On-line coupling of size exclusion chromatography and capillary electrophoresis via solid-phase extraction and a Tee-split interface

An on-line size exclusion chromatography (SEC)-solid-phase extraction (SPE)-capillary electrophoresis (CE) system using a Tee-split interface has been developed for the analysis of peptides in biological fluids. The SEC column fractionates the sample by molecular size and the low-molecular-weight fraction, which contains the peptides, is directed to a C(18) SPE microcolumn, where the peptides are trapped and concentrated. The SPE column is desorbed with 425 nL acetonitrile and the effluent is sent to the Tee-split interface, which hydrodynamically splits (1:40) the flow and, thus, allows appropriate injection of analytes into the CE system. The performance of the system is investigated by the analysis of enkephalins in cerebrospinal fluid (CSF). It is demonstrated that the SEC step efficiently removes potentially interfering proteins, permitting reproducible SPE and CE. The total system provides efficient separations of the enkephalins with plate numbers up to 100,000. Concentration limits of detection (S/N = 3) for the peptides are about 100 ng/mL for injection of 20 microL spiked CSF samples. Plots of enkephalin peak areas versus concentration showed good linearity over the 0.25-10 microg/mL range (R2 > or = 0.985). Repeatability of migration time and peak area was within 2% and 10% R.S.D., respectively.     

Discovery of early-stage biomarkers for diabetic kidney disease using ms-based metabolomics (FinnDiane study)

Diabetic kidney disease (DKD) is a devastating complication that affects an estimated third of patients with type 1 diabetes mellitus (DM). There is no cure once the disease is diagnosed, but early treatment at a sub-clinical stage can prevent or at least halt the progression. DKD is clinically diagnosed as abnormally high urinary albumin excretion rate (AER). We hypothesize that subtle changes in the urine metabolome precede the clinically significant rise in AER. To test this, 52 type 1 diabetic patients were recruited by the FinnDiane study that had normal AER (normoalbuminuric). After an average of 5.5?years of follow-up half of the subjects (26) progressed from normal AER to microalbuminuria or DKD (macroalbuminuria), the other half remained normoalbuminuric. The objective of this study is to discover urinary biomarkers that differentiate the progressive form of albuminuria from non-progressive form of albuminuria in humans. Metabolite profiles of baseline 24?h urine samples were obtained by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) to detect potential early indicators of pathological changes. Multivariate logistic regression modeling of the metabolomics data resulted in a profile of metabolites that separated those patients that progressed from normoalbuminuric AER to microalbuminuric AER from those patients that maintained normoalbuminuric AER with an accuracy of 75% and a precision of 73%. As this data and samples are from an actual patient population and as such, gathered within a less controlled environment it is striking to see that within this profile a number of metabolites (identified as early indicators) have been associated with DKD already in literature, but also that new candidate biomarkers were found. The discriminating metabolites included acyl-carnitines, acyl-glycines and metabolites related to tryptophan metabolism. We found candidate biomarkers that were univariately significant different. This study demonstrates the potential of multivariate data analysis and metabolomics in the field of diabetic complications, and suggests several metabolic pathways relevant for further biological studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0291-6) contains supplementary material, which is available to authorized users.     

Effects of intima stiffness and plaque morphology on peak cap stress

Background  

Rupture of the cap of a vulnerable plaque present in a coronary vessel may cause myocardial infarction and death. Cap rupture occurs when the peak cap stress exceeds the cap strength. The mechanical stress within a cap depends on the plaque morphology and the material characteristics of the plaque components. A parametric study was conducted to assess the effect of intima stiffness and plaque morphology on peak cap stress.     

Simulation of stent deployment in a realistic human coronary artery

Background  

The process of restenosis after a stenting procedure is related to local biomechanical environment. Arterial wall stresses caused by the interaction of the stent with the vascular wall and possibly stress induced stent strut fracture are two important parameters. The knowledge of these parameters after stent deployment in a patient derived 3D reconstruction of a diseased coronary artery might give insights in the understanding of the process of restenosis.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Olfactory sensitivity in tsetse flies: a daily rhythm

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U- shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.      

On-line coupling of solid-phase extraction and capillary electrophoresis for the determination of cefoperazone and ceftiofur in plasma

We present a method for determining two cephalosporins (cefoperazone and ceftiofur) in plasma by on-line solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) with a T-split interface. Using this interface, a part of the SPE elution plug containing the cephalosporins is injected while the rest of the sample is flushed to waste. SPE was carried out using a C(18) micro-precolumn and the cephalosporins presented good retention properties with breakthrough volumes above 1 ml. Using a desorption volume of 426 nl of acetonitrile, recoveries were 75 and 90%, for cefoperazone and ceftiofur, respectively. The resulting elution volume was about 1.8 microl. A deproteinization step was included prior to SPE for the analysis of plasma samples with recoveries of 90 and 57% for cefoperazone and ceftiofur, respectively. With UV detection at 254 nm, linear relationships between the injected concentration and peak area was measured between 10 and 500 ng ml(-1) for standards, and 200 and 1500 ng ml(-1) for plasma samples. Intra-day (n=5) and inter-day (n=5) peak area repeatability were lower than 12% RSD. The detection limits obtained for spiked plasma (100 ng ml(-1) cefoperazone and ceftiofur) are sufficient for applying the method to pharmacokinetic studies.     

Biochemical pathways analysis of microarray results: regulation of myogenesis in pigs

Background  

Combining microarray results and biological pathway information will add insight into biological processes. Pathway information is widely available in databases through the internet.     

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout

We have investigated the morphological effects of a genetic locus, Pgm1- t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.      

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.