An objective measure of reactive behaviour in horses

Several tests have been devised in an attempt to detect behaviour modification due to training, supplements or diet in horses. These tests rely on subjective observations in combination with physiological measures, such as heart rate (HR) and plasma cortisol concentrations, but these measures do not definitively identify behavioural changes. The aim of the present studies was to develop an objective and relevant measure of horse reactivity. In Study 1, HR responses to auditory stimuli, delivered over 6 days, designed to safely startle six geldings confined to individual stalls was studied to determine if peak HR, unconfounded by physical exertion, was a reliable measure of reactivity. Both mean (±SEM) resting HR (39.5 ± 1.9 bpm) and peak HR (82 ± 5.5 bpm) in response to being startled in all horses were found to be consistent over the 6 days. In Study 2, HR, plasma cortisol concentrations and speed of departure from an enclosure (reaction speed (RS)) in response to a single stimulus of six mares were measured when presented daily over 6 days. Peak HR response (133 ± 4 bpm) was consistent over days for all horses, but RS increased (3.02 ± 0.72 m/s on Day 1 increasing to 4.45 ± 0.53 m/s on Day 6; P = 0.005). There was no effect on plasma cortisol, so this variable was not studied further. In Study 3, using the six geldings from Study 1, the RS test was refined and a different startle stimulus was used each day. Again, there was no change in peak HR (97.2 ± 5.8 bpm) or RS (2.9 ± 0.2 m/s on Day 1 versus 3.0 ± 0.7 m/s on Day 6) over time. In the final study, mild sedation using acepromazine maleate (0.04 mg/kg BW i.v.) decreased peak HR in response to a startle stimulus when the horses (n = 8) were confined to a stall (P = 0.006), but not in an outdoor environment when the RS test was performed. However, RS was reduced by the mild sedation (P = 0.02). In conclusion, RS may be used as a practical and objective test to measure both reactivity and changes in reactivity in horses.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Metabolomic analysis characterizes tissue specific indomethacin-induced metabolic perturbations of rats

In this study, the promising metabolomic approach integrating with ingenuity pathway analysis (IPA) was applied to characterize the tissue specific metabolic perturbation of rats that was induced by indomethacin. The selective pattern recognition analyses were applied to analyze global metabolic profiling of urine of rats treated by indomethacin at an acute dosage of reference that has been proven to induce tissue disorders in rats, evaluated throughout the time-course of -24-72 h. The results preliminarily revealed that modifications of amino acid metabolism, fatty acid metabolism and energetically associated metabolic pathways accounted for metabolic perturbation of the rats that was induced by indomethacin. Furthermore, IPA was applied to deeply analyze the biomarkers and their relations with the metabolic perturbations evidenced by pattern recognition analyses. Specific biochemical functions affected by indomethacin suggested that there is an important correlation of its effects in kidney and liver metabolism, based on the determined metabolites and their pathway-based analysis. The IPA correlation of the three major biomarkers, identified as creatinine, prostaglandin E2 and guanosine, suggested that the administration of indomethacin induced certain levels of toxicity in the kidneys and liver. The changes in the levels of biomarker metabolites allowed the phenotypical determination of the metabolic perturbations induced by indomethacin in a time-dependent manner.

Development of an integrated metabolomic profiling approach for infectious diseases research

Metabolomic profiling offers direct insights into the chemical environment and metabolic pathway activities at sites of human disease. During infection, this environment may receive important contributions from both host and pathogen. Here we apply an untargeted metabolomics approach to identify compounds associated with an E. coli urinary tract infection population. Correlative and structural data from minimally processed samples were obtained using an optimized LC-MS platform capable of resolving ~2300 molecular features. Principal component analysis readily distinguished patient groups and multiple supervised chemometric analyses resolved robust metabolomic shifts between groups. These analyses revealed nine compounds whose provisional structures suggest candidate infection-associated endocrine, catabolic, and lipid pathways. Several of these metabolite signatures may derive from microbial processing of host metabolites. Overall, this study highlights the ability of metabolomic approaches to directly identify compounds encountered by, and produced from, bacterial pathogens within human hosts.