Live cells were imaged 48 h posttransfection in KR2 buffer on a Nikon inverted epifluorescence selleck microscope with a charge-coupled device camera. About 12�C15 cells were found to express Epac-camps, and 6�C10 cells/35 mm glass bottom dish were found to express both fluorescent proteins, indicating the ��-cells. Pancreatic ��-cells from multiple 35-mm dishes were selected, and fluorescence was recorded before and during stimulation with 100 nM exendin-4. The FRET ratio was recorded every 5 s. Dynamic changes in cAMP estimated from the change in FRET ratio was acquired by MetaFluor software (Universal Imaging). GLP-1R-cyan fluorescent protein and yellow fluorescent protein-SUMO-1 live cell FRET.

MIN6 cells were transfected with cyan fluorescent protein (CFP)-GLP-1R and yellow fluorescent protein (YFP)-SUMO-1 or conjugation-deficient YFP-SUMO��GG construct where a stop codon was introduced at G96 to remove the last four amino acids essential for covalent interaction in SUMO-1. The cells were imaged on a live cell Olympus wide-field IX-50 fluorescent microscope. FRET was calculated by CFP (donor) bleaching, and the images were acquired every 200 ms. Fluorescence decay was calculated for the entire cell using ImageJ software (NIH). Time constants were calculated using Graphpad Prism software. Cell-Surface Biotinylation For cell-surface biotinylation, MIN6 cells cotransfected with untagged SUMO and GLP-1R-GFP were biotinylated on a 10-cm plate for 45 min and lysed in RIPA buffer. Biotinylated receptor was purified according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL).

Biotinylated receptors at the membrane and nonbiotinylated receptors in the cytosol were detected by an anti-GFP monoclonal antibody (Roche Applied Science). Densitometric analysis was carried out using Image J software. Immunofluorescence MIN6 cells transfected with GLP-1R-HA and GFP-SUMO-1 were fixed in 4% PFA for 15 min, and permeabilization was not carried out to enable detection of only NH2-terminally HA epitope-tagged GLP-1R at the cell surface. The cells were incubated with rabbit HA antibody and stained with TRITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). Immunostained and live cell images were acquired on a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) with sequential scanning of GFP and mCherry signals.

cAMP Assay MIN6 Dacomitinib cells were transfected with GFP or GFP-SUMO and sorted according to GFP fluorescence 48 h posttransfection. Cells expressing GFP or GFP-SUMO were plated in 96-well plates and treated with 100 nM exendin-4 for 3 h with and without 100 ��M IBMX. Quantitative analysis of cAMP was done using a sensitive chemiluminescence-based cAMP-specific enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (Invitrogen).

b. brucei cells (strain Lister 427, cell line 449) were cultured up to the exponential growth phase and homogenates were obtained by grinding the washed parasite pellets with silicon-carbide abrasive grain (mesh 300) in disruption such buffer containing 250 mM sucrose, 25 mM Tris-HCl and l mM EDTA, pH 7.8 (STE buffer). Differential centrifugation [33] was performed as follows: the suspension was taken up in another 3 ml STE buffer and centrifuged at 30 g for 3 min. The supernatant, representing the cell homogenate, was centrifuged at 1,500 g for 10 min giving the nuclear fraction. The post-nuclear supernatant was then centrifuged at 5,000 g for 10 min giving the large-granular (mitochondria-enriched) fraction as pellet. This fraction was resuspended in 300 ��l STE buffer.

Oxygen consumption measurements Oxygen consumption was monitored in a thermostated vessel at 37��C with a Clark electrode (oxygraph). Measurements in permeabilized bloodstream-form T. brucei were done in buffer 1 (96.9 mM NaCl, 3.1 mM KCl, 5 mM MgCl2, 2 mM Na2HPO4, 90 mM Tris at pH 7.5). Batches of 2��107 cells were pelleted, washed in buffer 1 and incubated for 5�� on ice in 1 ml of buffer 1 containing 1 ng digitonin to permeabilize the cells. Subsequently the permeabilized cells were pelleted and washed twice with buffer 1 without digitonin after which the pellet was taken up in 1 ml buffer 1 and transferred to the oxygraph. Substrates and inhibitors were added as described in the text. 2 mM salicylhydroxamic acid (SHAM) was used to completely inhibit the T. b. brucei alternative oxidase (TAO) that is part of the GPO.

B6 and SHAM were dissolved in DMSO. The DMSO concentration used in these oxygen consumption measurements was less than 0.7%. Measurement of hydrogen peroxide production The method used to measure H2O2 production in T. b. brucei mitochondria is based on the fluorogenic probe 2��,7��-dichlorodihydrofluorescein diacetate (H2DCFDA) which emits an intense green fluorescence only after deacetylation and subsequent oxidation. ROS production by the mitochondrial fraction of bloodstream form T. b. brucei was measured in a 96-well microtiter plate using a fluorescence plate reader (Victor Wallace multiplate reader). In each well, 0.25 mg mitochondrial protein/ml and 5 ��M H2DCFDA to a final volume of 0.2 ml with 10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, pH 7.5 were present.

The reaction, performed at 25��C, was started by the addition of 10 mM G3P, in the presence and absence of 10 ��M B6. T. b. brucei growth conditions We used bloodstream and procyclic forms of T. b. brucei strain Lister 427, cell lines AV-951 449 [34], constitutively expressing the E. coli tetracycline (Tet) repressor gene via the chromosomally integrated plasmid pHD449 that also confers phleomycin resistance. Bloodstream forms were cultured in HMI-9 medium containing 10% heat-inactivated foetal calf serum (Invitrogen) and 0.

In control cells (in the absence of view more both zanamivir and bacterial neuraminidase), antigen-positive cells increased according to incubation times and the majority (70%) of the cells became positive at 12 hpi, indicating cell-to-cell spread of virus infection (Figure 5, top). In the presence of zanamivir, only small portion of cells (12%, Figure 5B) became antigen-positive at 12 hpi. In contrast, in the presence or absence of zanamivir, the number of positive cells at 4 hpi was the same. These results clearly suggest that the spread of infection was severely suppressed by zanamivir but the initial infection was not (Figure 5, middle). However, when V. cholerae RDE was present in addition to zanamivir, the majority of cells (68%, Figure 5B) were antigen-positive at 12 hpi, indicating that the presence of RDE diminished the inhibitory effect of zanamivir and restored the cell-to-cell spread of infection (Figure 5, bottom).

Figure 5 Bacterial neuraminidase restores the spread of infection from the inhibition by zanamivir. Inactivation of Hemagglutination Inhibition Activity of Saliva by Neuraminidase Treatment Hemagglutination activity of viruses reflects their receptor-binding activity. We detected significant inhibitory activity in human saliva against hemagglutination by influenza viruses. Saliva samples from three healthy donors were tested for hemagglutination inhibition (HI) activity against three strains of A (H3N2) virus, three strains of A (H1N1) virus and two strains of B virus (Table 2). HI titers against A type viruses varied considerably among donors.

H3N2 subtype viruses tended to be more resistant to saliva than H1N1 subtype viruses and saliva HI titers of Donor 3 were under the detection limit of two against H3N2 viruses. The saliva samples exhibited the highest HI titer of 4,096 against B/Johannesburg/99, which was the most sensitive to the inhibitory activity of saliva. Table 2 Hemagglutination inhibition activity of human saliva against influenza virus. Next, we tested effect of bacterial neuraminidase on saliva HI activity (Table 2). Saliva samples were incubated with V. cholerae RDE at 37��C for 16 h, followed by heating at 56��C for 30 min to inactivate the enzyme, and the remaining HI titers were determined. As shown in Table 2, the HI activity of saliva was completely inactivated by RDE treatment.

We confirmed that heating at 56��C for 30 min did not decrease the HI titer of saliva (data not shown), indicating that the HI ability of saliva was neuraminidase-sensitive and heat-stable. We also determined the HI titer of serum from AV-951 the three saliva donors after standard RDE treatment (data not shown). In contrast to saliva HI activity, serum HI activity was resistant to RDE treatment and HI titers against B/Johannesburg/99 virus were 2 to 10 fold lower than that of corresponding saliva, confirming that saliva HI activity is not due to serum antibodies against influenza virus.

Retrieval of tissue and clinical data was performed according to the regulations of the local institutional review board and data safety laws. The grade of the tumours was sellekchem obtained by a review of each case by a specialised pathologist. A total of 1407 colon cancers (1261 adenocarcinomas NOS, five medullary carcinomas, 119 mucinous carcinomas, five signet ring carcinomas, four other types), 559 stomach, 1527 lung (367 adenocarcinomas (AC), 13 adenosquamous carcinomas, 82 bronchioloalveolar carcinomas, 258 large cell carcinomas, 63 neuroendocrine carcinomas, 744 squamous cell carcinomas (SCC)) and 553 prostate carcinomas were then arrayed as described before (Bubendorf et al, 2001). Briefly, tissue cylinders with a diameter of 0.6mm were punched from representative tumour areas of each donor tissue block and brought into a recipient paraffin block.

Multiple 4��m sections of the resulting tissue microarray block were cut and mounted to an adhesive-coated slide system. Clinical data All relevant patient data were anonymised. Sex, age and stage according to the WHO/UICC 1997 were recorded in subsets of gastric, colon, lung and prostate cancers. In prostate cancers, the Gleason score was available. Grade was also known in colon and lung cancers. Additionally, the survival time was recorded in tumours from the colon, lung and prostate, but not stomach. Overall survival (OS) was calculated from the date of diagnosis until death from any cause or date of last contact for living patients. The cause of death was recorded in a subset of patients to calculate the tumour-specific survival.

Immunohistochemistry Standard indirect staining procedures were used for immunohistochemistry (ABC-Elite-Kit, Vector Laboratories, Burlingame, CA, USA). After heat-induced pretreatment (water bath, 30min, 99��C in target retrieval solution buffer (DAKO code S1699, DAKO, Glostrup, Denmark)) for antigen retrieval, a mouse monoclonal anti-Ep-CAM-antibody (ESA, clone VU-1D9, Novocastra, Newcastle upon Tyne, UK) was applied for 2h at a dilution of 1:50 at 37��C. The slides were then incubated with the secondary, biotinylated antibody. Osmium-enhanced diaminobenzidine was used as the chromogen. Counterstaining was carried out with Harris’ haematoxylin. Only fresh cut slides were stained simultaneously to minimise the influence of slide ageing and maximise repeatability and reproducibility of the experiment.

For negative controls, the primary antibody was omitted, as positive controls the internal normal tissues with known EpCam positivity were used. For each sample, staining intensity (0, faint to moderate, intense) and percentage of positive tumour cells was estimated. A case was considered Drug_discovery strongly positive if the antibody detected >70% positive cells, otherwise weakly positive, or negative if no cells were stained.

, 2005), voltage-dependent anion channel protein (VDAC) monoclonal antibody was purchased from Calbiochem (San Diego, CA), and phospho-threonine rabbit polyclonal Wortmannin DNA-PK antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA). The 125I-insulin radioimmunoassay kit was obtained from DPC/Siemens Healthcare Diagnostics (Deerfield, IL). INS-1 cells were obtained from the laboratory of Christopher Newgard at Duke University (Durham, NC). Screening for Inhibitors of PTPMT1. The Prestwick library of approximately 1000 small molecules dissolved in Me2SO (Prestwick Chemical Inc., Washington, DC) was screened by using the substrate O-MFP in an in vitro assay (Gottlin et al., 1996) in a 384-well plate format with a Beckman Biomek FX robot (Beckman Coulter, Brea, CA) under conditions where the Z�� score for the assay (Zhang et al.

, 1999) was more than 0.5. In brief, the assay was carried out in a total reaction volume of 40 ��l in a solution consisting of 0.1 M sodium acetate, 0.05 M bis Tris, 0.05 M Tris at pH 5.5, 50 ��M O-MFP substrate, 50 ��M test compound, 5% Me2SO, and 44 nM PTPMT1. The reaction was initiated by addition of enzyme, allowed to proceed for 40 min at room temperature, and then quenched with 40 ��l of 0.4 M NaOH. Product formation was determined by reading fluorescence emission at 520 nm. The 10 compounds showing the most potent inhibition were then subjected to a second round of screening using compound concentrations of both 50 and 5 ��M.

In these assays, product formation was monitored by both fluorescence emission at 520 nm and absorbance at 477 nm to ensure that decreased fluorescence indicated decreased product formation and true inhibition, rather than quenching of the fluorescence signal. Kinetic Studies. After the initial screen, it was determined that the optimal pH for PTPMT1 phosphatase activity with the substrate O-MFP was 7.0. Hence all kinetic experiments were carried out at this pH. Concentration that inhibits response by 50% (IC50) determinations for inhibition of PTPMT1 were carried out by using 44 nM enzyme in a total reaction volume of 100 ��l at pH 7.0 in the buffer system described above. VHR phosphatase assays were carried out by using 21.8 nM enzyme in the same buffer system, except that the pH was adjusted to 6.5, and �� protein phosphatase and T-cell PTP assays were carried out in buffers provided by the supplier using 44.

8 and 7.2 nM enzyme, respectively. All assays were carried out with 50 ��M O-MFP as the substrate at the optimal pH for each enzyme, and product formation was monitored continuously by fluorescence with initial velocity readings used for the analysis. Kinetic characterization of the inhibition Brefeldin_A of PTPMT1 by alexidine dihydrochloride was carried out with 88 nM enzyme to optimize signal and in a total reaction volume of 100 ��l at pH 7.0. PTEN assays were carried out as described previously (Maehama et al., 2000) by using 0.

The unique feature of nuclear translocation of hCAR1+A seems to contribute to its xenobiotic obviously activation in immortalized cells. Nevertheless, it is difficult to explain the robust activation of hCAR1+A entirely by this relatively moderate nuclear accumulation. Results from chromatin immunoprecipitation assay indicated that the enhanced recruitment of hCAR1+A to the promoter region of CYP2B6 gene upon CITCO treatment may also account for the optimal xenobiotic response of hCAR1+A in vitro. In addition, ligand-independent coactivator assembly has been established as a foundation for the intrinsically high activity of CAR (Tzameli et al., 2000; Li et al., 2008). In contrast, the relatively low basal interaction between hCAR3 and SRC-1 was significantly enhanced in the presence of CITCO (Auerbach et al.

, 2005). Our mammalian two-hybrid and GST pull-down assays showed that, similarly to hCAR3, the hCAR1+A also exhibits low basal and high CITCO-inducible interactions with SRC-1 and GRIP-1, suggesting alanine alone seems adequate to confer this distinctive nature of protein-protein interaction to hCAR3. Taken together, we demonstrate in this report that a single amino acid residue, alanine, within the unique hCAR3 insert is sufficient to convert the constitutively active hCAR1 to the xenobiotic-responsive hCAR3, while maintaining the chemical specificities correlate to the reference hCAR1.
the pulmonary vascular endothelium forms a continuous, semipermeable dynamic barrier for water, solutes, and plasma proteins between the intravascular space and underlying tissues.

Impairment of the barrier function results in vascular leakage and pulmonary edema formation. Inflammatory cytokines (22, 36), viruses (17, 30), biophysical forces such as stretch and shear stress (25, 29), and hypoxia (3, 7, 27, 40) can lead to increased endothelial cell permeability, thus disrupting the segregation between blood plasma and interstitial fluid. Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in stabilizing this barrier function and preventing pulmonary damage. This class B G protein-coupled receptor binds peptides of the calcitonin gene-related peptide (CGRP) family, and its activation results in a marked increase in cytosolic cAMP and subsequent activation of protein kinase A, supposing a coupling to adenylyl cyclase (19, 33).

Notably, CRLR alone exhibits little affinity to its ligands and becomes ligand selective only when associated Anacetrapib with one of the three receptor activity-modifying proteins, RAMP1�C3. CGRP activates the CRLR/RAMP1 complex, adrenomedullin (AM) appears to act as an agonist for CRLR/RAMP2 and RAMP3 (8), and intermedin (IMD; synonym adrenomedullin-2) signals primarily through CRLR associated with either RAMP1 or RAMP3 but also CRLR/RAMP2 heteromers (9, 33). The pronounced protective pulmonary effects of AM in inflammation and hypoxia are well documented.

05).Soil pH was recorded potentiometrically using 1n KCl extraction, mobile selleck chemical P2O5 and K2O (mgkg?1 of soil)��by the Egner-Riehm-Domingo (A�CL) method [48]. Soil gravimetric moisture was also continuously recorded using probe (HydroSense Campbell CS-620), and soil bulk density was measured with meter (Fieldscout SC900 Spectrum Technologies) [49].2.2. Experiment SetupField test area of each fertilizing treatment was 10m2 (2 �� 5m). The N (ammonium saltpeter 34.4% N) and NPK (ammonium saltpeter 34.4% N + granulated superphosphate 19% P2O5 + potassium chloride 60% K2O) application scheme of 9 treatments in 2 replications (n = 18) of semi-natural sward (>20yrs abandoned former sown sward): control (0); N60; N120; N180; N240; N180P120; N180K150; N60P40K50; N180P120K150.

Investigated cultural pasture (CP) was fertilized with N180P120K150 sum year rate. P and K were applied before plant vegetation in early spring, and N fertilizer was applied two times: end of April and after 1st cut (beginning of July) in all grasslands. Fresh mass (FM) weighting (g 0.2m?2 per treatment, n = 20) and drying (105��C) were used to determine grassland productivity (gm?2) and obtain dry materials (DM, %). Grassland botanical composition was determined on harvested vegetation.GHG (CO2, N2O, and CH4) emissions were monitored by the static chamber method [50] using opaque circular chambers (0.05m?3), with 6 replicates per treatment (n = 60). Cylindrical steel collar (20cm high and 43cm diameter) was inserted into the soil to a depth of 6cm. Two collars and chambers were placed in each treatment.

The collar frames remained in the soil and were open to the atmosphere between samplings, except when removed for tillage and sowing. During the measurements, the chambers were closed with an airtight lid simultaneously Cilengitide in all treatments. Chamber air was sampled 3 times in one-hour interval period. Gas fluxes were measured on 4 different dates in grasslands. The measurements were carried out 2 or 3 weeks after fertilizer application every month between June and September in the absence of frost stress. The gas samples were analyzed in the laboratory by nondispersive infrared gas analyzer (MGA3000; ISO 9001:2000) calibrated separately for each gas using ML-800 gas standard (2atm) in accordance with LST/ISO: 1401: 2005. Gas samples were analyzed on the same day evaluating volume concentrations (ppm) of trace gases. Daily net exchange (mgh?1m?2) of CO2, CH4, and N2O in agroecosystem was calculated by integrating the 60-minute fluxes determined by the meteorological measurements over each day.

2.6. Determination selleck chemicals Lapatinib of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)The MICs of the extract against the susceptible Listeria isolates were determined using the broth microdilution assay method of EUCAST [21] and carried out in sterile disposable flat-bottomed 96-well microtiter plates. Twofold serial dilutions using sterile distilled water were carried out from 10mg/mL of the stock plant extract to make 9 test concentrations ranging from 0.039 to 10mg/mL. Double strength Mueller-Hinton broth (100��L) was introduced into all the 96 wells and 50��L of the varying test concentrations of the extracts were added in decreasing order along with 50��L of the test organism suspension.

Column 1 was used as the sterility wells containing 100��L of sterile distilled water in addition to the 100��L of Mueller-Hinton broth, column 2 was used as the positive control wells containing 100��L of the broth, 50��L of ciprofloxacin, and 50��L of the test organism, column 3 was used as the negative control wells containing 100��L of the broth, 50��L sterile distilled water, and 50��L of the test organism whilst columns 4 to 12 were used as test wells containing 100��L of the broth, 50��L of the test extract concentration, and 50��L of the test organism. The plates were then incubated at 37��C for 18�C24h. Results were read visually by adding 40��L of 0.2mg/mL of ��-iodonitrotetrazolium violet (INT) dissolved in sterile distilled water into each well [4]. A pinkish coloration is indicative of microbial growth because of their ability to convert INT to red formazan [23].

The MIC was recorded as the lowest concentration of the extract that prevented the appearance of visible growth of the organism after 24h of incubation [21].The method of Sudjana et al. [24] was used to determine the MBC from the MIC broth microdilution assays through subculturing 10��L volumes from each well that did not exhibit growth after 24h of incubation and spot inoculating it onto fresh Mueller-Hinton agar plates. The plates were incubated for 48h after which the numbers of viable colonies were counted. The MBC was defined as the lowest concentration killing more than or equal to 99.9% of the inoculum compared with initial viable counts [24].2.7. Rate of Kill AssayThe time-kill assay was done according to the method of Odenholt et al. [25] following the descriptions of GSK-3 Akinpelu et al. [26]. The selected test Listeria isolates, namely, L. ivanovii (LEL 18), L. grayi (LAL 15), L. monocytogenes (LAL 8), and L.

WB smears were stained with selleck chem a hematoxylin-eosin-based rapid stain (Pan��tico r��pido, Laborclin, Brazil) and observed by microscopy (100X objective, under immersion oil). The M- and G-enriched samples were obtained from 4mL of WB with the SepCell kit (LGC Biotecnologia, Brazil), according to the manufacturer’s instructions. The BC fraction was collected from 1mL blood that was centrifuged at 12,000g for 10min.2.3. DNA ExtractionFrom each dog, a sample of blood was collected, and the DNA was extracted. Four milliliters of blood were extracted with sodium citrate and 1mL without sodium citrate. The DNA samples from the WB (200��L), BC (50��L), M (50��L), G (100��L), and C (50��L) fractions were extracted with a commercial kit (Invisorb Spin Blood Midi kit; INVITEK), following the manufacturer’s instructions.

The DNA from 21 WB, 19 G and 19M, 20 BC, and 15 C samples was used in the nPCR to amplify the E. canis and A. platys 16S rRNA sequences.2.4. Nested PCR (nPCR)The first round of PCR used 0.5 to 1.0��g of the genomic DNA, and the primers ECC and ECB were designed to amplify a 478 base-pair (bp) fragment of the Ehrlichia 16S rRNA [13]. The second round of PCR used a 1.0��L aliquot of the first reaction as a template and the EHCA sense/EHCA antisense [14] and EHPL sense/EHPL antisense (Jo?o Pessoa Ara��jo Jr.: pers. comm., 2010) primers, which were designed to amplify a 389bp fragment for E. canis and 384bp fragment for A. platys, respectively. Separate reactions were used to detect each species individually. The primers are described in Table 1.

The primer design was confirmed with the software Primer 3 (http://fokker.wi.mit.edu/primer3/input.htm). The reaction mix contained 1X reaction buffer (50mM KCl, 20mM Tris-HCl (pH 8.4), and 0.1% Triton X-100), 1.75mM MgCl2, 0.2mM dNTP mix, 1��M PCR primers, 0.625U Taq DNA polymerase, and autoclaved ultrapure water to a final volume of 25��L. The thermocycle was as follows: 94��C for 10 minutes followed by 40 cycles at 94��C for 60 seconds, 60��C for 60 seconds, 72��C for 60 seconds, and a final step of 72��C for 4 minutes before holding at 4��C. Ultra-pure autoclaved water was used as negative control in each PCR batch. The genomic DNA from confirmed E. canis and A. platys cases was used as positive controls for the E. canis 16S rRNA and A. platys 16S rRNA genes, respectively. Ten microliters of the final products were electrophoresed at 90 volts for approximately 1 hour in 1.5% agarose gels containing ethidium bromide in Drug_discovery Tris-Borate EDTA (TBE). The E. canis and A. platys reactions were positive when a 389 or a 384bp fragment was detected, respectively.Table 1The primer sequences for the 16S rRNA gene used to detect the E. canis and A. platys by the nPCR reactions.2.5.

Figure 12Results of shaking table test: (a) sine wave 2Hz, (b) sine wave 4Hz, (c) random case 1, and (d) random case 2.3.2. Time Synchronization TestAnother shaking test verified the time synchronization algorithm used in this study. Figure 13 shows the configuration of the time synchronization test using a shaking table. In this test, three laptop PCs, one master and two slaves, Dasatinib side effects were used, and the typical specifications as follows: Lenovo R61, Intel Core Duo 2.4MHz, 160 of HDD, 2GB of RAM, Acer Asprire 5580, Intel Core 2-Duo 1.66MHz, 160GB of HDD, 3GB of RAM, Lenovo X201 Tablet, Intel Core i7-640LM 2.13MHz, 320GB of HDD, 4GB of RAM. Figure 13Experimental setup for the time synchronization test.Lenovo X201 Tablet was used as the master PC. Two subsystems simultaneously tracked the same target panel at a distance of 16 meters.

All the subsystems were connected to master PC via a wireless LAN access point placed 10 meters away from all the PCs. The distance between the systems was determined considering the performance of telescopic lenses and camcorders. The software, including the time synchronization algorithm, was installed on both the master and the slave PCs.The system time of the slave PCs were synchronized based on the internal time clock of the master PC. For verifying the accuracy of the salve PCs’ internal time clocks, each slave PC generated voltage signals through its serial port based on its internal clock. An oscilloscope connected to the serial ports of the slave PCs monitored the voltage signals generated by the slave PCs, and the time lag between the two slave PCs was determined by referring to the voltage signals.

The time lag of the subsystems were checked every 60s and found a time delay of 1.54ms initially after the time synchronization process. The time delay increased with time due to differences in the accuracy of the internal time clocks of the slave PCs, as shown in Figure 14(a). In this experiment, the time lag increased 1.66ms an average of every 60s. Thus, to avoid increasing in the time lag, the time synchronization process was Batimastat performed every 60s. Figure 14(b) compares the displacements measured from the LVDT with those obtained from the system with the periodical time synchronization process. With sinusoidal excitation of 1Hz, the displacements obtained from the system using different Laptop configurations were in good agreement with the LVDT measurements.Figure 14Result of the time synchronization test: (a) time lag between the subsystems and (b) displacement with an excitation frequency of 1Hz.4. Conclusions In this study, an advanced synchronized multipoint vision-based system using an image processing technique has been successfully developed for a real-time dynamic displacement measurement.