrome. AMPK is a highly conserved serine/threonine protein kinase that serves as an energy sensor in metabolism. It is activated under conditions that induce stress, such as exercise, ischemia, hypoxia and glucose deprivation, which are accompanied by an increasing cellular AMP/ATP or ADP/ATP ratio. AMPK is also regulated by cytokines including leptin, adiponectin, resistin, ghrelin, interleukin-6 and ciliary neurotrophic factor (CNTF). Once activated, AMPK stimulates catalytic pathways that generate ATP, such as glucose uptake and fatty acid oxidation, whereas it inhibits ATP-consuming anabolic pathways, including the synthesis of hepatic triacylglycerol, cholesterol, protein and glycogen in vitro and in vivoin vivoAMPK is a heterotrimer that consists of a catalytic α subunit and two regulatory subunits, β and γ, which have multiple isoforms  and are expressed differently in various tissues and subcellular locations. The heterotrimeric complex is required for maximum enzymatic activity ob/ob mice. However, it only activates AMPK heterotrimers containing the β1 isoform in vivo efficacy and functional study.There are several small-molecule allosteric AMPK activators, for example, A-769662, which was identified by Abbott db/db mice. Our results suggest that this novel AMPK allosteric activator may represent a promising therapeutic approach for treating type 2 diabetes mellitus and metabolic syndrome.We identified ZLN024 as a novel AMPK allosteric activator that has no effect on mitochondrial function or the ADP/ATP ratio. ZLN024 provided metabolic benefits in L6 myotubes and primary hepatocytes by activating AMPK, and it reduced glucose intolerance and fatty liver characteristics in diabetic 2, 1 mmol/l DTT, 2 µmol/l biotin-SAMS, 2 µmol/l ATP and 7.4×103 Bq/well [γ-33P]ATP. The reactions were initiated by the addition of 50 nmol/l recombinant AMPK protein to the reaction solutions, followed by incubation at 30°C for 2 hr. The reactions were then terminated by the addition of 40 µl of stop solution containing 80 µg streptavidin-coated SPA beads per well, 50 mmol/l EDTA and 0.1% Triton X-100 in PBS, pH 7.5, followed by incubation for 1 hr. Finally, 160 µl of suspension solution containing 2.4 mol/l CsCl, 50 mmol/l EDTA and 0.1% Triton X-100 in PBS, pH 7.5, was added to the reaction solution to suspend the SPA beads completely. The SPA signals were measured in a Wallac Microbeta plate counter  30 min later.Before the SPA assay, 200 nmol/l recombinant AMPK protein , α1(1–335), α1(1–312)) was constructed, expressed, purified and fully phosphorylated as described previously 2 and 10 mmol/l pNPP; the PP2Cα enzyme concentration was 400nmol/l.Recombinant human protein phosphatase-2Cα (PP2Cα) was obtained from Abcam . PP2Cα activity was measured using pNPP as a substrate. The activity was measured in a SpectraMAX 340 plate reader at 37°C and 410 nmol/l for 120 sec. The assay was carried out in a 96-well plate in a final volume of 100 µl containing 50 mmol/l Tris-HCl, pH 7.6, 2 mmol/l DTT, 10 mmol/l MnCl3H(N)]-palmitic acid  to a final concentration of 250 µmol/l and 5.55×104 Bq per well in DMEM. After incubation with the compound for 4 hr in differentiated L6 myotubes, sample from each well was added to charcoal slurry for centrifugation and then the radioactivity was measured.The assay was initiated by adding  acetate incorporation into lipids. Rat hepatocytes were treated for 20 hr with ZLN024 at the concentrations described in FBS-free DMEM with or without 10 nmol/l insulin, followed by 4 hr incubation in fresh medium containing 0.1 µCi/ml sodium [14C] acetate (3.7×103 Bq/ml). Thereafter, cells were washed three times by ice-cold PBS and analyzed as previously described Fatty acid synthesis in rat primary hepatocytes was assayed by measuring [Recombinant adenovirus expressing dominant negative forms of AMPK, AMPKα1 (D159A) and AMPKα2 (K45R) (α1/α2-DN), were constructed by using pAdEasy system . L6 myotubes were infected with adenovirus expressing control GFP reporter protein or α1/α2-DN, at the 4th day after differentiation before compound treatment.db/db  mice were bred at the Shanghai Institute of Materia Medica .The animals were maintained under a 12 hr light–dark cycle with free access to water and food. Animal experiments were approved by the Animal Care and Use Committee, Shanghai Institute of Materia Medica. At 8 weeks of age, male db/db mice were randomly assigned to the various treatment groups by body weight and glucose levels (n = 6–8). The treatment groups for the 5-week chronic study were as follows: vehicle (0.5% methylcellulose), ZLN024 (15 mg/kg) and metformin (250 mg/kg). The treatments were orally administered once daily. The body weights and food intake were measured daily. After 5 weeks of treatment, the mice were killed after a final dose, and the tissues were collected for further analysis.C57BKS This assay was described previously The dephosphorylation of AMPK was performed as described previously These assays were described previously t-test. The differences among multiple groups were compared by one-way ANOVA, followed by a LSD comparison. P<0.05 was regarded as statistically significant.The results are presented as the mean ± SEM. The differences between the two groups were analyzed using Student’s Km values of the biotin-SAMS and ATP substrates toward recombinant AMPK α1β1γ1 were determined to be 48 µmol/l and 37 µmol/l, respectively (data not shown), which is in accordance with previous reports Because AMPK is a promising therapeutic target, the availability of simple, sensitive, and cost-saving assays suited to high-throughput screening is crucial in discovering small-molecule AMPK activators. To meet this need, we have developed a novel assay for AMPK allosteric activator screening based on scintillation proximity assay (SPA) technology, which is currently being widely applied to the screening of various kinases 50 of about 1–2 µmol/l , with a novel structure . ZLN024 5 µmol/l . ZLN024 3 µmol/l .After observing the allosteric activation of the AMPK heterotrimer by ZLN024, we investigated its effect in L6 myotubes. ZLN024 stimulated the phosphorylation of AMPK, and phosphorylation of the well recognized AMPK downstream Acetyl coenzyme A carboxylase (ACC) in a concentration-dependent manner . The inc50 values of 0.92 µmol/l and 2.23 µmol/l, respectively. The maximal activation of these truncations was 2.3-fold and 2.8-fold, respectively, which was greater than the activation of the heterotrimers , increasing the concentration of ZLN024 to 10 µmol/l produced a small (1.3-fold) but significant additional activation . In the m-chlorophenylhydrazone (CCCP), which is a mitochondrial chemical uncoupler, decreased the MMP and increased the ADP/ATP ratio. These results suggest that ZLN024 does not interfere with mitochondrial function, and it increases the phosphorylation of AMPK by a direct allosteric interaction and not through the ADP/ATP ratio.Changes in mitochondrial oxidative phosphorylation coupling and the cellular energy state could affect the AMP/ATP or ADP/ATP ratio, thereby leading to cellular AMPK activation LKB1 and CaMKKβ have been identified as AMPK upstream kinases in cells, and the predominant upstream kinase in L6 myotubes is LKB1. To investigate the requirement for the upstream kinase in cellular AMPK stimulation by ZLN024, we utilized HeLa cells that were deficient in LKB1. ZLN024 stimulated the phosphorylation of AMPK and ACC in HeLa cells in a concentration-dependent manner after ZLN024 treatment for 3 hr ; the incBecause we demonstrated that ZLN024 activates AMPK and its pathway in L6 myotubes, and the activation of AMPK in muscle has been hypothesized to stimulate glucose uptake and fatty acid oxidation Since activation of AMPK is known to regulate lipid and glucose metabolism in liver, the effects of ZLN024 on fatty acid synthesis and gluconeogenesis were evaluated in primary rat hepatocytes We next evaluated the glucose production and expression of gluconeogenic genes. As expected, glucose production was obviously repressed by ZLN024 in a concentration-dependent manner . After ain vivo. C57BKS db/db mice were administered a 15 mg/kg/day dose of ZLN024 by daily gavage for 5 weeks; 250mg/kg/day metformin (Met) was used as a positive control. During the treatment period, there was no significant alteration in food intake and body weight compared with the vehicle group  , whereas the metformin group exhibited no decrease . BecauseAMPK has been speculated to activate catabolic pathways that generate ATP while deactivating anabolic pathways that consume ATP We next analyzed the expression of genes related to fatty acid oxidation and mitochondrial biogenesis in the abdominal muscle. ZLN024 treatment significantly upregulated genes related to fatty acid oxidation, such as CPT-1, medium chain acyl CoA dehydrogenase (MCAD) and LCAD. Genes involved in mitochondrial biogenesis, such as peroxisome proliferators activated receptor γ coactivator α (PGC-1α), cytochrome c (cyto-c) and ATPase , were aldb/db mice. AMPK phosphorylation was not affected; however, ACC phosphorylation was increased in both tissues  To further investigate the potential mechanism of action of ZLN024, we ruled out the potential influence of ZLN024 on the cellular ADP/ATP ratio. Many AMPK activators work by regulating mitochondrial function and reducing ATP generation, which is related to the MMP. ZLN024 had no effect on the MMP or the ADP/ATP ratio, which suggests that cellular AMPK activation occurs primarily through allosteric activation and protection of dephosphorylation. We then observed a robust stimulation of the AMPK pathway in L6 myotubes and rat primary hepatocytes by ZLN024. The stimulation mostly abrogated by treatment with the adenovirus infection of AMPK α1/α2-DN, which confirmed that the activity of ZLN024 requires a functional AMPK pathway.in vitro findings, we sought to test the effects of ZLN024 in vivo. We assessed the chronic effects of ZLN024 in db/db mice. ZLN024 improved glucose homeostasis, as evidenced by an improvement in glucose tolerance. The prolonged activation of AMPK leads to increased insulin sensitivity in vivo, fatty acid synthesis was reduced by ZLN024 treatment in hepatocytes.Based on our in vitro and in vivo. Our findings provide another tool for understanding the allosteric activation of AMPK and suggest that the direct activation of AMPK is a promising approach for discovering novel therapies for the treatment of type 2 diabetes mellitus and metabolic diseases.In summary, we have identified a novel allosteric AMPK activator ZLN024, which exerts beneficial metabolic effects Figure S1ZLN024 does not affect AMPK α1 truncations dephosphorylation by PP2Cα. Effect of AMP, A-769662, ZLN024 on AMPK α1(1–394) (n = 2) (A) and AMPK α1(1–335) (n = 2) (B) dephosphorylation by PP2Cα. The ratio of the phosphorylation level to the protein level of AMPK was determined. NC = negative control, PP2Ca was not added. *, P<0.05, **, P<0.01 compared with the DMSO group.(TIF)Click here for additional data file.Figure S2ZLN024 decreases gluconeogenesis in rat primary hepatocytes. (A) Treatment with ZLN024 for 5 hr decreases glucose production in hepatocytes; metformin (1 mmol/l) was used as a positive control (n = 3). (B) The effects of treatment with ZLN024 for 21 hr on the gene expression of PEPCK and G6Pase (n = 3). #, P<0.1, *, P<0.05, **, P<0.01 compared with the untreated.(TIF)Click here for additional data file.Figure S3Chronic effects of ZLN024 on diet, weight and fasting blood glucose in db/db mice. Eight-week-old db/db mice were gavaged with vehicle (0.5% methylcellulose), ZLN024 (15 mg/kg/day) or metformin (250 mg/kg/day) (n = 6–8) for 5 weeks. (A) Body weight. (B) Food intake. (C) Fasting blood glucose levels after 4 weeks of treatment; the mice were fasted for 6 hr. #, P<0.1, *, P<0.05, **, P<0.01 compared with the vehicle group.(TIF)Click here for additional data file.Table S1The sequences of oligonucleotide primers. The detail primer sequences for real-time PCR, all samples were run in duplex and normalized to actin expression.(DOCX)Click here for additional data file.


The present study reports an easy and efficient method for obtaining adult mesenchymal precursors from different adult mouse tissues.in vitro by defined morphology and by a specific gene expression profile and surface markers.We describe the isolation and expansion of mesenchymal precursors from skin and lung by a non-enzymatic method. Skin and lung mesenchymal precursors isolated by a modified explant technique were characterized Our results show that these precursors express stem cell and mesenchymal surface markers as well as epithelial markers. However, they are negative for markers of endothelium, cardiac and skeletal muscle or adipose tissue, indicating that they have initiated commitment to the tissues from which were isolated. These precursors can migrate without any stimulus and in response to stimuli as SDF1, MCP1 and TNFα and can be differentiated into epithelial lineages. Based on the properties of these precursors from adult tissues, we propose their use as tools for regenerative biomedicine. Regenerative medicine research has attracted increasing interest in recent years, but the clinical applications and outcomes require further analysis Cohnheim 130 years ago Friedenstein et al. in vitro, together with their relative ease of expansion in culture and their immunologic characteristics, make MSCs and the related MSC-like cells promising sources of stem cells for tissue repair. More recently skin has attracted attention as is a highly accesible tissue and is considered a stem cells reservoir for numerous cell lineages [LOOSEST The presence of non-hematopoietic stem cells in bone marrow was first suggested by the observations of Here, we present a minimally invasive non-enzymatic method for isolating similar mesenchymal precursors from skin or lung adult tissues. Independently of the source, we found that a modification of the explant technique Mesenchymal precursor cells (MPCs) were isolated from adult tissues by the explant method 3 flasks were washed with PBS and detached by incubation in trypsin-EDTA solution for 30 seconds at 37°C. Cells were centrifuged for 5 min at 250 g at 4°C. Cells were plated in E-Plates 16  at 255×103 cells/cm2 (500000 cells/ml) in 100 µl complete medium, and incubated for at least 30 min in a culture hood at room temperature. Control wells only contained 100 µl cell-free complete medium. E-plates were inserted into the cradle pockets of the Real-Time Cell Analyzer (RTCA) DP Analyzer, and the background impedance of each well was measured. The number of proliferative cells was measured as Cell Index (CI), a dimensionless parameter derived as a relative change in measured electrical impedance to represent cell status. . The RTCA DP Analyzer, placed in a standard cell-culture incubator, monitored cell number in each well every 15 min (quadruplicate measurements) over 48 h. MPCs showed a normal cell growth profile. Data were analyzed with the RTCA software .MPC clones maintained in 75 cmAdult MPCs were detached from culture flasks with 5% EDTA on ice and incubated with pre-conjugated antibodies against surface markers for 25 min at 4°C. Surface markers analyzed were Sca1-PECy7, c-kit-PE, CD29-Fitc, CD34-PE, CD44-PECy5, CD45-v450, CD73-PE, CD90.2-Fitc and CD105-PE . Cells were washed and labelled samples were analyzed in a BD FACS CantoII cytometer . At least 10000 events were counted for each sample. All nine different MPCs clones were characterized by flow cytometry. Data analysis was performed with BD FACS DIVA .Total RNA was extracted from MPCs with Trizol reagent  and quantified spectrophotometrically . RNA integrity was monitored by ethidium bromide staining after agarose gel electrophoresis. RNA (1 µg) was reverse-transcribed to cDNA using the iScript cDNA Synthesis kit, with RNAase H+ and random hexamer primers included . PCR was performed using a Veriti 96w thermal cycler  and PCR reaction mixtures contained cDNA templates, primers and 5′MasterMix  in a final volume of 25 µl. Each PCR reaction was performed in triplicate, and each PCR also included no-template negative controls. Primers were designed using the Prime3 online program, checked with the Basic Local Alignment Search tool from NCBI, and made by Sigma . Primer sequences and amplification product lengths are shown in Adult MPCs were lysed directly with Laemmli buffer. Lysates were resolved by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane . Membranes were probed with rabbit anti β-actin, rabbit anti α-tubulin and rabbit anti β1-integrin antibodies , and signal developed with enhanced chemiluminescence reagent .Adult MPCs were grown on 0.1% gelatin-coated coverslips, washed with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were maintained with donkey serum 1% for 1 h. Cells were incubated with each antibody for 1 h. For β actin detection, 1 µg/ml rabbit anti β-actin; for α-tubulin detection, 1 µg/ml rabbit anti α-tubulin; and for β1-integrin detection, 1∶500 rabbit anti β1-integrin . Cells were incubated with 1∶500 goat Alexa488 conjugated anti-rabbit secondary antibody. Cells were then stained with 300 nM DAPI for 30 min. Cells were maintained at room temperature during the process. Confocal images were obtained with a Leica DM2500– TSC.SPE microscope.4 cells/well) in MIX medium, composed of small airway basal medium  supplemented with 10 µM all-trans retinoic acid  and 50 µM keratinocyte growth factor . Control cells were incubated in complete medium. Cultures were maintained for 3 or 7 days before analysis. To induce adipogenic differentiation, MPCs were cultured in serum-free DMEM/F12 medium (1∶1) supplemented with 10 µg/ml transferrin, 15 mM NaHCO3, 15 mM HEPES, 33 µM biotin, 17 µM pantothenate, 1 nM insulin, 20 pM triiodothyronine, 1 µM cortisol, plus antibiotics. Accumulation of triglycerides in adipocytes was visualized by staining formalin-fixed cells with Oil Red O. In order to measure the osteogenic potential of the MPCs, 2×104 cells were incubated in complete expansion medium until a confluent layer was achieved and then osteogenic medium was added, containing IMDM supplemented with 9% FBS, 9% HS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 ng/mL L-thyroxine, 20 mM β-glycerol phosphate, 100 nM dexamethasone, and 50 µM ascorbic acid. Medium was refreshed every 3–4 d. After 15 d of culture cells were fixed in 10% formalin and stained with 10% Alizarin Red. To measure the chondrogenic differentiation of ASCs, 5×105 cells were incubated in 500 µL of complete chondrogenic medium (CCM), containing IMDM, 100 nM dexamethasone, 50 µg/mL ascorbic acid, 40 µg/mL L-proline with ITS + supplement, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 ng/mL TGFβ-3 and 100 ng/mL BMP-2. The medium was changed three times a week. After 18 d, cells were stained with Toluidine Blue sodium borate. Finally, differentiation into smooth muscle cells was induced by treatment with TGFβ1 and BMP-4, while co-culturing MPCs with C2C12 myoblasts induced differentiation into skeletal muscle cells. All experiments were done with three different MPCs clones and results are expressed as percentage of differentiation capacity for each treatment.To study epithelial differentiation, adult MPCs were plated on gelatin-free P6 culture plates (103 cells/cm2 (600000 cells/ml) in 100 µl complete medium, which we determined as the optimal density to ensure confluent monolayers. The CIM-Plates were inserted into the cradle pockets of the RTCA DP Analyzer, and the background impedance of each well was measured after 1 h. The RTCA DP Analyzer, placed in a standard cell-culture incubator, monitored the migrated cell number in each lower chamber well every 15 min (quadruplicate measurements) over 36 h. Data were analyzed with the RTCA Software.The upper chamber wells of CIM-Plates 16  were precoated with fibronectin, added to wells at 10 µg/ml (30 µl) and removed after 30 min. The lower chamber wells were filled with 160 µl culture medium, allowing a clearly-defined meniscus to form. SDF1, MCP1 and TNFα  were added to the lower chambers at a final concentration of 100 ng/ml. CIM-Plates were equilibrated for at least 30 min in a culture hood at room temperature. Trypsin-EDTA-dispersed MPCs (100 µl in complete medium) were then plated in the fibronectin-precoated upper chamber wells at 306×10t test. One-way or two-way ANOVA were used as required by the assay. Differences between groups were considered statistically significant at P<0.05.All data are represented as mean ± SEM from 3–9 independent experiments. Comparisons between two groups were done by Student’s Mesenchymal precursors from different adult tissues were isolated using the explant method Similar outcomes were obtained with explants from the other control adult tissues (data not shown), and the mesenchymal cells obtained from cardiac, skeletal muscle and adipose tissue had similar properties to those described in previous studies 4/well in complete medium, and cells were counted automatically every 15 min over 48 hours for each well , containing ATRA, KGF and SABM  confirmed the capacity of MPCs to differentiate further toward an epithelial fate. MPCs switch off nestin expression after 3 days of treatment. This timing is consistent with the observed induction of epithelial genes in embryonic bodies Effective cell therapy strategies require the implanted cells to be able to migrate to sites of damage, in order to avoid invasive delivery procedures. Skin and lung MPCs both showed an ability to migrate under basal conditions and in response to SDF1, MCP1 and TNFα, described as signals that induce migration of different mesenchymal cells This study presents a modified explant technique method for isolating homogeneous committed mesenchymal precursor cells from skin and lung. This MPCs population express mesenchymal markers and show pre-commitment to epithelial lineages. The ease of obtaining and expanding these cells, especially from skin, together with their migratory and differentiation capacity, mark them as attractive candidates for translation to clinical strategies employing autologous cells for regenerative therapy. The similar gene and surface marker expression in skin and lung MPCs highlights that similar kinds of precursors can be isolated from different tissues using a simple and standardized procedure. Nonetheless, the specific responses of skin and lung MPCs to migratory stimuli suggests that precursors derived from a specific tissue might not be suitable for the therapeutic replacement of epithelial cells in other tissues.Figure S1Differentiation properties of MPCs. Percentages of skin and lung MPCs clones that differentiate into adipose, chondrogenic, osteogenic or muscle tissues in three independent experiments .(TIF)Click here for additional data file.