Rapid detection of sulfamethazine and ofloxacin residues in duck meat using synchronous fluorescence spectroscopy coupled with chemometric methods.

Rapid detection of antibiotic residues in duck meat is of great significance for strengthening food safety and quality supervision of duck meat and fighting against inferior products in the duck meat market. The objective of the current paper was to evaluate the potential of synchronous fluorescence spectroscopy (SFS) coupled with chemometric methods for the rapid detection of sulfamethazine (SM2) and ofloxacin (OFL) residues in duck meat.The SFS spectral data from duck meat containing different concentrations of SM2 and OFL were preprocessed by baseline offset. The detection conditions, including the adding amounts of ß-mercaptoethanol solution and o-phthalaldehyde solution, as well as the reaction time, were optimized by a single factor experiment for obtaining a better detection effect, and their optimal values were 400 µL , 25 µL , and 40 min, respectively. By comparing 2 chemometric models based on peak-height algorithm and peak-area algorithm, the prediction model based on peak-height algorithm was a better quantitative model with correlation coefficient for the prediction set (Rp) of 0.9031 and 0.9981, the root mean error for the prediction set (RMSEP) of 7.9509 and 0.5267 mg/kg, recovery of 81.7 to 155.1% and 96.4 to 111.2%, and relative standard deviation (RSD) of 4.1 to 6.7% and 2.9 to 6.8% to predict SM2 and OFL residues in duck meat, respectively. Overall, the results of this investigation showed that SFS technique was an effective and rapid tool for the detection of SM2 and OFL residues in duck meat.

Evaluation of surface plasmon field-enhanced fluorescence spectroscopy for rapid measurement of progesterone concentration in bitches.

OBJECTIVE: To compare progesterone (P4) concentrations measured with surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and chemiluminescence immunoassay (CLIA) in serum and plasma samples of client-owned bitches of various ages and breeds and to determine reference ranges for P4 concentrations at various stages of the estrous cycle. SAMPLES: 102 serum samples and 104 plasma samples. PROCEDURES: In experiment 1, 1 aliquot each of serum and plasma was analyzed for P4 concentration by use of SPFS incorporated in a veterinary-specific point-of-care immunologic analyzer and CLIA. In experiment 2, serum collected from bitches in various stages of the estrous cycle was analyzed for P4 concentration by use of SPFS to establish reference ranges for each stage. RESULTS: In experiment 1, P4 concentrations measured by SPFS and CLIA were highly correlated (serum, r = 0.966; plasma, r = 0.968). In experiment 2, ranges of serum basal (proestrous) P4 concentrations (n = 114) and P4 concentrations at the estimated time of ovulation (76), during pregnancy or diestrus (107), and during the prepartum period (50) measured with SPFS were 0.42 to 1.46 ng/mL, 3.69 to 7.85 ng/mL, 11.73 to 28.24 ng/mL, and 1.54 to 3.22 ng/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Because serum and plasma P4 concentrations measured with SPFS were highly correlated with those measured with CLIA and ranges of serum P4 concentrations measured with SPFS for each of phase of the estrous cycle were well-defined for the large sample size, veterinarians may be able to accurately use this veterinary-specific point-of-care immunologic analyzer with SPFS methodology to determine P4 concentrations of bitches in their daily practice.

Effect of pH on heat-induced interactions in high-protein milk dispersions and application of fluorescence spectroscopy in characterizing these changes.

This study investigated casein-whey protein interactions in high-protein milk dispersions (5% protein wt/wt) during heating at 90°C for 1.5 to 7.5 min at 3 different pH of 6.5, 6.8, and 7.0, using both conventional methods (gel electrophoresis, physicochemical properties) and fluorescence spectroscopy. Conventional methods confirmed the presence of milk protein aggregates during heating, similar to skim milk. These methods were able to help in understanding the denaturation and aggregation of milk proteins as a function of heat treatment. However, the results from the conventional methods were greatly affected by batch-to-batch variations and, therefore, differentiation could be drawn only in nonheated samples and samples heated for a longer duration. The front-face fluorescence spectroscopy was found to be a useful tool that provided additional information to conventional methods and helped in understanding differences between nonheated, low-, and high-heated samples, along with the type of sample used (derived from liquid or powder milk protein concentrates). At all pH values, tryptophan maxima in nonheated samples derived from powdered milk protein concentrates presented a blue shift in comparison to samples derived from liquid milk protein concentrates, and tryptophan maxima in heated samples presented a red shift. With the heating of the sample, Maillard emission and excitation spectra also showed increases in the peak intensities from 408 to 432 and 260 to 290 nm, respectively. As the level of denaturation increased with heating, a marked differentiation can be seen in the principal component analysis plots of tryptophan, Maillard emission, and excitation spectra, indicating that the front-face fluorescence technique has a potential to monitor and classify samples according to milk protein interactions as a function of pH and heat exposure. Overall, it can be said that the pattern of protein-protein interactions in high-protein dispersions was similar to the observation reported in skim milk systems, and fluorescence spectroscopy with chemometrics can be used as a rapid, nondestructive, and complementary method to conventional methods for following heat-induced changes.

A rapid method to quantify casein in fluid milk by front-face fluorescence spectroscopy combined with chemometrics.

Casein in fluid milk determines cheese yield and affects cheese quality. Traditional methods of measuring casein in milk involve lengthy sample preparations with labor-intensive nitrogen-based protein quantifications. The objective of this study was to quantify casein in fluid milk with different casein-to-crude-protein ratios using front-face fluorescence spectroscopy (FFFS) and chemometrics. We constructed calibration samples by mixing microfiltration and ultrafiltration retentate and permeate in different ratios to obtain different casein concentrations and casein-to-crude-protein ratios. We developed partial least squares regression and elastic net regression models for casein prediction in fluid milk using FFFS tryptophan emission spectra and reference casein contents. We used a set of 20 validation samples (including raw, skim, and ultrafiltered milk) to optimize and validate model performance. We externally tested another independent set of 20 test samples (including raw, skim, and ultrafiltered milk) by root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and relative prediction error (RPE). The RMSEP for casein content quantification in raw, skim, and ultrafiltered milk ranged from 0.12 to 0.13%, and the RPD ranged from 3.2 to 3.4. The externally validated error of prediction was comparable to the existing rapid method and showed practical model performance for quality-control purposes. This FFFS-based method can be implemented as a routine quality-control tool in the dairy industry, providing rapid quantification of casein content in fluid milk intended for cheese manufacturing.

A mechanism underlies fish GRP78 protection against Pb2+ toxicity.

Heavy metal exposure impacts basic cellular processes and results in serious toxicological effects. Pb2+ can activate the response to endoplasmic reticulum (ER) stress by protein denaturation, changing intracellular calcium homeostasis, and inducing cell death. As an ER retention protein, 78-kDa glucose-regulated protein (GRP78) can relieve the Pb2+-induced ER stress and enhance cell viability. We previously showed that heavy metal ions such as Pb2+ etc. are harmful to fish cell lines in a time- and dose-dependent manner. The phenomenon is accompanied by the increasing accumulation of grass carp GRP78 (CiGRP78), which can protect the cells from heavy metal ion cytotoxicity. Here, we investigated the mechanism in which CiGRP78 exerted its protective function. Using metal ions affinity elution method and fluorescent spectral analysis, we showed that CiGRP78 could respectively form a complex with Calcium, Lead and Cadmium ions, especially with Lead ion in vitro. However, another ER retention protein CiGRP94 could not bind to Pb2+, highlighting the functional differentiation might exist in CiGRP78 and CiGRP94 in regulating heavy metal cytotoxicity. Our results suggested that CiGRP78 might increase cellular tolerance to Pb2+ via the direct interaction with it.

Photodynamic detection of canine mammary gland tumours after oral administration of 5-aminolevulinic acid.

5-Aminolevulinic acid (5-ALA) is widely used in photodynamic detection (PDD) and therapy. We evaluated the pharmacokinetics of 5-ALA-induced porphyrins and its effectiveness in PDD in dogs with mammary gland tumours (MGTs) following oral administration. Healthy dogs and those with MGTs (nine each) were orally administered 4 mg kg-1 5-ALA. Protoporphyrin IX (PpIX) was not detected in the plasma of healthy dogs but it peaked in dogs with MGT at 2 h after 5-ALA administration. In the PDD study, 16 dogs with MGT were orally administered 40 mg kg-1 5-ALA, and MGT but not normal tissue showed red fluorescence after 2-4 h. Photon counts were 6635-63 890 and 59-4011 (median, 19 943 and 919) for MGT and non-tumour tissues, respectively. Cell density strongly correlated with PpIX photon counts of MGT tissue of the dogs (R = 0.743, P = 0.0009). We suggest that 5-ALA-PDD might be an effective diagnostic tool for MGTs.

Measuring embryo metabolism to predict embryo quality.

Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo's immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.

The use of native fluorescence analysis of synovial fluid in the diagnosis of medial compartment disease in medium- and large-breed dogs.

We assumed that proteins are most likely responsible for synovial fluid fluorescence and that changes detected in fluorescence intensity are most likely the result of changes in the concentration of fluorescent proteins. Synchronous fluorescent matrices from synovial fluid samples were measured in the excitation wavelength range of 200-350 nm using a luminescence spectrophotometer. The synchronous matrix of synovial fluid consists of 2 dominant fluorescent centers (F1 and F2) in the ultraviolet region. The fluorescence intensities of both centers were significantly higher in pathological samples, with p = 0.001 (a 59% increase of the median value) for the F1 center and p = 0.002 (a 52% increase of the median value) for the F2 center. Receiver operating characteristic analysis confirmed that synovial fluid autofluorescence is a significant predictor of medial compartment disease in dogs, with the area under the curve at 0.776 (F1) and 0.778 (F2). We did not detect any differences in the autofluorescence of synovial fluid between male and female, or any breed-based changes. No position changes of fluorescent centers were recorded in the synovial fluid in diseased dogs compared with healthy dogs. The synovial fluid metabolic fingerprint of canine patients with medial compartment disease differed from that of healthy dogs. Our study demonstrated the feasibility of synovial fluid fingerprinting to identify disease-specific profiles of synovial fluid metabolites.

Prediction of the concentration of standardized ileal digestible amino acids in distillers dried grains with solubles.

Values for the concentration of standardized ileal digestible (SID) CP, Lys, Met, Thr, and Trp from 34 sources of distillers dried grains with solubles (DDGS) were obtained from a series of 5 experiments with the objective of predicting the concentration of SID AA from physical and chemical assays. The concentration of NDF, ADF, hemicellulose, acid detergent insoluble CP (ADICP), and KOH soluble protein (SolCP) were measured and calculated in all DDGS sources. Likewise, particle size was measured and color of each source of DDGS was determined with a Minolta colorimeter and HunterLab spectrometer and was expressed as lightness (L*), redness (a*), and yellowness (b*). The HunterLab spectrometer also provided optical density that was recorded between 400 and 700 nm. Front face fluorescence was measured at 360 nm excitation and recorded from 380 to 600 nm. Multiple linear regression and principal components analyses were performed to predict the concentration of SID AA among DDGS sources, and predicted means as well as predicted residual sums of squares (PRESS) were calculated to estimate accuracy and precision of the model. Some correlations (P < 0.05) were observed between ADF, hemicellulose, ADICP, and SolCP with SID CP and AA but were generally low (r < 0.51). There was a greater association (R(2) = 0.40; P < 0.05) between L* and SID Lys among DDGS sources when L* was less than 50 than when samples had L* values greater than 50. In addition, a* was negatively correlated (P < 0.05) with SID CP (r = -0.41), Lys (r = -0.59), and Met (r = -0.50) whereas b* tended to be positively correlated (P < 0.10) with SID Lys (r = 0.31) and Trp (r = 0.30) and was correlated (P = 0.05) with SID Met (r = 0.43) and Thr (r = 0.36). There were no correlations between NDF or particle size with SID CP and AA. Optical density, along with CP, was highly predictive of SID Lys (R(2) = 0.97; PRESS = 0.05), Thr (R(2) = 0.94; PRESS = 0.06), and Trp (R(2) = 0.93; PRESS = 0.004) but not SID Met (R(2) = 0.39; PRESS = 0.12). Front face fluorescence was also highly predictive of SID Lys (R(2) = 0.99; PRESS = 0.07), Met (R(2) = 0.95; PRESS = 0.05), Thr (R(2) = 0.99; PRESS = 0.008), and Trp (R(2) = 0.99; PRESS = 0.006). In conclusion, correlations between ADICP, SolCP, NDF, particle size, and color measurements with SID AA concentrations were poor, but optical density and front face fluorescence methods appear to provide good predictions of SID AA concentrations in DDGS. However, these prediction equations need to be validated using samples of DDGS from a separate data set.

Oxidative mechanisms of fish hepatocyte toxicity by the harmful dinoflagellate Cochlodinium polykrikoides.

Harmful Algal Blooms caused by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish worldwide. In this research, we investigated the cytotoxic mechanisms of aqueous extract of C. polykrikoides on isolated Rainbow trout (Oncorhynchus mykiss) liver hepatocytes. Algal extract exposure with isolated trout hepatocytes caused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, lysosomal membrane rupture, collapse of mitochondrial membrane potential, ATP depletion and increase in ADP/ATP ratio, cytochrome C release into the hepatocyte cytosol, and activation of caspases cascade. Anti-oxidants, free radical scavengers, mitochondrial permeability transition (MPT) pore sealing agents, microsomal oxidases inhibitors, ATP generators and lysosomotropic agents protected fish hepatocytes against C. polykrikoides. Fish hepatocyte toxicity was also associated with mitochondrial and lysosomal membrane injury. These events caused cytochrome C release from the mitochondrial intra-membrane space into cytosol. The cytochrome C release could trigger activation of caspase-3 and apoptosis.

Validation of a spectrophotometer-based method for estimating daily sperm production and deferent duct transit.

The objectives of the present work were 3-fold. First, a new method for estimating daily sperm production was validated. This method, in turn, was used to evaluate testis output as well as deferent duct throughput. Next, this analytical approach was evaluated in 2 experiments. The first experiment compared left and right reproductive tracts within roosters. The second experiment compared reproductive tract throughput in roosters from low and high sperm mobility lines. Standard curves were constructed from which unknown concentrations of sperm cells and sperm nuclei could be predicted from observed absorbance. In each case, the independent variable was based upon hemacytometer counts, and absorbance was a linear function of concentration. Reproductive tracts were excised, semen recovered from each duct, and the extragonadal sperm reserve determined by multiplying volume by sperm cell concentration. Testicular sperm nuclei were procured by homogenization of a whole testis, overlaying a 20-mL volume of homogenate upon 15% (wt/vol) Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY), and then washing nuclei by centrifugation through the Accudenz layer. Daily sperm production was determined by dividing the predicted number of sperm nuclei within the homogenate by 4.5 d (i.e., the time sperm with elongated nuclei spend within the testis). Sperm transit through the deferent duct was estimated by dividing the extragonadal reserve by daily sperm production. Neither the efficiency of sperm production (sperm per gram of testicular parenchyma per day) nor deferent duct transit differed between left and right reproductive tracts (P > 0.05). Whereas efficiency of sperm production did not differ (P > 0.05) between low and high sperm mobility lines, deferent duct transit differed between lines (P < 0.001). On average, this process required 2.2 and 1.0 d for low and high lines, respectively. In summary, we developed and then tested a method for quantifying male reproductive tract throughput. This method makes the study of semen production amenable to systems biology.

New insights into the characterization of the binding of tetracycline analogues with lysozyme: a biophysical study.

Tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC) are the most common members of the widely used veterinary drug tetracyclines, the residue of which in the environment can enter human body, being potentially harmful. Lysozyme is a monomeric protein widely distributed in the nature including human beings, having many physiological and pharmaceutical functions. The aim of this study was to examine the interaction of lysozyme with the three tetracyclines (TC, CTC and OTC) through spectroscopic and molecular modeling methods. The experimental results revealed that all the three tetracyclines (TCs) can interact with lysozyme with one binding site to form TCs-lysozyme complex, mainly through electrostatic forces with the affinity order: CTC>TC>OTC. The binding of TCs can cause conformational and some microenvironmental changes of lysozyme. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that all the three TCs can bind into lysozyme cleft and interact with the key active-site residues Glu 35 or Asp 52, resulting in competitive inhibition of lysozyme activity. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TCs with lysozyme in vivo.

The two faces of DOC.

Dissolved organic carbon (DOC), through its ability to complex metals and thereby reduce their bioavailability, plays a major role in ameliorating metal toxicity in natural waters. Indeed DOC is a key variable in the Biotic Ligand Model (BLM) for predicting metal toxicity on a site-specific basis. However, recent evidence indicates that all DOCs are not alike, but rather heterogeneous in their ability to protect organisms against metal toxicity, at least in fresh water. The degree of protection appears to correlate with optical properties, such that dark, aromatic-rich compounds of allochthonous origin, with greater humic acid content, are more effective in this regard, particularly against Cu, Ag, and Pb toxicity. The specific absorption coefficient of the DOC in the 300-350nm range (SAC(300-350)) has proven to be a simple and effective index of this protective ability. PARAFAC, a multivariate statistical technique for analysis of excitation-emission fluorescence spectroscopy data, also holds promise for quantifying the humic-like and fulvic-like fluorophores, which tend to be positively and negatively correlated with protective ability, respectively. However, what has been largely missing in the toxicological realm is any appreciation that DOC may also affect the physiology of target organisms, such that part of the protection may occur by a mechanism other than metal complexation. Recently published evidence demonstrates that DOC has effects on Na(+) transport, diffusive permeability, and electrical properties of the gills in fish and crustaceans in a manner which will promote Na(+) homeostasis. These actions could thereby protect against metal toxicity by physiological mechanisms. Future research should investigate potential direct interactions of DOC molecules with the branchial epithelium. Incorporation of optical properties of DOC could be used to improve the predictive capabilities of the BLM.

Molecular and biochemical biomarkers in environmental monitoring: a comparison of biotransformation and antioxidant defense systems in multiple tissues.

The cytochrome P450 pathway and antioxidant responses are known for their responsiveness to environmental pollutants and are frequently used as biomarkers at the transcriptional, translational and catalytic levels. Although molecular responses are often assumed to reflect similar changes in enzyme function, several factors can influence intracellular effects, including mRNA stability and protein turnover, signal sensing and transduction, post-translational modifications of proteins, and multiple mode of action of chemicals in complex mixtures. The aim of this study was to use experimental data for a general discussion on the importance of mechanisms modulating transcriptional and catalytic responses of these pathways, and the resulting implications for environmental monitoring. The European eel Anguilla anguilla was selected as fish model to compare the effects of polluted sediments on gene expression and functional levels of cytochrome P450, glutathione S-transferases, UDP-glucoronosyl transferases, catalase, glutathione peroxidases, superoxide dismutase, glutathione, glutathione reductase, glucose 6-phosphate dehydrogenase and γ-glutamylcysteine ligase in the liver and gills. The overall results confirmed significant changes in gene transcription related to biotransformation and oxyradical metabolism, but also supported the evidence of a frequent dissociation between mRNA expression and protein activity. More similar trends of variations and exposure-dependent relationships was observed in the liver for transcriptional and catalytic responses of those pathways closely regulated by specific interactions between substrate, transcription factors, gene and metabolizing protein (i.e. phase I and phase II). On the other hand, the lower metabolism and the cellular machinery of gill cells may prevent elevated transcriptional responsiveness to be translated to an adequate functional response of a protein. Relationships between transcriptional and catalytic effects were often inconsistent for antioxidant responses confirming the complexity of interactions between exposure to chemical pollutants and regulation of oxidative stress responses. Oxidative stress responses may not necessarily be associated with transcriptional variations of genes, but rather with post-translational modifications of proteins. These mechanisms are just beginning to be revealed in marine organisms, but their characterization will be fundamental for better understanding of the implications of variations in gene expressions according to system, tissue, intensity and duration of exposure.

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes.

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Evaluation of tramadol and its main metabolites in horse plasma by high-performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques.

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O-Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the micro-receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high-performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA-ESI-MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O-desmethyltramadol (M5). LC/PDA-ESI-MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N-desmethyltramadol (M2), and N,N-didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI-MS/MS techniques in a search for tramadol metabolites in horse plasma samples.

An excitation wavelength-scanning spectral imaging system for preclinical imaging.

Small-animal fluorescence imaging is a rapidly growing field, driven by applications in cancer detection and pharmaceutical therapies. However, the practical use of this imaging technology is limited by image-quality issues related to autofluorescence background from animal tissues, as well as attenuation of the fluorescence signal due to scatter and absorption. To combat these problems, spectral imaging and analysis techniques are being employed to separate the fluorescence signal from background autofluorescence. To date, these technologies have focused on detecting the fluorescence emission spectrum at a fixed excitation wavelength. We present an alternative to this technique, an imaging spectrometer that detects the fluorescence excitation spectrum at a fixed emission wavelength. The advantages of this approach include increased available information for discrimination of fluorescent dyes, decreased optical radiation dose to the animal, and ability to scan a continuous wavelength range instead of discrete wavelength sampling. This excitation-scanning imager utilizes an acousto-optic tunable filter (AOTF), with supporting optics, to scan the excitation spectrum. Advanced image acquisition and analysis software has also been developed for classification and unmixing of the spectral image sets. Filtering has been implemented in a single-pass configuration with a bandwidth (full width at half maximum) of 16 nm at 550 nm central diffracted wavelength. We have characterized AOTF filtering over a wide range of incident light angles, much wider than has been previously reported in the literature, and we show how changes in incident light angle can be used to attenuate AOTF side lobes and alter bandwidth. A new parameter, in-band to out-of-band ratio, was defined to assess the quality of the filtered excitation light. Additional parameters were measured to allow objective characterization of the AOTF and the imager as a whole. This is necessary for comparing the excitation-scanning imager to other spectral and fluorescence imaging technologies. The effectiveness of the hyperspectral imager was tested by imaging and analysis of mice with injected fluorescent dyes. Finally, a discussion of the optimization of spectral fluorescence imagers is given, relating the effects of filter quality on fluorescence images collected and the analysis outcome.

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA).

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils.

Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.