Sedimentation Rate in Sheep

In the present work, nanohydroxyapatites (nHAp) doped with copper and/or zinc ions were investigated for the assessment of its antibacterial activity and biocompatibility. Three forms of material with diverse surfaces were tested: nanopowder in colloidal suspension, galactose hydrogel (3,6-Anhydro-α-l-Galacto-β-d-Galactan) scaffold and pellet. The structural and morphological properties of the obtained biomaterials were comprehensively determined by using: XRPD, FT-IR, SEM-EDS, AAS, XPS and EPR techniques. The antimicrobial active ions, mostly Cu2+, were successfully released from the apatite structure despite the material being suspended in the porous galactose hydrogel matrix. The colloidal solutions of nanohydroxyapatites on bacterial viability revealed moderate activity of Cu2+-doped materials against Escherichia coli strain and significant activity against Pseudomonas aeruginosa strain. The comparative study of bacterial attachment to the hydrogel and pellet surface indicated that hydrogels were more prone to be colonized by both tested strains. Moreover, an additive of the Cu2+ ion modified bacterial attachment and biofilms forming on nHAp:Cu2+ and nHAp:Cu2+-Zn2+ materials. In the case of hydrogels, the biofilms were scattered while these forming on other materials were more clumped. The cytotoxicity evaluation of tested biomaterials showed biocompatible properties of both nanomaterial colloidal solutions as well as galactose hydrogel eluates toward normal mouse osteoblast cell lines (7F2) and human chondrocytes (TC28A2) and osteosarcoma cell line (U2OS). The biocompatibility of tested materials was additionally confirmed by conducting a hemolysis assay which showed full hemocompatibility of nanopowder colloidal solutions and galactose-based materials. Furthermore, unaltered red blood cell morphology was visible after a short and long time of incubation with the obtained biomaterials by using confocal laser scanning microscopy (CLSM). The comparison research provided data of 7F2, TC28 and U2OS cell attachment to the galactose hydrogel surface.

Accurate predictions of drug uptake transporter involvement in renal excretion of xenobiotics require determination of in vitro transport kinetic parameters under initial-rate conditions. The purpose of the present study was to determine how changing the incubation time from initial rate to steady state influences ligand interactions with the renal organic anion transporter 1 (OAT1), and the impact of the different experimental conditions on pharmacokinetic predictions. Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1) and the Simcyp Simulator was used for physiological-based pharmacokinetic predictions. Maximal transport rate and intrinsic uptake clearance (CLint) for PAH decreased with increasing incubation time. The CLint values ranged 11-fold with incubation times spanning from 15 s (CLint,15s, initial rate) to 45 min (CLint,45min, steady state). The Michaelis constant (Km) was also influenced by the incubation time with an apparent increase in the Km value at longer incubation times. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 s or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide, whereas indomethacin was less potent, and probenecid (~2-fold) and telmisartan (~7-fold) more potent with the longer incubation time. Notably, the inhibitory effect of telmisartan was reversible, albeit slowly. A pharmacokinetic model was developed for PAH using the CLint,15s value. The simulated plasma concentration-time profile, renal clearance, and cumulative urinary excretion-time profile of PAH agreed well with reported clinical data, and the PK parameters were sensitive to the time-associated CLint value used in the model.

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.

This in vitro study was conducted to analyze the formation of biomarker for DNA damage 8-hydroxy-2'-deoxyguanosine (8-OHdG) due to Bisphenol A and metal ion Cu (I) exposure in the presence of H2O2 as oxidizing agent on 2-deoxiguanosine via Fenton-Like reaction in non-physiological conditions (temperature 60°C and pH 8.4). Samples were incubated in numerous incubation time (7 hours and 12 hours) and further analyzed by using UHPLC reverse phase with mobile phase sodium phosphate buffer and methanol (85:15) with UV-Vis detector at wavelength 254 nm. The results showed that 8-OHdG can be formed by reacting Bisphenol A, Cu(I) and H2O2. Mostly, samples with longer incubation time (12 hours) have higher 8-OHdG concentration than shorter incubation time (7 hours). 8-OHdG concentrations in all samples were above LOD value and mostly were above LOQ value. Only one mixture that have 8-OHdG levels below LOQ value, i.e. dG, Cu(I), and H2O2. The highest 8-OHdG concentrations found on the complete mixture of reagents (dG, Bisphenol A, Cu(I), and H2O2) with longer incubation time (12 hours) with concentration equal to 92.438 ppb.

We present an integrated experimental-computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m(2)/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8mPa (0.018 dyn/cm(2)). In the non-perfused pellets, the lowest oxygen concentration was 0.003mM (0.3% tension) and the maximal oxygen flux was 102.4 nmol/m(2)/s. A local correlation was observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results show up-regulation of hyaline matrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications.

The acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability. Besides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry. It was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface. EPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage

Experiment was conducted to investigate if seed priming with polyethylene glycol (PEG) activator solutions affects the viability and vigour of deteriorating wheat (Triticum aestivum L. cv. Caxton) seed lot. Seeds were subjected to PEG priming solutions with varying osmotic potentials (-1, -2, -3 MPa) at temperatures of 15 or 20°C for 6, 12, 18 or 24 h and compared to the non-primed control. Highest germination percentage at first and final count, length of plumule and dry weight of seedling were all associated with Treatment 1 MPa/20°C/6 h (92%, 94%, 9.2 cm, 0.0133 mg, respectively) compared to the control (82.5%, 86%, 7.8 cm, 0.0112 mg, respectively). The best values of coefficient of velocity of germination (CVG), mean germination time (MGT) and germination rate index (GRI) were associated with Treatment 2 MPa/15°C/24 h. There were significant interactions between the factors under study and whilst most positive effects decreased with incubation time the opposite was true at 15°C Treatment 2 MPa where an initial decrease in germination after 6 h was restored with longer incubation times. Significant correlations were found between most of the characteristics under study although these did not always account for a high percentage of variation but CVG and MGT were very highly correlated. It was concluded that, 6 h in Treatment 1 MPa PEG at a temperature of 20°C resulted in significantly improved germination percentage whilst 24 h Treatment 2 MPa at 15°C was optimal for the highest CVG and MGT. The highest speed of germination was not associated with the highest germination percentage.

Quinoline antimalarials decrease the rate of β-hematin formation

Research on microfluidic devices for biological analysis has progressed sufficiently to be developed into point-of-care diagnostics products. The goal of this thesis is to improve multiple aspects of capillary-driven microfluidic devices. In particular, the objective is to provide devices with a fast time to result, that are simple to use (one-step), that can be portable, that accept a variety of samples, that operate reliably, that provide a range of detection signals, that are mass manufacturable at lost cost, and that are able to detect medically relevant biological molecules. First, we survey the evolution of microfluidic research into portable medical diagnostic devices. By looking at several gaps and opportunities in current medical diagnostics, we provide an overview of research topics that have the potential to shape the next generation of point-of-care diagnostics. Specifically we explain technologies in the order of sample interacting with different components of a device. We investigate the materials, surface treatments, sample processing, microfluidic elements (such as valves, pumps and mixers), receptors and analytes and the integration of these components into a device that might conceivably leave the laboratory for the hands of consumers. The knowledge of what is important in a point-of-care diagnostics device was used to develop a proof of concept. One of the main challenges is to make microfluidics easy to use by incorporating reagents and microfluidic elements. We integrated a number of functional elements on a chip such as a sample collector, delay valves, flow resistors, a deposition zone for detection antibodies (dAbs), a reaction chamber sealed with a polydimethylsiloxane (PDMS) substrate, and a capillary pump and vents. We further incorporated capture antibodies (cAbs), detection antibodies (dAbs) and analyte molecules for making one-step immunoassays. The integrated microfluidic chip requires only the addition of sample to trigger a sequence of events controlled by capillary forces to detect C-reactive protein (CRP), a general inflammation and cardiac marker, at a concentration of 1 ng mL-1 within 14 min using only 5 µL of human serum. The proof-of-concept is extended to easily modify several assay parameters such as the flow rates and the volumes of samples for tests, and the type of reagents and receptors for analytes. The multiparametric microfluidic chip is capable of analyzing 20 µL of human serum in 6 parallel flow paths in a range of flow rates with filling times from 10 minutes to 72 minutes. The asymmetric release of dAbs in a stream of human serum is compensated by a Dean flow mixer. Sample is equally split into 6 reaction chambers connected to flow resistances that vary flow rates, and the kinetics of capture of analyte-dAb complexes. The increased incubation time leads to a fourfold increase in detection signal in the reaction chamber with the longer incubation time. Furthermore, integrating reagents and controlling their release is essential for simple and accurate point-of-care diagnostic devices. We developed reagent integrators (RIs) to release small amounts of dried reagents (ng quantities and less) into microliters of sample. Typical RIs are composed of an inlet splitting into a central reagent channel, with a high hydraulic resistance, and two diluter channels. Reagents spotted in the central channel reconstitute in sample during filling and merge at the end of the RI with a dilution factor corresponding to the relative hydraulic resistance of the channels forming the RI. RIs are simple to integrate in lateral flow assays and provide a great degree of control over reagent integration and dissolution. Finally, the one-step capillary-driven microfluidic chips have the ability to not only detect a variety of proteins, but also to detect nucleic acids for molecular diagnostics. These devices, especially if manufactured in low cost plastic and used with portable fluorescence readers, have the potential to identify a wide variety of health conditions and to enable truly decentralized medical diagnostics.

Oxysterols are cholesterol-oxygenated derivatives generated in the organism and also present in foods because of cholesterol oxidation during processing and storage. They are the natural ligands of liver X receptors (LXRs) and are generally recognized as hypocholesterolemic and anti-inflammatory molecules although this latter property is still controversial. Most oxysterol studies have been performed in macrophages, whereas the effects of oxysterols in neutrophils are poorly known. In this study, human neutrophils were exposed to two different oxysterols, 7-keto-cholesterol (7-k-chol) and 25-hydroxy-cholesterol (25-OH-chol), and their possible participation in inflammatory process was evaluated. Human neutrophils were incubated with 7-k-chol and 25-OH-chol, and ROS production, translocation of the NADPH oxidase cytosolic components, hemoxygenase-1 (HO-1) expression and lysozyme secretion were analyzed. An increase in ROS production was observed within a short period of time (minutes) with both molecules. These oxysterols also stimulated the cellular membrane translocation of the NADPH oxidase cytosolic components, p47phox and p67phox. On the other hand, HO-1 expression, a cytoprotector enzyme, is inhibited in human neutrophils upon oxysterols treatment. Moreover, both oxysterols were associated with high lysozyme enzyme secretion at 5 and 18h of incubation. The present paper describes for the first time that two oxysterols (7-k-chol and 25-OH-chol) enhance the ROS production within a short period of time in human neutrophils, stimulate the translocation of the cytosolic components of NADPH oxidase to the cellular membrane and increase lysozyme secretion. These data suggest that both oxysterols are able to activate pro-inflammatory effects in human neutrophils which contrasts with the role assigned to the oxysterols when they act through LXR at long time of incubation.

1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system.

This work aimed to evaluate the Caco-2 cells as a model to study the epithelial transport of intestinal lipid extracts subjected to invitro digestion, to establish a standard protocol for the determination of bioaccessibility and bioavailability of fatty acids in meat matrix, especially in those enriched with ω-3 (eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)). Samples were first subjected to invitro digestion, and then, the intestinal extract was incubated with Caco-2 cells. A first trial was conducted to select the most influencing variables on the fatty acid transport during Caco-2 cell incubation: fat quantity on the intestinal extract, incubation time, and shaking. Then, a second experiment was carried out to determine the influence of these variables, being the fat quantity and the incubation time the most influencing factors on the transport and bioavailability of fatty acids. The effect of shaking was not so marked but seemed to improve the bioavailability of saturated fatty acids. This study also allows establishing the most suitable conditions: intestinal extracts with 30 mg of fat, longer incubation times (8 h), and shaking, achieving active and passive fatty acid transport without compromising the integrity of the Caco-2 cell monolayer. The accurate results obtained for major and minor fatty acids, especially EPA and DHA are remarkable, due to the interest in these bioactive compounds. Thus, this study provides a combined protocol based on static invitro digestion followed by Caco-2 cell incubation to assess the bioaccessibility and bioavailability of fatty acids in meat samples.

Impact of time between repeated sperm freezing cycles on sperm quality

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay's performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify alpha,beta-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4'-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.