Cognitive dysfunctions are directly correlated with Aβ oligomers in Tg2576 mice, which start at around 6 months old and are stable until 14 months old [29]. Thus, we first evaluated cognitive Selleck OSI 906 functions in both non-tg (n = 18) and Tg2576 mice (n = 24) at the age of 12 months. After the behavioral test, mice were divided into two groups to be treated with rSeV-LacZ or rSeV-Aβ. There is no difference between the two groups in behavioral scores at the age of 12 months. To evaluate the effect of vaccine treatment, each group (rSeV-LacZ-treated non-tg mice, n = 9; rSeV-Aβ-treated non-tg mice, n = 9; rSeV-LacZ-treated

Tg2576 mice, n = 10; rSeV-Aβ-treated Tg2576 mice, n = 14) was subjected to behavioral tests at the age of 15 months. All tests were done according to the methods described previously [30]. 24 h after 10 min-training session following 3 day-habituation, each mouse was placed back into the same box in which one of the familiar objects used during training was replaced with a novel one. The animals

were then allowed to Ulixertinib solubility dmso explore freely for 10 min and the time spent exploring each object was recorded. The exploratory preference (%), a ratio of the amount of time spent exploring any one of the two objects (training session) or the novel object (retention session) over the total time spent exploring both objects was used to measure cognitive function. Each mouse was placed at the center of the apparatus and allowed

to move freely through the maze during an 8-min session, and the series of arm entries was recorded visually. Alternation was defined as successive entry into the three arms on overlapping triplet sets. The % alternation was calculated as the ratio of actual alternations to the possible alternations (defined as the number of arm entries minus two) multiplied by 100. The Morris water maze test was conducted in a circular pool (1.2 m in diameter) with a hidden platform (7 cm in diameter) filled with water at a temperature of 22 ± 1 °C. The mice were given two trials Sodium butyrate (one block) for 10 consecutive days during which the platform was left in the same position. The time and distance taken to reach to the escape platform (escape latency and distance moved) was determined in each trial by using the Etho Vision system (Brainscience Co. Ltd., Osaka, Japan). Three hours after the last training trial, the platform was removed, and mice were allowed for 60 s to search the removed platform. For measuring basal levels of freezing response (preconditioning phase), mice were individually placed in a neutral cage for 1 min, and then in the conditioning cage for 2 min. For conditioning, mice were placed in the cage, and an 80 dB tone was delivered for 15 s. During the last 5 s of the tone stimulus, a foot shock of 0.

It wasn’t feasible to select a marker compound for 3rd group for subsequent tentative identification. Therefore the compounds present in Group 1 and 2 were used to compare degradation rate based on the marker MLN8237 nmr compounds. For formulae generation, the isotopic pattern of unknown compound, relative high atom number and low mass error limits were used. Based on these

factors MassHunter software generated several formulae which has been sorted out by MGF score. Molecular formulae presented in Table 2 (along with predicted abundances) and 3 had the highest score and lowest error calculated by the software. A compound search for the above candidates was performed using online databases and available literature. The metabolites which were identified by comparing standard mass spectra and fragmentation pattern and found only in fresh juice are given in Table 3. Degradation rate of important and known metabolites were explored using total abundance of metabolites present in different sample (Fig. 4). A supervised pattern recognition method was used to discriminate and classify the stem juice samples. The result in terms of classification abilities of the samples showed 88.888% accuracy (Table 4). The classification ability was observed to be slightly lower due to incorrect assignment of one sample of Group 3 in may

be due to extensive degradation in Group 2. The same has been confirmed by comparing the abundances of ions of identified compounds in juice (Fig. 4) where Group 2 showed very low abundance as compared to Group 1. The UPLC–QTOFMS is advanced technique used extensively for diseases diagnostics, drug MG-132 chemical structure discovery and human nutrition. In this study, the technique has been successfully used to explore the stability of untreated stem juice of stems of T. cordifolia stored at 0 °C. The reported medicinally important compounds i.e. jatrorrhizine,

mangoflorine, second manisperine, columbamine, berberine and tinosporoside were identified using standard mass spectra from literature and comparing the mass fragmentation patterns. Manisperine is the alkaloid, first time reported from T. cordifolia. There abundance comparisons showed complete degradation of some compounds after one month storage. As consumers continue to seek products with improved medicinal value and functionality, the stabilizers for medicinal juices should be used judicially. It is also advisable to use the fresh juice of T. cordifolia instead of stored one, as degradation starts immediately in the juice contents even if stored at 0 °C. At the same time, considering the encouraging results obtained in this study, the application of UPLC–QTOFMS to detect stability of herbal products seems to be a very promising approach. All authors have none to declare. Authors are thankful to CCRAS, Department of AYUSH, Government of India to support the study. “
“Curcuma longa L.

Vers la fin mars 2014 était signalée la réapparition du virus Ebola dans une épidémie émergente en Guinée [1] :

dès le 24/03/2014, les autorités signalaient 49 cas, parmi lesquels 29 décès. D’emblée, plusieurs éléments originaux soulevaient interrogations mais aussi inquiétudes : non seulement c’était la première fois que cette infection, habituellement observée en Afrique Centrale, apparaissait en Afrique de l’Ouest mais surtout, à côté des cas initiaux signalés dans le Sud-Est du pays (Gueckedou), d’autres cas étaient repérés très vite dans la capitale Conakry. L’atteinte d’une grande ville laissait d’emblée supposer que le phénomène infectieux, contagieux, serait beaucoup plus difficile à contrôler. Depuis, en dépit des mesures prises (peut être insuffisantes Rapamycin nmr ou mal appliquées), l’épidémie s’est étendue à une vitesse variable, s’accélérant à partir du mois de juin pour s’accroître en juillet et août, avec à nouveau une accélération en septembre [2] : au-delà de la Guinée,

le Liberia et la Sierra Léone [3] étaient concernés ; actuellement, de façon plus modérée, le Nigeria est touché à son tour ; on note aussi un cas sénégalais isolé à partir d’un malade venu de Guinée. Au 17/09/2014, 4985 cas étaient recensés, parmi lesquels Selleck GDC-0068 2461 décès, soit une mortalité de 50 %. À noter qu’un signalement en République Démocratique du Congo serait dû à un virus Ebola différent. Au total, en ce début septembre, nous en sommes à plus de 4000 cas et plus de 2000 décès. Quoiqu’il en soit, le non-contrôle de l’épidémie et le risque d’extension à travers des frontières difficiles à contrôler, et donc poreuses, inquiètent l’OMS et la communauté internationale [4]. La prise de conscience des autorités, certes accrue, ne suffit pas à maîtriser une épidémie qui mobilise aujourd’hui des organisations

humanitaires et préoccupe davantage nos politiques. Le virus Ebola est connu depuis 1976, où il fût responsable d’épidémies au Nord Zaïre et au Sud Soudan, créant la panique et de nombreux décès. Il emprunta alors son nom à une rivière zaïroise [5] and [6]. Des petits foyers épidémiques apparurent ensuite en différents pays (Zaïre, Gabon, Côte d’Ivoire, Congo…), à chaque fois en zone forestière, faisant de nombreuses Tolmetin victimes, notamment parmi les soignants. À chaque fois, la poussée s’éteignait en quelques semaines avec la mise en place de mesures d’hygiène. Le virus Ebola est un filovirus (famille des filoviridés), proche du virus Marbourg. Les filovirus sont des virus enveloppés, se présentant en long filament (d’où leur nom) et comportant un certain nombre de sous types antigéniquement différents : Ebola Zaïre, Ebola Soudan, Ebola Reston… Le responsable actuel, Ebola Guinée, appartient à un CLADE* différent mais avec de fortes identités avec les Ebola de République Démocratique du Congo et du Gabon. Le réservoir de virus, longtemps demeuré inconnu, est très vraisemblablement, une fois encore, la chauve-souris frugivore [7].

Passive surveillance systems are able to identify safety signals, but are subject to known limitations, due to underreporting, delayed reporting and a lack of denominator data. Active surveillance in a defined PR-171 mw cohort of vaccines can complement passive surveillance by overcoming problems of delayed and underreporting and enabling calculation of adverse event rates. Recent studies internationally have emphasised the importance

of active surveillance to detect important signals early so that appropriate investigations can be launched and necessary actions taken [8] and [9]. Internationally the usefulness of Patient Reported Outcomes (PROs) utilising available internet tools has been increasingly recognised. There is evidence that in relation to adverse events PROs can identify real-world signals earlier and in higher volume, accurately characterise the signals, allow a focus on specific events

or populations of interest, and permit ongoing efficient safety monitoring [10]. The finding that there was a significantly higher rate of reactions in participants who received IIV in the previous year deserves further investigation as it has not been a consistent finding in previous studies [3]. The initial practice visit by Vaxtracker staff of this pilot phase could be replaced by a brief diagrammatic user guide or online web PF01367338 demonstration to further improve efficiency and reduce the cost of the roll out phase. We estimate that once established the ongoing human resources to operate the system are not great as survey results provide sufficient information for assessment and very few respondents require subsequent telephone clarification of clinical details or support. After the Vaxtracker survey was completed by respondents, case review and data analysis for signal detection quickly take place. The automatic management of survey dispatch and return of completed surveys and email alerts has allowed for the efficient and

prompt review of AEFIs and rapid data analysis and rate calculation. It is essential to reassure the community of vaccine safety and to prompt Phosphoprotein phosphatase early investigation should severe reactions occur or if there is an unexpected increase in the frequency of clinical events [11]. The Vaxtracker active surveillance system achieved encouraging completion rates. These were found to be higher where parents received both mobile phone and email reminders. Feedback and a certificate of appreciation were provided to all General Practice clinics that enrolled participants. Respondents who reported serious AEFI were contacted by telephone to discuss their report, ensure that appropriate clinical management had occurred if required and enquire whether symptoms had resolved. There was no formal feedback to respondents in this pilot but plans are underway to make Vaxtracker safety data available to the public on a website as the programme is expanded.

3 bacterial expression vector. pPACIB.3 is an “in house” developed plasmid for bacterial periplasmic expression of recombinant proteins via an ompA leader

sequence. The tryptophan promoter and a terminator Epacadostat chemical structure sequence from the T4 phage ensure high expression levels and the vector provides expressed proteins with six-histidine tags at their C-terminus. The hrVEGF molecule was purified from bacterial periplasm using conventional IMAC procedures [16]. The recombinant P64K protein derived from Nm was supplied by the Development Department of the CIGB. P64K is produced routinely to be used as a vaccine carrier protein [17]. Clinical grade preparations (0.8 mg of protein/0.5 mL per vial) of VSSP were supplied by the Center for Molecular Immunology of Havana. The VSSP preparation is obtained by physical disorganization of outer membrane vesicles of Nm and further re-association and stabilization with the inclusion of GM3 gangliosides. VSSP induces the activation of CTL responses to peptides and proteins, and can also stimulate the humoral response to different antigens [18], [19] and [20]. The

oil-based adjuvant was obtained from Seppic (France). Emulsification was done as recommended by the supplier using two syringes, a connector, and 100 syringe passes. Animals were randomly assigned to five groups of five animals each and given: (a) six subcutaneous http://www.selleckchem.com/products/erastin.html injections of 100 μg of the recombinant protein pP64K-hVEGFKDR− mixed with 200 μg of VSSP (hereafter denominated CIGB-247), in weekly or biweekly schedules,

or (b) six intramuscular injections of CIGB-247 in a volume of 0.1 mL, mixed with 0.1 mL of montanide ISA 51, in a biweekly schedule. Control (placebo) animals received only Tris 10 mM. The rats assigned to the weekly schedule received three additional injections of CIGB-247 25 days after the sixth immunization. A week after the last booster the animals were euthanized, sera and plasma collected and their organs processed for histopathology. Platelet rich and platelet depleted plasma were obtained as described [21]. Animals (three per group) were given: (a) six subcutaneous very injections of CIGB-247, in weekly or biweekly schedules, or (b) six intramuscular injections of CIGB-247 in a volume of 0.1 mL, mixed with 0.1 mL of montanide ISA 51, in a biweekly schedule. Control animals (two animals) received only Tris 10 mM. The animals assigned to the weekly schedule received an additional booster 21 days after the sixth immunization and were euthanized a week later. Sera were collected and organs dissected and fixed in 10% formalin for histological evaluation. Animals were screened first for antibodies to P64k and VEGF proteins and considered naive with respect to both antigens when specific antibodies were undetectable by ELISA (titer <1:50) (see methods below). Monkeys were subsequently ranked by weight and age and then randomly assigned to three groups of three animals each.

The antisera were tested at two different dilutions, 1:8 and 1:16. Fig. 5 shows the number of CFUs recovered after incubation of pneumococci with peritoneal cells in the presence of sera at the dilution of 1:16 with the exception for Strain P 1079 in which the anti-PspA 94/01 opsonophagocitic http://www.selleckchem.com/products/Bortezomib.html activity was observed only at a dilution of 1:8. The anti-PspA 245/00 antisera (clade 1) was able to reduce the number of CFUs recovered in at least 40% for strains bearing PspA clade 1 and 30% for strains containing clade 2 PspA, reaching a maximum of 50% in strains of the same clade. Furthermore, sera from

mice immunized with PspA 94/01 (clade 2), was able to mediate killing of at least 30% of the bacteria expressing PspAs clade Pomalidomide mouse 1 or 2. The only exception was that of strain P278, for which the reduction in CFU recovered was only 17%. The maximum reduction induced by anti-PspA 94/01 antisera was 46 and 63% for strains bearing PspA 1 and 2, respectively. The CFU reduction mediated by anti-PspA 245/00 and 94/01 was statistically significant when compared to serum from mice receiving Aluminum hydroxide (except

for strain P 278). Both sera induced similar degrees of bacterial phagocytosis among pneumococci bearing family 1 PspAs, since there were no statistically significant differences between the effect induced by anti-PspA 245/00 and anti-PspA 94/01 antisera. Microscopical analysis of the samples revealed the interaction between the phagocytes and the pneumococci incubated with both sera (Fig. 6). In the control group, after incubation of the cells with bacteria previously treated with non-specific

antibodies, no interaction was observed, as depicted by the mononuclear cell in Fig. 6A. On the other hand, incubation of the cells with a PspA clade 1 expressing strain, previously opsonized with anti-PspA 94/01 (clade 2), induced a strong interaction between the bacteria and the peritoneal cells, as demonstrated by the pneumococci-covered macrophage in Fig. 6 B. Noteworthy is the ability of the anti-PspA 94/01 antibodies to mediate phagocytosis of a pneumococcal strain expressing a heterologous PspA, a strong indication of cross-protection. A similar TCL result was obtained when cells were cultured in the presence of the pneumococcal strain P 69, containing PspA clade 1, previously incubated with anti-PspA245/00, also clade 1; Fig. 6C and D shows a large number of internalized bacteria in a macrophage and a neutrophil, respectively. PspA is a promising vaccine candidate against pneumococcal disease; however, it is structural and serological variability could limit the coverage of a PspA-based vaccine. Therefore, understanding the nature of PspA’s variability has been the focus of many studies regarding anti-pneumococcal vaccine development. Hollingshead et al. [12], grouped most PspAs into two major families, 1 and 2, which were subdivided into 5 clades.

08. The results obtained by laser light scattering tests were higher than those observed by SEM that was related selleck kinase inhibitor to hydrodynamic diameter of swollen polymeric

nanoparticles in water.10 Drug loading and entrapment efficiency for all samples are shown in Table 1. The choice of the method to produce nanoparticles is strongly dependent on the identity of the drug that is going to be encapsulated. Hydrophobic water-insoluble drugs are more efficiently encapsulate by the simple ESE or nanoprecipitation.11 The main problem in the preparation of carvone and anethole loaded nanoparticles was volatility of them. So in this study a method with the shortest time of process to achieve the nanoparticles with lowest evaporation carvone and anethole was assessed. In ESE method, evaporation of organic phase takes a long time (about 3 h) and probably we lose a lot of carvone and anethole. The highest drug loading in this method was 0.29% for anethole and 0.33% for carvone. Hence, nanoprecipitation method without evaporation and freeze drying steps was applied and antimicrobial test was examined in suspension form of nanoparticles. The highest drug loading in this method was 14.73% for anethole and 13.64% for carvone. Some of advantages associated with this method

like: large amount of toxic solvents are avoided, small particle size with narrow size distribution are obtained, and without the use of external energy source.12 The main problem with the nanoprecipitation is the frequent agglomeration of particles due to find more the lack of a stabilizer. This can be solved using efficient stirring, by slow addition of the organic phase to the aqueous phase, and by selection of an adequate solvent system.12 The high DCM/acetone volume ratio in the organic phase of ESE method led to an improvement in entrapment efficiency but this improvement was not so Fossariinae significant (2.9% for anethole and 3.35% for carvone). Rapid diffusion of acetone into the outer phase may be the reason for such low entrapment efficiency. The high polymer/drug concentration in the injection phase with the low ratio of water: DMSO led to a significant improvement in

entrapment efficiency of nanoprecipitation method (87.3% for anethole and 68.2% for carvone). The in vitro release behavior of the two essential oil-loaded nanoparticles is summarized in the cumulative percentage release shown in Fig. 3. The initial burst release was detected for both nanoparticles during the first 6 h. The carvone-loaded nanoparticles showed a higher burst release (36%) compared with the anethole-loaded nanoparticles that release only 16% during the same time period. The ether group of anethole makes it more lipophil than carvone that leads to more encapsulation of anethole and takes longer time to diffuse from nanoparticles to the buffer phosphate medium. The initial burst could be ascribed to antimicrobial agent distributed at or just beneath the surface of the nanoparticles.

0; 0.01 M) (B) in a gradient mode. The solvent program was set as follows: (Tmin/A:B; T0/60:40; T8.0/60:40; T10/50:50; T13/60:40; T16/60:40). The flow rate of 1.0 ml/min, column temperature

at 25 °C, injection volume of 20 μl and wavelength of 280 nm were found to be suitable to achieve the separation of paliperidone and its degradation products. Validation of the optimized LC method was done with respect to various parameters outlined in ICH guideline MLN2238 cell line 13 and was extended to LC–MS2 studies. The chromatographic conditions used for LC–MS analyses were the same as that for LC–PDA analyses, except that injection volume was 10 μl. LC–MS studies were carried out using positive as well as negative atmospheric pressure chemical ionization (+APCI and −APCI) modes in the mass range of 50–2000 m/z. High purity helium was used as carrier gas and nitrogen was used Cisplatin solubility dmso as nebulizer. The operating conditions for LC–MS scans of drug and degradation products in both the ionization modes were optimized as follows: Rf loading: 80%; capillary voltage, 80 V; syringe volume, 250 μL; spray chamber temperature, 50 °C; nebulizer pressure, 35 psi; drying gas temperature, 300 °C; drying gas pressure, 10 psi; vaporizer gas temperature, 350 °C; vaporizer gas pressure, 20 psi; spray shield voltage (±), ±600.0 V. Specificity is the ability of the analytical method to measure the analyte concentration accurately

in presence of all potential degradation products. Specificity of the method towards the drug was studied by determination of purity for drug peak in stressed sample using a PDA detector. The study of resolution factor of the drug peak from the nearest resolving degradation product was also done. Drug as well as degradation product

peaks were found to be pure from peak purity data. Also, the resolution factor for the drug from degradation peak was greater than 3. Peak purity and resolution factor data is given in Table 4. Linearity test solutions were prepared from stock solution at seven concentration levels of analyte (5, 50, 100, 200, 400, 600, 800 μg/ml). The peak area versus concentration data was performed by least squares linear regression analysis. The calibration curve was drawn by plotting paliperidone those average area for triplicate injections and the concentration expressed as a percentage. Linearity was checked over the same concentration range for three consecutive days. Good linearity was observed in the concentration range from 5 to 800 μg/ml of paliperidone. The data was subjected to statistical analysis using a linear regression model; the linear regression equation and correlation coefficient (r2) were y = 1.0617x + 2.6806 and 0.9995, respectively. These results indicate good linearity. The LOD and LOQ for PPD were estimated at a signal-to-noise ratio of 3:1 and 10:1, respectively. The LOD and LOQ were 0.32 μg/ml, 0.99 μg/ml, respectively.

16 In the present study, total flavonoid, total phenolic contents and radical scavenging activities of 6 selected medicinal plants were assessed. In this study, out of 6 medicinal plants tested, P. amarus had the maximum phytochemical and antioxidant activity followed by L. aspera. Still extensive studies are needed to evaluate the phytochemical and pharmacological activities of specific lead compounds in order to use these plants as a probable source for the potential natural antioxidants. All authors have none

to declare. selleck screening library The authors are very thankful to The Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India for supporting

this research through DST-FIST and UGC-SAP funds “
“Skin and skin structure infections (SSSIs) are infections which include skin, and range from minor pyodermas HSP inhibitor to severe necrotizing infections.1 and 2 Among the gram-positive organisms, particularly Staphylococcus aureus and gram-negative organisms are common causes of SSSIs. Gram-positive organisms, predominantly Staphylococci and Streptococci, are responsible for the majority of bone and joint infections (BJIs). The treatment of SSSIs and BJIs remains difficult to treat because of increasing resistance to commonly used antibiotics for the treatment of these infections. 3, 4, 5 and 6 Moreover the emergence of extended spectrum-β-lactamase (ESBL) and metallo-β-lactamase (MBLs) 7, 8 and 9 is making it difficult to treat BJIs and SSSIs caused by gram-negative and gram-positive infections. Resistance being the first cause of failure of therapy particularly in Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella

pneumoniae, Klebsiella oxytoca, Escherichia coli and S. aureus. 10 In view of the increasing failure rate of β-lactams including carbapenems, there is a need of a new antibiotic/combination of antibiotics which can work more efficiently against ESBLs and MBLs. Therefore, we have designed a new antibiotic adjuvant entity of Ceftriaxone-sulbactam-with adjuvant disodium edetate (Elores) (US patent no 8273732). Bumetanide The in vitro, preclinical, microbiological and molecular studies have demonstrated it to be more effective than penicillins, cephalosporins, beta-lactam and beta-lactamase inhibitor combinations including piperacillin + tazobactam, cefoperazone + sulbactam, amoxicillin + clavulanate.11, 12 and 13 Therefore, present study was planed to study randomized, open label, prospective, multicenter comparison of Elores versus ceftriaxone in the treatment of SSSIs and BJIs. Current study is approved by DCGI and has been performed in accordance with GCP guidelines.

Repeatability was assessed by measuring the lysates six times by one technician on one day. The mean repeatability CV of all laboratories ranged between 8% and 19% for the three lysates (Table 2). The intermediate precision was assessed by measuring the three lysates six times on six separate days by two technicians. The mean intermediate precision CV for all laboratories

VE-821 ranged between 25% and 40% for the three lysates (Table 2). Finally, the reproducibility was determined by calculating the average of the intermediate precisions from all laboratories (Table 2 and Supplementary Fig. 1a). This resulted in overall CV values of 25%, 12% and 15% for the mock, H3N2, and Con A lysates, respectively. Importantly, each lab could significantly distinguish between low (mock), intermediate (H3N2) and high (Con A) granzyme B levels (data not shown). In conclusion, when taking into account a threshold of approximately 30% as the acceptable upper limit for the CV [34] and [36], the granzyme B assay showed acceptable variability as determined by repeatability, intermediate precision and reproducibility [34] and [35]. For the ultimate application of the granzyme B assay in large scale vaccine trials, we determined the overall robustness of the 5-FU datasheet assay by using samples of PBMC for validation. Each research group performed the standard

procedure as described above on four different days with the same batch of frozen PBMC from two donors. Each laboratory could clearly distinguish between the high Rebamipide (donor 1) and low (donor 2) responder (Fig. 2b). The intra-laboratory robustness for H3N2 stimulation showed a mean CV of 33%; 95% confidence interval (CI), 18–48. The inter-laboratory robustness for H3N2 stimulation showed a mean CV of 29%; 95% CI, 28–30 (Table 3). Collectively, these data indicate

that the granzyme B assay is a robust assay capable of generating similar responses between different laboratories. Detection of cytokines by the multiplex assay was validated by the supplier. We tested applicability of the assay by determining the parameters specificity, reproducibility and robustness following stimulation of PBMC as described above. To determine whether the cytokine assay can specifically measure each cytokine in samples of cell culture supernatants, the bulk Con A supernatant was diluted and analyzed (Table 1). Two-fold dilution of the Con A supernatant resulted in a mean recovery of 92%. Ten-fold dilution of the Con A supernatant resulted in a mean recovery of 84%. These data indicate that the cytokines can be measured specifically in samples of cell culture supernatants harvested after stimulation. Reproducibility of the cytokine assay was assessed by all four laboratories with the same batch of supernatant derived from PBMC stimulated with mock, H3N2, or Con A. The supernatants were tested three times on three separate days by each laboratory.